Advances in Applied Microbiology, Volume 12

ADVANCES IN

Applied Microbiology VOLUME 12

CONTRIBUTORS TO THIS VOLUME

M, C. Allwood MiloB Kulhanek P. Margalith T. L. Miller D. Perlman G. P. Peruzzotti A. D. Russell Y. Schwartz Anthony J. Sinskey R. Steel A. Taylor Thomas Kennedy Walker Daniel I. C.Wang

ADVANCES IN

Applied Micro biol ogy Edited by D. Perlman School

of P h a r m a c y

The University of Wisconsin Madison, Wisconsin

VOLUME 12

@

1970

ACADEMIC PRESS, N e w York a n d London

COPYRIGHT

8 1970, BY ACADEMIC PRESS, INC.

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LIST OF C O N T R I B U T O R S

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

M. C. ALLWOOD,' Department of Pharmaceutics, Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardif, Great Britain (89) MILOS KULHANEK,Research Institute for Pharmacy and Biochemistry, Prague, Czechoslovakia (11) P. MARGALITH,Department of Food and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel (35)

T. L. MLLLER,The Upjohn Company, Kalamazoo, Michigan (153) D. PERLMAN,School of Pharmacy, The University of Wisconsin, Madison, Wisconsin (277) G. P. PERUZZOTTI,School of Pharmacy, The University of Wisconsin, Madison, Wisconsin (277) A. D. RUSSELL,Department of Pharmaceutics, Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cardiff, Great Britain (89) Y. SCHWARTZ,Department of Food and Biotechnology, Technion - Israel Institute of Technology, Haifa, Israel (35) ANTHONY J. SINSKEY,Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (121) R. STEEL,The Upjohn Company, Kalamazoo, Michigan (153) A. TAYLOR, Atlantic Regional Laboratory, National Research Council of Canada, Halifax, Nova Scotia (189)

THOMASKENNEDY WALKER,Emeritus Professor of The University of Manchester Institute of Science and Technology, Manchester, England (1) DANIELI. C. WANG, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (121)

'Present address: Department of Pharmacy, The University, Nottingham, Great Britain. V

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PREFACE The breadth of topics discussed in this volume is as broad as microbiology itself- manufacture of ascorbic acid using microbial transformations; flavoring materials produced in fermented foods; bioengineering techniques useful in large-scale production and recovery of microbial cells and metabolites; toxic metabolites (including antibiotics) from microbial species; and microbial metabolites as pharmacologically active agents are all frontiers of microbiology currently receiving attention. Some of them are “new,” others are as old as the discipline itself but are being approached from new angles. In keeping with the practice started in Volume 11,we have included a historical essay, and were fortunate to have Professor T. K. Walker’s summary of the philosophy and achievements of the Manchester School which he headed for many decades when the interest in applied microbiology shifted from the alcohol-producing fermentations to the use of microbial systems for the production of special metabolites and for transformation of organic substances. Much can be learned by study of the research patterns in certain areas of applied microbiology. We plan to include historical essays in our future volumes so that those who are now responsible for planning and organizing research programs can better understand how those who preceded us solved some of the problems similar to those w e now face.

D. PEJXMAN Madison, Wisconsin March, 1970

vii

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CONTENTS

LIST OF CONTRIBUTORS.............................................................................. PREFACE................................................................................................... CONTENTS OF PREVIOUS VOLUMES..............................................................

V

vii xiii

History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester THOMASKENNEDY WALKER. Text................................................................................................

1

Fermentation Processes Employed in Vitamin C Synthesis MILO$ KULHANEK I. Reichstein’s Synthesis ....... 11. Newer Processes for Vitamin C Preparation ........................................ 111. Conclusion ...................................................................................... References ......................................................................................

11 17 28 28

Flavor a n d Microorganisms P. MARGALITH AND Y. SCHWARTZ

I. General Introduction .. ...... ..... ... 11. The Contribution of Microorganisms to the Production and Development of Flavor in Traditional Foods ....................................... 111. Concluding Remarks ........................................................................ References ...........

36 40 74

83

Mechanisms of Thermal Injury in Nonsporulating Bacteria M. C. ALLWOOD AND A. D. RUSSELL I. Introduction

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

11. Original Approaches to the Problem ... ............ 111. Thermosensitivity of Various Bacteria.. ...............................................

IV. The Causes of Thermal Inactivation of Vegetative Bacteria by Moist Heat ............ ..................................................... V. Repair of Thermal Injury .................................................................. VI. Conclusions ..................................................................................... VII. Summary.... References

ix

...

89 90 93

112 114 115 116

X

CONTENTS

Collection of Microbial Cells DANIEL I .

I. I1. I11. IV V. VI . VII .

.

c. WANG AND

ANTHONY J . SINSKEY

Introduction ............................................................................. Centrifugation .................................................................................. Filtration ......................................................................................... Flocculation ............................................................................. Foam Fractionation .......................................................................... Miscellaneous Recovery Systems ....................................................... Summary ................................ .................................... References ..........................

121 122 132 141 143 146 150 150

Fermentor Design

.

R . STEEL AND T L . MILLER I

.

Introduction

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

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

I1. Requirements ..................................................................................

................................................ .................................................... ................................................ . Agitator Shaft Seals ......................................... Aseptic Operations .......... Air Filtration ...................................................................... Mechanical Defoamers ..................................................... X . Antifoam or Nutrient Addition ........................ XI . Insfmmentation ............................................................................... XI1. Continuous Fennentors .... References ...................................................................................... Appendix: Addresses of Equipment Suppliers .....................................

111. IV. V VI . VII . VIII . 1x.

Fennentor Geome

153 154 155 157 157 159 161 164 169 171 172 178 184 187

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

I. I1. I11. IV V

. .

Introduction ........... ................................................................. Production of Peptide-Lactones ..................................... ............ Chemistry of Peptide-Lactones .......................................................... Toxicology of Peptide-Lactones ......................................................... Conclusions ....................................................... ......... .... References ......................................................................................

189 190 192 239 263 263

Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. PERLMAN AND G . P . PERUZZOTTI I. Introduction .................................................................................... I1 Types of Pharmacological Activity Reported .......................................

.

277 278

CONTENTS

111. Summary .........................................................................................

xi

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

288 288

AUTHOR INDEX.......................................................................................... SUBJECT INDEX .........................................................................................

295 319

References

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CONTENTS OF PREVIOUS VOLUMES A Commentary on Microbiological As-

Volume 1

saying

Protected Fermentation

F. Kavanagh

Milo; Herold and Jan NeEasek

Application of Membrane Filters

The Mechanism of Penicillin Biosynthesis Arnold L. Demain

Richard Ehrlich Microbial Control Brewery

Preservation of Foods and Drugs by Ionizing Radiations

Rudolph J . Allgeier and Frank M . Hildebrandt

David Pramer Microbial Synthesis of Cobamides

The Microbiological Transformation of Steroids

D. Perlman Factors Affecting the Antimicrobial Activity of Phenols E. 0.Bennett Germfree Animal Techniques and Their Applications Arthur W. Phillips and James E. Smith Insect Microbiology S . R. Dutky

T. H. Stoudt Biological Transformation o f Solar Energy William J. Oswald and Clarence G.

Golueke SYMPOSIUM ON ENGINEERING ADVANCES IN FERMENTATION PRACTICE Rheological Properties of Fermentation Broths

Fred H . Deindoerfer and John M . West

The Production of Amino Acids b y Fermentation Processes

Fluid Mixing in Fermentation Processes

Shukuo Kinoshita

J . Y . Oldshue

Continuous Industrial Fermentations Philip Gerhardt and M . C . Bartlett

Scale-up of Submerged Fermentations

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

Volume 2

W .H . Bartholemew Air Sterilization

Arthur E. Humphrey Sterilization of Media for Biochemical Processes

Lloyd L. Kempe

Newer Aspects of Waste Treatment

Nandor Porges Aerosol Samplers

the

Newer Development in Vinegar Manufactures

The State of Antibiotics in Plant Disease Control

AUTHOR INDEX- SUBJECT INDEX

in

Gerhard J . Hass

W . Dexter Bellamy

Davidson

Methods

Fermentation Kinetics and Model Processes

Fred H. Deindoerfer

Harold W . Batchelor

xiii

xiv

CONTENTS OF PREVIOUS VOLUMES

Continuous Fermentation W. D. Maxon Control Applications in Fermentation George J . Fuld AUTHOR INDEX

- SUBJECT INDEX

Volume 3

Preservation of Bacteria by Lyophilization RobertJ.Heckly

Sphaerotilus, Its Nature and Economic Significance Norman C . Dondero Large-Scale Use of Animal Cell Cultures Donuld j . Merchant and C . Richard Eidam Protection Against Infection in the Microbiological Laboratory: Devices and Procedures MarkA. Chatigny Oxidation of Aromatic Compounds by Bacteria Martin H . Rogof

AUTHOR INDEX

- SUBJECT INDEX

Volume 4

Induced Mutagenesis in the Selection of Microorganisms S. 1. Alikhanian The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . J . Babel Applied Microbiology in Animal Nutrition Harlow H . Hall Biological Aspects of Continuous Cultivation of Microorganisms T. Holme Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C . Morris Submerged Growth of Plant Cells L. G. Nickell AUTHOR INDEX-SUBJECT

INDEX

Volume 5

Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, jr., and Robert F . Pittillo

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrien Albert

The Classification of Actinomycetes in Relation to Their Antibiotic Activity Ello Baldacci

Generation of Electricity by Microbial Action J . B. Davis

The Metabolism of Cardiac Lactones by

Microorganisms and Biology of Cancer G . F. Gause

Microorganisms Elwood Titus

the

Molecular

Intermediary Metabolism and Antibiotic Synthesis J . D. Bu’Lock

Rapid Microbiological with Radioisotopes Gilbert V. Levin

Determinations

Methods for the Determination of Organic Acids A. C . H d m e

The Present Status of the 2,bButylene Glycol Fermentation Sterling K. Long and Roger Patrick

CONTENTS OF PREVIOUS VOLUMES

Aeration in the Laboratory W . R. Lockhart and R. W . Squires

xv

AUTHOR INDEX- SUBJECT INDEX Volume 7

Stability and Degeneration of Microbial Cultures on Repeated Transfer Fritz Reusser Microbiology of Paint Films Richard T. Ross The Actinomycetes and Their Antibiotics Selman A. Waksman Fuse1 Oil A. Dinsmoor Webb and John L. lngraham AUTHOR INDEX-SUBJECT

INDEX

Volume 6

Global Impacts of Applied Microbiology: An Appraisal Carl-Goran Heden and Mortimer P. Starr

Microbial Carotenogenesis Alex Ciegler Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander Cold Sterlization Techniques John B. Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes D. Perlman Development of Coding Schemes for Microbial Taxonomy S . T. Cowan Effects of Microbes on Germfree Animals Thomas D. Luckey

Microbial Processes for Preparation of Radioactive Compounds D. Perlman, Aris P. Bayan, and Nancy A. Giuffre

Uses and Products of Yeasts and Yeastlike Fungi Walter J . Nickerson and Robert G. Brown

Secondary Factors in Fermentation Processes P. Margalith

Microbial Amylases Walter W. Windish and Nagesh S . Mhatre

Nonmedical Uses of Antibiotics Herbert S . Goldberg

The Microbiology of Freeze-Dried Foods Gerald J . Silverman and Samuel A. Goldblith

Microbial Aspects of Water Pollution Control K. Wuhrmann Microbial Formation and Degradation of Minerals Melvin P. Silverman and Henry L. Ehrlich Enzymes and Their Applications Irwin W . Sizer

A Discussion of the Training of Applied Microbiologists B. W . Koft and Wayne W . Umbreit

Low-Temperature Microbiology judith Farrell and A. H . Rose AUTHOR INDEX- SUBJECT INDEX Volume 8

Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain Genetics in Applied Microbiology S . G. Bradley

xvi

CONTENTS OF PREVIOUS VOLUMES

Microbial Ecology and Applied Microbiology Thomus D. Brock The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondoinestic Water Supplies Cecil W . Chniribers and Norman A . Clorke The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kolliris Oral Microbiology Heiner Hofrtiuri

Microbiological Aspects of the Formation and Uegmdation of Cellulosic Fibers L. JuraSek, 1. Ross Colvin, and D. R. Whitaker The

Biotransformation of Lignin to Hiiiiius-Facts and Postulates R. T . Oglesby, R. F . Christmuia, and C . H. Driver

Bulking of Activated Sludge Wesley 0. Pipes Malo-lactic Fermentation Ralph E. Kurikee AUTI IOW INI>l$X- SUBJECT INDEX

Media and Methods for Isolation and Enumeration of the Enterococci Puul A. Hartniun, George W . Reinbold, und Devi S . Sarusiuut Crystal-Forming Bacteria Pathogens Martin H . Rogoff

Cellulose and Cellnlolysis Rrigitta Norkruns

as

Insect

Mycotoxins in Feeds arid Foods Emunuel Borker, Nirio F. Insalata, Colette P . Levi, and John S . Witzemail AUTHOR INDEX- SUBJECT INDEX

Volume 10

Detection of Life in Soil o n Earth and Other Planets. Introductory Remarks Robert L. Sturkey For What Shall Wc Search? A l l m H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets G. Stotzky

Volume 9

Experiincnts and Irlstrumentatiou Extraterrestrial Life Detection Gilbert V. Levin

The Inclusion of Antiinicrolhl Agents in Pharmaceutical Products A. D. Russell, June Jenkins, and I . H . Harrison

Halophilic Bacteria D. J . Kushizer

Antiserum Production in Experimental Animals Richard M . Hyde Microbial Models of Tumor Metabolism G. F. Guuse

for

Applied Significance of Polyvalent Bacteriophages s. G. Bradley Proteins and Enzymes as Taxonomic Tools Edward D.Gurber und John W . Rippuri

xvii

CONTENTS OF PREVIOUS VOLUMES

Micotoxins Alex Ciegler uiad Eiwind R. Lillelaoj Transformation of Organic Compounds by Fungal Spores Claude Vezinu, S . N . Sehgal, and Kortar Singh Microbial Interactions in Continuous Culture Henry R . Bungay, 111 u n d Mary Lou Bungay Chemical Sterilizers (Chemosterilizers) Paul M . Borick Antibiotics in the Control of Plant Pathogens M . J. Thirumalachur AUTIIOR INDEX-SUBJECT

INDEX

Structure-Activity Relationships of Semisynthetic Penicillins K . E . Price Resistance to Antimicrobial Agents J. S . Kiser, G. 0. Gale, and G. A. K e m p

Micromonospora Taxonomy George L u e d e m a n n Dental Caries and Periodontal Disease Considered as Infectious Diseases W i l l i a m Gold

The Recovery and Purification of Biochemicals Victor H . E d w a r d s Ergot Alkaloid Fermentations W i l l i a m J. Kelleher

CUMULATIVE AUTHOR INDEX-CUMULA- The Microbiology of the Hen’s Egg TIVE TITLEINVEX R. G. Board Volume 11

Successes and Failures in the Search for Antibiotics Selman A. W a k s m a n

Training for the Biochemical Industries I . L. Hepner AUTHOR INDEX-SUBJECT

INDEX

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

THOMASKENNEDY WALKER]

I am compelled to introduce a bold personal note into this account of the establishment of a department for the teaching and practice of biochemistry because it was my privilege not only to initiate the activities but also to plan and direct them over a period of 34 years ( 1925- 1959). At the time of my appointment as Lecturer in fermentation processes in the Faculty of Technology of the University of Manchester,2 early in 1925, I had already been engaged for 10 years in postgraduate research work. This period had been spent as follows: (1) I worked for 2 years as one of the assistants to Dr. Chaim Weizmann (who later became the first President of Israel) on pilot-plant trials and afterward in running, on a technical scale, his acetonebutanol fermentation process. This work was carried out in distilleries in London and Greenock and also at the Royal Naval Cordite Factory, Poole, Dorset. While in London I worked for a time at the Lister Institute where, with other chemists, I attended a course of instruction in general bacteriology, to prepare for future work with the acetone-butanol organism. (2) At the Research Department of The Royal Arsenal, Woolwich, for a 2-year service, I assisted in the elaboration of various new processes from the laboratory scale to that of pilot-plant working. (3) Three years of research in pure organic chemistry under the direction of Professor Arthur Lapworth, F.R.S. was spent in the Chemistry Department of the University of Manchester. I was awarded Ph.D. at the end of 1921. (4)From early 1922 until appointed Lecturer in 1925, I acted as private assistant to Professor F. L. Pyman, F.R.S., in the Faculty of Technology of Manchester University. Together we studied the bacteriostatic and bitter-flavored substances present in the resin of 'Emeritus Professor of The University of Manchester Institute of Science and Technology, Manchester, England. 2The former Faculty of Technology of the University of Manchester, which was housed in The Manchester College of Technology, has expanded greatly in recent years and is now designated The University of Manchester Institute of Science and Technology.

1

2

T. K. WALKEH

hop-cones. The investigation was sponsored by the Research Fund Committee of the Institute of Brewing. Upon becoming a Lecturer I was able to continue my interest in the hop investigation. Professor Pyman appointed Mr. J. J. H. Hastings to work with me on this particular project. When Pyman left the University in 1927 the conduct of the hop research was entrusted to me which I continued to direct for the Institute of Brewing until 1948. After planning and executing a considerable program of highly original work, Mr. Hastings left in 1932 to take up a post on acetonebutanol production with Commercial Solvents Inc.; thereafter, Dr. Alan Parker was m y colleague in the hop inquiries until 1939. In 1925 there was already included in the syllabus of Pyman’s Department of Applied Chemistry a course of studies in the science of brewing, and students reading for the degree of B.Sc. Tech. in Chemistry could, at that time, offer Brewing as a special subject in the final examination for this degree. Four laboratories were at my disposal in 1925. They consisted of (a) a general teaching laboratory, about 40 x 30 feet in area; ( b ) a research laboratory capable of accommodating three workers; (c) a laboratory for equipment in which were housed 2 polarimeters, an incubator, an electric-oven for dry sterilization, 4 balances, and 6 microscopes; and (d) a large room about 40 x 40 feet in which there was a model brewery containing a copper plant capable of dealing with 100 gallons of liquid. These instruments and plant, together with a nonpressure steam sterilizer and the usual laboratory glassware, constituted the only equipment available to me for carrying out teaching and research. Fortunately, at this time, the Institute of Brewing allocated to me a grant of R5OO which enabled the purchasc of two modern electrically heated incubators, a modern centrifuge, a gasheated pressure sterilizer, and apparatus for determination of pH values. After buying this equipment there were sufficient fiinds left to pay for an enamel-lined iron experimental fermentation vessel of 50-gallon capacity. This was made to my design and in it mashes could be sterilized at pressures up to 40 p.s.i. if necessary. There was always available an ample supply of chemicals, special reagents, and solvents, but funds for the purchase of the more expensive types of equipment were lamentably short during the next 20 years. It would not be untrue to say that my financial allocation for this purpose seldom amounted to more than &?,lo0in any one fiscal year. I am sure it will be understood that this set-up which, by modern standards, was very limited circumscribed to no little extent the field

DEVELOPMENT OF A SCHOOL OF BIOCHEMISTRY

3

in which biochemical researches could effectively be undertaken. Nevertheless, 40 years ago at a time when microanalysis, ultraviolet and infrared spectroscopy, Craig countercurrent separation, chromatography, ionophoretograms, and X-ray diffraction diagrams had not yet been evolved as means for the rapid identification of metabolites, there was, in fact, a wide area remaining for the study of certain features of the metabolism of bacteria and mold fungi by methods which, for their performance, demanded nothing more complicated than the use of bacteriological glassware and ordinary laboratory apparatus, including numerous flasks, conical and otherwise. At any rate, so far as I personally was concerned, such items had perforce to be my principal working tools until the period which followed World War 11. At about 1925 the term “chemical microbiology” had not come into general use, if, in fact, it had been coined at all. One of my difficulties in those early days was to make contact with a sufficient number of workers interested in chemical processes, brought about b y the agencies of nonpathogenic microorganisms, so that some exchange of ideas of mutual interest could take place. There was plenty of bacteriological work in progress in the medical schools of the universities and in the laboratories of hospitals, but naturally it was of a specialized nature and related to problems in medicine. Outside the great school of biochemistry built up by Sir F. Gowland Hopkins at Cambridge it was only in the laboratories of Sir Arthur Harden at the Lister Institute, in those of the Department of Brewing at the University of Birmingham, in the laboratories at the Rothamsted Experimental Station, and in one or two other institutions such as that at Reading where research in agricultural science was in progress, that biochemists could be found who were able to give informed opinions on questions relating to metabolic processes in nonpathogenic bacteria and mold fungi. Another factor which, in some cases, actually interfered with the progress of chemical microbiology in the United Kingdom in the earlier years of this century, was the assumption on the part of certain leading medical bacteriologists that mere chemists who had not been through a course in medical bacteriology were not qualified to work with microorganisms. I can recall one case in Manchester where this attitude hindered the commencement of an investigation by a biochemist who had not a medical degree. His study carried no medical interest and did not require the use of pathogenic organisms. I might add, the late Professor A. J. Kluyver of Delft, who was for many years

4

T. K. WALKER

one of Europe’s leading authorities on the metabolism of yeasts, molds, and bacteria, once told me in conversation in 1931 that this attitude on the part of some medical bacteriologists could be found also in Holland. It will be recalled that an outstanding case of this kind was the adverse reaction of French medical authorities of an earlier period to the pioneering experiments of the great Pasteur. For my part, I am pleased to state that I did not meet with this sort of thing in the course of my early work at Manchester for I received every encouragement and help from Professor H. S. Raper, at that time Dean of the Medical School, and from Professor H. B. Maitland, Head of the Public Health Department in the University. When first I received instruction and training in general bacteriology there was not a very large range of textbooks available and I had to rely principally on two British standard works for medical students, one by Muir and Ritchie, the other by Hewlett, Percival’s “Agricultural Bacteriology,” several American textbooks similar to that of Yercival, Henneberg’s “Handbuch der Garungsbakteriologie,” Neumann and Lehmann’s work in German on bacterial classification, and an excellent textbook on biological chemistry and microorganisms by the veteran French biochemist, G. Bertrand. In order to obtain the fullest information regarding developments in my particular field I made it my business to attend numerous meetings and symposia dealing with biochemistry, in general, and microbiology, in particular, both in the United Kingdom and on the Continent of Europe. I paid the expenses of these travels out of my own bank account; the University had not yet begun to finance such expeditions by members of its staff. I established contact with Neuberg and Nord in Berlin, Fernbach and Schoen in Paris, Chrzaszcz in Poznan, Kluyver in Delft, and Bernhauer in Prague. I visited the laboratories of all these men with the exception of that of Fernbach. With all of them I had very useful conversations and received much helpful advice, freely given. To F. F. Nord, in particular, I became indebted for help and encouragement received at those early meetings, and I have continued to enjoy his friendship for the past 40 years. I visited Cambridge often and there again I was given much valuable information, particularly by Dr. Majory Stephenson, Dr. Malcolm Dixon, and Dr. J. H. Quastel, all of whom worked in the Department of Professor F. Gowland Hopkins. I also paid occasional visits to the Lister Institute where I was afforded the privilege of seeing something of the work being carried out by Sir Arthur Harden and Professor R. Robison.

DEVELOPMENT OF A SCHOOL OF BIOCHEMISTRY

5

At that time I studied the origins of bacteriology and of biochemistry and became acquainted with the work of the old masters, notably van Leeuwenhoek, Lavoisier, Berzelius, Liebeg, Pasteur, HoppeSeyler, Moritz Traube, Winogradsky, and Beijerinck. I believe that in teaching biochemistry, the processes of respiration should be approached, in lectures, by first giving students an outline of the work and views of these men. Discussion of the Liebeg-Pasteur controversy and an account of the viewpoints of Hoppe-Seyler and Moritz Traube on oxidation and reduction in living cells should certainly precede description of the early work on phosphate ester formation in sugars. The experiments of Buchner, Harden and Young, Neuberg, and Meyerhof could be described before relating how Warburg and Wieland clashed in their opinions concerning the precise mechanisms by which oxidation proceeds in living matter and how Kluyver and later Szent-Gyorgyi, showed the manner in which some of these apparently opposing points of view could be reconciled. Finally, descriptions of the later work of Meyerhof, the work of Nord, of Szent-Gyorgyi, and of Krebs can be given in order to show to students the essentials of our present-day views on respiration and its concomitant involvements. In my opinion, a program of lectures in which the above-mentioned topics are dealt with in the chronological order I have indicated, provides a student with a thorough background from which he can then draw when faced with problems in fermentation processes. The research topics other than the hop investigations, which we started on metabolic processes in mold fungi arose out of discussions which I had in 1926 with my first Ph.D. student, Vira Subramaniam from the Indian Institute of Science, Bangalore, who told me of his interest in the question as to how organic acids are formed in green plants, for example, tartaric acid in tamarinds. As it happened, one of my friends, Professor F. Challenger, who was at that time Senior Lecturer in organic chemistry in Manchester University, was also interested in this topic, particularly in the mode of synthesis of citric acid in Aspergillus niger. So, for a period of about 3 years we collaborated in work on the citric acid problem and certain related matters. It proved in every way an enjoyable and rewarding partnership and led to six joint communications. The work with humulone and lupulone from hop resin drew our attention to the bacteriostatic activity associated with the presence of the 1,3-diketo system in some classes of organic compounds. This led us to study the effects consequent upon attaching this system to the nucleus in certain phenols.

6

T. K. WALKER

The work for the Institute of Brewing provided opportunities to visit breweries in different parts of the country and these visits revealed the nature of some of the problems which have to be faced from time to time by those engaged in brewery operations. On many occasions I was consulted about infections which had occurred in yeast and in beer. Arising out of these spoilage problems grew my interest in the bacteriology and biochemistry of Candida species, of Acetohacter species, and of Lactobacillus species and, for many years, work with strains of these organisms occupied some of my time, particularly, from 1940 onward. (Publ. Nos. 65-109 inclusive.) Early in the course of World War I1 it became a matter of great urgency to set up the production of penicillin in the United Kingdom on a scale to meet the rapidly increasing demands of the medical services. This called for mycologists who were also experienced organic chemists; the number of them was woehlly small. During the War ten such scientists who worked on the technical production of penicillin were men who had received their mycological training in our department. The most senior of these were J. J. H. Hastings, who at that time was employed with The Distillers’ Company, and A. Parker and C. R. Bond, who were with Imperial Chemical Industries. After the termination of World War I1 the acquisition of funds eased and much new equipment was obtainable. The number of students increased and, whereas, before the war I had only the services of one demonstrator, I was now able to secure the help of lecturers. At this stage, Dr. (now Professor) U. J. D. Hockenhull joined me and, for a time, before leaving to take up an appointment with Glaxo Laboratories, collaborated with me to extend the scope of the lecturing syllabus. Later, Dr. A. N. Hall afforded an immense amount of help both in lecturing and in the direction of research. He continued in collaboration with me until 1957 when he left Manchester to work with the late Dr. Jackson Foster at Austin, Texas. In 1948, I was requested by Sir Charles Harington, F.R.S., of the Medical Research Council, to undertake on behalf of its Committee on Chemical Microbiology an investigation into the question of producing edible fats by the agency of mold fungi. At that time there was somewhat of a world shortage of fats, and the Committee was anxious to explore the possibilities of obtaining additional supplies of edible fats, should there ever again be the danger of an emergency such as that caused by enemy submarine activity. In this undertaking I was fortunate to have the cooperation of Dr.

DEVELOPMENT OF A SCHOOL OF BIOCHEMISTRY

7

Malcolm Woodbine, who was seconded from the Ministry of Food and came to Manchester. This program, carried out under the aegis of the Medical Research Council, forms the subject matter of communications numbered 42-64, inclusive. Dr. Jose Garrido, a senior member of the staff of the University of Madrid, worked with us on the mycological fat question for more than 2 years, and other senior workers who participated in these studies were Dr. A. M. Gad and Dr. K. Naguib from Cairo University and Dr. J. Singh from Panjab University, Hoshiapur, India. At an early stage in this work Dr. Woodbine and I established that a number of mold species, other than those studied for fat-forming capacity b y earlier workers, were promising agents for our purpose and, ultimately, some high yields of fat were obtained under defined conditions of cultivation. Some of these specimens of fat were examined by Dr. T. Moore and his colleagues at Cambridge University and were found to be free from toxic substances when fed to rats and to dogs. Several of the specimens of mold fat were shown also to be high in content of linoleic acid, a point distinctly in their favor as possible edible substances. In the years 1925 to 1958, inclusive, 70 research workers studied in our laboratories. Of this number 33 obtained the postgraduate degree of M.Sc.Tech., while 32 were awarded Ph.D. Of this group of 70 workers 28 were either foreign students or came from British Dominions. The inquiries carried out by workers in the department over the period under review, have formed the subject matter of about 190 publications in scientific journals of repute. These activities were recognized by the University of Manchester. On the initiative of Dr. B. V. Bowden (now Lord Bowden) supported by Professor H. N. Rydon, head of the Department of Applied Chemistry, the Senate in 1954 set in motion steps to create a Chair of Industrial Biochemistry. At that time I had the title of Reader in Fermentation Processes and the University conferred on me the honor of the appointment of the first holder of the new Chair. By 1950 the threats of Joseph Stalin had really awakened Britain to the menace inherent in the imposing technological developments proceeding in Russia, and University extensions were actively pushed forward throughout the entire United Kingdom. As a result, I was given a whole floor in a separate building at Manchester. This quadrupled my former floor space. Augmented Treasury grants to the Universities began about this time and in 1956 the sum of g19,OOO was

8

T. K. WALKER

allocated to me for equipment for my new premises. Consequently, I was able on retirement at the end of 1958 to leave a department fully equipped in every respect for the prosecution both of teaching and research in microbiology. I had at that time a staff of three lecturers and several demonstrators and technicians. In this account of developments at Manchester I have dealt with some of the early difficulties caused by lack of laboratory accommodation and funds. Perhaps I may now review some abstract matters such as changes in my thinking on programs as these progressed. On looking back on past inquiries most of us can recall some cases of experiments based on a train of reasoning or on an assumption which, in the light of knowledge acquired later, can be seen to have been faulty in some respect. Reviewing some of the things I did as a beginner very many years ago, I realize now that my training initially as an organic chemist had left with me a tendency to assume, somewhat too readily, that mechanisms by which certain substances can be produced by chemical reaction under mild conditions in uitro, might also b e those by which these substances are formed in living cells. Of course, I was not alone in this respect, others working on mold metabolism 40 years ago speculated in this manner at one time or another. However, I did come to realize the danger of carrying such comparisons too far and, to guard against this in the case of beginners in research, I made arrangements whereby those working in our laboratories could attend lectures in the Botany Department of the University and also in physiological chemistry in the University Medical School. I found also that reading the work of Pasteur and particularly his publications relating to his controversy with Liebeg, proved a healthy corrective to a tendency to adopt in chemical microbiology a too purely mechanistic outlook. In the Pasteur-Liebeg controversy, echoes of the teachings of Descartes, on the one hand, and of the Vitalists, on the other, are clearly to be detected. Indeed, as late as the beginning of this century what perhaps might be designated as a neovitalistic viewpoint was held by Professor Benjamin Moore of Liverpool University, who believed in what he termed “Biotic Energy”; a form of energy which he conceived to be in operation only in living cells. Now I would like to remark on the relation of fundamental work in chemical microbiology to the application of microorganisms in industry. Few programs of scientific work, certainly not those in microbiology, can be pursued profitably beyond a certain point by a sci-

DEVELOPMENT OF A SCHOOL OF BIOCHEMISTRY

9

entist dwelling mentally in an ivory tower of his own construction. Sooner or later his stream of ideas will dry up or end in a stagnant morass unless recharged from outside sources. This has been selfevident from the first among those practicing such disciplines as metallurgy and the various branches of engineering, but in my own time I have known not a few chemists and a good sprinkling of physicists also, who were pure chemists and pure physicists to a degree which made them recoil instinctively at the very mention of the word “applied.” So far as bacteriologists and mycologists are concerned, in recent years the increasing interest in scientific control in agriculture and the need in medicine for supplies of antibiotics in considerable quantities have proved potent factors in bringing about the construction of strong bridges between research laboratories and the sites of large-scale operations elsewhere. Such a bridge provides for two-way traffic as I have found in several instances. For example, I have already stated elsewhere how studies which we made of the processes going on in technical vinegar acetifiers brought to light Acetobacter spp. revealing unusual features. One of these proved to be a cellulose-producer which permitted studies of the process of cellulose formation from a relatively large number of substrates. This same organism yielded under special conditions of cultivation “cellulose-less” mutants which became the starting point for new inquiries into bacterial nutrition and intermediary metabolism. To quote yet another case of a fundamental investigation which stemmed from examination of a technical process, the isolation from cider of a spoilage organism (a Lactobacillus spp.) which gave rise to viscosity in the beverage, provided us with an agent for the production of a new and interesting dextran of low molecular weight. I would like to end these reminiscences with a few remarks about personal relationships in a research school. When choosing men for postgraduate research I did not make the standard of their primary degree qualification the sole criterion of suitability; a man with a good second class higher (honors) degree was as acceptable to me as a man with a “first,” and I enlisted for research, on different occasions, students who had graduated at the ordinary or pass level, when these had given evidence in a short period of probationary work that they were 1ikely to develop well under further training. When I was a beginner in research I learned a great deal from Weizmann, Lapworth, and Pyman by seeing them at work at their benches in their own laboratories, and with my own students I made it a practice, in the earlier years, to work alongside them for several hours at

10

T. K. WALKER

least in each week. This can be done when an establishment is small and the work not too diversified, and it can provide a way of getting to know a beginner and realize difficulties h e may be experiencing. Observed faults can be checked and, what is perhaps even more important, a beginner can be given encouragement which will increase his self-confidence and often enable him to overcome his difficulties and rise above his self-imagined limitations. This remark may seem to be very much an expression of the obvious but, nevertheless, it will bear stating, for it concerns a matter which is not always apparent to those directing operations. On two occasions workers who had just successfully completed P1i.D. courses told me: “If I had not felt that you had confidence in me I could never have done it.” In order to ensure the maintenance of a high standard in the M.Sc. and Ph.D. graduates who left our Department I never relied on the services of merely two or three individuals as external examiners year after year but, over the years, for the 32 Ph.D. candidates awarded degrees, the oral examinations were conducted by 15 professors. Within the period 1925-1958 workers came from India, Pakistan, Egypt, Jugoslavia, Poland, Norway, Spain, Uruguay, United States, and Canada. I bclieve I am correct in claiming that all in their subsequent careers have won recognition for themselves in their respective fields: Fifteen of them have become university professors. Other old students have become readers or senior lecturers in universities or have attained important posts in research institutes or in industrial corporations.

Fermentation Processes Employed in Vitamin C Synthesis

MILOS

KULHANEK

Research Institute for Pharnzucy and Biochemistry, Prague, Czechoslovakia I. Reichstein’s Synthesis ....... .......................... A. Sorbose Fermentation. .......................... B. Problems of Further Simplification of Reichstein’s Synthesis ............................... 11. Newer Methods for Vitamin C Preparation ..................... A. Calcium 5-Keto-~-gluconate.................................... B. Hydrogenation of Calcium 5-Keto-D-gluconate to a Mixture of Calcium D-Gluconate and ...................... Calcium L-Idonate ..... C. Separation of Reduction Mixture ............................. D. Hydrogenation of Calcium 5-Keto-D-gluconate to Calcium L-Idonate ......................................... E. Dehydrogenation of L-Idonic Acid to e-Keto-~-idonic Acid F. Possible Ways of Ob from 2,5-Diketo-D-gluconic Acid .............................. G. Preparation of L-Ascorbic Acid from S-Keto-~-idonicAcid .............................................. 111. Conclusion References

11 12

16 17 19

20 20

26 27

Vitamin C, L-ascorbic acid, has been manufactured for 30 years by chemical industry on a continuously increasing scale. Within the period 1918-1925, vitamin C was isolated from lemons as the socalled “reducing factor,” and later from capsicum fruit, adrenals, etc. (1). Its structure has been established by Hirst (2) and Micheel and Kraft (3). The first syntheses of vitamin C were published in 1933 independently by both Reichstein and associates ( 4 ) and by Haworth et al. (5). Industrial production of the vitamin had been based on Reichstein’s procedure. Although this procedure is very economical, much time has been devoted, following its first application, to studies of other methods of vitamin C synthesis, involving 1 or 2 fermentation stages (similar to Reichstein’s procedure). Some of them were comprehensively described by Razumovskaya (6); these will also be discussed in the present survey. I. Reichstein‘s Synthesis In Reichstein’s synthesis D-glucose is chemically hydrogenated to 11

12

MILUS KULIIANEK

produce D-sorbitol. Instead of using crystalline glucose, the contemporary methods allow direct hydrogenation of deionized enzymatic hydrolyzate of starch which is obtained by the action of mold glucamylase (7).Nonisolated sorbitol thus produced, in the form of a 20% or more concentrated solution, is then subjected to biochemical dehydrogenation, by Acetobacter suboxydans, to yield L-sorbose. The isolated crystalline 1,-sorbose is first protected from too advanced oxidation by condensation with acetone to form 2,3,4,6-diisopropylidene-L-sorbose (so-called diacetonesorbose). This is chemically oxidized to diacetone-2-keto-~-gulonicacid, which, after hydrolysis, enolization, and lactonization yields L-ascorbic acid. Detailed descriptions of this process are available in fairly recent papers (8,9). It has been stated that the production of 1 kg. of vitamin C requires 2-4 kg. of glucose. The peak yield attained, i.e., 1kg. of pure product obtained from 2 kg. of glucose, proves the high degree of economy of this method. A.

SOHBOSEFEHMENTATION

Biochemical dehydrogenation of D-sorbitol to L-sorbose was discovered by Bertrand (10) who isolated the “sorbose bacterium,” now named Acetobacter xylinurn, from fermenting juice of mountain ash berries. Surface fermentation using Acetobacter xylinurn yielded, after about 6 weeks, approximately 4 0 4 0 % sorbose. Dehydrogenation of sorbitol to sorbose b y the action of this and other species of Acetobacter was studied during subsequent years by several authors (1 1-1 7). Acetobacter suboxydans, discovered later (13),yielded, after 7 days of surface fermentation, 80-90% sorbose ( 18). Wells and associates (19) were the first to employ the more modern submerged process in sorbose fermentztion. They carried out the fermentation, using a medium containing 20% sorbitol and 0.5% yeast extract, in a rotating drum with an air overpressure of 30 p s i . over periods of 33-45 hours, and obtained a 98% yield. They succeeded in isolating more than 80% of the sorbose that was contained in the fermented medium. For the preparation of inoculum they used a rnediurn containing 10% sorbitol, 0.5% yeast extract, 1%glucose, and 3.1% calcium carbonate. On pilot plant scale a 98% fermentation yield, calculated for the sorbitol brought in, was attained in 14 hours with a 10% solution, in 24 hours with a 20% solution, and in 40 hours with a 29% solution (20). A defoamer, e.g., 0.08% octadecanol (21), was sometimes added into the rotating drum. A further development in production equipment was represented

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

13

by conventional-type vat fermentors equipped with means for the dispersion of air (porous stones, perforated pipes, etc.). Since nickel at that time was supposed to be highly toxic for Acetobacter suboxydans, the equipment was constructed of high-purity aluminum or nickel free stainless steel. The culture was agitated by an air stream, mechanical stirring being considered unnecessary at that time. Defoamers such as 0.1% octadecanol, soya bean oil, liquid portion of lard were used. For this purpose activated charcoal was also recommended (22). The whole production process, including analytical checking and isolation procedures, has been described by Lockwood (23). Later on, sorbose was produced in cylindrical fermentors, equipped with mechanical stirrers and aeration devices, similar to those commonly used in the production of antibiotics (24,25). These fermentors are usually made of common stainless, i.e., chromium-nickel steel. The problem of nickel sensitivity of the commonly used Acetobacter strains was studied in considerable detail with particular regard to the presence of nickel in sorbitol, of which the latter is produced at the present time solely by catalytic hydrogenation using Raney nickel catalyst. In the past the nickel present in sorbitol was removed by using disodium hydrogen phosphate (9,30); at present, if necessary, it is removed with the use of catexes' (26). The removal of nickel by precipitation with raw protein contained in the nourishing additives, e.g., corn steep liquor, is advantageous. The precipitate formed by boiling is removed b y filtration or centrifugation, thereby substantially reducing the nickel content (27). Another approach to the problem of reducing the amount of nickel present in sorbitol is to adapt the culture used (28). For Acetobacter melanogenum, the upper limit of nickel tolerated in the fermentation medium is 10 mg./liter (29), whereas Acetobacter suboxydans is successfully employed for industrial-scale fermentation of a 20% sorbitol solution which contained 24 mg. nickel/l liter. I n laboratory experiments, this latter organism was successfully adapted to tolerate as high a concentration of nickel as 600 mg./l liter of fermentation medium (30). Acetobacter suboxydans, the organism most frequently used for dehydrogenation fermentation, requires for growth (aside from assimilable sources of carbon, organic nitrogen, and mineral salts) pantothenic, p-aminobenzoic, and nicotinic acids; it does not require 'Catex (cation-exchange resin); anex (anion-exchange resin); ionex (ion-exchange resin).

14

MILO$

KULIIANEK

riboflavine and biotin (31). Sorbitol serves as the source of carbon; other nutrients are supplied by dried yeast extract [0.5% is added as a rule; Muller (32) prefers 0.1-0.3%], yeast autolyzate, or corn steep liquor. Industrial production of sorbose requires cheap materials, such as corn steep liquor, a decoction of waste brewers’ yeast, acidic yeast hydrolyzate (33), and alfalfa extract (34).As a rule, 0.3%of corn steep liquor is added (20) but our results prove that its content in the medium may be reduced to as little as 0.1% The medium is adjusted to pH 5-6 (35). More detailed studies of sorbose fermentation conditions showed that organic sources of nitrogen may be partly replaced by ammonium sulfate, phosphate, or nitrate (36-38). Dehydrogenation activity of cells grown in media with a high content of organic nutrients (in which the growth rate is higher) is inferior to that of cells grown in less nutrient media (39-41). Amounts of phosphates to 10-50 mg./liter were found to be optimal (42). It was found earlier that the sorbitol concentration in the medium may be as high as 35% (18,43,44);in such a case the sorbose content attainable per 100 ml. of medium is 28 gm. after complete fermentation. On production scale, sorbitol solutions containing, as a rule, 20 gm./100 ml. were used; in such cases the inoculum could be cultivated in the same medium. Whenever possible, the inoculum used should be taken from the preceding batch at the moment when the culture achieves the highest activity. Fresh inoculum is prepared only in cases of contamination or where decreased activity of the culture is observed. The amount of inoculum to be added varies between 5 and 20%; Lockwood (23) uses 3%. Use of small amounts of inoculum extends the duration of fermentation. If media with higher sorbitol concentration are to be used, it is advantageous to begin the fermentation in a less concentrated medium (e.g., 10-20 gm./100 ml.), and to enrich it by gradually adding concentruted solution of sorbitol (if necessary, with respective nutrients added) until the total amount of sorbitol added corresponds to an initial concentration of 28 gm.1100 ml. of medium (20,41). Both the preparation of inoculum and the proper fermentation require intense aeration (45-47); fermentation inay be accelerated by increasing the air pressure in the fermentor. Substitution of air b y oxygen under increased pressure substantially shortened the duration of fermentation using both Acetobacter xylinum and A. suboxydans (48). According to Mikhlin (49),oxygen content elevated over its usual percentage in the air inhibited the activity of Acetobacter melanogenum. Sorbose fermentation is interrupted as soon as the concentration of

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

15

reducing sugars, calculated as sorbose, reaches about 96-99% of refractometrically estimated dry sugars in the solution, of which the latter is filtered or contrifugated and the clear liquid then thickened under reduced pressure (at a temperature not exceeding 50°C) to crystallization. Deionization of the filtrate before thickening leads to increased yield of the isolation (50).A paper by Stickdorn et al. (51) recommends crystallization at pH 3 to obtain an increased yield and better quality of sorbose. The following yields have been published: 70% of sorbitol used (20, 52-54), and more recently, about 82% (27); and with previous deionization, an 87% yield (50). Quite recently, Wakisaka and associates (55)described a yield amounting to about 89% of the expected theoretical value of substrate sorbitol in molar bases, with the isolated sorbose achieving at least 99% purity. They performed fermentation of 700 liters of a 20% w./v. solution of technical grade sorbitol, containing 0.2% w./v. corn steep liquor, pH 6.0, which had been previously sterilized for 20 minutes at 120°C. A 1000-liter fermentor of nickel-free stainless steel was used, with an agitation of 200-240 r.p.m., an aeration of 0.5 vol. per min. 2.11 kp. cm.? pressure, and 30°C temperature. Fermentation using Acetobacter suboxydans (Shionogi) was completed within 24 hours. The authors used about 6% of inoculum prepared in an analogous seed tank by about a 2O-hour fermentation of a medium containing 10% w./v. sorbitol and 0.5% w./v. corn steep liquor. Also recently the continuous process of sorbose fermentation has been investigated (32,5649) but no reports have as yet been published on its industrial-scale realization (60). In our laboratory we found that in sorbose fermentation, apart from the production of sorbose itself, other reducing sugars are produced in small amounts as side metabolites. These were identified as D-fructose and 5-keto-~-fructose(2,5-~-threo-diketohexose) (61-64). The feasibility of dehydrogenation of D-sorbitol to D-fructose was proved by experiments using a cell-less extract of Acetobacter suboxydans (65,66). The possibility of 5-ketofmctose formation had already been apparent on the basis of manometric work in which, under certain conditions, a cell suspension of Acetobacter suboxydans consumed 2 atoms of oxygen per each molecule of sorbitol, and 1atom of oxygen per each molecule of sorbose, respectively (67,68). Later, 5ketofructose was proved to be produced from D-fructose (69-86,172) or L-sorbose (87,88) by dehydrogenation action of various Acetobacter species, or from either of these ketohexoses by chemical oxidation (89,90).

16

MILO$

KULHANEK

In sorbose fermentation, 5-ketofructose may be produced by further dehydrogenation of primarily arising fructose or sorbose (87).In our experiments, 12 of 53 collection strains of Acetobucter species proved to be capable of performing a complete fermentation of 20% w./v. sorbitol medium. In all cases, more or less extensive formation of 5-ketofructose was proved to occur toward the end of fermentation. Formation of small amounts of D-fructose was likewise shown to proceed with all the abovementioned strains except Acetobacter albidus CCM 2365; in this latter case the absence of fructose is explainable since the strain in question possesses a marked capability of dehydrogenating fructose to 5-ketofructose. We succeeded in establishing conditions of sorbose fermentation under which the formation of 5ketofructose is practically coinpletely inhibited, but failed to find fermentation conditions inhibiting the foiination of fructose. OF FURTHERSIMPLIFICATlON REICHSTEIN’SSYNTHESIS

H. PHOBLEMS

OF

Work aimed at further simplification of Keichstein’s synthesis has been directed mainly toward finding a process which would allow direct oxidation of sorbose to 2-keto-~-gulonicacid. Initially, direct chemical oxidation of sorbose to 2-keto-~-gulonicacid gave yields of 15-20% because of concomitant side reactions (91-93); newer patents describe processes which use slow air oxidation, catalyzed by platinum, with stated yields of 6045% (94). With regard to the stated yields of only theoretical interest, for the time being, have been studies of direct bacterial oxidation. A Pfizer patent (95)uses 0.5-2% solutions of sorbose that are oxidized by selected strains of the genus Pseudomonas in a weakly alkaline medium. The process lasts 50-70 hours; no yield has been stated. Other papers state that after a 5-day fermentation of 2% solution of sorbose b y a Pseudomonas sp. mutant, about 16% conversion of e-keto-~-idonicacid takes place, 0.8% sorbose remaining in the solution (96). Japanese Takeda patents (97) use selected species of the genera Acetohacter or Pseudomonas for oxidation of up to 5% sorbitol solutions directly to S-keto-~-idonic acid; this acid may be, if convenient, separated from the solution with the aid of an anex, esterified without previous isolation, and converted to L-ascorbic acid. Yields of isolated 2-keto-~-idonicacid reach about 8% of the sorbitol used; the fermentation lasts ~ i pto 150 hours.2 *See also recent papers (185-189).

17

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

II.

N e w e r Processes for Vitamin C Preparation

Newer processes of vitamin C production are based on the work done by Bernhauer's team on biochemical oxidation of aldoses and aldonic acids. Bernhauer's team was the first to produce, by way of fermentation, calcium 5-keto-D-gluconate (5-keto-~-idonate)(98)and calcium 2-keto-D-gluconate (99). Originally, this work followed that done by Pasternack on catalytic hydrogenation of calcium 5-keto-Dgluconate to a mixture of calcium D-gluconate and calcium L-idonate (100). Despite variations in details, the preparation of vitamin C by these methods may be represented as in Fig. 1. Coo

H-

Ca

I H0-C-H

H-C-OH

H-C-OH

n 0 - L %no-&--HI n-c-on H-C-OH H-C-OH I

CH,OH (1)

H-C

-OH CH,OH

(11)

H-C-OH

~H_HO-&-HI H- C-OH

HO-C

I

COOH

F O

c=o

-2~_Ho-&n

I

/H

COOH I

I

c- on

CH,OH

(IV)

0

n-c

H- C- OH I HO-C-H I CH,OH

I

HO- C-H CH,OH

(V)

(VI)

c=o C8

cH,on

(rn) tZHl\-ZH+

CI o o 5

n-Y-oH HO-C--H H-A-onI

H-C-OH cH,on (VII)

COOH

c= 0 -2H HO-C-H

-HO

H- CI OH H-C-

OH CH,OH

(VIII)

HO-C

n- c n- c-on CH,OH

(nr)

Glucose (I) is converted by biochemical dehydrogenation in the presence of calcium carbonate to calcium 5-keto-~-gluconate(111); D-gluconic acid (11) is an intermediate product. According to the original procedures, calcium 5-keto-~-gluconate(111) was catalytically hydrogenated to a mixture of calcium D-gluconate (VII) with calcium L-idonate (IV) in a 1:l ratio. Of this mixture, further termed the " reduction mixture," only the L-idonate component is able to be used for further preparation stages of L-ascorbic acid (VI); the D-gluconate (VII), processed analogously, yields isoascorbic (D-araboascorbic) acid (IX) that possesses only about 1/20 of the biological activity of L-ascorbic acid (VI) (101).Therefore the reduction mixture has to be processed further in order to separate out either hexonate, or, at least, to isolate the L-idonate component from the mixture. Several separation processes have been used.

18

MILOS KULHANEK

1. Chemical separation of calcium L-idonate (IV) from calcium Dgluconate (VII) is feasible over the slightly soluble dibenzal-L-idonic acid (X) or the likewise slightly soluble binary salt which consists of cadmium (11) L-idonate and cadmium (11) chloride or bromide. This second process offers fair yields of either hexonate but is very laborious. 2. Calcium D-gluconate (VII), present in the reduction mixture, is dehydrogenated, using a suitable strain of Acetobacter suboxydans, back to the slightly soluble calcium 5-keto-~-gluconate(111);this salt is returned into the process while calcium L-idonate (IV) remains in the solution. Yields obtained from this relatively simple process are, however, unsatisfactory. 3. The reduction mixture is directly dehydrogenated by bacterial strains capable of selective dehydrogenation of either hexonic acid in position 2. It was found that, in this case, D-gluconate is first dehydrogenated to 2-keto-~-gluconate(2-keto-D-mannonate) (VIII) which is totally degraded in the further course of the process, while 2-keto-~idonate (2-keto-~-gulonate)(V) remains in the solution. This otherwise very simple process of isolating the L-idonate component [the separation directly yielding the intermediate product (V)] has, however, the disadvantage of losing one-half of the material at the third stage of synthesis. The separation of the reduction mixture, or the isolation of the Lidonate component from the reduction mixture, represent the most difficult stage of the entire process. The loss of one-half of the material at the third stage of the synthesis, or the necessity of returning half the material into the process are reasons why none of these methods of vitamin C preparation has been considered suitable for practical use. In recent years, Japanese workers have been studying ways of directly obtaining calcium L-idonate (IV) from calcium 5 - k e t o - ~ gluconate (111) by catalytic hydrogenation in a weakly alkaline medium or by microbial reduction, thus eliminating the most difficult stage of the synthetic process. Calcium L-idonate (IV) obtained by one of the described processes is then converted by bacterial dehydrogenation to calcium 2-keto-~idonate. From the fermentation medium, crystalline S-keto-~-idonic acid (V) is isolated and converted, either directly or over its methyl ester, to L-ascorbic acid (VI). The following sections present a more detailed description of individual stages of the synthetic process.

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

A.

CALCIUM

19

5-KETO-D-GLUCONATE

Calcium 5-keto-~-gluconate(111) was prepared for the first time by biochemically using Bacterium xylinum, in stationary culture in the presence of calcium carbonate, from calcium D-gluconate with a 50% yield (98). With Bacterium gluconicum, Acetobacter suboxydans, and other Acetobacter species (99, 102-105) a 50% yield was also reported from calcium D-gluconate, aside from the 30%yield of calcium 2-keto-D-gluconate (99). Later on, Bernhauer reported a yield of about 70% from calcium D-gluconate, using the same species (106). In manometric experiments with Acetobacter suboxydans it was found, on the basis of oxygen consumption, that a roughly quantitative conversion of D-glucose to calcium 5-keto-D-gluconate is possible (107). Stubbs and associates (108), using Acetobacter suboxydans in an aerated rotating fermentor and 10% glucose solution with added calcium carbonate, found, after a 33-hour fermentation, that the yield of calcium 5-keto-D-gluconate reached 90% of the expected theoretical value depending on the glucose used. They found that dehydrogenation proceeds in two phases. First, glucose (I) is dehydrogenated to D-gluconic acid (11) that, after neutralization by the calcium carbonate present, is further dehydrogenated to calcium 5-keto-D-gluconate (111).Recently (109, 110), the role played by the pH of the medium was established. In the absence of calcium carbonate a quantitative dehydrogenation of glucose to gluconate may occur, but further dehydrogenation to 5-keto-D-gluconate is conditioned by the presence of calcium carbonate. According to enzymological studies (111, 112), the primary products of glucose dehydrogenation are either the y- or the a-lactone of gluconic acid. In shaker experiments using Acetobacter suboxydans, yields of 5keto-D-gluconic acid from 10% glucose solution after 7 days are reported to reach about 85% of the theoretical yield (113), and about 90% relative to glucose employed (109). In our laboratory, a fair yield of calcium 5-keto-D-gluconate was obtained directly from a glucose solution prepared by enzymatic hydrolysis of starch using mold glucamylase. Aside from the proper conditions of fermentation preparation of calcium 5-keto-~-gluconate,the choice of the bacterial strain plays a decisive role. Since the first discovery that Acetobacter suboxydans produces, besides 5-keto-D-gluconate, also 2-keto-D-gluconate from calcium D-gluconate (99), a number of studies have been dedicated to this problem (104, 105, 110, 114-118). To obtain a fair yield of cal-

20

MILOS KULHANEK

cium 5-keto-D-gluconate, a strain must be chosen that does not produce 2-keto-D-gluconate (113). The course of fermentation has to be supervised, by using, among other means, chromatographic analysis

(1 13,119).

H. HYDROGENATION OF CALCIUM 5-KETO-D-GLUCONATE TO A MIXTUREOF CALCIUM D-GLUCONATE AND CALCIUM L-IDONATE Pasternack ancl Brown (100) were the first to describe catalytic hydrogenation of calcium 5-keto-~-gluconate(111). The substance, slurried with water with added Raney nickel, was hydrogenated with hydrogen at 100 kp./cm.' at 60°C. After 4 hours they obtained a mixture of calcium D-gluconate (VII) with calcium L-idonate (IV) and recommended its use for preparation of concentrated injection solutions of calcium. They mentioned, also, the possibility of isolating L-idonic acid from the reduction mixture over dibenzal-L-idonic acid (X) according to Van Ekenstein and de Bruyn (120). Gray (121) converts calcium 5-keto-~-gluconateto ammonium salt prior to hydrogenation. Recently this reaction was described as a part of a fermentation process of vitamin C preparation (122).Under atmospheric or increased pressure, calcium 5-keto-~-gluconateyielded 90% of R reduction mixture containing 47% calcium L-idonate and 43% calcium n-gluconate. At the pressure of 100 kp./cm.2 at 8O"C, complete reduction may be reached within 1M -2 hours (123). C. SEPARATION OF REDUCTIONMIXTURE

1 . Chemicnl Processes a. Intermediate: Dihenznl-L-idonic Acid

The fact that L-idonic acid is condensed with benzaldehyde in the medium of concentrated hydrochloric acid, producing the sparingly soluble 2,3,4,5-dibenzal-~-idonic acid (X), has been known for a fairly long time (100,120,124)(Fig. 2 ) . COOH I

,o-c-n I / c,n,cH n-c-o 'o-h-n cn,oH (X)

CHC,H,

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

21

Since D-gluconic acid, under analogous conditions, does not form any condensate with benzaldehyde, the process described below was employed for isolation of L-idonic acid from the reaction mixture (100, 125). The mixture was first brought to solid crystalline form by repeated trituration with methanol. Subsequent condensation with benzaldehyde in the medium of concentrated hydrochloric acid produced dibenzal-L-idonic acid that, after hydrolysis by dilute sulfuric acid, was neutralized to calcium L-idonate in a yield of about 65% of its content in the reduction mixture. Japanese authors (126)performed the condensation in a medium containing sulfuric acid; no yield is mentioned. This process was described again recently (123). This process allows preparation of pure L-idonic acid but is expensive because of the use of benzaldehyde, of which, only about 65% is recoverable. Isolation of D-gluconic acid from the acidic mother liquor remaining after the crystallization of dibenzal-L-idonic acid is not profitable.

b. Intermediate: Binary Salt L-Idonic acid may be separated from D-gluconic acid, in the form of the sparingly soluble binary salt of cadmium(I1) L-idonate, with cadmium(I1) chloride or bromide since no analogous crystalline salt is formed with cadmium(I1) D-gluconate under the given conditions. A binary salt with cadmium(I1) bromide [(C,HI1O7)2Cd* CdBrp * HzO] has been described in an earlier paper (127). In our laboratories, L-idonic acid was isolated from the reduction mixture in the form of this salt. It was found also thatcadmium(I1) chloride, which is cheaper, forms an analogous sparingly soluble binary salt, viz., (C6H,,07),Cd CdCl, * 2 H 2 0 (125). L-Idonic acid was isolated from the binary salt by precipitation of cadmium in the form of sulfide or carbonate, and the removal of hydrogen chloride from the mixture of L-idonic acid with hydrogen chloride thus obtained by addition of silver carbonate or b y protracted vacuum distillation. Silver carbonate removes hydrogen chloride completely; vacuum distillation removes a major part of it. Calcium L-idonate, prepared by neutralization of the residual solution of L-idonic acid by calcium carbonate, then contains a small percentage of calcium chloride that, however, does not interfere with further fermentation processing to calcium 2-keto-~idonate. Total yield of calcium L-idonate reached in these processes exceeded the 90% calculated in relation to the calcium L-idonate present in the reduction mixture. This process may be further simplified by using catexes for removing cadmium from a dilute solution of

-

22

MIL&

KULHANEK

the binary salt, and anexes for removing hydrogen chloride from the solution of L-idonic acid and hydrogen chloride. Advantages of this procedure are the high yield of the product, easy recoverability of cadmium salts, and the possibility of obtaining D-gluconic acid from the mother liquor remaining after the crystallization of the binary salt; a disadvantage is its considerable laboriousness.

2. Bacterial Dehydrogenation of’ C u k i u m D-G~uconnteto Calcium 5-Keto-D-gluconute Gray (121, 128) described isolation of calcium L-idonate (IV) from the reduction mixture by reconversion of the calcium D-gluconate (VIII) present to the sparingly soluble calcium 5-keto-D-gluconate (111) by dehydrogenation using Acetohacter suboxydans. Calcium 5-keto-D-gluconate (111) was first converted to the corresponding ammonium salt, the latter was hydrogenated under pressure in the presence of Raney nickel, and the reduction mixture was then reconverted to calcium salts. These operations seem to be aimed only at circumvention of Pasternack’s patent (100). Glucose (I) (in an amount corresponding to 23% of the original weight of the calcium 5-keto-~-gluconate)and calcium carbonate were added to the reduction mixture and dehydrogenation using Acetobacter suboxydans was performed to yield calcium 5-keto-~-gluconate(111), that deposited from the solution and was returned into the process. The remaining calcium L-idonate (IV) was dehydrogenated, with the aid of Pseudomonas mildenbergii, to calcium 2-keto-~-gulonate(2-ketoL-idonate). The overall effect is expressed so that 75 parts of glucose yields 45 parts of calcium e-keto-~-idonate,i.e., about 50% of the theoretical yield. Our attempts to reproduce this procedure, however, brought only low yields. A recent paper (129) reports the following yields obtained by this process using Acetobacter suboxydans: conversion of calcium gluconate (VII) to calcium 5-keto-D-gluconate (111), yield 70%;reduction mixture (IV -tVII) to 35% of theoretical yield of calcium 5-keto-D-gluconate (111) besides 95% of theoretical yield of calcium L-idonate (90%of theoretical yield of the isolated substance).

3. Dehydrogenation of the Reduction Mixture to Calcium 2 - K e t o - ~ idonate In our laboratory it was found that if the reduction mixture (IV+ VII) is subjected to biochemical dehydrogenation, using a suitable strain of Pseudomonus aeruginosu, then the fermentation proceeds so that the content of reducing sugars, corresponding to the sum of calcium salts of both 2-ketohexonic acids (V VIII) produced by hydrogena-

+

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

23

tion, first increases from zero to a certain maximum (about 50% of the theoretical value). This first maximum is due to the formation of calcium S-keto-~-gluconate (VIII). In the further course of the process, the content of reducing sugars decreases to a certain minimum (about 20% of the theoretical value) because the primarily formed calcium 2-keto-~-gluconateis, in the further course of the fermentation, degraded to nonreducing substances, the degradation being finished by a total dissimilation to water, carbon dioxide, and calcium carbonate. The minimum having been reached, the content of reducing sugars begins to increase again to a second maximum (about 55% of the theoretical value). In this second phase, dehydrogenation of calcium L-idonate proceeds; it does not start before the dehydrogenation of calcium D-ghconate is finished. If the fermentation is stopped at this second maximum, then after precipitation of calcium and concentration of the filtrate through evaporation, 2-keto-~-idonicacid (V) is obtained in a fair yield. Although it was evident that this method has the inherent disadvantage that one-half of the material is automatically lost, and at the third stage of synthesis at that, a procedure was elaborated, involving submerged fermentation of a 4% solution of the reduction mixture, that produces crystalline 2-keto-~-idonicacid in a 90% yield calculated with regard to calcium L-idonate present in the reduction mixture used (130). Working independently on our work, Yamazaki (131) observed the above described two phases in the fermentation of the reduction mixture in surface cultivation of a Pseudomonas strain; in a later paper he described the isolation of crystalline sodium 2-ketoL-idonate in 31-40% yields, using Pseudomonas ftuorescens (132). Later on, he reported that the yield after 70-hour submerged fermentation, using the same species, amounted to 52.5% of crude crystalline 2-keto-~-idonicacid, a calculation based on the mixture of hexonates (133). The appropriate patent (134) states that a yield of 200 gm. of 2-keto-~-gulonicacid obtained by fermentation of a 5% solution of a mixture containing 250 gm. of L-idonic and 250 gm. of D-gluconic acids, gave about 80% of the theoretical yield. The conjecture, based on these results, that this procedure enabled the Japanese industry to sell, since the year 1956, vitamin C considerably below the world market prices (8), must be considered unsubstantiated for reasons given in the preceding paragraph. Later work based on the same principle used a bacterium named by this author Pseudomonas chromospirans, or C yanococcus chromospirans (135, 136), or also Pseudomonas cychro (137), apparently belonging to the Pseudomonas aeruginosa strains which dehydrogenate

24

MILO$

KULHANEK

hexonatcs (130).From a 3% solution of the reduction mixture, after a 26-hour submerged fermentation, 66% of the theoretical yield of calcium e-keto-~-idonatewas isolated (138). Improved preparation of the inoculum made possible a quantitative removal of calcium Dgluconate from 10% solutions of the reduction mixture within 22-30 hours, whereas in 20% solutions of the reduction mixture, aside from calcium S-keto-~-idonate, calcium 2-keto-D-gluconate was a1so always present (139). All this work was later described in a cumulative paper as a so-called new biosynthesis of vitamin C (140),and the pertinent methods were patented (137, 141). Another patent (142) protects the process of isolation of e-keto-~-idonicacid from the solution obtained after fermentation of the reduction mixture b y the process described above (130).

D. HYDROGENATION O F CALCIUM 5-KETO-D-GLUCONATE CALCIUML-IDONATE

TO

In recent years, several papers were published, dedicated to the conversion of calcium 5-keto-~-gluconateto calcium L-idonate. Successful realization of this direct conversion would overcome the most difficult stage of the production of vitamin C involving calcium 5-ketoD-gluconate, viz., separation of calcium n-gluconate from calcium L-idonate, both produced by hydrogenation of calcium S-keto-~gluconate; the entire procedure would be substantially simplified as a consequence. These procedures employ hydrogenation of calciuni 5-keto-Dgluconate in a weakly alkaline medium, evidently producing a salt of the enolic form of the 5-keto-~-gluconicacid (XI), probably the 5,e-enediol (XII) (143) that is hydrogenated to a salt of L-idonic acid (XIII, Fig. 3). A 30% slurry of calcium 5-keto-~-gluconate,whose pH had been adjusted to 8.6 by sodium hydroxide, was hydrogenated in COOH

COONa

Ho-6-n I H-c-on

n-c-OH -

c=o cnp (XI)

COONa I

I

n-c-oH +NaOH

I

H0-c-H

H-eon I

n-c-on +ZH __t

I

HO-C-H I

H.- C- OH I

II

Ho-c-n I m,on

(XII)

(XIII)

C-OH

CHOH

the presence of Raney nickel at 80°C and 84 kpdcm.', reaching a 94% yield of calcium L-idonate (144). Another procedure (145) starts from a 33% solution of 5-keto-~-gluconicacid that, after adjustation of the

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

25

pH to 8.6, is allowed to enolize by standing for 48 hours at 25°C; the enolic acid is then reduced to L-idonate, in the presence of Raney nickel, at 80°C and at hydrogen pressure of 90 kp./cm.' reaching a roughly quantitative yield. According to a further patent (146), 40 gm. of moist calcium 5-keto-D-gluconate (containing 26.3% of water) is mixed with 100 nil. of water, the pH is adjusted to 10.0 by sodium hydroxide, and hydrogenation is performed in the presence of a nickel-kieselguhr catalyst at 90°C and 80 kp./cm.' hydrogen pressure. Twenty-five grams of calcium L-idonate are obtained (85% of the theoretical yield). Still another paper states a yield of 94-95% of calcium L-idonate (147). Another patent (148) performs pressureless hydrogenation in the presence of dimethylamine (pH 7.6) with a quantitative yield. Attempts to reproduce these procedures, however, have been unsuccessful. In our experimental studies, at best, we succeeded in obtaining a reduction mixture containing more than 60% of calcium L-idonate (149), in rough agreement with another patent issued at that time ( 150). In hydrogenation of calcium 5-keto-D-gluconate the calcium Lidonate present is assayed on the basis of isolated dibenzal-L-idonic acid that, however, does not arise in a quantitative yield. Qualitatively, D-gluconic acid may be differentiated from L-idonic acid by paper chromatography after previous lactonization with the aid of methanol (151). With regard to possible formation of methyl esters, however, lactonization by boiling with hydrochloric acid is more expedient (149,152). Of theoretical interest only is microbial hydrogenation of a 1% slurry of 5-keto-~-gluconateto L-idonate. Mostly, however, only a low degree of conversion is attained (e.g., 10%)(152-154).

E. DEHYDROGENATION OF L-IDONIC ACID 2-KETO-L-IDONICACID

TO

2-Keto-~-idonic(2-keto-~-gulonic)acid (V) is prepared by biochemical dehydrogenation of L-idonic acid (IV). Biochemical dehydrogenation in position 2 of the epimer of the latter acid, i.e., L-gulonic acid (XIV), is more difficult (155) (Fig. 4). The preparation of 2-keto-~-idonicacid from the reduction mixture is described in an earlier section of this paper. In the text following, procedures used for dehydrogenation of isolated L-idonates are described. Biochemical dehydrogenation of L-idonic acid to S-keto-~-idonic

26

MILOS KULHANEK YOOH HO-C-H I

HO-C-H I H-C-OH I

HO

C I

H

CH,OH

(XIV)

acid was first performed with the aid of Pseudomonas mildenbergii (121), and later with Cyanocnccus chrornospirans (135,136).In a 5% solution of calcium L-idonate, the following yields, expressed in percentage of theoretical yields of isolated calcium e-keto-~-idonate, were obtained with various microorganisms: with Pseudomonas fluorescens, 7040%; with Pseudomonas aeruginosu, 60-70%; and with Acetobacter suboxydans melanogenunz, SO-SO% (157). Further work described the use of Pseudomonas sp., similar to Pseudomonas jluorescens, isolated from the soil ( 1 58); a cellular suspension of Pseudomonas jluorescens (159); Micrococcus aurantiacus ( 1 60); and a mixture of two Pseudomonas spp. with a practically quantitative yield in a 2% solution of sodium L-idonate (161); Pseudomonas 2-keto-~-gulonicum, with the aid of which a 15% solution of calcium r>-idonate was fermented in the course of 50 days to calcium 2-ketoL-idonate, yielding 65% of isolated substance (162). Also from Lgulonate, 2-keto-~-idonicacid was obtained with the aid of Pseudomonas aeruginosa with a 44% conversion degree after 8 days (156). At the time when the preparation of 2-keto-~-idonicacid using the genus Pseudomonas had not yet been known, a procedure was proposed, consisting of epimerization of L-idonic acid, obtained according to Gray (121), in pyridine medium to L-gulonic acid, and of dehydrogenation of the latter to S-keto-~-idonicacid with the aid of Acetobacter suboxydans (128). OBTAINING 2-KETO-L-IDONIC ACID FROM 2,5-DIKETO-D-GLUCONIC ACID 2,5-Diketo-D-gluconic acid [(XV); Fig. 51 was obtained in the form of its calcium salt by bacterial action upon D-glucose, in the presence of calcium carbonate, or upon calcium D-gluconate. Takahashi and Asai (163-164) probably were the first to note its presence in oxidative fermentation of D-glucose with the aid of Bacterium hoshigaki var. glucuronicum, supposing, however, that the product obtained was glucuronic acid. Bernhauer’s team that obtained a similar substance, using Bacterium gluconicum, from glucose with calcium carbonate

F. POSSIBLE

WAYS OF

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

27

FOOH

co I

HO-C-H

~

1

H-C-OH 1

co I

CH,OH

(XV)

added, or from calcium D-gluconate besides calcium 5-keto-Dgluconate (103), and later also in addition to calcium 2 - k e t o - ~ - g h conate (99), supposed that the substance in question was 6-aldehydogluconic (L-guluronic) acid. Later the authors found that the substance is produced especially by Acetobacter melanogenum (104,165,166). Katznelson and associates (167-1 69) later identified the substance as 2,5-diketo-~-gluconicacid and proved its formation from glucose via gluconate and 2-ketogluconate. It is produced also by other Acetobacter spp. (116,170,171) and by a species of the genus Pseudomonus (172).The pathways of its dissimilation were also investigated (173-1 80). Its presence was proved after chlorine oxidation of methylP-D-glucopyranoside (181). It is a rather unstable compound whose solutions spontaneously turn brown on standing, especially at pH values higher than 4.5 (182). Although its presence in solution is easily detected by paper chromatography, the preparation of a pure substance is difficult. Up to the present time, it has been isolated only by precipitation of its calcium, barium, or potassium salts by alcohols (172,183). 2,5-Diketo-~-gluconicacid might be converted to 2-keto-~-gulonic acid by selective and stereospecific reduction. Actually, however, catalytic hydrogenation using Raney nickel produced, under consymption of 1 molar equivalent of hydrogen, a mixture of 2 - k e t o - ~ gluconic acid and 2-keto-~-gulonicacid, in which mixture the former acid was predominant (172).

G. PREPARATION OF L-ASCORBIC ACID FROM 2-KETO-L-IDONIC ACID L-Ascorbic acid is obtained from 2-keto-~-idonicacid by enolization and lactonization. This process is amply described in literature, and particularly in numerous patents. Some procedures employ methyl S-keto-~-idonate,obtained by esterification of free acid by anhydrous methanol in the presence of sulfuric acid, as an intermediate product. This methyl ester, in some cases without isolation, is then converted in an alcoholic medium to L-ascorbic acid (184).

28

MIL@

KULHANEK

Direct conversion of 2-keto-L-idonic acid to vitamin C in a strongly acidic medium, with a reported yield of 76% (133),seems to be more advantageous. 111.

Conclusion

Available information indicates that vitamin C is produced in all countries by the Reichstein’s method and that it has been successively improved. This method would be fiirther substantially simplified if the problem of direct oxidation of sorbose to 2-keto-~-gulonicacid were solved chemically or biochemically. With regard to the high effectiveness of the fermentative preparation of sorbose, the direct biochemical oxidation of sorbitol to 2-keto-~-gulonicacid seems to be less promising. Of the newer procedures of vitamin C preparation, the direct biochemical dehydrogenation of a mixture consisting of L-idonate and D-gluconate (obtained by catalytic hydrogenation of calcium 5-keto-~-gluconate)to 2-keto-~-idonic (2-keto-L-gulonic) acid seems to be, comparatively, the most advantageous one for the time being. As to the economic aspect, however, this procedure cannot compete with Reichstein’s synthesis. Newer procedures for preparation of vitamin C would become more interesting if siiccesshl realization of stereospecific hydrogenation of 5-keto-~-gluconic acid to L-idonic acid, or partial a ~ i dstereospecific hydrogenation of 2,5-diketo-D-gluconic acid to 2-keto-~-gulonic(2-keto-~-idonic) acid, could be done under economically acceptable conditions.

REFERENCES

1. A. Szcnt-Gyorgyi, Biocheni.J. 22, 1387 (1928). 2. E. I,. Hirst, Chern. (?. Ind. 11.221(1933). 3. F. Micheel, and K. Kraft, Z. Physiol. Chem. 222,235 (1933). 4. T. Reichsteiri, A. Criissner, and R. Oppenaner, Notzcre 132, 280 (1933); ibid. Hela. Chim. Actci 16, 561, 1019 (1933); T. Heichstein and A. Griissner, ibid 17, 311 (1934). 5 . R. G. Atilt, L). K. Bnird, H. C. Carrinyton, W. N. Haworth, R. Herbert, E. L. Hirst, E. G. V. Percival, F. Smith and M. Shcey,]. Chem. Soc. p. 1419 (1933); D. K. Baird, W. N. Haworth, R. W. Herbert, E. L. Hirst, F. Srnith, and M. Stacey, /. Chern. Soc. p. 62 (1934). 6 . Z. G . Razumovskaya, Mikrobiologiya 31,172 (1962). 7 . M. KulMnek, M. Tadra, and V. Mansfeld, Czech. Patent 124,995. 8. P. Rumpf, and S. Marlier, Bull. Soc. Chim. France p. 187 (1959). 9. Z. BudgSinsky, and M. Protiva, “Synthetische Arzneimittel.” Akademie Verlag, Berlin, 1961.

FERMENTATION PROCESSES USED IN VITAMIN C SYNTHESIS

29

10. G. Bertrand, C o m p t . Rend. 122,900 (1896). 11. R. Sazerac, C o m p t . Rend. 137,90 (1903). 12. H. J. Waterman, Zentr. Bakteriol. Parasitenk. A b t . IZ 38,451 (1913). 13. A. J. Kluyver, and F. J. d e Leeuw, Tijdschr. Vergelijk. Ceneesk. 10, 170 (1924). 14. F. Vissert Hooft, Dissertation, Delft, 1925. 15. K. Maurer, and B. Schiedt, Biochem. Z . 271,61(1934). 16. K. Bernhauer, and B. Garlich, Biochem. Z. 280,375 (1935). 17. J. Boeseken, and J. L. Leefers, Rec. Trau. C h i m . 54,861 (1935). 18. E. I. Fulmer, J. W. Dunning, J. F. Guymon, and L. A. Underkofler,]. Am. C h e m . SOC. 58,1012 (1936). 19. P. A. Wells, J. J. Stubbs, L. B. Lockwood, and E. T. Roe, Ind. Eng. C h e m . 29,1385 (1937). 20. P. A. Wells, L. B. Lockwood, J. J. Stubbs, E. T. Roe, N. Porges, and E. A. Gastrock, Ind. Eng. C h e m . 31,1518 (1939). 21. E. Delvaux, and R. Welvaert, Bull. Assoc. Ancien h t u d . Brass. Uniu. Louuain 41, 36 (1945). 22. I . T. Strukov, and V. T. Plotnikova, USSR Patent 67,565. 23. L. B. Lockwood, In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.). Chem. Publ., New York, 1954. 24. I. R. Shenvood,Australian C h e m . Inst. J. Proc. 14,221 (1947). 25. K. Bernhauer, Ergeb. Enzymforsch. 11,151 (1950). 26. A. Hersiczky, Sturke 18,249 (1966). 27. L. B. Lockwood, Methods Carbohydrate C h e m . 1,151 (1962). 28. Z. G. Razumovskaya, and R. E. Konikova, Uch. Zap. Leningr. Gos. Uniu. Ser. Biol.Nauk 41,23 (1956). 29. T. Elsisser, J. Huber, and H. Hilscher, Z . Allgem. Mikrobiol. 2,249 (1962). 30. V. BGhal, Kuusny Prumysl9,224 (1962). 31. L. A . Underkofler, A. C . Bantz, and W . H. Peterson,]. Bacteriol. 45,183 (1943). 32. J. Muller, Zentr. Bakteriol. Parasitenk. Abt. IZ 120,349 (1966). 33. S. M. Zhdan-Pushkina, Mikrobiologiya 24,545 (1955). 34. E. I. Fulmer, A. C. Bantz, and L. A. Underkofler, Iowa State Coll. J. Sci. 18, 369 (1944). 35. C. Widmer, T. E. King, and V . H . Cheldelin,J. Bacteriol. 71,737 (1956). 36. N. M. Mityushova, Mikrobiologiya 22,249 (1953). 37. S. M. Zhdan-Pushkina, Uch. Zap. Leningr. Gos. Univ. Ser. Biol. Nauk 41, 49 (1956). 38. Z. G. Razumovskaya, and V. V . Averyanova, Uch. Zap. Leningr. Gos. Uniu. Ser. Biol. Nauk 41,31(1956). 39. Z. G. Razumovskaya, and S. M. Zhdan-Pushkina, Mikrobiologiya 25,16 (1956). 40. S. M. Zhdan-Pushkina, and R. A. Krenova, Mikrobiologiya 32,711 (1963). 41. Z. G. Razumovskaya, Tr. Inst. Mikrobiol. Akad. Nauk S S S R , 6,46 (1959). 42. S. A. Shchelkunova, Mikrobiologiya 32,529 (1963). 43. E. I. Fulmer, and L. A. Underkofler, Iowa State Coll.J. Sci. 21,251 (1947). 44. G. Matsakura, M. Kudaka, S. Takahashi, and T. Asai, J. Agr. C h e m . SOC.J a p a n 23, 223 (1949). 45. S. M. Zhdan-Pushkina, Mikrobiologiya 24,447 (1955). 46. Z. G. Razumovskaya, and N. M. Mityushova, Mikrobiologiya 24,265 (1955). 47. R. B. Epshtein, Pishcheuaya Prom. 2,27,25 (1947). 48. M. Domodaran, and S . S. Subramonyan,]. Sci. Ind. Res. (India) 10B, 7 (1951).

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86. Y. Yamada, K. Aida, and T. Uemura,J. Biochem. 61,636 (1967). 87. 0. Terada, S. Suzuki, and S. Kinoshita, Agr. Biol. Chem. (Tokyo) 25,871 (1961). 88. K. Sato, Y. Yamada, K. Aida, and T. Uemura, Agr. Biol. Chem. (Tokyo) 31, 640 (1967). 89. F. Micheel, and K. Horn,Ann. 515, l(1935). 90. R. A. Whiting, and R. A. Coggins, Chem. iL Znd. p. 1925 (1963). 91. W. N. Haworth, British Patent443,901. 92. Swiss Patent 183,450 and 188,019. 93. U S . Patent 2,190,377 and 2,189,778; German Patent 692,897. 94. K. Heyns, and H. Paulsen,Aduan. Carbohydrate Chem. 17,170,182 (1962). 95. H. T. Huang, U.S. Patent 3,043,749. 96. R. P. Tengerdy,j. Biochem. Microbiol. Technol. Eng. 3,241,255 (1961). 97. British Patent 994,119; French Patent 1,376,741. 98. K. Bernhauer, and K. Schon, 2. Physiol. Chem. 180,232 (1929). 99. K. Bernhauer, and B. Gorlich, Biochem. 2.280,367 (1935). 100. R. Pasternack, and E. V. Brown, U.S. Patent 2,168,897 and 2,168,878. 101. W. W. Pigman, and M. L. Wolfrom, Aduan. Carbohyd. Chem. 2,79 (1946). 102. S. Hermann, Biochem. Z. 214,357 (1930). 103. S. Hermann, and P. Nueschul, Biochem. 2.233,129 (1931). 104. K. Bernhauer, and K. Irrgang, Biochem. Z. 280,360 (1935). 105. K. Bernhauer, and H. Knobloch, Naturwissenschaften 26, 303, 308, and 819 (1938). 106. K. Bernhauer, “Garungschemisches Praktikum.” Springer Verlag, Berlin, 1939. 107. A. J. Kluyver, and A. G. J. Boezardt, Rec. Trau. Chim. 57,609 (1938). 108. J. J. Stubbs, L. B. Lockwood, E. T. Roe, B. Tabenkin, .ind A. Ward, Ind. Eng. Chem. 32,1626 (1940). 109. M. Yamazaki,J. Agr. Chem. Soc,Japaia28,748 (1954). 110. J. A. Fewster, Bi0chem.J. 63,26P (1956). 111. K. Okamoto,J. Biochem. (Tokyo)53,348 (1963). 112. R. Weimberg, Biochim. Biophys. Acta 67,359 (1963). 113. S. Khesghi, H. R. Roberts, and W. Bucek,Appl. Microbiol. 2,183 (1954). 114. E. Riedl-Tfimova, and K. Bernhauer, Biochem. 2,320,472 (1950). 115. J. Frateur, P. Simonart, and T. Coulon, Antonie uan Leeuwenhoek J. Microbiol. Serol. 20,111 (1954). 116. M. Ameyama, and K. Kondo, Bull.Agr. Chem. Soc.Japan22,373 (1958). 117. J. A. Fewster, Biochem.J. 68,19P (1958). 118. D. Kulka, and T. K. Walker,Arch. Biochem. Biophys. 50,169 (1954). 119. K. Macek, and M. Tadra, Chem. Listy 46,450 (1952). 120. W. A. Van Ekenstein, and C. A. L. d e Bruyn, Rec. Trau. Chim. 18,305 (1899). 121. B. E.Gray,U.S.Patent2,421,611. 122. M. Yamazaki, and T. Miki,J. Fermentation Technol. (Japan)31,39 (1953). 123. R. M. Alieva, Vestn. Leningr. Uniu. Ser. Biol. 4,48 (1963). 124. E. Seebeck, E. Sorkin, and T. Reichstein, Helu. Chim.Acta 28,934 (1945). 125. B. Gorlich, and M. Kulhanek, Collection Czech. Chem. Commun. 31, 1407 (1966). 126. S. Teramoto, and I. HoriJ. Ferment. Technol. 28,143 (1950). 127. E. Fischer, and I. W. Fay, Chem. Ber. 28,1975 (1895). 128. B. E. Gray, U.S. Patent2,421,612. 129. M.Kudaka, H. Aida, and K. Miyainoto, Hakko Kyokuishi 11,251 (1953). 130. M. Kulhanek, Chem. Listy 47,1071 (1953).

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Flavor a n d Microorganisms

P. MARGALITHAND Y. SCHWARTZ Department of Food and Biotechnology, Technion -Israel Institute of Technology, Haifa, Israel I. General Introduct‘ .... .... 11. The Contribution Pr and Development of Flavor in Traditional Foods ............ A. Baked Products ....... B. Fermented Beverages ............................................ C. Dairy Products ...................................................... D. Pickles ................................................................. E. Oriental Foods ........ 111. Concluding Remarks .................................................... References ..................................................................

36

40 40 45

64 68

72 74 83

The spectacular increase in world population has led to a major effort, on a national and international basis, for the increased production of foods. Although substantial advances have been made in pi-oducing nutritionally valuable food ingredients of low cost, such as protein from microbial sources (Champagnat et al., 1963; Fiechter, 1967) and fishmeal concentrate, the acceptance of these foods in the needy developing countries where undernourishment is frequently encountered is far from satisfactory; one of the major disadvantages of these unconventional foods are their poor taste. Many workers have pointed out that after all the nutritional requirements of foods have been met with, the incorporation of suitable flavoring material into these “synthetic foods” have become imperative (Hornstein and Teranishi, 1967). There have been spectacular advances in the characterization of flavor. Gas-liquid chromatography and mass spectroscopy have opened new fields in the analysis of flavor and flavoring materials. It seems, however, that analytical devices and instrumentation are stil€ lagging behind the discriminatory power of human taste. Microbiology has played a prominent role in the production of fermented foods and beverages. The production of alcohol has been considered the major contribution of yeasts, while that of the lactic acid bacteria was mainly concerned with the transformation of sugars into lactic acid. The contribution of microorganisms to the development of flavor was not as widely investigated and only fragmentary information is available. With the increase in knowledge of the chemical nature of flavor it was discovered that microorganisms not only 35

36

P. MARGALITH AND Y. SClIWARTZ

contribute to taste in fermented products, but may be also employed for the specific production of food additives with flavor-enhancing properties on an industrial scale (Kuninaka, 1966; Solms, 1967). In this survey an attempt will be made to review the information available on the contribution of microorganisms to flavor as well as the fermentative production of some major flavor enhancing materials. However, it must be pointed out that the subject of microbial food spoilage is outside the scope of this review.

I.

General Introduction

In the interest of the general reader, a brief introduction to the nature of flavor will be made. Moncrieff (1951) defines flavor as “a complex sensation comprising taste, odor, roughness or smoothness, hotness or coldness, and pungency or blandness.” Although taste and odor are generally considered as main attributes of flavor, it should be remembered that flavor is mainly the psychological attitude of the human senses, and not the inherent properties of any material (Hornstein and Teranishi, 1967). Taste is the sense peculiar to the mouth and tongue. Historically a large number of components of taste have been discerned, although today it is generally agreed that taste comprises four basic taste qualities, i.e., sweet, salty, sour, and bitter (von Skramilk, 1921). However, niodifications of these basic qualities are frequent; expressions such as metallic, alkaline, and fatty tastes may be encountered. The sensation of taste is achieved by the sense buds distributed all over the tongue and some restricted areas in the buccal cavity. Their number is estimated to be between 9000 and 10,000. Taste buds are located in tiny papillae in the anterior part of the tongue, whereas in the posterior portion they line the sides of minute trenches. Each taste bud consists of about 10 to 15 taste cells from which very minute villi protrude. The taste buds are innervated b y nerve fibers arising from the subepithelial plexus. For a detailed description of the anatomy of these tissues the reader is referred to a histological text (e.g., Amerine et al., 1965). Although it was previously assumed that there exist different taste buds or receptor cells that respond to different taste stimuli according to the four basic taste qualities, this has been recently shown not to be the case. In fact, all taste buds respond to many of the taste principles, although quantitatively they are not alike. However, it is the integrated taste response of a multitude of taste buds that create the sensation of taste ( P f a b a n n , 1964; Beidler, 1966). There have been many efforts to explain the mechanism by which

FLAVOR AND MICROORGANISMS

37

taste active materials cause the sensation of taste. Beidler (1966) has shown that the initial step in the taste stimulation is a weak adsorption of the active material onto the receptor site on the surface of the taste cell. This in turn causes a depolarization in the charge of the nerve fiber leading to the formation of a nerve impulse. The involvement of enzymatic reactions in the creation of the taste stimulus has been suggested by Duncan (1963). One of the basic features in the evaluation of taste activity, is the determination of the threshold value of the specific material, i.e., the minimum detectable concentration of the test substance. For comparison the following threshold values of common taste materials will be shown below: Material

Threshold value (%)"

~~

Sodium chloride Hydrochloric acid Sucrose Quinine

0.25 0.007 0.5 0.00005

Howell, 1922.

Since similar taste qualities may be achieved by compounds of different chemical structure, a brief discussion of the correlation between structure and sensation is pertinent. Sourness is achieved only by acids, most of their activity being probably due to the dissociation of hydrogen ions (Amerine et al., 1965). The sensation of saltiness is produced by many low molecular salts, both cations and anions contributing to a different extent to this sensation. As the molecular weight of the salt increases, in addition to saltiness other qualities arise, primarily bitterness. Thus, the saltiness decreases in the order: KCI, KBr, KI, while the bitterness increases accordingly (Moncrieff,

1951). While sourness and saltiness are produced by distinct groups of chemical compounds, bitterness and sweetness occur in a large variety of unrelated compounds. From the chemical point of view bitterness and sweetness seem to be very close. In fact there are a number of compounds which are structurally very close but cause distinct taste qualities. For example, 2-amino-4-nitropropoxybenzene is about 4000 times sweeter than sucrose, 4-amino-2-nitropropoxybenzene is tasteless, while 2, 4-dinitropropoxybenzene is bitter (Hornstein and Teranishi, 1967). Also, the taste stimulus is greatly affected by isomeric changes. Thus, o-nitrobenzoic acid is very sweet, m, slightly

38

F. MARGALITH AND Y. SCHWARTZ

sweet, and p , is bitter (Moncrieff, 1951). On the other hand, completely different molecules can produce a similar taste stimulus. Sucrose, saccharine (o-sulfonbenzimide), and cyclamate (cyclohexyl sulfamate) are widely known examples. OCH,CH,CH,

@

NH2

NO2

(2- Amino-l-nitropropoxybenzene)

o-”

OCH,CH&€&

NH2

(4-Amino-2-nitropropoxybenzene)

OCH,CH,CH, +NOZ

NO,

(2,4-Dinitropropoxybeneena)

Recently, the effect of the optical configuration on the taste activity of a number of compounds has been studied. The L-isomer of glucose has been found not to be sweet but slightly salty (Boyd and Matsubara, 1962). The anomers of mannose show a different taste activity, a-D-mannose being very sweet while p-D-mannose is very bitter (Steinhardt et aZ., 1962). Different tastes have been attributed to the stereoisoniers of amino acids. For example, L-isoleucine is bitter, whereas the D-isomer is sweet (Berg, 1953). The threshold value of flavoring material is considerably affected b y the presence of various promoting or depressing substances. For example, sucrose has a pronounced effect on the threshold value of sodium chloride. In a mixture of both ingredients, the presence of sucrose at a concentration of below 6% reduces the threshold for salt, while above this value, the sensitivity to salt is diminished (Bujas, 1934). On the other hand, the sweetness of sucrose was pronouncedly depressed by the presence of citric acid at very low concentrations (Pangborn, 1961).Clearly, the flavoring activity of a certain substance may be subjected to the enhancement or depression of other compounds even at very minute concentrations. In practice, a flavor enhancer may be defined as a seasoning material that improves the flavoring properties of a particular food product, sharpening and emphasizing flavors already present, without adding flavors of their own (Kurtzman and Sjiistriim, 1964; Stier et UZ., 1967). One of the early products of fermentation to be commercially promoted for the enhancement of flavor, was monosodium glutamate (MSG). This product is now widely used in food technology to emphasize meaty and other flavors. The exact nature of the activity of MSG is still unknown, although several controversial hypotheses have been suggested (Amerine et al., 1965). The picture was further

FLAVOR AND MICROORGANISMS

39

complicated when the flavor-enhancing activity of certain nucleotides was discovered. This may be due to a demasking action on certain receptors, thus exposing more sites to flavor sensation (Beidler, 1966). It seems that not only do these products affect foods by emphasizing present flavors, but also they indirectly influence their taste activity by changing the viscosity of certain foodstuffs (Wagner et al., 1963; Caul and Raymond, 1964). As pointed out earlier, flavor comprises a number of factors. So far attention has been given mostly to taste. Obviously, odor plays a prominent part in the sensation of flavor. In order that odor be perceived, molecules have to be accessible to the olfactory receptors. In other words, a certain degree of volatility of odoring materials is implied. The olfactory receptors or odor cells are located in the olfactory region in the roof of the nasal cavity. There are about 10 to 20 million cells, from each, minute cilia protrude into the cavity. Axons from the olfactory cells constitute the olfactory nerve that leads to the olfactory bulb of the brain. There are many theories to explain the mechanism of olfaction; Moncrieff (1951) mentions twenty-four of them. Today, the most commonly held view on the mechanism of olfaction is that of Davies and Taylor (1954), which in essence applies to the induction of the odor stimulus, a mechanism similar to that suggested by Beidler for the perception of taste. There have been many attempts to classify odors and to establish their basic components. Man can distinguish between an enormous number of different odors. However, Amoore (1952) believes only in seven primary odors: (1) etheral, (2) camphoraceous, (3) musky, (4) floral, (5) pepperminty, (6) pungent, and (7) repulsive. Other odors are considered to be the product of a combination of the primaries. He further believes that the sites of olfaction for different basic odors are distinct (Amoore et at., 1964). The sensitivity to odors is much more pronounced than that of taste. Some threshold values will be given for comparison (Moncrieff, 1951): Diethyl ether Vanillin

1 mg./m.3 of air mg./111.~of air

The different sensitivities of taste and odor can be demonstrated by ethyl alcohol with a threshold for odor of about 4 mg./liter air; while the threshold for taste would approach 130 mg./liter water.

40

P. MARGALITH AND Y .

SCIIWAHTZ

Little can be said about the correlation of the chemical structure of odor active compounds and odor perception. Similarly to taste, there are substances of completely different structures that elicit similar odor sensations, while others, with similar chemical constitutions produce completely different odors. The reader is referred to Moncrieff (1951) for a complete discussion of the problems related to chemical constitution and odor. Stereochemical aspects in the physiology of odor perception have been recently suggested by Amoore and Venstrom (1966). In dealing with flavor it is necessary to define the techniques of flavor evaluation. As previously stated, flavor is primarily the reaction of an individual to a certain compound via his sensory organs. Hence, the evaluation of flavor is greatly influenced by the person exposed to it. Age, sex, and culture as well as mood and alertness, all affect the results of a certain flavor test. There are a number of tests for the sensory evaluation of flavor, some more suitable for a certain case than others. Generally these tests are carried out by a panel of people employing one or more of the following methods: (1) difference; (2) rank order; (3) scoring; (4)descriptive; (5) acceptance and preference. For a complete evaluation of these sensory tests in the description and evaluation of flavor, the reader is referred to a number of texts (Arthur D. Little, Inc., 1958; Amerine et at., 1865; Moncrieff, 1966). A statistical treatment of results is usually employed in order to reach valid conclusions. Coming hack to the main topic of this review it seems appropriate to recall the classic paper by Omelianski who as early as 1923 collected information on the “aroma-producing microorganisms” - a designation today commonly used by microbiologists. In this paper Omelianski describes a long list of organisms related to the production of odor, from the sweetish scent that resembles limetree flowers of Pseudomonas p~ocyaneato the pungent odor of sweat formed by a culture of Bacillusfitzianus. For the sake of piquancy we would like to mention an unusual case, when Omelianski isolated the so-called Bacterium esteroaroma ticum from rabbit brain which, in culture, produced a pleasant fruity aroma resembling the odor of apples! II.

The Contribution of Microorganisms to the Production a n d Development of Flavor in Traditional Foods

A.

BAKEDPRODUCTS

Bread is probably the most common product of fermentation en-

FLAVOR AND MICROORGANISMS

41

countered in every day life. The flavor of bread is, however, the result of both the fermentation process in the dough and the crust formation during baking. In addition, the ingredients of the dough contribute to the final flavor of the baked product. In ancient times and practically up to the beginning of this century, the fermentation process of the dough consisted of a spontaneous fermentation. Part of the successful dough leading to the desirable product was then conserved and used for successive dough fermentation, thus constituting the barm of the sour dough or “sauerteig” of the bakery. Evidently, the fermentation taking place in the dough was due to the activity of a mixed population differing from place to place, and the ingredients employed for any particular bread. Various attempts have been made to analyze the microbial flora in the rising dough. Representatives of the genera Escherichia, Aerobacter, and lactic acid bacteria have been described. A number of organic acids including lactic and acetic acid as well as ethanol, diacetyl, and acetone were found to contribute to the specific flavor of sauerteig (Tanner, 1944). Brewers’ yeast were also employed for dough raising, leading to an excellent bread with a sweet and nutty flavor in the hand of the competent baker (Fance, 1960). When eventually the nature of fermentation became understood and the propagation of bakers’ yeast made their use increasingly popular, the old process of sauerteig was slowly abandoned. Modern bakery equipment as well as obvious sanitary regulations made the exclusive use of bakers’ yeast a necessity. Nevertheless, even today some bakeries employ the sauerteig process for the production of the characteristically flavored rye bread (Fance, 1960). In discussing the nature of dough raising as a spontaneous process, it is pertinent to mention briefly the problem of leavened and unleavened bread on the occasion of the Passover festivities celebrated by the Hebrews. The Law of Moses forbids the use of leavened bread during Passover. A practical point is however, how to define what may be considered unleavened bread. The Sages of the Babylon Talmud (Pessachim 46a) decided that leavening starts within the time required for walking 1 mile, i.e., approximately 18 minutes after the flour was brought in contact with water. Incidentally, this time evaluation is very close to the generation time of a rapidly dividing bacterium, e.g., Escherichia coli. Modern breadmaking employs several different processes for the production of bread. Three main types are currently distinguished. (1) The “Straight-Dough”Method. This involves only one mixing of all ingredients and a fermentation period of 2-4 hours before the

42

P . MARGALI’I’EI AND Y. SCHWARTZ

dough is divided and molded. This method however results in a nonuniform loaf and a less soft product. The quickest of the straightdough methods would be the “no-time dough,” where no fermentation time is allowed after mixing the ingredients. The dough is immediately molded and processed. This however results in a bread with poor flavor, in spite of the comparatively high concentration of yeast, which contributes to a yeasty taste of the bread but does not improve its flavor. This method is limited for emergency baking only. (2) The “Sponge and Dough” Method. This involves two distinct operations; first about 60% of the flour and water and the whole load of yeast are mixed and allowed to ferment for 3-5 hours; after that the sponge is mixed with the remaining ingredients. After an additional rest period the dough is molded and processed. Such loaves are usually of better quality in both physicaI appearance and taste. ( 3 ) The “Pre-ferment” Method. This is a comparatively recent technique developed for the use in continuous mixing. The pre-ferment consists usually of a mixture of yeasts, sugar, salts or milk, and water. The suspension is thus held for a number of hours, usually 6 at 100”F., with agitation, before it is mixed with the flour (Miller arid Johnson, 1958; Matz, 1960; Skovholt, 1964). Yeasts fermenting sugars produce ethanol and large amounts of carbon dioxide. The primary role of such a fermentation is, of course, the buildup of the voluminous texture of the loaf d u e to the format’ion of cells contained by the stretched gluten of the flour. Microbial activity in dough is, however, not limited to the metabolism of yeasts since it is well known that any commercially produced compressed yeast contain large amounts of a variety of bacteria. White (19S4) claims that compressed yeast may tolerate up to lo7bacteria per gram. This number is likely to increase during dough making as a result of reproduction and infection from other ingredients as well as bakery utensils. Moreover, this accidental bacterial population constitutes an important part of bread flavor. Carlin (1958)has shown that dough raised with a pure culture of bakers’ yeast yielded a loaf of bread with poor taste. However, most investigations dealing with the evolution of flavor during dough raising do not distinguish between the part played by the yeast and that of other microorganisms in the formation of taste and odor. A great number of different chemical entities were shown to be involved in the constitution of bread flavor. Most of these compounds are organic acids, alcohols, carbonyl compounds, and esters. Wiseblatt (1960) using a gas chromatographic procedure, showed that fermented dough made up in the laboratory, contained the following

FLAVOR AND MICROORGANISMS

43

acids in decreasing amounts: acetic, butyric, isovaleric, and caproic acids. In an examination of the organic acids in a “straight dough,” in addition to acetic acid, only traces of propionic acid were found (Wiseblatt and Kohn, 1960). In “pre-ferments” a large variety of organic acids were detected comprising, in addition to the aforementioned, formic, isobutyric, valeric, crotonic, isocaproic, heptylic, caprylic, pelargonic, capric, lauric, myristic, and palmitic acids (Hunter et al., 1961). Lactic and pyruvic acid were found by other workers (Cole et al., 1962; Johnson et al., 1958). Most of these acids were claimed to be produced during the first hours of the fermentation of the pre-ferment mixture. Most of the nonacid fractions of dough flavor constituents were studied during recent years in various preparations of pre-ferments. Cole et al. (1962, 1963) studied the kinetics of ethanol production in these systems. n-Propyl, isobutyl, amyl, isoamyl alcohols, as well as 2J-butanedio1, and 2-phenylethyl alcohols were also found in preferment liquids (Smith and C o h a n , 1960). These workers believe, however, that lower alcohols from pre-ferments were not involved in the ultimate flavor of baked bread. Carbonyl compounds formed during panary fermentation received the attention of many bread chemists. Kohn et al. (1961) investigated the carbonyl compounds from straight dough prepared with compressed yeast and with a special yeast preparation with a very low bacterial count, and made some very important observations with regard to the role of the different groups of organisms in the production of carbonyl compounds during fermentation. The following carbony1 compounds were analyzed: 2-hexanone7n-hexanal, isovalerylaldehyde, n-butyraldehyde, acetone, and acetaldehyde. A total of about 400 p.p.m. of carbonyl compounds were found in both preparations, with very little difference in the qualitative and quantitative spectrum of the individual carbonyl compounds. It was concluded that bacterial activity contributes little, if at all, to the formation of these compounds, which probably are derived from the activities of the yeast. Miller and co-workers (1961) who studied the carbonyl content of pre-ferments, found in addition to the above compounds also: formaldehyde, isobutyraldehyde, methylethyl ketone, nvaleraldehyde as well as 2-methyl-l-butanal, even though no attempt was made to study the origin of these compounds. Linko et al. (1962) extended this list of carbonyls detected in pre-ferments by adding propionaldehyde and n-hexaldehyde. Other workers identified acetoin (acetylmethylcarbinol) and diacetyl in pre-ferments (Smith and C o h a n , 1960). It seems, however, that the amount of carbonyl com-

44

P. MARGALITH AND Y . SCHWAHTZ

pounds detected in pre-ferments are very inferior to those of ordinary dough. Linko et al. (1962) further examined the effect of various bacterial cultures added to pre-ferments on their carbonyl content. It was found that of all the cultures studied only Pediococcus cerevisiue increased considerably the amount of propionaldehyde and acetone . The presence of esters in pre-ferments has been studied b y a number of workers. Obviously, ethyl esters predominate. Ethyl formate, ethyl acetate, ethyl lactate and 1,Spropanediol monoacetate have been detected in pre-ferments (Johnson et al., 1958; Smith and CofFman, 1960). However, there is little indication what part, if any, microorganisms take in the formation of these esters. It should be pointed out that compounds produced by different organisms during the panary fermentation, contribute only in part to the final flavor of bread owing to their great volatility and their disappearance during baking. Other flavor components are produced during crust formation to which microbial activities can contribute only indirectly, e.g., amino acids liberated during proteolysis of dough ingredients, will enhance the browning reactions taking place during baking. Also, Wiseblatt and Zoumut (1963) have shown that the reaction product between proline, abundant in flour protein, and dihydroxyacetone derived from the panary fermentation, is a compound with a characteristic crackerlike flavor that may constitute a major component of the organoleptic qualities of baked products. A number of interesting experiments have been carried out by different workers aiming at the flavor improvenient of baked products by the introduction of pure cultures of various bacteria isolated from different sources, which may be involved in the enhancement of bread flavor. Robinson et al. (1958) and Miller and Johnson (1958)examined the effect of pure cultures when added to the pre-ferment. These cultures were isolated from pre-ferments, aged sponge dough, and from dairy sources. Organoleptic examinations of bread, baked with such pre-ferments were carried out. It was found that certain bacteria improved the taste and odor characteristics of bread, especially Lactobacillus bulguricus 09, L . pluntarum, and L. breuis. However, no analysis of flavor components of such loaves were recorded. Similarly, Carlin (1959) investigated the flavor fortification of straight and sponge dough by the introduction of various bacteria from the genera Leuconostoc and Lactobucillus, previously isolated from commercial yeast, and found a significant improvement of bread flavor. Thus, it can be said that although modern bread making has abandoned the old pro-

FLAVOR AND MICROORGANISMS

45

cedure of sauerteig in favor of the compressed yeast method, the contribution of the bacterial flora to the development of the characteristic bread flavor has been well documented. The more recent attempts to introduce pure bacterial cultures to the yeast mix may therefore be regarded as a semisynthetic version of the original sauerteig procedure.

B. FERMENTED BEVERAGES 2 . Brewing and Flavor

Modern beer making has led to a change in the production of beer, not only with regard to the amounts of beer brewed in various countries but also in the quality of its taste and aroma; unfortunately, not always for the better. The introduction of the continuous fermentation of beer has so far only aggravated this problem. A better knowledge of the factors involved in the production and enhancement of beer flavor is therefore imperative. It is evident that the flavor of beer is d u e to a number of different factors originating in the raw materials used, e.g., the type of malt, nature and amount of adjuncts, hops etc., type of fermentation, aging, and processing. Since we are mainly concerned with the contribution of microorganisms to flavor, the reader is referred to a number of textbooks and more recent reviews for further information on the nonmicrobial factors involved in the formation of beer flavor (Hind, 1950; DeClerk, 1958). The primary contribution of brewers’ yeast to the taste of beers is, of course, ethanol. Although the ethanol content varies with the wort employed in the fermentation of the different beer types (ca. 2-6% w./w.) it is more likely that the characteristic flavor of a certain beer is due, to a greater extent, to other products of fermentation. It is generally agreed that higher alcohols (fuse1 oil), aldehydes, ketones, lower fatty acids, and esters, etc., determine the flavor quality of a certain brand. Employing conventional methods of analysis, Hartong (1963) suggested an analytical profile of beer aroma composed of the following groups: (a) higher alcohols, esters, aldehydes, and volatile acids, occurring at mg./liter concentrations, yielding a more or less favorable beverage; (b) diacetyl, hydrogen sulfide, and mercaptans appearing at pg./Iiter quantities that confer an unpleasant aroma to the product. Recent advances in the application of gas-liquid chromatography has greatly advanced our knowledge of the minor components of beer and their relationship to its flavor. Bavisotto and Roch

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P. MAHCALITH AND Y. SCHWARTZ

(1959) were probably the first to have studied the fermentation of beer employing gas chromatography. Following the formation of fusel oil components it was found that isoamyl alcohol could be identified very soon after the pitching of the wort, reaching maximal values at the time of the major fermentation period, i.e., within 3-5 days, comprising about 60 to 70% of the total fusel oil (Harold et al., 1961; Arkima and Shito, 1963). Pfenninger (1963) analyzed a large number of Swiss beers and found the fusel oil content to vary between 73 and 129 mg./liter. Further, a strong correlation between ethanol production and fusel oil formation could be established, thus confirming the metabolic relationship between these products of yeast activity. A number of interesting points with regard to the nature of beer fermentation and its effect on the formation of fusel oil were raised by Hough and Stevens (1961) who found that top beer always contained higher values of higher alcohols that bottom beer. Further, it was found that fermentations carried out at higher temperatures yielded higher levels of fusel oil. Similar results were obtained by Drews et al. (1964) who examined a large number of German lager beers. A change in temperature of fermentation from 7.5” to 1O.o”C. led to an increase in the higher alcohol content of the final product from 59 to 77 p.p.m. It was further claimed that agitation during beer fermentation increased the level of higher alcohols. Skating and Venema (1961) consider that in rating the characteristic beer flavor, the relative amounts of the alcohols is of importance. Analyzing Dutch beers, the amyl alcohols, i.e., isoamyl and optically active amyl alcohols, were followed in importance by phenethyl alcohol, isobutanol, n-butanol, n-propanol, isopropanol, n-hexanol, and furfury1 alcohol. The occurrence of aromatic alcohols in beer has been studied using gas chromatographic methods by Drews and co-workers (1965), who found in addition to phenethyl alcohol, also tryptophol and tyrosol. The concentration of aromatic alcohols varied in the German beers examined, according to the composition of the wort and temperature of the fermentation. Highest levels of phenethyl alcohols (44p.p.m.), tryptophol (4 p,p.m.), and tyrosol (28 p.1J.m.) were found in 3 Pilsner beers obtained by an “intensive fermentation.” It is generally agreed that the biogenesis of higher alcohols during beer fermentation follows the Ehrlich scheme for the transformation of amino acids or their precursors into alcohols via deamination and decarboxylation. The importance of nitrogen constituents of the medium on the regulation of the biosynthesis of higher alcohols via the Ehrlich scheme, has been discussed at length by Ayrapaa (1963).

FLAVOR AND MICROORGANISMS

47

Relatively little work has been done so far on the effect of yeast species and varieties on the final composition of flavor-determining components of the beer fermentation. Jenard and Devreux (1964) examined a number of strains of Saccharomyces cerevisiae and S. carlsbergensis and found significant variations between the formation of higher alcohols at the stage of the main beer fermentation. Bottom beer brewed with different yeast strains produced total higher alcohols in the range of 59 to 90 p.p.m., whereas top yeast yielded values ranging between 101 and 180 p.p.m. Drews et al. (1964) studied the effect of the flocculent nature of certain yeasts on the formation of higher alcohols and found that powdery yeast gave less n-propanol and more 2-methyl-l-butanol than flocculent yeasts. Ingraham and co-workers (1961) prepared a number of auxotrophic yeast mutants and studied their fermentation metabolites. Significant differences were found between the yields of the different alcohols. A leucineless mutant could not synthesize isoamyl alcohol, while an isoleucineless strain could not form 2-methylbutanol. A triple auxotroph requiring leucine, isoleucine, and valine would not produce isoamyl, 2-methylbutanol, and isobutanol but formed large amounts of nbutanol, which the wild type produces only in trace amounts. These observations may become of great importance in future studies on the development of better flavor under controlled beer fermentation. The glycerol content of various beers was investigated by Enebo (1957) who found it to vary between 1500 and 2000 mg./liter although little is known on the factors affecting glycerol production during brewing. A number of organic acids comprising acetic, formic, lactic, and traces of other acids, were found in beer. The origin of these compounds has not yet been established although there is good reason to assume that they are derived mainly from the activity of various contaminants during the initial fermentation. Ethyl acetate is the most abundant ester in beer followed by smaller amounts of ethyl formate, isoamyl acetate, and other esters. It is assumed that yeasts take an active part in the formation of these esters (Harold et al., 1961; Kepner et al., 1963; Masschelein et al., 1965). West et al. (1951)analyzed a large number of American beers and gave an average of 40 p.p.m. for total esters in beer; while Jenard and Devreux (1964) give the maximum of 27 p.p.m. for bottom beer and 82 p.p.m. for top beer. A “fruity” flavor has been described for beers with high ethyl acetate and isoamyl acetate levels (Hartl, 1964; Masschelein et al., 1965; Gilliland and Harrison, 1966). Acetaldehyde is considered to be a product of leakage of the alcoholic fermentation and attains considerable levels in beers. Maximum

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P. MARGALITH AND Y. SCHWARTZ

values of acetaldehyde have been recorded during the first days of the main fermentation. The “green” off-flavor of beer has been attributed to high acetaldehyde levels. The disappearance of this off-flavor during the latter part of the fermentation and storage has been correlated to a decrease in acetaldehyde (Enebo, 1957; Bavisotto and Roch, 1960; Sandegren and Enebo, 1961). Although diacetyl occurs in beer only at very low concentrations, it may be the cause of various off-flavors. West and co-workers (1952) analyzed a large number of beers with normal and off-flavor. They found that diacetyl at concentrations above 0.5 p.p.m. is responsible for these off-flavors. Although diacetyl is considered to be a normal product of yeast alcoholic fermentation, the excessive formation of diacetyl leading to the well known off-flavors are probably due to the activity of contaminants such as Pediococcus, Aerobacter, etc. (Sandegren and Enebo, 1961). However, Czarnecki and Van Engel (1959), and Gilliland and Harrison (1966) pointed out that high values for diacetyl in beer could be obtained also when respiratory-deficient mutants of S . cerevisiae were used. Gjertsen e t nl. (1964) believe that more diacetyl is produced during the fermentation with flocculent yeast than with powdery strains. Brenner et al. (1963) claim that the nutritional composition of the wort has a significant effect on the diacetyl formation during fermentation. A number of methods have been suggested to reduce the diacetyl content of beers in order to reach the organoleptically acceptable levels of below 0.2-0.3 p.p.m. From the present standpoint the most interesting method would be the practice of “Krausening” which leads to a more reductive environment (Sandegren and Enebo, 1961; Lawrence, 1964)The biogenesis of diacetyl in beer has been studied by a number of workers. West et al. (1952) considered dimethylene glycol to be the precursor of acetoin (acetylmethylcarbinol), and diacetyl. However, Sandegren and Enebo (1961) in a later report, suggested that acetoin is derived from the condensation of 2 moles of acetaldehyde; acetoin being oxidized to form diacetyl. A similar mechanism was suggested b y Antoniani (1961). On the other hand, Owades et aZ. (1959) and Yoshizawa (1964) suggest a scheme in which alanine serves as initial source for the formation of pyruvate which yields a-acetolactic acid and acetoin. Aerobic conditions favor the transformation of acetoin into diacetyl (Owades et al., 1959). In practice, acetoin in European beers was found within the range of 3.1 and 14.6 p.p.m. A number of factors seem to affect the formation of acetoin. Lower vitamin levels in wort as well as a deficiency in Mg ions seem to favor acetoin formation (Yoshizawa, 1964). An inositol-

FLAVOR AND MZCROORGANISMS

49

deficient strain was found to produce much higher concentrations of this compound (Lewin and Smith, 1984). Sulfur-containing volatiles are usually present in most beers at very low concentrations (p.p.b.) and are generally considered to be produced during the active part of the yeast fermentation. These compounds comprise hydrogen sulfide, mercaptans, thioformaldehyde, dithioformaldehyde, and thioacetone (Hashimoto and Kuroiwa, 1966). An interesting feature of yeast activity and its detrimental effect on beer flavor has been recently reported. Among the phenolic compounds, ferulic acid seems to be very common in grain mash and final beer. Certain yeasts seem to be involved in the decarboxylation of ferulic acid and the formation of 4-methylguaiacol and 4-vinylguaiacol which gave a pungent off-flavor to the beverage (Brumsted et al.,

1965):

(4-Methylguaiacol) OH

(Ferulic acid)

Recent advances in brewing technology and the new practice of continuous fermentation have led a number of workers to study the effect of batch versus continuous processes on the formation of taste and aroma of beers. Hudson and Stevens (1960) found that continuously fermented top beers did not differ significantly from those produced by conventional methods. However, in the case of the more common bottom fermentation Sandegren and Enebo (1961) found a number of interesting differences between the two kinds of fermentation. Beer produced by the continuous process contained higher nonvolatile acids and was of lower pH, showed greater accumulation of acetaldehyde, a higher fatty acid content, more higher alcohols,

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P. MARGALITH AND Y. SCHWARTZ

comprising higher values of phenylethyl alcohol, and apparently also a higher potential for the formation of sulfhydryl compounds. Nordstrom (1965) also found an increased production of volatile esters in the continuous process. In general, it is considered that the continuous fermentation of bottom beer bestows a more alelike flavor to the final product (Sandegren and Enebo, 1961). An interesting case of flavor formation in beer is the use of Brettanomyces and Lactobacillus pastorianus in the secondary fermentation for the production of the Lambic-type beer. The formation of the characteristic flavor is mainly due to the increase in acidity and was found to be optimal with a wort of comparatively high specific gravity (Gilliland, 1961). However, little is known on other flavoring compounds formed during such a secondary fermentation. In summary it may be said that microorganisms determine to a great extent the characteristic flavor of the final product. Recent advances in flavor analysis have opened new fields for the characterization of various beer types, although no definite correlations have so far been laid down.

2. Vinijication and Flavor Making wine is a very old practice. To cover all the technological developments which led to modern wine making is outside the scope of the present review. We shall limit ourselves to microbial activities concerned with wine making and their contribution to flavor. There are many textbooks and reviews which deal with this problem at varying lengths so that much of the material will be only briefly summarized or referred to. Similarly to brewing the taste and aroma of a bottle of wine is derived from a number of factors affecting the final product: raw materials, technology of vinification, and processing. However, wine making differs from brewing not only in raw materials and fermentation technology, but also in another aspect, i.e., in the less rigid control and hence much greater variability in all the stages of wine making. Climatic differences and a variety of agrotechnical factors have a pronounced effect on the nature of the chemical composition of must. It is, therefore, not surprising that many workers believe that the major contribution to flavor is due to the nature of the grape variety and climatic conditions of the corresponding vintage and only to a lesser part to the character of the microorganism involved during vinification. However, in recent years and with the advent of modern analytical approaches, the importance of the microorganism both dur-

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ing the main vinification and the secondary fermentation processes have been newly emphasized. The main product of vinification is of course ethanol. Although wine is characterized primarily by its alcohol content (that varies according to the sugar content of the must and fermentation technology, but may reach much higher concentrations than in beer and other alcoholic beverages), it is the emphasis on diminution of other bouquet components that determine the importance of ethanol to the perception of flavor. Thus, it has been recently shown by Rankine (1967) that the threshold values of a large number of higher alcohols, the importance of which is well known with regard to flavor evaluation, is increased over a hundredfold as compared to the values obtained in distilled water for the corresponding alcohols. Most of the higher aliphatic alcohols discerned in beer fermentation take part also in the higher alcohol composition of wine. The most important.ingredients of the fusel oil are: is0 and active amyl alcohols, isobutanol, and n-propanol. The most objectionable, with regard to taste and odor, is that of isoamyl alcohol. The average fusel oil content of wines has been examined by a large number of workers. In general the higher alcohol content of red wines is higher than that of white wines. Guymon and Heitz (1952) give a mean value of 250 p.p.m. for white Californian table wines versus 287 p.p.m. for red table wines, while Peynaud and Guimberteau (1962) find 309 p.p.m. and 394 p.p.m. (mean values) for the corresponding types of a large number of French wines. Although it is generally agreed that the fermenting yeast is responsible for the formation of the higher alcohols (Guymon, 1966), until the last decade comparatively little was done to examine the fusel oil-producing capacity of various yeast strains involved in vinification. However, as pointed out by Webb (1967) “the group of alcohols is usually present at concentrations low enough so that the sensory impression is not unfavorable.” The capacity of various yeasts to produce fusel oil components has been examined by a number of workers. Webb and Ingraham (1963) in their exhaustive review on the problem of fusel oil, summarize earlier observations and state that although various yeast genera comprising a number of wild yeasts, produce different amounts of fusel oil, little variation in the formation of fusel oil could be found in the naturally occurring wine yeasts. Nevertheless, Webb and Kepner (1961) noted considerable differences in the composition of fusel oil of wines that were fermented with three different wine yeasts with the same grape juice and under identical conditions of vinification.

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The highest level of n-propanol was found in the wine fermented with a Jerez yeast (20.2 weight % of total fuse1 oil) as compared to 2.6% of wine fermented with Montrachet yeast. Amy1 alcohols were highest in Montrachet fermented wine (94.7%) and lowest with Burgundy yeast (69.4%);Montrachet yeast produced the largest amount of isobutyl alcohol (16.5%). The most recent analysis of the ability of various species of Saccharomyces to produce higher alcohols has been reported by Rankine (1967). Vinification was performed in a number of grape juices employing different yeasts and the formation of alcohols was analyzed for each species. Significant differences were found between the different organisms. The following ranges were observed. (Means from different grape juices with same yeast.) n-propyl alcohol: minimum 13 p.p.m, (S. carlsbegensis No. 731) maximum 106 p.p.m. ( S . cereuisiae No. 350) isobutyl alcohol: minimum 9 p.p.m. (S. fructuum No. 138) maximum 34 p p m . (S. cheunlieri No. 317) amyl alcohols: minimum 115 p.p.m. ( S . cereuisiae No. 213) maximum 262 p.p.m. (S. cereuisiae No. 727)

The production by the various yeasts relative to one another seems to be consi5tent so that it would be justified to designate certain yeasts as high or low producer of some of the higher alcohols. Furthermore, it may be concluded that different strains of the same species seem to differ substantially in their capability to produce these alcohols. Guymon (1966) has also shown that mutants of S. cereuisiae would produce different amounts of the various higher alcohols according to their blocks in the biosynthetic pathways of their corresponding precursors. This seems to be in perfect agreement with results obtained in a similar approach with beer-fermenting yeasts (Ingraham et al., 1961). The reader is referred to Webb and Ingraham (1963)and Guymon (1966) for further information on our present knowledge on the biogenesis of various higher alcohols in wine fermentation. It is evident that organic acids have a very important place in the formation of aroma of wine. Recent analyses employing gas-liquid chromatography have revealed a large number of acids during vinification and maturation. Van Wyk and co-workers (1967)have published the acid content of a methylene chloride extract of White Riesling and found: acetic, n-butyric, n-caproic, n-caprylic, n-capric, 9-decenoic, and succinic acids as major constituents. Formic, propionic, isobutyric, 2-methylbutyric, isovaleric, lactic, 2-hydroxyisocaproic, n-pelargonic, and malic acids were found in smaller amounts. However, no distinction between the origin of these acids, if derived from the grape

FLAVOR AND MICROORGANISMS

53

juice or formed during vinification, has been suggested. Volatile acids, primarily acetic acid, are produced during bacterial-free alcoholic fermentation in considerable amounts. Amerine and Cruess (1960) give the values of 0.03-0.05% in different wines as compared to 0-0.02% in the respective musts. The excess of acetic acid, over 0.05% is an indication of bacterial spoilage. However, a number of yeasts are known to possess a greater volatile acid-producing ability. Cappucci (1948) claimed to have found up to 0.369%volatile acidity in normal fresh wines and attributed this to the activity of apiculated yeasts and certain Zygosaccharomyces. Although wine spoilage is generally considered to be due to the formation of excess acetic acid, it has been shown experimentally that the organoleptic deterioration of wine is not due to the accumulation of volatile acids, but rather to the formation of high levels of ethyl acetate (Amerine, 1954; Ribereau-Gayon and Peynaud, 1961). With regard to the biogenesis of acetic acid during the yeast fermentation, it seems that, oxidation-reduction potentials are involved in the oxidation of acetaldehyde to acetic acid and its reduction. It has been pointed out that during the initial stages of fermentation, levels of acetic acid are considerably higher than toward the final stages (Joslyn and Dunn, 1941; Ribereau-Gayon and Peynaud, 1946). However, no definite evidence has been put forward for the biochemical pathways and enzymes that perform these reactions. Other volatile acids, such as formic, propionic, and butyric acids, are probably not derived from the yeast fermentation. In spite of the pungent odor of these compounds their occurrence of, only in very minute amounts, in normal wine may contribute favorably to the taste of this beverage (Webb, 1967). Among the nonvolatile organic acids produced during vinification, lactic acid occupies a very important position, both from the practical standpoint and biochemical interest. Lactic acid occurs in all wines that have undergone the vinification process. Wines that have had no bacterial contamination, contain usually only very low levels, up to 0.06% of lactic acid, which has been recently shown to be predominantly of the D(-) isomer (Peynaud et al., 1966). In a later work (Peynaud and co-workers, 1967) the formation of lactic acid by various yeasts has been examined under controlled conditions of vinification. It was found that there exists considerable variation among the species of Saccharomyces. Saccharomyces rosei gave the lowest level 101-135 mg./liter, while most of the saccharomycetes produced around 200400 mg./liter. An interesting exception was found with S. veronae which produced up to 1800 mg./liter and was further distinguished by

P. MARGALITH AND Y. SCHWARTZ 54 the fact that most of the acid formed was of the L(+) isomer. The contribution of lactic acids formed during yeast vinification to the flavor of wine, has so far received little attention. The most important feature of lactic acid in wines, with all its organoleptic implications is, however, not due to the activity of yeasts but to the formation of lactic acid by certain groups of bacteria. Since the early years of wine microbiology (see Ribkreau-Gayon and Peynaud, 1961, for an excellent historical review) it was known that certain bacterial processes lead to a change in the acidity of wines after the alcoholic fermentation was completed. A reduction of acidity is of great importance in many cases of table wines that are produced from juices particularly rich in total acidity. According to Amerine and Cruess (1960) almost half of the acidity in various musts is due to the presence of L-malic acid (0.1-0.8%). Malic acid, a relatively strong dicarboxylic acid is readily attacked by many lactobacilli, which thus reduce the acidity of the wine by the production of the weak, monocarboxylic lactic acid according to:

NADH,

, lactic dehydrase +&

5

7% CH(OH)COOH

Most of the lactic acid thus produced has been shown to be of the isomer (Peynaud et d., 1966). Various workers have studied the biochemical mechanisms involved in the decarboxylation of malic acid and the reduction of the intermediate pyruvic acid to form the lactic acid. A number of coenzymes and cofactors have been described (Ochoa, 1951; Jerchel et al., 1956).An important point is sometimes overlooked. Thermodynamically, the reaction that leads to the decrease in malic acid is an energy consuming process, hence the malolactic fermentation requires the presence of a source of energy (Schanderl, 1943). However, as pointed out by Radler (1958) the amounts of carbon compounds that drive this reaction are minute and affect little, if at all, the composition and flavor qualities of the wine. This is in sharp contrast to the reduction of malic acid, which may totally disappear from the wine (Pilone and Kunkee, 1965). Although the involvement of lactobacilli in the malo-lactic fermenL(+)

FLAVOR AND MICROORGANISMS

55

tation has been known for a long time, the exact nature of the microorganism was studied little until the early fifties. Ribereau-Gayon and Peyiiaud (1961) describe in detail most of the work done until 1960. Hornofernientative and heterofermentative Lactobacillus (L. plantarum, L . delbriikii, L. brevis, and L. hilgardii) as well as homofermentative Pediococcus, ( P . cerevisiae) and heterofermentative Leuconostoc (L. citrovorum) have been described in wines that had undergone the malo-lactic fermentation (Lambion and Meskhi, 1957; Ingraham et al., 1960; Fornachon and Lloyd, 1965). Although the malo-lactic fermentation can be easily demonstrated under laboratory conditions, it occurs frequently but not always under commercial conditions. This is probably due to the inability of the lactic culture to become established owing to a number of factors such as high sulfitage, high alcohol content, and early separation of wine yeasts by racking which thus deprives the wine from the growth factors required by the lactobacilli, etc. (Fornachon, 1957). This author also stressed the importance of the p H as observed with Australian wines. It seems that not only is the development of the malolactic culture sensitive to low p H values (lower limit 2.9-3.0), but the nature of the lactic culture that becomes established differs with the pH. Other factors which control the outcome of the malo-lactic fermentation have been recently reviewed by Kunkee (1967a,b). The time interval between the alcoholic fermentation and the onset of the malo-lactic activity was found to be about 3 weeks (Rice, 1965). Frequent failures in the commercial application of the malo-lactic fermentation have led many workers to attempt the reduction of acidity in wine by introducing an abundant starter population of a selected lactic organism. French authors, who strongly advocate the use of the malo-lactic induction by massive inocula, expect its largescale application to be realized in the very near future. RibereauGayon and Peynaud (1961) suggest the use of homofermentative lactobacilli for this purpose. This is in accordance with the widely accepted view that heterofermentatives produce considerable quantities of flavor affecting volatiles, such as acetoin and especially diacetyl, which may become deleterious to wine bouquet (Vaughn and Tchelistcheff, 1957). However, American enologists ?re not in complete agreement with these observations. Pilone et al. (1966), who made a careful study of experimental vinification of Californian wines employing pure lactic cultures and modern procedures of analysis, found that malo-lactic reduction of acidity is carried out by various lactic organisms, with a slight increase in volatile acidity, and changes

56

P. MARCALITH AND

+

Y. SCHWARTZ

in the acetoin diacetyl values (control 7.5-8.4 p.p.m. versus 4.913.9 p.p.m. in the malo-lactic wines) which do not support the conclusion that homofermentatives are less productive with regard to diacetyl and acetoin. A number of other compounds seem also to be affected by the malo-lactic fermentation. Significant higher values have been found for ethyl lactate in the malo-lactic wines, while hexyl alcohol appears to diminish. The importance of these changes with regard to flavor has not yet been elucidated. However, as pointed out earlier (Pilone and Kunkee, 1965), while organoleptic tests revealed significant differences between normal and malo-lactic wines, differences between wines produced with a number of different lactic cultures could be distinguished only by some of the members of the taste panel. Considerable quantities of succinic acid are known to be formed during the alcoholic fermentation. Levels of up to 0.15%have been recorded in wines (Amerine and Cruess, 1960). Little is known about their specific contribution to the taste of wine. Recently some information became available regarding the contribution of various microorganism to the formation of ketonic acids in fermented juice. LafonLafourcade and Peynaud (1966) found up to 785 mg./liter of pyruvic acid and up to 146 mg./liter of a-ketoglutaric acid in wines of different vintages. Yeasts related to the vinification process seem to have different abilities with regard to the production of these keto acids. Low values were obtained with wild yeasts, such as KZoeckera apdcuZata, Hansenula anomda (less than 100 and 30 p.p.m., respectively) while strong alcohol-producing yeasts formed higher levels; in the case of Schixosaccharomyces pombe up to 830 and 284 p.p.m., respectively. The effect of these acids on the formation of wine bouquet has not yet been studied. The importance of esters formed during vinification to the formation of wine aroma has been studied over many years. Ethyl acetate is the most prevalent ester in fermented must and, as pointed out by Amerine (1954), constitutes the only ester important from the standpoint of taste and aroma. Other esters such as ethyl caproate, heptanoate, caprylate, pelargonate, isoamyl acetate, and others have been identified in various wines. Van Wyk et al. (1967) enumerate a total of 22 esters found in Riesling wine. The content of ethyl acetate in wine varies according to the grapes employed and the vinification process. Ribkreau-Gayon and Peynaud (1961) who analyzed the composition of wines during several decades found wines to contain generally between 44 and 176 mg./liter ethyl

FLAVOR AND MICROORGANISMS

57

acetate; seemingly, wines can tolerate up to 200 mg./liter of ethyl acetate, and above this value appears to give a spoiled character to the wine (Amerine and Cruess, 1960).It is now generally agreed upon that the formation of esters is due to the metabolic activities of microorganisms and little, if at all, to chemical esterification. RibbreauGayon and Peynaud (1961) distinguish between 5 different groups of microorganisms with regard to their production of ethyl acetate. First, the true wine yeasts of the genus Saccharomyces which never produce more than 50 mg./liter. Second, yeasts from the group of: Candida pulcherima, Kloeckera africana, and Brettanomyces which may enhance the ethyl acetate content of wine up to 110 mg./liter, however, these are of little practical importance. Third, .Saccharomycodes ludwigii which differs from the other yeasts in the fact that its esterogenic activity is greatly enhanced under anaerobic conditions. Grape juice fermented with S . ludwigii has a very pronounced odor of this ester (up to 200 mg./liter). Fourth, the abundant group of apiculated wild yeasts of the Kloeckera-Hanseniaspora type which produce over 300 mg./liter of the ester and fifth, the group that comprises yeasts such as Pichia spp. and more important, Hansenula anomala which apparently has the strongest esterogenic activity. The latter was found to produce up to 900 mg./liter of ethyl acetate and thus may be detrimental to the process of vinification. With the exception of Saccharomyeodes ludwigii all esterogenic activity is promoted by contact with air. Under these conditions various species of Acetobacter may proliferate with a concomitant increase in the formation of ethyl acetate. Tabachnick and Joslyn (1953) have studied the kinetics of ethyl acetate formation and found that levels of the ester decrease with time, under aerobic conditions. This seems to be in accordance with the observation of Wahab et al. (1949) who found, that on aging, the ester content of wines decreases substantially with a constant improvement of wine bouquet. Ough and Amerine (1967) report the optimal temperature for ester formation to be in the vicinity of 20°C. Since early days of wine microbiology it was known that glycerol is a constant by-product of the alcoholic fermentation. Its sweet taste and viscous character undoubtedly has a pronounced effect on the character of the fermented product, especially what the enologist calls “body.” Others believe that glycerol imparts smoothness and ameliorates the burning taste of alcohol (Hickinbotham and Ryan, 1948; Hinreiner et al., 1955). Glycerol in wines may be detected organoleptically at 0.9% in white wine and 1.3% in red wine (Hinreiner

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et al., 1955).The usual concentration of glycerol in wines, as given by Amerine (1954), is in the range of 0.5-1.5% and therefore may be regarded as a significant constituent of wine flavor. The effect of temperature of vinification, the concentration of sulfite, sugar, and other factors have been studied by a number of workers (Venezia and Centilini, 1941; Hickinbotham and Ryan, 1948; Uchimoto and Cruess, 1952; Gentilini and Cappelleri, 1959). In the case of botrytized grapes, unfermented juices may contain already considerable quantities of glycerol due to the metabolic activity of the fungus Botrytis cinerea. (For a summary of the information on B. cinerea see Ribereau-Gayon and Peynaud, 1964.) A number of workers have studied the glycerol-forming ability of various yeast species and strains. Interestingly, strong fermenting yeasts show considerable similarity in their glycerol production under experimental conditions. Usually levels of 0.6-0.9% are found. Zygosaccharomyces acidifaciens seems to be outstanding with respect to its glycerol-prod\icing capacity, since it was found to yield over 1.5% under similar conditions (Ribereau-Gayon and Peynaud,

1964). Do iiorrnal wine yeasts contribute to the level of carbonyl compounds during vinification? There seems to b e little agreement with regard to the quantities of acetaldehyde found in wines. Ribkreau-Gayon and Peynaud (1961) give the range of 40-120 p.p.m., while Ough and Amerine (1967) found much lower values, 6-49 p.p.m. These differences may be due to different analytical procedures, but also may depend on the concentration of sulfite during vinification. Sulfite, that entraps acetaldehyde, prevents its reduction and thus leads to higher levels. However, as pointed out by Hinreiner et al. (1955), at these concentrations acetaldehyde in wine contributes little to the organoleptic qualities of the beverage, the threshold levels being 100-125 p.p.m. in red and white table wines, respectively. Acetoin and diacetyl are found at much lower concentrations. Fornachon and Lloyd (1965) give the values of 0.7-0.9 pap.m. and 0.1-0.3 p.p.m., respectively, for table wines, as against the much higher levels found by Ribereau-Gayon and Peynaud (1961) 4-25 p.p.m. and 0.5-2.5 p.p.m., respectively. Clearly, various factors such as temperature of vinification, aeration, and agitation strongly affect the levels of these carbonyl compounds (Crowell and Guymon, 1963; Ough and Amerine, 1967). Carefully controlled experiments conducted by Guymon and Crowell (1965) have shown that both sugar solutions and grape juice inoculated with a pure culture of S. cerevisiae (Montrachet strain) produced acetoin and diacetyl, reaching a maximum of 25-100 p.p.m.

FLAVOR AND MICROORGANISMS

59

(combined) about midway through the vinification process, while declining strongly toward the end of the fermentation. As expected, fortified wines yielded much higher values (up to 236 p.p.m.). From the organoleptic standpoint, both acetoin and its reduction product 2,3-butylene glycol, have apparently little importance, although the former is considered to be the precursor of its oxidized form diacetyl, which may be detected at very low levels. According to Amerine (1954) diacetyl in wines may be detected already at 2-4 p.p.m. levels, which as pointed out earlier, may be reached in certain normal wine fermentations. Whether very low diacetyl levels formed during vinification may contribute favorably to the characteristic qualities of certain wines is still a matter of controversy. Hydrogen sulfide and mercaptans in fresh wines may be frequently encountered (Amerine and Cruess, 1960). Considering the very low threshold values of these compounds [0.1-1.0 p.p.m. for hydrogen sulfide, according to Staudenmayer (1961)], their occurrence in wine are of primary importance in the sensory evaluation of this beverage. Various workers have attempted to reveal the origin and mode of formation of these compounds during vinification. It is generally assumed that most of the hydrogen sulfide is derived from sulfur occurring on sprayed grapes or used in the wineries, and to much lesser extent due to the reduction of sulfur dioxide or bisulfite (Rentschler, 1951). However, other workers also consider organic compounds such as sulfur-containing amino acids, metabolized during vinification, to contribute significantly to the accumulation of reduced sulfur compounds (Benvegnin et d.,1951; Woll, 1955). From our standpoint, however, it would be interesting to elucidate the contribution of yeasts employed during the vinification process, to the formation and accumulation of such undesirable by-products of fermentation. Much of the information available today on this problem is due to the thorough investigation carried out by Rankine (1963). In a number of carefully controlled experiments followed by gas-liquid chromatographic analysis, it was found that the accumulation of H2S during fermentation is strongly correlated to the decrease in the oxidation-reduction potential during the alcoholic fermentation. The yeast varieties employed in these experiments displayed considerable variation with regard to the HzS-forming ability. Values of 0.0 to 7.0 p.p.m., in the absence of any inorganic sulfur compounds, and up to 12 p.p.m. in the presence of elemental sulfur, were obtained. Rankine concludes that most of the hydrogen sulfide in wines is derived from the reduction of elemental sulfur and only to a lesser extent to other inorganic and organic compounds. Ethanethiol, which is probably the

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most deleterious compound, is considered to be formed from H2S and ethanol, although the mechanism of its production has still to be worked out. In spite of the occurrence of different yeasts with different hydrogen sulfide-forming abilities, and the practical aspects which may be derived from this fact, it is not known whether under commercial conditions of vinification, yeast are directly responsible for the sulfur reductive reaction or only indirectly through the low E h values developed during fermentation. One of the most interesting cases in which a microbial process leads to the transformation of a fermented product into a beverage of higher organoleptic and commercial qualities, is the flor type sherry. This wine originated in the region of Jerez de la Frontera where the normal alcoholic fermentation (14.5-15.5% volume alcohol-for the characteristic fino-type) is followed by a secondary process carried out in the so-called solera-system. There, in a number of barrels filled only about 80% of their volume, the wine undergoes an oxidative process owing to the development of a certain film yeast-theflor yeast. Fresh wine is introduced into such a system, transferred from one layer of barrels to a successive one, about six times, until the characteristic flavor of the flor sherry is acquired. For a detailed description of the process see Bobadilla (1943) and Schanderl (1959). Recent advances in wine technology have introduced other methods of sherry production with the aim of eliminating the time consuming solera procedure, by either promoting the flor yeast under submerged culture condition with occasional agitation and aeration or by the “baked process” without the intervention of the microbial stage (Ough and Amerine, 1960). There is little doubt, that this very complicated wine with its distinct aroma, as produced by the traditional Spanish solera system, is a result of the activity of the typical flor yeast. There exists some argument with regard to the speciation and nomenclature of this yeast. Some workers believe flor yeast to belong to a special species: Saccharomyces beticus (Marcilla et al., 1936) or s. fermentati (Rankine, 1955; Castor and Archer, 1957) while others believe that the flor yeast is but the oxidative stage of a normal wine yeast, of which many strains form the pellicle stage when cultured under suitable conditions (Schanderl, 1959). The chemical transformations that the sherry wine undergoes during the oxidative stage are very complex and have been studied by a large number of enologists. The principal flavor components characteristic of sherry wine are aldehydes and acetals. Acetaldehyde is quantitatively the most important. During “sherryzation,” amounts of

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over 300 p.p.m. aldehydes may be formed during commercial production (Marcilla et al., 19361, while Amerine (1958) reported much higher levels of about 1000 p.p.m. under experimental condition employing the Californian method of submerged culture. Considering the fact that normal wines contain only minor amounts of aldehydes, it is clear that these compounds contribute significally to the formation of the sherry wine aroma. However, there is still a scarcity of information with regard to the optimal concentration of aldehydes in sherry for the consumers’ market. A blending procedure for the production of sherry with conventional concentrations of acetaldehyde has been proposed by Webb et al. (1964) for the Californian types of submerged sherry. Acetals are the product of condensation of aldehyde with two molecules of alcohols and the elimination of water: CHSCHO

+

2C2H5OH

(Acetaldehyde)

(Ethanol)

OCzH, / CHsCH \ OCzHs

+

HzO

(Diethyl acetal)

Although there are indications of the occurrence of acetals in wine grapes and normal wines (Kepner and Webb, 1956; Webb and Kepner, 1957; Lipis and Mamakova, 1963) few quantitative data are available. However, it is assumed that the “sherryzation” process increases significantly the amounts of acetals present in sherries (Webb et al., 1964), and thus, contribute considerably to the specific character of these wines. With the advent of gas chromatographic analysis further insight has been gained with regard to the qualitative constitution of the acetal compounds. Diethyl acetal was found in higher amounts in flor sherry than in the submerged culture. Galetto et al. (1966) detected eight other acetals: ethyl-active amyl, ethyl-isoamyl, ethylpentyl, isoamyl-pentyl, diactive amyl, active amyl-isoamyl, active amyl-pentyl, and diisoamyl acetal. Although it has been proposed to evaluate the organoleptic qualities of sherries according to their acetal constituents, there is little evidence that the acetals are formed during the microbial stage, since similar amounts of diethyl acetal could be demonstrated in “baked” sherries (Webb et al., 1964). Both flor sherry and the “baked” product have been shown to contain acetoin in the usual range of 10-32 p.p.m. (Lukas, cited in Schanderl, 1959; Guymon and Crowell, 1965). However, the submerged product is outstanding in its acetoin content which may be as high as 350 p.p.m. Undoubtedly, the aerated culture leads to a higher

62

P. MARCALITH

A N D Y. SCHWARTZ

metabolic activity of the flor yeast as compared to that of the solera system. The effect of flor yeast on the accumulation and degradation of alcohols during “sherryzation” has been studied in various systems. Ethanol was found in some cases to decrease somewhat (Schanderl, 1959; Webb et al., 1966) which, at least in part, may be attributed to evaporation. Glycerol has been found to decrease considerably. Marcilla et ul. (1936) claim a 50% reduction of the glycerol content after the completion of the flor process. Similar results were obtained in the wbmerged culture sherry (Amerine, 1958). In addition to the customary fuse1 compounds, benzyl alcohol, and 2-phenylethyl alcohol were found in flor sherry of Spanish origin (Webb et al., 1967). Numerous publications deal with the occurrence of other alcohols, acids, and esters as determined b y gas-liquid chromatography. Lactones, ethyl-.l-hydroxybutyrate, ethyl pyroglutnmate, diethyl siiccinate, diethyl nialate, and 2-phenylethyl caproate were identified. It is suspected that the presence of acetate and caproate esters of 2-phenylethyl alcohol have a special place in the formation of the typical sherry flavor (Webb and Kepner, 1962; Webb et al., 1964; Rodopulo et ul., 1967). However, information on the quantitative arid organoleptic aspects of the sherry aroma components is still fragmentary. It should be pointed out that the contribution of flor yeast to the totii’l aroma edifice of shcrry can be assessed only if uoniparative analyses are carried out with fresh wine, before and after the termination of the sherry process. Comparatively little has been published on the production of flavor in nongrape alcoholic beverages. Apple cider is the best known example of such fruit juice that undergoes an alcoholic fermentation ( 5 4 % v./v., ethanol). Pollard et u1. (1966) reviewed the flavor components of apple cider and pointed out that the most important change during the processing is the transformation of the fruity acid character of the juice to the softer mature flavor of the cider. It has been shown earlier (Whiting and Coggins, 1960) that these chaliges are partly d u e to bacterial activities during or after the yeast fermentation. Lactic acid bacteria may attack sugars in nonsulfited juices with pH above 3.8. Concurrently, malic acid is converted to lactic acid in a manner similar to the malo-lactic fermentation in wine. Also the yeast fermentation contributes to the general increase in cider acidity due to the significant formation of succinic acid. (This is obscured in the grape juice fermentation owing to the changes in the tartaric acidity in the C02-saturated environment.) There was little difference between the yeast strains employed (Thoukis et ul., 1965).

FLAVOR AND MICROORGANISMS

63

Another component of microbial origin associated with the desirable flavor of cider is the relatively high level of fusel oil. Although some of these higher alcohols may be present in certain varieties of apple juices, the bulk of this fraction is derived from the yeast fermentation. In comparing the content of higher alcohols of apple juice and cider, it was found that with the exception of n-butanol, alcohols increase significantly during the fermentation, primarily isobutyl, is0 and active amyl alcohols as well as 2-phenylethanol. In a series of laboratory experiments it was shown that the formation of apple juice by its natural microflora usually resulted in a much higher fusel oil content than when the fermentation was carried out with a culture yeast used in this process: 197-335 p.p.m. versus 151-167 p.p.m., respectively; in the case of 2-phenylethanol 127-254 p.p.m. as against 33-65 p.p.m. with the culture yeast. Usually the fusel oil content of ciders are intermediate between that of low gravity beers and wines, which would correlate with their respective ethanol content. However, in the case of cider the scented character of some of these alcohols may constitute an important part of its aroma. Taste panels have shown that u p to 200 p.p.m. of higher alcohols were considered essential to typical cider aroma (Pollard et al., 1966). Sake is the most widely known fermented beverage of the Far East. Production involves a mixed fermentation of rice, employing Aspergi2lus oryzae for the transformation of the starch into fermentable sugars, as well as a yeast for the alcoholic fermentation, and some bacteria, such as Lactobacillus saki. The characteristic Sake flavor arises thus directly or indirectly from the constituents of rice. Recent analytical studies have shown that most of the fermentation byproducts detected in the conventional alcoholic fermentations such as fusel oil, acids, and carbonyl and phenolic compounds, have also been detected in rice wine (Yamamoto, 1961; Komoda et al., 1966; Owaki, 1967). The analysis of Sakk flavor, however, reveals little of the changes that occur due to the chemical activities of each organism involved in the mixed fermentation. A significant increase in the content of tricarboxylic acids as a result of fungal metabolism has been demonstrated during the initial stages of the fermentation (SakC-Koji). Recently more information has become available on the formation of phenolic compounds during the Sake fermentation. Yamamoto et al. (1961) identified ferulic acid, vanillin, and vanillinic acid in SakC, while in the Sakk-Koji only ferulic acid could be demonstrated. It was assumed, therefore, that the transformation of ferulic acid, which constitutes a minor component of the plant material, was carried out by the microorganisms involved in the later stages of the SakC fermen-

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P. MARGALITH AND Y. SCHWARTZ

tation. In a later work, Omori and co-workers (1968) describe the metabolism of ferulic acid by a pure culture of a Sake yeast under laboratory conditions. It was found that both p-hydroxybenzoic acid and vanillic acid were formed. When the substrate was changed into vanillin, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, and vanillic acid could be identified. It was concluded that vanillin may be formed as an intermediate in the degradation of ferulic acid, followed by the demethoxylation of vanillin. p-Hydroxybenzoic aldehyde and acid have also been shown to be formed from amino acids like tyrosine by a number of yeasts. However, it is known that vanillin is a more important component of Sake flavor, contributing to its light and sweet fragrance (Yamamoto et al., 1966).

C. DAIRYPRODUCTS The contribution of microorganisms to the development of specific flavors associated with milk products is widely known, although de$,tailed studies to provide deeper insight into the chemical entities involved in many processes are still needed. Milk products such as sour milk, yogurt, cream, and cream cheeses as well as butter, which undergo practically no aging processes, have taste characteristics due in part to the lactic acid produced during the manufacturing. The typical dairy aroma of these products is developed by a specific aroma-producing microflora, predominantly from the heterofermentative group of Leuconostoc organisms. M’ilk citrate is femiented by Leuconostoc dextranicum or L . citrouorum to give diacetyl (2,3-butanedione) as well as acetylmethylcarbinol and 2,3-butanediol. Some acetic and propionic acids are also produced. The important contribution of diacetyl to flavor is now well recognized and detailed information on the handling of such lactic cultures in relation to their aroma-producing capacities may be found in various textbooks on dairy microbiology (Foster et al., 1957; Hammer and Babel, 1957). The biogenesis of diacetyl has been amply discussed by Lindsay (1966). The formation of diacetyl, however, is not limited to the leuconostocs only. It has been shown that certain streptococci ferment citrate, producing considerable quantities of diacetyl (Swartling, 1951). A serious drawback in the propagation of these aroma bacteria would be the formation of great amounts of acetaldehyde, which impairs flavor and contributes to the so-called “green flavor” of dairy products (Keenan et al., 1966). In pure culture experiments it was

FLAVOR AND MICROORGANISMS

65

found that the greatest amount of acetaldehyde was produced by Streptococcus diacetilactis, followed by S . cremoris and S . Zactis. The advantage of S . diacetilactis in producing diacetyl in the absence of acid-producing organisms (Pack et al., 1968) is thus overshadowed by its abundant acetaldehyde production, which has been shown to attain 6 to 10 times the level of diacetyl (Keenan et al., 1966).According to Lindsay et al. (1965) the flavor balance of butter cultures was found to be closely related to the diacetyl: acetaldehyde ratio. Desirable full flavored cultures exhibited ratios from 3:l to about 5:l. Streptococcus thermophilus is known to produce diacetyl in yogurt, while Lactobacillus bulgaricus has been shown to form acetaldehyde in amounts adequate for the typical yogurt flavor when cultivated in the presence of S . thermophilus (Schulz and Hingst, 1954). Acidophilus milk which is widely advocated for the therapeutic use in certain gastric disorders differs from yogurt in its lesser “buttery” and more astringent flavor. These differences are probably due to the different metabolism of L. acidophilus although the nature of the flavoring components found in acidophilus milk has not yet been worked out (Davies, 1963). The contribution of lactic organisms and other microflora to the flavor of dairy products that are regularly aged is much more difficult to assess. Various kinds of cheeses have been analyzed for their microflora and flavoring components but it is extremely difficult to distinguish between the role of the living microbial population, the oxidative processes affecting various milk ingredients or their enzymic transformations, and the contribution to the development of cheese flavor during maturation by autolytic enzymes originating in the microflora of the product. However, it is clear that microorganisms decide the course of evolution of cheese flavor. This could be shown by the technique of Mabbitt and co-workers (1955) who obtained cheddar cheese by an aseptic procedure using 8-gluconic acid lactone as acidulant. Such cheese was devoid of any cheese flavor (Reiter et al., 1966). Although these experiments repudiate the early doubts by Mabbitt (1961) as to the contribution of microorganisms to cheddar flavor, it cannot be stated with certainty what starter cultures are most suitable. Various streptococci, such .as S . cremoris and S. lactis, are generally used in starter cultures, But lactobacilli as well as micrococci and other bacteria increase in number during maturation. Using the aseptic technique, the Reading group has demonstrated that cheeses made with starter organisms alone, had a distinct flavor although they did not develop the fullness of flavor obtained with

66

P. MAHGALITI-1 A N D Y . SCHWAHTZ

ordinary cheddars. Hence, starter organisms should be considered responsible only in part for the development of cheese flavor (Reiter et al., 1967). Undoubtedly, proteolysis has its share in the formation of cheese taste. Amino acids and their degradation products such as tyramine, cadaverine, putrescine, histamine, tryptamine, ammonia, and hydrogen sulfide, have been claimed to contribute to the flavor of cheese. Today, these substances are more considered to be of secondary importance, providing the background for the typical flavor (Reiter et al., 1967). Although rennet is the main source of proteolytic enzymes, nonstarter cheeses failed to develop any flavor resembling that of normal cheese. Hence, such proteolysis cannot be responsible for cheese flavor (Mabbitt et al., 1955). That proteolysis may play an important part in the off-flavor of milk products has been indicated b y various workers. Bitter flavor has been shown to be due to “bitter” strains of S. cremoris which seem to form a peptide that causes the bitter sensation. Although this peptide is probably derived from milk casein, its mode of formation is yet unclear (Emmons et al., 1960; Czulak and Shimmin, 1961). It is generally considered that the free fatty acids (FFA) are associated with flavor intensity of cheeses (Patton, 1963).Acetic, propionic, butyric, and higher fatty acids have been identified. Microorganisms may produce lower fatty acids from carbohydrates, but it seems more likely that the greater part of the FFA are derived from milk fat through lipolysis (Reiter et al., 1966). Although lipolytic organisms such as micrococci and gram-negative bacteria may contribute considerably to the FFA components of flavor, the weak lipolytic activities of the abundant starter organisms should not be overlooked. Since cheese flavor was shown to develop in starter cheeses even under aseptic conditions, after all the starter organisms had perished, it is evident that autolysis during ripening plays an important role in the buildup of flavor in aged dairy products. Also here the contribution of milk lipases must be tiiken into consideration (Reiter et al., 1967). Off-flavors frequently encountered in the dairy industry, have drawn the attention of many investigators with technological and analytical interest. Gas-liquid chromatography of volatiles produced b y organisms that were previously found to be related to “maltiness” has shown that malty strains of Streptococcus lactis var. maltigenes produce a variety of compounds, including 2-methylpropanol and 3-methylbutanol, but that the typical malty aroma was due principally to aldehydes, mainly 3-methylbutanal (Morgan et al., 1966). Veda-

FLAVOR AND MICROORGANISMS

67

muthu and co-workers (1966a,b), in a series of papers dealing with the problem of the so called “fruity” off-flavor of Cheddar cheese found no difference in the sugar-protein degradation potential of lactic cultures leading to normal and fruity products, respectively, but observed a considerable difference in the carbonyl production of starters. High values were found with cultures consisting of S. lactis and S. diucetiluctis that led to fruitiness, while cultures of S. cremoris and Leuconostoc sp., that produced less than 20 p.p.m. carbonyl compounds, gave normal flavor cheeses with good closed texture. Acetaldehyde, pyruvic acid, and diacetyl were the chief carbonyl compounds investigated. Thus, ripened cheeses may suffer from offflavors similar to those of nonaged dairy products (see p. 64). A new type of off-flavor in cheese that was due to the undesirable activities of bacteria, has been recently described in Dutch Gouda cheeses (Badings et al., 1968). It was found that a microflora closely related to the group of Lactobacillus plantarum and L. casei was involved in the production of a certain phenolic off-flavor, the responsible component of which was identified as p-cresol. It was found that these undesirable organisms were derived from a contamination of inadequately filtered rennet. The chemistry of flavor in mold-ripened cheeses is even more complex. In addition to the processes leading to the formation of normally ripened cheeses, the metabolism of molds developing on, or within such cheeses contributes to the characteristic flavor. Roquefort, Gorgonzola, and other blue-veined cheeses are ripened with the aid of Penicillium roqueforti, that in addition to the softening of the curd and the formation of various protein breakdown products, shows a very high lipase activity that leads to the rapid production and accumulation of a number of fatty acids (mainly caprylic acid, but also capric and caproic acids). These fatty acids seem to be involved in the formation of methyl ketones, which probably are one of the most important components of cheese flavor. There have been indications with regard to the formation of such methyl ketones by the microflora involved in the ripening process of Cheddar cheese (Walker and Harvey, 1959).However, this has been later shown not to be the case, since the precursors of methyl ketones were found to be in the milk and an overall similarity could be demonstrated between the yields of methyl ketones by distillation of fat from milk and that obtained from Cheddar cheese (Lawrence, 1963; Hawke, 1966). In the case of mold-ripened cheeses, the fungal production of methyl ketones seems to be of primary importance from the organoleptic standpoint. Patton

68

P. MARGALITH AND Y. SCHWARTZ

( 1950, 1951) has isolated 2-pentanone, 2-heptanone, and 2-nonanone from mold-ripened cheese, emphasizing the importance of 2-heptanone to the characteristic cheese flavor. A comparison of the methyl ketone-producing capacity of a number of molds has been published recently by Kubeczka (1968). The formation of these ketones is supposed to be the result of the following enzymic reactions: (1) The liberation of fatty acids from milk triglycerides; (2) oxidation of the free fatty acids to a-keto acids; and (3) decarboxylation of a-keto acids to methyl ketones (Hawke, 1966). Gehring and Knight (1963) maintain that the formation of methyl ketones from fatty acids is confined to the fungal spores and is not a function of mycelial cells. The ripening of Camembert cheese involves apparently not only the surface growth of P . camemberti, but also that of various film yeasts and Geotrichum spp., that reduce the acidity of the cheese surface before the main mold becomes established. Information on the chemical compounds involved in Camembert maturation and the specific contribution of each group of organisms to the development of the characteristic Camembert flavor is still very meager. The same may be said about microorganisms involved in the specific flavor of slimesurface cheeses, such as Brick, Limburger, and others.

D. PICKLES The action of lactic acid bacteria on vegetable material is utilized for the purpose of preservation of nutritionally important ingredients as well as for the production of different types of foodstuffs with a special appeal to the consumer. From the microbiological point of view, this type of fermentation differs from that of the dairy industry not only in the nature of the raw materials, but also in the fact that pickling involves an environment characteristic in its high saline content. Since the early days of food microbiology and u p to recent times, shidies on the nature of cucumber pickling, sauerkraut and olive fermentation, etc., have concentrated on the microorganisms involved in the process, and the transformation of fermentable sugar into lactic acid. Good and bad fermentations have been evaluated by the appearance of the final product and its taste characteristics. In considering the place of the microorganism in the formation of the characteristic flavor of fermented plant material, we are confronted with a number of difficulties due to the complex nature of these processes. That lactic acid is not the only factor in the formation of pickle-flavor can be easily assessed by the fact that nonfermented pickles prepared with

FLAVOR AND MICROORGANISMS

69

similar amounts of such acid and salinity can be easily distinguished from fermented ones. However, during fermentation it is rather difficult to distinguish between the interaction of the microbial systems involved in the process and that of the enzymes that are present in the plant material. This difficulty is further aggravated by the fact that all vegetables employed for the lactic fermentation carry a very heavy load of microorganisms which do give way to a succession of lactic organisms, but may also affect the final organoleptic qualities of the product. In order to establish the part played by the different types of organisms, two pieces of information would be invaluable: (1) the approximate chemical composition of the flavor components of each type of fermented products, and (2)the share of each of the microorganisms involved in the lactic fermentation, when these are carried out under pure culture conditions. Although these prerequisites seem to be almost unattainable, recent studies have at least begun to conbate upon these approaches. An attempt to correlate the nonlactic acid components of pickles with flavor quality of the fermented product, has been made by Christensen and co-workers (1958), who analyzed the acetic acid composition of fermented vegetables. In using a setup which permitted pure culture fermentations, it was found that homofermentative lactics produced only minor amounts of acetic acid, independently of the sugar concentration available; while with heterofermentatives the amount of acetic acid formed during the fermentation increased with the available sugar concentration. Considerable variations have been encountered with the different strains employed. In a later paper, Pederson et al. (1962) emphasized the importance of the velocity of the fermentation process to the organoleptic qualities of the product. In a comparison of the fermentation of Yugoslavian cabbage, it was found that whole cabbage kraut was superior to that of shredded kraut, the former developing a mellow flavor, while the latter showed a more pungent, acid taste. This was probably due to the rapid fermentation which took place in the shredded cabbage, leading to higher volatile acidity. Recently Hardlicka et al. (1967) studied the formation of carbonyl compounds in kraut. Acetaldehyde and diacetyl seem to be formed during the first days of fermentation, declining thereafter. A new approach to the study of the changes that take place during vegetable fermentations was that of Vorbeck et al. (1963) and Pederson and co-workers (1964). These workers concentrated on the behavior of the lipid fraction during fermentation. It was found that in kraut the amount of free fatty acids (FFA) increased considerably during

70

P. MARGALITH AND Y. SCtIWAHTZ

the lactic fermentation. On the other hand the amount of unsaponifiable matter and fatty acids of both acetone soluble and insoluble lipid fractions decreased during the fermentation. A general increase in the shorter fatty acids was observed. The presence of longer-chain fatty acids in the nonesterified fatty acid fraction of the fermented material that were absent in the raw material, hus been attributed to the unsaponifiable fraction, and not only to the hydrolysis of lipids during fermentation. Although lactics are not considered lipolytic bacteria, the activity of lipolytic enzymes during autolysis may be involved. Indeed, recently Oterholm et al. (1968) have demonstrated the occurence of a number of lipolytic endoenzymes in many lactic acid bacteria. Also, it is not clear to what extent plant lipolytic enzymes may take part in this transformation. The chemical changes taking place during kraut fernientation have, however, not been discussed from the point of view of flavor formation or organoleptic evaluation. More pertinent data were obtained by the study of the cucumber ferinentation (Pederson et al., 1964). There was a general increase in the concentration of free fatty acids, neutral fats, and unsaponifiables. Gas-liquid chromatography of the methyl esters showed that a sharp increase in linolenic acid took place in the normal product, while in that of “bloaters” an increase in oleic acid was evident. Furthermore, the disappearance of tridecenoic acid from cucumbers and appearance of caproic, caprylic, and capric acids were noted. A striking characteristic of the cucumber fermentation was the decrease in the phospholipid fraction, down to 10%of the original. This, however, was not commensurate with the increase in FFA, neutral fat, and unsaponifiables. An active synthesis is, therefore, postulated to take place during the lactic fermentation. Incidentally, the breakdown of phospholipids during cucumber pickling has been noted earlier by Keil and Weyrauch (1937), who noted the accumulation of acetylcholine and lactylcholine in fermented foods and ascribed it to the activity of Bacterium acetylcholini which was later identified as a strain of Lactobacillus plantarum (Rowatt, 1948). Although details on the chemical transformations that take place during vegetable fermentations are accumulating rapidly, information on the chemical nature of the flavor of fermented olives is still very meager. Only the off-flavor leading to the malodorous “zapatera” fermentation has received considerable attention. The first off-odor to appear in zapatera has been described as “cheesy,” developing later into a foul, fecal stench. This is accompanied by a continuous loss of acidity. Delmouzos and co-workers (1953) have shown that at least

FLAVOR AND MICROORGANISMS

71

part of the odor results from the volatile acids that develop in the brine; these include formic, propionic, butyric, valeric, caproic, and caprylic acids. In contrast, normal brines contain acetic, lactic, and sometimes succinic acids. Plastourgos and Vaughn (1957)believe that the propionic acid is produced by Propionibacterium pentosaceum and P . zeae. These organisms were shown to develop similar cheesiness in uitro. However, these authors believe that the propionic acid fermentation can be considered only the first stage in the malodorous fermentation, to be followed by other anaerobic organisms. The preservation of meat products, like sausages, ham, and bacon, through the action of salts (NaCl, NaN03, and NaN02), and their subsequent maturation are generally referred to as meat curing. The carbohydrate-rich substrate of vegetable pickling is thus replaced by the proteinaceous material subjected to curing. This, however, leads to a completely different microflora, comprising a large number of genera and species (Jensen, 1954). While one of the major purposes of meat curing is the stabilization of color through the action of nitrite and the formation of nitric oxide hemoglobin, the contact of meat with such a diversified microflora, both under conditions of brine and dry curing, must affect the organoleptic qualities of the meats. Although several attempts have been made to study the effect of a number of parameters on the selective enrichment of microorganisms in brines (Deibel et al., 1961; Shank et al., 1962) comparatively little is known on the specific effect of the microorganisms that may be involved in the formation of the characteristic flavor. Only limited information on the incorporation of specific “aroma cultures” during the curing processes is available from literature. Thus, Niniivaara (1955) attempted the introduction of a Micrococcus into raw sausage. McLean and Sulzbacher (1959) reported the use of a Pseudomonas in a meat curing brine. Deibel, Wilson, and Niven (1961) used a Pediococcus as a starter in the preparation of summer sausages. A commercial preparation under the name of “Equinibe” has been marketed in France, probably a culture of Vibrio costicolus (Ribeiro, 1964) in order to improve color and flavor of ham. As Ingram (1966) pointed out, the main result of such practices was to minimize the chances of spoilage by alien species, although no high grade of flavor quality could be achieved. Knowledge on the contribution of microorganisms to the curing processes of meat is, thus, again fragmentary owing to the fact that so very little is known of the chemical entities involved in meat flavor. More recent work seems to provide new information on these aspects (Tarr, 1966).

72

P. MARCALITH AND Y. SCHWARTZ

E. ORIENTALFOODS The current international effort to provide new foods to needy populations has drawn the attention of many workers to the vast array of fermented foods which for centuries have served many nations in the Far East. Various plant and animal materials are being used for ;he fermentative processes employed in the preparation of these foods. For an exhaustive review on the nature and mode of preparation of such foods, the reader is referred to the excellent surveys by Hesseltine (1965), Hesseltine and Wang (1967), Amano (1961), and Saisithi et aZ. (1966). The main purpose in the fermentative production of these foods is to provide and enhance their organoleptic qualities. Fermentation adds to the flavor of these foods. Sometimes these flavors become so strong as to provide the raw material for the blending of other foodstuffs. Another advantage of the microbial process is to mask or even to destroy a number of repulsive components occurring in the raw material. Changes in the flavor qualities due to microbial activity in a number of well known oriental foods will be briefly discussed. Fish fermentation is one of the oldest processes which varies greatly according to the type of fish employed and local practice. For example, in Thailand Nam-pla is prepared from various small fish which are mixed with salt (25%) and fermented at about 40°C in sealed tanks for about 6 months until most of the material is liquefied. The filtrate is then ripened for a number of months under the sun. The finished product is a dark brown liquid with a distinct sharp flavor (Visco and Fratoni, 1963; Saisithi et al., 1966). Clearly, the flavor of this fish sauce is the result of the microbial activity of a number of halophilic microorganisms. Among these coryneform bacteria, streptococci, micrococci, and staphylococci as well as a Bacillus sp. were isolated. Quantitative information on the occurrence of these organisms and their respective proteolytic action is still unavailable. Most of the organisms were shown to produce volatile acids. Jones (1961) claimed that the flavor of the fish sauce arises in part from glutamic and other amino acids like proline and histidine, liberated during proteolysis. Saisithi and co-workers (1966), however, believe that the distinct flavor of the fish product is a blend of organic acids and a number of amines like glutamine, histamine, glucosamine, and trimethylamine. Low molecular weight volatiles, such as methyl ketones and other carbonyl compounds have been suggested by others (Yanagihara et al., 1963; Yurkowski, 1965). Clearly, the relationship between the flavor of the fermented fish product, microbial activity, and the autolytic action of the fish tissues warrants more extensive investigations.

FLAVOR AND MICROORGANISMS

73

The production of soya sauce is usually preceded by the preparation of koji which serves as a starter for the main fermentation. Koji is prepared by the fermentation of wheat bran and soybean flour cultured with the mold Aspergillus oryzae or A. soya in trays or small containers. Proteolysis is promoted by the incubation at suitable temperatures. It is not clear to what extent the degree of proteolysis affects the organoleptic qualities of the final product (Sugita, 1956). Asao and Yokotsuka (1957) emphasize the importance of phenolic compounds during the koji fermentation. It was found that most of the phenolic compounds such as 4-ethylguaiacol and vanillic acid increased gradually before spore formation of the mold, while that of ferulic acid diminished and vanillin disappeared (see also phenolic compounds in Sake, p. 63). The koji is then mixed with equal amounts of salt brine to form the mash (moromi). During a prolonged incubation period a lactic fermentation sets in, to be followed by a yeast alcoholic fermentation. A remarkable decrease in the concentration of malic and citric acids, probably due to microbial activity, has been observed during the initial stages of the mash fermentation (Ueda et al., 1958). After some aging, the liquor is separated by pressing, and constitutes the shoyu. The dark brown liquid is very salty and has a distinct sharp flavor. According to Yokotsuka (1960), the flavor of shoyu is very complex. In addition to salt, a comparatively high concentration of protein breakdown products such as peptones, peptides, and free amino acids, especially glutamic acid, may be considered as principal flavoring materials. In addition, organic bases, derived from the breakdown of nucleic acids, organic acids, such as acetic, lactic, succinic, and pyroglutamic acids, as well as a number of alcohols and esters have been mentioned to contribute to the flavor of shoyu (Shigemi and Michiyo, 1966). It is evident that most of the characterization and analysis of the distinct shoyu flavor have dealt little with the source of each flavoring component or their respective biochemical pathways. Obviously, a considerable number of these compounds are derived from the different fermentation processes in the koji or later during the fermentation of the mash. However, information on the specific action of microorganisms in this complex process is still very meager. Recent studies on the nature of shoyu flavor have shown that a number of phenolic compounds may be considered to take part in the formation of such flavor (Yokotsuka et al., 1967a,b; Asao et al., 1967); 4-ethylguaiacol, 4-ethyl phenol, and 2-phenylethanol were shown to be involved. Torulopsis spp. (especially T . uersatilis) were

74

P. MARGALITH A N D Y. SCHWARTZ

found to be responsible for the transformation of ferulic acid into alkyl phenols, while most of the yeasts isolated from the shoyu process (such as Saccharomyces rouxii) produced 2-phenylethanol (P-phenylethyl alcohol). It was found that high quality shoyu contained 0.5 to 2.0 p.p.m. 4-ethylguaiacol.

Ill.

Concluding Remarks

Although this review does not pretend to be an exhaustive survey of flavor in fermented products, an attempt has been made to expose the industrial microbiologist who usually is not concerned with food production, to various aspects of taste and aroma in a number of processes that are controlled by the activities of microorganisms. Since the early days of applied microbiology until today, when practically all problems of flavor in foodstuffs are studied by means of gas-liquid chromatography techniques, a wealth of analytical data has accumulated which permit a deeper insight into the contribution of the different microbial populations to the buildup of flavor in fermented food products. In the present chapter reference has been made only to processes where microbial activity is taking place in situ during the manufacturing. In a subsequent chapter an attempt will be made to review recent advances in the fermentation of various chemical compounds which are used in concentrates or even pure form in order to enhance the flavor qualities of foodstuffs which normally are not subjected to a fermentation process during their manufacturing.

75

FLAVOR AND MICROORGANISMS

TABLE I CHEMICAL COMPOUNDS INVOLVED IN THE PRODUCTION OF FLAVORBY MICROORGANISM Formula

Compound Alcohols Ethanol

CH3CH20H

Glycerol

CH,OH I CH. OH

I

CH20H n-Propanol

CH3CH2CH2 OH

Isopropanol

(CH,), CHOH

n -Bu tanol

CH~CHICH~CH~OH

Isobu tanol (2-methyl- 1-propanol)

(CH,)2CHCH,0H

2,3-Butanediol(2,3-butylene glycol) (dimethylene glycol)

CH3CH2 (OH)CH2 (OHKH,

Acetoin (Acetylmethylcarbinol)

CHI CH(OH)COCH,

Active amyl alcohol (2-methyl-I-butanol)

CH3CH2CHCH,OH

I

CH3 Isoamyl alcohol (3-methyl-1-butanol)

(CH,),CHCH,CH,OH

n-Hexanol

CH3 CH2 CH2 CHI CH2 CHI OH

Furfuryl alcohol

Benzyl alcohol

2-Phenethyl alcohol (2-phenylethanol)

Tryptophan01

CH2CH20H

(continued)

P. MARGALITH AND Y. SCHWARTZ

76

TABLE I (Continued) Compound

Formula

Aldehydes, Ketones, and Acetals Formaldehyde

HCHO

Acetaldehyde

CH3CH0

Propionaldehyde

CHI CHzCHO

Acetone

CH3COCH3

n-Butyraldehyde

CH3CHzCHzCH0

Isobutyraldehyde

(CH,)aCH2CH0

Diacetyl (2,3-butanedione)

CH3COCOCH

Methylethyl ketone

CH~COCH~CHJ

2-Methyl- 1 -butanal

CH3CH2CHCH0

1

CH3

n-Valerylaldehyde

CH3CHaCHzCHzCHQ

lsovalerylaldehyde (3-methyl- 1-butanal)

(CHI )zCHCH2CH0

2-Pentanone n-Hexaldehyde 2-Heptanone 2-Nonanone Diethyl acetal (Acetal)

Ethylpentyl acetal

Ethyl, active amyl acetal

Ethyl, isoamyl acetal

77

FLAVOR AND MICROORGANISMS

TABLE I (Continued) Formula

Compound Isoamyl, pentyl acetal

CH,CH,

Diactive, amyl acetal

CH,CH(

,OCHi CH, CH(CH3 )Z OCHZCH,CHZCHZCHS OCH1CHCH,CH3 LH, OCH,CHCH,CH, I

CH, OCH,CHCH,CH, I CH,CH CH3 ‘OCH~CH,CH(CH,), /

Active amyl, isoamyl acetal

/

OCH, CHCH,CH, I CH3 OCH2CH,CH,CH,CH,

Active amyl, pentyl acetal

CH,CH,

Diisoamyl acetal

,OCHzCHiCH(CH,), CHJCH \OCH~CH,CH(CH,),

Acids

Formic

HCOOH

Acetic

CH,COOH

Propionic

CH,CH,COOH

Lactic

CH,CH(OH)COOH

Pyruvic

CH,CG€OOH

n-Butyric

CH3CH2CH,COOH

Isobutyric

(CH,),CHCOOH

Succinic

HOOCCH,CH,COOH

Malic

HOOCCH,CH(0H)COOH

n-Valeric

CH,CH,CH, cn,coon

lsovaleric

(CH,IICHCH,COOH

2-Me thylbutyric

CH3CHaCH(CH3 )COOH

(continued)

P. MARGALITH AND Y. SCHWARTZ

78

TABLE I (Continued) Compound Acetolactic (I-

Ketoglutaric

Formula CHI COCH, CH(0H)COOH HOOCCH, CH, COCOOH

nCaproic

CH, (CH,)4COOH

Citric

HOOCCH, C(OH)CH1COOH

I

COOH

Isocaproic

(CH3 ),

2-H ydroxyisocaproic

(CH, ), CHCH,CH(OH)COOH

n-Heptylic

CH3(CHl)s COOH

nCaprylic

CH3(CH,)6COOH

Pelargonic

CH,(CH,),COOH

nCapric

CH,(CHl ),COOH

9-Decenoic

CH,=CH(CH, ),COOH

Lauric

CH, (CH,)ioCOOH

Tridecanoic

CH,(Cli,),

Myristic

CH,(CHl),2COOH

Palmitic

CH,(CH2 ),,COOH

Oleic

C H ~ ( C H ~ ) ~ C H = C H ( C),COOH HZ

Linoleic

CH,(CH, )a (CH,CH=CH), (CH, ),COOH

Linolenic

CH3(CH2CH=CH)~(CH1 ),COOH

CHCH~CH,COOH

I COOIi

Amino acids and amines

Trimethylamine

Putrescine

Cadaverine

NH2CH,(CHz)iCHzNHa NH,CH,(CH1)3CH1NH2

Clutarnic acid

HOOC(CHz)2CH(NH, )COOH

Glutainine

HOOC(CH, ),CH(NH, )CONHI

FLAVOR AND MICROORGANISMS

TABLE I (Continued) Formula

omcooH nco0,, H

Pyroglutamic acid

Proline

H

Histidine

Histamine

Glucosamine

Ho OH

HO OH H

Tryptamine

NH,

a

CH,CH,NH,

Eaters

Ethyl formate

HCOOCH2CH3

Ethyl acetate

CH3COOCH2CH3

2-Phenylethyl acetate

CH~COOCHZCH,C,HS

1,3-Propanediol monoacetate

CH,COOCH,CH,CHzOH

lsoamyl acetate

CH3COOCH2CHzCH(CH3 )i

(continued)

P. MARGALITH AND Y. SCIiWARTZ

TABLE I (Continued) Formula

Compound Ethyl lactate

CHI CH(OH)COOCH, CH,

Ethyl Chydroxybutyrate

(HO)CHtCH, CHI COOCH, CHS

Diethyl succinate

CH2COOCHzCH3 I CH2COOCHzCH3

Diethyl malate

(OH)CHCOOCH2CH3 I Cl12C00Cti,CH3

H Ethyl pyrodutamate

O

r

a

c

H

3

2-Phenylcthyl caproate

CHJ(CH2),COOCHlCH,C~H~

Acetylcholine

CH3COOCHzCHzN(CH3

Lactylcholine

CH3 CH(0H)COOCHZ CHI N(CH3 )3

Phenol compounds

p-Cresol

4-Ethylphenol

+

+

81

FLAVOR AND MICROORGANISMS

TABLE I (Continued) Compound

Formula

on

Q I

p-Hydroxybenzaldehyde

on I

p-Hydroxybenzoic acid

Q COOH

OH

I

Tyrosol

OH

4Methylguaiacol

Qocn3

OH

4-Ethylguaiacol

3”:”. (continued)

P. MARGALITH AND Y. SCHWARTZ

TABLE I (Continued) Compound

Formula OH I

4-Vinylguaiacol

CH=CIla

OH I

Vanillin

CHO

Vanillic acid

COOH

OH

I

Ferulic acid

qoCH CH=CHCOOH

Sulfur compounds

Hydrogen sulfide

Has

Thioformaldehyde

HCHS

Dithiofonnaldehyde

(HCHSh

Thioacetone

CH3CSCH3

Ethanethiol

CH3CH2SH

FLAVOR AND MICROORGANISMS

83

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Mechanisms of Thermal Injury in Nonsporulating Bacteria

M.

c. ALLWOOD' AND A.

D. RUSSELL

Department of Pharmaceutics, Welsh School of Pharmacy, University of Wales Institute of Science an.d Technology, Cardiff,Great Britain I. Introduction ..................... 11. Original Approaches to the 111. Thermosensitivity of Various Bacteria ............................ A. Thermophilic Bacte B. Psychrophilic Bacte C . Bacterial Spores.. ... ............................ IV. T h e Causes of Thermal Bacteria by Moist Heat ................................................ A. Survival Curves of Heated Bacteria ..... B. Possible Types of Damage to Bacterial C e V. Repair of Thermal Injury VI. Conclusions ................................................................. VII. Summary ..................................................................... References ..................................................................

I.

89 90 93 93 94 95

97 98 103 112 114 115 116

Introduction

It is surprising, in an era in which far-reaching discoveries as to the structure, functions, and biosynthetic processes of the bacterial cell have been made, to find that the primary damage induced in nonsporing bacteria exposed to moist heat is still unknown. However, although moist and dry heat have been used as methods of sterilization for a considerable period of time, the reasons for this lack of knowledge are not, perhaps, too difficult to assess: the fact that vegetative bacteria were killed at low, and spores at higher, temperatures was, until fairly recently, sufficient cause for using thermal processes without the necessity for carrying out critical experiments to determine the nature of the damage inflicted on the bacteria. This could be one reason for the oft-repeated statement that moist heat kills vegetative bacterial cells by causing an intracellular coagulation of protein material; while such coagulation undoubtedly takes place to varying degrees, it is not unreasonable to propose that this effect masks other, more delicate, changes in the bacterial cell which could be induced before coagulation becomes apparent. It would appear that investigations have been concerned more with studies of loss of 'Present address: Department of Pharmacy, T h e University, Nottingham, Great Britain.

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viability, i.e., death rates, rather than with attempts to elucidate the reasons for such death. Two pleas for a more critical approach to an understanding of the mechanisms of thermal injury have been made (Wood, 1956; Hansen and Rieman, 1963). Before venturing upon a discussion of the possible causes of thermal injury in vegetative bacteria and the evidence available to support the present concept of thermally-induced death of bacteria, a brief description of the original approaches to the problem, followed by a sunirnary of other relevant topics that have provided information to describe heat-labile components in bacterial cells, will be presented.

II.

Original Approaches to the Problem

More than 50 years ago, Eijkman (1908) reported that the longer an organism was subjected to treatment with moist heat, the greater was the period necessary for recovery. H e introduced the’ concept of injury, which was responsible for an increase in the time required before the organism was able to grow and resume cell division following exposure to sublethal heat treatment. An alternative explansat’ion was later suggested by Burke (1923) who postulated that the delay in growth following heat treatment was due to the presence of a fraction of the culture that was more heat resistant. It was suggested that this resistance was a consequence of more stable cell walls and this property reduced the rate of reproduction of the heat-resistant members of the population. The lethal action of moist heat on vegetative bacterial cells was considered as early as 1910. Chick and Martin (1910) studied the kinetics of the coagulation of proteins and a similarity was noted between protein coagulation and the disinfection of bacteria by moist heat (Chick, 1910). It was shown that the rate of death of a population of vegetative bacteria proceeded logarithmically and the conclusion was drawn that such a process occurred as a reaction of the first order. Chick further pointed to an analogy between disinfection by hot water and the coagulation of protein: “The disinfection of bacteria by heat in the presence of water exhibits a striking analogy with the behavior of some proteins under similar conditions and leads to the inference that disinfection of bacteria by this means is due to hydration of their constituent proteins.” The logarithmic rate of death of a population of bacteria was due to temporary and rhythmic changes in their resistance, which, by analogy, was due to temporary changes in the energy content of their constituent proteins. It was not d u e to individual variation in the resistance of members of the population.

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This important work proposed a mechanism by which moist heat kills bacteria and illustrated the logarithmic rate of death, which has also been found to be a result of a large number of other chemical and physical treatments lethal to microorganisms. The phenomenon of bacterial resistance to moist heat was studied by Fay (1934). It was reported that a number of microorganisms showed increased resistance to heat in the presence of high concentrations of sugar. The conclusion reached was that the permeability of individual cells played an important part in regulating the rate of cell death. The recovery phenomenon was also considered. It was shown that the presence of high concentrations of sugar in the recovery medium had no effect on survival. Hershey (1939) described a series of experiments in which the increase in the lag phase of growth of bacteria following exposure to moist heat was studied in order to elucidate the physiological significance of the delay in multiplication following thermal injury. It was shown that the period of latency following heat exposure was progressively lengthened as the temperature was increased and this extension of the lag phase of growth was a direct effect of injury and not, as previously suggested, merely the result of the selection of resistant individuals inherently slower to develop. During this lag phase, there was a regeneration of respiratory function. It was concluded that this increase in respiratory activity during the extended lag phase, and the ability of heat-injured bacterial cells to produce colonies on agar, were manifestations of normal biological growth and not as a consequence of any reactivation of metabolic functions in the heat-injured cells. The growth of the survivors was identical with that of untreated cells. The concepts of injury and repair were misleading and loss of viability was an all-or-none response, whatever the criterion used for its determination. Considerable work has been carried out to show that the recovery of heat-treated bacteria could be affected by the growth medium in which they were placed subsequent to their exposure to sublethal heat treatment. Chambers et al. (1957) suggested that the number of survivors could be increased by the presence of cell metabolites in the recovery medium. Nelson (1943) showed that heat-treated cells of S taphlococcus uureus were more exacting in their requirements for growth compared with untreated cells. It was also clear that factors other than the chemical constitution of the recovery medium were important, and these included the temperature of incubation and the pH of the medium. It was later shown that the oxidation-reduction

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potential of the recovery medium could also influence the recovery of heat-damaged S. uureus (Nelson, 1944). The proposal that particular chemical changes induced by physical agents lethal to bacteria could be responsible for the death of the organism was made b y Rahn (1945). It was evident that although certain physical treatments, such as moist heat or exposure to irradiation, resulted in the loss of cell viability, the ultimate causes of death must be due to fimdamental chemical changes at particular sensitive sites within the cell. Thus, death by exposure to dry heat was probably due to oxidation (Rahn, 1945), while death caused by exposure to moist heat, requiring a lower temperature, occurred by a different mechanism. Rahn suggested that death might be due to coagulation of important cellular proteins. It would seem unlikely that death was due to inactivation of enzymes, unless there existed a particularly heat-sensitive enzyme, because the logarithniic rate of death was possible only if loss of viability was due to the destruction of a single molecule within the cell (however, this appears to ignore the possibility that the logarithmic order of death may be a nianifestztion of an inherent biological variation within the population). Also, it appeared that most bacterial enzymes were stable at temperatures considerably higher than those lethal to the bacteria themselves. The factors that controlled the thermal inactivation of bacteria included the time of contact, the composition and pH of the suspending rnenstruum, the number of cells present, and the inherent resistance of the microorganism. Gaughran (1947)discussed the unusually high resistance of bacterial spores and the existence of thermophilic bacteria able to grow and divide at temperatures above 60°C. The reviewer agreed with Rahn (1945), concluding that death caused by moist heat was unlikely to be due to enzyme inactivation. However, it was pointed out that the alternative proposed mechanisms of thernial injury and death should be approached with caution. The lack of ability of heat-treated cells to multiply could not be the only criterion for death. Thus, the inactivation of a single gene essential for the normal reproductive fiirictioning of the cell, producing a “sterile mutant,” was consistent with a logarithmic rate of death. It may be implied that thermophilic bacteria possess a heat-stable genetic structure in addition to more heat-stable enzyme complexes. Alternative explanations to account for the existence of thermophilic bacteria were proposed. Such types may possess an efficient method for replacing compounds destroyed by heat, or are composed of a type of lipid of an unusually high melting point.

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The extreme sensitivity of biologically active material to environmental changes is universal and microorganisms are remarkable, therefore, in the tolerance that they exhibit to changes in their physical environment, including temperature. Many aspects of thermobiology have been studied (see Rose, 1967). In the field of microbiology, research has illustrated that bacteria show a wide divergence in their ability to grow over a large range of temperature. This has created an arbitrary classification of bacteria into three types: psychrophiles, mesophiles, and thermophiles, according to their ability to grow at low, “normal,” and high temperature ranges, respectively. It has also been found that many bacteria possess the ability to withstand high temperatures over limited periods without affecting their normal growth and reproductive functioning. Such resistance to thermal stress was found to vary greatly with different types, and an examination of the lethal effects of high temperature has become of considerable interest in attempting to define (i) reasons for the tolerance of certain bacteria to high temperatures, (ii) the thermal inactivation of psychrophiles, (iii) the factors responsible for the unique heat resistance of bacterial endospores, and ( i v ) the causes of inactivation of vegetative bacteria by moist heat. These studies have developed rapidly of late because of the practical importance of moist heat in sterilization and pasteurization processes. The first three of these problems will be discussed briefly to show how these studies have indicated the possible sites in the bacterial cell that are thermolabile. The last problem, the inactivation of vegetative bacteria, will then be discussed in detail as will the possible sites that could be affected by exposure to moist heat leading to cell death. 111.

Thermosensitivity of Various Bacteria

A. THERMOPHILIC BACTERIA The observation that certain types of bacteria are capable of normal growth at temperatures above 65°C. has stimulated research to explain this extraordinary phenomenon. Stanier et al. (1963) suggested that thermophilic bacteria contained enzymes with greater heat stability, compared with mesophiles, which influenced their optimum growth temperature and thermal sensitivity. This conclusion was drawn from the knowledge that heat-sensitive mutants of certain thermophiles have been studied that owe their sensitivity presumably to the production of a more labile enzyme. The stability of thermophiles to moist heat must also be influenced b y other species of pro-

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tein in the cell and ribonucleic acid (RNA), the biological activities of which are destroyed between 50" and 90°C. (Eigner et al., 1961). Gaughran (1947) discussed the high heat tolerance of certain bacteriai and concluded that it was not necessarily due to the greater stability of the constituents of the cell but rather to their tremendous capacity for replacing compounds destroyed by heat. This was also inferred by Christopherson and Hensel (1955). However, the thermal stability of enzymes and intracellular material has been related to the high growth temperature of these microorganisms (Allen, 1953; Koffler, 1957). Saunders and Campbell (1965) reported that the temperature of denaturation of ribosomes isolated from vegetative cells of Bucillus stearothermophilis was markedly higher than that of ribosomes of Escherichia coli. Pace and Campbell (1967) have confirmed that the ribosomes of a number of thermophiles are more heatstable, and suggest that this stability is a consequence of the interaction of RNA and ribosomal protein, and not because of a higher guanine and cytosine content of the RNA (often associated with the heat stability of RNA). Stenesh and Yong (1967) have reported that the ribosomes of thermophilic and rnesophilic bacteria appear to be similar, although the former were more stable to heat. The enhanced stability of ribosomes from thermophilic bacteria could be as a result of an additional component present in the ribosomes (Friedman et al., 1967). Two other relevant observations have recently been reported: Forrester and Wickens (1966) have found that the cell walls of a thermophilic bacterium contain an unusually high lipid content, and Arnelunxen (1967) concludes that the physical state of the enzyme molecules is at least one aspect of their thermal stability in bacteria able to grow at high temperatures.

H. PSYCHROPHIIXBACTERIA Heater and Vanderzant (1957) have made a study of the recovery of psychrophilic bacteria after heat treatment and have found that a proportion of the population shows an increased sensitivity to the recovery medium. Hagen et al. ( 1 9 ~ studied ) the thermally-induced death of a psychrophilic organism and concluded that death was not caused by cell wall breakdown or damage to the cytoplasmic membrane, because leakage and lysis followed death during exposure to 35°C. Lysis of the cells may have been the result of the release of an autolytic enzyme by heat treatment. Purohit and Stokes (1967) recently suggested that the enzymes of psychrophiles are more heat-labile than those of mesophiles and

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reported a number of such enzymes that were inactivated more rapidly than similar enzymes of E . coli. Studying the influence of moderate temperatures on a marine psychrophile, Vibrio marinus, Morita (1965) found that a process of heat shock caused inactivation of the metabolic processes involved in oxygen uptake, possibly due to biochemical lesions in this metabolic pathway. Langridge and Morita (1966) confirmed that many enzymes in this organism became more heat labile following growth above 20°C. Detailed studies were made of thermally-induced death in the organism above its maximum growth temperature, 20°C. (Robison and Morita, 1966; Haight and Morita, 1966). It was suggested that thermal stress brought about many changes contributing to the loss of viability, including enzyme inactivation, membrane damage, and nucleic acid breakdown. It was also found that cytological abnormalities were caused by exposure to moist heat, and Hagen et al. (1964) showed that the heat treatment of a marine psychrophile caused lysis of the cells, indicating the thermal sensitivity of the cell walls of psychrophilic bacteria. Cousin (1967) has suggested that the increased thermal sensitivity of a mutant of E. coli is due to the low-temperature inactivation of an enzyme controlling an energy-yielding reaction. A heat-sensitive strain of Proteus vulgaris contains a thermolabile enzyme responsible in some way for cell synthesis (Harmon and Peron, 1967). Clearly, a number of biochemical differences between psychrophilic and thermophilic bacteria have been reported that could be responsible for the extremes in sensitivity of these types of organism to temperature. These studies have provided evidence that particular sites within the bacterial cell are sensitive to thermally-induced lesions which could be responsible for inactivation of bacteria exposed to moist heat. C. BACTERIALSPORES

The fact that bacterial endospores are able to withstand temperatures higher than the most heat-resistant vegetative bacterial cells has considerable biological implication, one of which is the reason for their stability at temperatures far in excess of those usually associated with the stability of living material. An elucidation of this phenomenon may also suggest possible changes induced in the vegetative parent cells to produce these heat-stable forms. Gaughran (1947) attempted to explain the heat resistance of bacterial spores. It was noticed that there had been attempts to correlate the moisture content of the spores with thermal stability, but the

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assumption that vegetative cells had a higher moisture content than spores had been disproved by Virtanen and Pulkki (1933). Permeability had also been suggested as a controlling factor in thermal resistance, together with the high calcium content of endospores. A high bound-water content could also increase the resistance of spores and could act as a mechanism preventing the coagulation of spore protein material. Maxwell Savage (1959) pointed out that spores were not killed in the presence of steam by the latent heat available, but by the toxic effect of water at high temperatures, suggesting that protein denaturation could be its major lethal influence. The thermal inactivation of spores during heat sterilization could be due, however, to an oxidative process (Bmch, 1963) but this has not been verified by later studies (Pheil et aZ., 1967). Thimann (1963) pointed out that the temperature coefficient (Qlo)for the inactivation of bacterial spores is very high (e.g., Qlo = 9-11 for Bacillus anthracis spores). Since the only process with such a high temperature coefficient was the heat coagulation of proteins, it was reasonable to deduce that the destruction of bacteria1 endospores b y moist heat is caused by protein coagulation (Thimann,

1963). The spore coat consists of an inner mucopeptide layer surrounded by a protein layer of high cysteine content. The cortex layer is rich in calcium and dipicolinic acid. Murrell (1964) has suggested a number of theories to explain the heat resistance of bacterial endospores: (1) The impermeability of the spore coat. (2) Enzymes in the spore are i n an inactive and stable form, due possibly to their ionic binding. (3) The dehydration of protein material within the spore increases its stability. The importance of a reduced atmospheric moisture content was stressed b y Russell (1965), although its influence on the heat stability of spores was difficult to assess. The presence of a small amount of water in the atmosphere appeared to be optimum for their thermal resistance (Murrell and Scott, 1966). Also the presence of the calcium salt of dipicolinic acid appeared to play a part in the thermal resistance of spores. The fact that the spore coat contained a high content of disulfide bonds may also be a contributing factor, although this may be of less importance than originally thought (Hitchens et al., 1966). Reports of dry heat-induced mutations occurring in bacterial spores have recently been made. Chiasson and Zamenhof (1966) showed that mutations could be induced in spores of Bacillus suhtilis by exposure

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to temperatures above 105°C. and occurred at a maximum frequency at 115°C. Similar results were obtained by Northrop and Slepecky (1967). It was suggested that mutation could be caused by the elimination of a number of cytoplasmic factors and, above lOWC., to depurination. The DNA of spores may differ in physicochemical properties from that of vegetative cells. Thimann (1963) has also listed possible reasons for the heat resistance of bacterial endospores, which included their lack of moisture, the presence of colloids in spores, the presence of calcium dipicolinate, and of heat-stable enzymes. The reasons for the thermal stability of spores are still undefined, although it seems probable that a number of the factors discussed may be responsible for this phenomenon. The contractile cortex theory (see Murrell, 1964) must also be considered. It may serve to indicate likely biologically heat-sensitive sites within living systems that are prone to thermal inactivation. IV.

The Causes of Thermal Inactivation of Vegetative Bacteria by Moist Heat

The main section of this essay will be devoted to a consideration of the biochemical basis of the mechanism of thermal injury leading to the loss of viability of vegetative bacteria following exposure to moist heat. A more scientific and biochemical approach to this problem has been considered only in the last decade, and especially during the last 5 years. The problem was expressed admirably by Wood (1956) who wrote: “It is not surprising that a great deal of controversy should have arisen as to how micro-organisms are killed by both high and low temperatures . . . so few of the many studies on temperature inactivation of cells have been carried out to elucidate the mechanism of temperature action.” At the present time, there are a number of possible mechanisms proposed to explain the lethal influences of hot water on vegetative bacteria and these were listed by Strange and Shon (1964): (1) Coagulation of protein. (2) Inactivation of enzymes. (3) Disruption of cellular lipids. (4) Damage to the genetic apparatus. (5) Breakdown of RNA. These and other possibilities will be discussed in the light of available evidence. The warning given by Mitchell (1951) should, however, be borne in mind “. . . It is characteristic of an economically built structure that the margin of safety of all its components should

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be the same. We would therefore not expect any one factor to be responsible for thermal inactivation.” Before proceeding to a detailed account of thermal damage in bacteria, an analysis will be made of the different survival curves obtainable during exposure of a suspension of bacteria to moist heat, and their possible significance in thermal inactivation studies. A.

SURVIVAL CURVES OF

HEATEDBACTERIA

A considerable amount of effort has been spent on studying the shape of timelsurvivor curves. Frequently, but not invariably, the loglo of the number of bacteria surviving a heat treatment gives a straight line when plotted against the period of treatment [Fig. 1, curve (b)]. Thus,

n/no= exp(-k’ t ) where k’ is the slope of the survival curve, no is the initial number of viable bacteria, n the number of bacteria surviving a n exposure time t , and e is the natural logarithmic base. However, survivor curves of different shapes may also be obtained, e.g., as shown in Fig. 1,curves (a) and (c). In relation to the exponen-

Time

Fig. 1. Different types of timelsurvivor curves for heated bacteria.

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tial type [curve (b)], curve (a) denotes a situation in which either cell clumping occurs or there is a need for a certain amount of heat damage to take place before the ability of the cells to recover is destroyed; curve (c) denotes that bacteria of unequal heat resistance are present in the treated culture. According to classic theories, the cause of the inactivation (cell death) in curves (a)and (c) being due to a single event is eliminated, and such curves therefore represent “multiple effect” or “cumulative” damage to the bacteria. With a straight line response [curve (b)], the bacterial population is homogeneous in respect to its sensitivity to heat; moreover, the death of a particular cell is not influenced by the number of other cells remaining viable, and the chance of a cell being killed at any time is independent of the previous history of heat exposure. Exponential death of heated bacteria could b e due to a single event (although this may not imply that such death is a monomolecular reaction) or it could be cumulative, in that several events on the same site could lead to inactivation. According to Hansen and Rieman (1963), the differently shaped timelsurvivor curves obtained when cultures of different ages are heated suggests that the shape is determined by a nonuniform distribution of heat resistance, so that the cause of death is accumulative damage to the cells; this consequently implies that the most heatresistant bacteria will survive longest. The implications of the exponential rate of death of vegetative bacteria have been discussed by many workers. Chick (1910)believed that the process of logarithmic death was explained by supposing that death was due to heat coagulation of cellular protein.materia1, which had been shown to follow first-order kinetics (Chick and Martin, 1910). Rahn (1945) considered that exponential death reflected a fundamental process common to all disinfectants, which could be explained in chemical terms. Jordan et al. (1947) examined variations in death rates of E . coli with time at 53°C. The constant death rate of microorganisms during exposure to heat was apparent assuming that the penetration of heat into the cells was almost instantaneous. Their results showed that the early stage of the time/survival curve often appeared to show a slower rate of kill, compared with the later part of the curve. Schmidt (1957), in a discussion of the exponential death of microorganisms exposed to moist heat, questioned the often held assumption that this reflected a unimolecular reaction. Wood (1956) implied that the exponential death was indicative of a single killing event. Sites within the cell

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that could he inactivated by a single event included the denaturation of protein and the inactivation of particular enzymes, the inactivation of deoxyribonucleic acid (DNA) or lipid degradation. Wood (19%) confirmed that the heat inactivation of many viruses occurred exponentially and was indicative of the destruction of a single functional unit within the virus particle. As the same was true in bacteria, a single event occurring in the microbial cell must be responsible for death. Three possibilities were suggested: (1) The change in a single molecule is responsible for an autocatalytic reaction causing changes in many other molecules and hence cellular inactivation. ( 2 ) An internal change in cell organization spontaneously exposes the cell to the injurious effects of heat. (3) The inactivation of a single unit in the cell essential for cellular function, caused by a single molecular change. Wood (19%) suggested that the third type of event was the simplest, and further pointed out that such a model could be most readily interpreted if it were assumed that thermal inactivation resulted from damage to the nuclear material. Generalized protein denaturation or enzyme inactivation a 5 a cause of thermally-induced death was difficult to apply to the concept of first-order kinetics, Leincke and White (1959) reported the effect of culture age on the heat resistance of E . coli. It was found that most of the cultures studied showed a concave-up type of death curve and this indicated that the cultures contained cells of different ages, accounting for variation in the heat resistance of individual bacteria in the culture. Oginsky and Unibreit (1959) have pointed out that it iS not known whether exponential death curves are an expression of the macrornolecular reaction of protein denaturation or of a subsequent phenomenon. The first-order reaction concept of thermal death has been questioned by Hansen and Rieman (1963). It was suggested that death could be due to chance contact with the lethal agent at a “sensitive spot” within the cell, or, alternatively, to variation in cellular resistance. The relationship between an increase in the rate of death of cells with increasing temperature was shown by the thermal-death time curve, in which a graph of the logarithm of the rate of death (the Dlo-value) plotted against temperature gave a straight line. The slope of the curve (the z-value) represented the increase in temperature required to reduce the Dlo-vdue to one-tenth of its original value. Thomas et al. (1966) also showed that the graph of the Dlo-value plotted against temperature was linear over a limited range, suggesting that this was indicative

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of death being caused by one mechanism within the cell, although this did not imply that such a mechanism was a unimolecular reaction. A similar study has been made in our laboratory over a wider range of temperature. The organism employed was S. aweus and suspensions were stored in water at different temperatures. The concentration of cells was ca. lo9 viable cellslml.; viable counts were carried out by the pour plate method (see Allwood and Russell, 1967a). The results are shown in Fig. 2. It would appear that the graph is not linear when

I

O' 30 6OoC 55°C

31

50°C

,

I

f

32

33

34

VAO x lo4 37°C

45°C

32°C

20°C

Fig. 2. Arrheriius plot of log,, of inactivation rate against 1/A" X 10' for S . aweus NCTC 6571.

results are taken over such a wide range of temperature. There is a clear change in the slope of the curve between 45" and 50°C., and is suggestive of a fundamental change in the causes of death of this organism. Above this critical temperature there is a proportional increase in the rate of death per unit increase in temperature. Clearly, the cause of death may also depend on the temperature to which cells are exposed. Turri et al. (1964) have examined the thermal inactivation of the bacteriophage K12 of E . coEi, and found that the inactivation curve followed two-component kinetics, each component representing a first-order reaction. It was suggested that the inactivation process was due to denaturation of the protein component of the phage particle.

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Later, Kota and Mikolajak (1967)found a similar result with a bacteriophage of Streptococcus cremoris and proposed that death was due to two processes. These were the denaturation of the protein overcoat and denaturation of the RNA core. Such an inactivation must have been as a result of large macromolecular changes in the phage protein and nucleic acid. Dimmock (1967)also considered that inactivation of virus activity b y heat could be caused by damagc to both RNA and protein moieties in the virus, the stability of each varying independently with temperature. Jockusch (1966) proposed that the thermal sensitivity of tobacco mosaic virus could be related to a mutation in the coat protein cistron which was responsible for the production of a heat-sensitive mutant. Such sensitivity appeared to be d u e to changes in the protein structure of the mutant and suggests the importance of the protein coat in determining the heat-sensitivity of the virus. This short discussion of the heat inactivation of virus particles has served to show how early approaches to the problem concerned with an analysis of the thermal inactivation curves have led to a more basic biochemical study of the problem. Two recent reviews have discussed the exponential rate of death of vegetative bacteria exposed to moist heat. Ingraham (1962) pointed out that the implication must be drawn that a bacterium has a certain probability of dying at a particular moment during lethal heat exposure, regardless of the prior period of time to which the organism has been exposed. That the loss of essential enzyme activity explained exponential death seemed unlikely, particularly since it had been shown that there was little correlation between loss of enzyme activity and of cell viability. However, logarithmic death indicated a single lethal event occurring in the cell, although one wondered whether the loss of a single molecule in the cell could be lethal. It would appear, according to Ingraham, that a more reasonable explan at'ion was that lethality was a result of the inactivation of a particular gene essential to the cell, or to the destruction of a structure such as the cell membrane. Meynell and Meynell (1965) suggested that the exponential death curve might be explained by two different hypothetical models. Either killing was random, possibly due to a single lethal event, or death was a result of deterministic events in the cell. The latter explanation appeared to be more feasible to explain thermal death in which a large number of molecules would presumably be involved. The slope of the death curve was attributed to variation in the susceptibility of the organisms within the population. Such variation could be due to age differences of the individual cells of the population, or to inherent

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nonuniformity,in the heat resistance of the cells. An apparent variation could also arise from the release of material from dead cells which protected survivors. At variance with this suggestion was that of Christopherson and Hensel (1955), that it was feasible for thermal death to be the result of a single event, involving damage to a “control centre” protein. Therefore, exponential death of a population was an indication that only one species of molecule was involved. In summary, the death of a population of vegetative bacteria exposed to moist heat invariably occurs exponentially, although deviations from such a postulate are encountered. It must be concluded that the biological, i.e., biochemical, basis of this phenomenon is difficult to explain in terms of single events occurring within the microbial unit. It would seem more feasible to assume that such a result is due primarily to inherent variation in the susceptibility of the members of the population to heat. Thus, it would appear that useful though it may be, a consideration of time/survivor curves alone provides no real answer to the causes of thermal injury in bacteria. For this a more biochemical approach is required. Having stated this, however, we also wish to suggest that the thermal death rate of bacterial cells taken from different stages of synchronously multiplying cultures might yield valuable information, particularly if used in conjunction with cell analysis, since it should theoretically be possible to link loss of viability with changes in cellular nucleic acid, protein, etc., in these homogenous populations.

B. POSSIBLE TYPESOF DAMAGE TO BACTERIAL CELLS 1 . Cell Walls The bacterial cell wall is responsible for maintaining the rigidity of the organism, and for protecting the delicate structures which are found beneath it. Chemically, the wall differs as between, in general, gram-positive and gram-negative bacteria: staphylococci, for.example, possess a considerable amount of mucopeptide and teichoic acids in their walls (Salton, 1964), whereas the walls of gram-negative bacteria, such as E . coli (Weidel et al., 1960) and Proteus vulgaris (Burge and Draper, 1967) are three-layered, in which the mucopeptide comprises one part, and the interpenetrating lipopolysaccharide and lipoprotein layers the remainder. It would, therefore, be expected that different bacteria would respond in differentways to heat treatment. The lipid components of the cell walls of gram-negative bacteria would seem to be a particularly vulnerable point of thermal attack; consequently, subsequent treatment of the organisms with lysozyme

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would enable the enzyme to attack the mucopeptide with resulting

cell lysis. The cell walls of these bacteria are, in fact, stripped off at temperatures between 75" and 100°C., and trypsin is active against E . coli only when the cells have been treated at these temperatures (Salton, 1953; Razin and Argamon, 1963).There was no lysis, however (this was also observed by Harries and Russell, 1967), presumably became of iritracellular protein coagulation. Lysozyme was found to have no effect on E. coli cells which had previously been heated at 50°-60"C. (Harries and Russell, 1967), and this would denote either that lysozyme had been unable to reach its mucopeptide substrate or that varying degrees of change in intracellular protein had prevented lysis. Penicillin-induced spheroplasts, in which the rnucopeptide layer is depleted (Salton and Shafa, 1958), presumably as a result of the interference by the antibiotic of a cross-linking reaction (Wise and Park, 1965), are of use in these studies: spheroplasts of E. coli (Hugo and Russell, 1961; Russell and Harries, 1968) and of Serrutia marcescens (Furr, 1966) are not lysed by high temperatures, but there is an increase in extinction at 500 m p of the spheroplast suspensions as a result of their exposure to nioist heat; this may be indicative of changes in intracellular protein (Russell and Harries, 1968). The outer layers of the cell wall play some part in maintaining cell form in Pseudomonas aeroginosu (Eagon and Carson, 1965) and, presumably, in other gram-negative bacteria; their removal from mucopeptidedepleted structures should thus lead to a subsequent effect of heat on the cytoplasmic membrane with ensuing lysis. The fact that this does not occur provides evidence for the contention that at temperatures of ca. 50"-6O"C., cell wall injury (lipid or mucopeytide) is either negligible or nonexistent. Lennox (1960) concluded that, because boiled cells maintained their cell wall antigenicity, the cell walls of gram-negative bacteria were unaffected by high temperatures. Evidence contrary to this conclusion has been found. The thermoresistance of L-forms of gramnegative bacteria has been found to be less than whole cells (Shaklovskii, 1965). It is therefore apparent that heat may cause changes in the cell walls of gram-negative bacteria, although it may not result in cell lysis but rather a weakening of the lipid layer. In a study of isolated cell walls of Proteus vulgaris (Burge and Draper, 1967), it was found that the kipopolysaccharide component of the cell wall decreased in size when heated at either 50" or 100°C. and such changes were riot reversible. The cell walls of many psychrophilic bacteria have been found to

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possess cell walls that are very sensitive to moist heat (Robison and Morita, 1966; Hagen et al., 1964).This may be significant in determining their sensitivity to the physical environment. The cell walls of staphylococci are unaffected by heat treatment up to 100°C. (Salton, 1953) and this has recently been confirmed in studies involving electron micrographs of ultrathin sections of whole cells of heated suspensions of S. aureus in this laboratory (Allwood and Russell, 1969a). This is not, perhaps, surprising in view of the remarkable mechanical strength of the wall of this organism. Their thermal stability has also been reported by Warren and Grey (1963). Thus, with the few bacteria studied to date, it is unlikely that thermally-induced injury and death occur as a result of damage to the cell wall. It is, however, tempting to speculate that the composition of the cell wall does influence the susceptibility of an organism to moist heat, since the greater its mechanical strength, the more it might act as a “thermoresistant barrier” around the underlying structures.

2. Cytoplasmic Membrane The bacterial cytoplasmic membrane is a delicate, semipermeable structure which is situated beneath the cell wall, and which is lipoprotein in nature. It is responsible for controlling the passage of solutes into, and of waste and other products out of, the cell. Thus, methods for detecting membrane damage involve either the measurement of various substances released from the cells or the determination of the intracellular penetration of substances which are normally excluded from cells with an undamaged membrane. Mitchell (1951) suggested that the osmotic barrier of bacterial cells might b e damaged by heat treatment. There appeared to be a link between loss of viability and the leakage of small molecules from the cell which would occur, at least partially, as a result of damage to the cytoplasmic membrane. It has also been reported that material which leaked from heated cells of S. aureus absorbed light at 260 m p and gave the orcinol reaction for pentoses (Califano, 1952). Leakage was presumably due to destruction of the osmotic barrier of the bacterial cell and the consequent escape of material from the cell cytoplasm, formed by an “uncoupling reaction” of heat (Beckett et al., 1959). The term “uncoupling reaction” implied the breakdown of protein, polypeptides, and nucleic acids into smaller components. Pethica, (1958) considered that leakage was a manifestation of mem-

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brane damage. Strange and Shon (1964) showed that a close relationship existed between leakage of RNA-like material from cells and death of Aerobacter aerogenes stored at 47°C. However, an examination of membrane changes was not undertaken and it was concluded that the loss of 260 mp-absorbing material was due, primarily, to RNA breakdown. Many reports have been made of the leakage of different materials from bacterial cells, although this may not necessarily be attributed to primary membrane damage. Iandolo and Ordal (1966) have shown that there is a rapid loss of 260 mp-absorbing material, potassium (K+) ions, and amino acids from S. aureus stored at 55°C. in buffer. It was considered that membrane damage may have been caused by exposure to high temperature. The leakage of 260 mpabsorbing material from E . coli was determined during storage in water at different temperatures by Russell and Harries (1967). A correlation was found between the rate of leakage and loss of viability of the organism. Macleod et aE. (1966) proposed that the boiling of a suspension of A. aerogenes caused damage to the permeability barrier of the cell. Amino acids are also released, presumably from the soluble pools, and so is protein material (Allwood and Russell, 196713, 1968). Thus, the question arises as to whether such leakage is responsible for death, or whether it is the result of some other primary changes induced elsewhere in the cell. It seems reasonable to infer that leakage is a secondary effect, occurring as a result of cellular degradation and/ or membrane damage, but that in gram-positive organisms, at least, a loss of soluble pool constituents occurs leading to starvation. This primary effect leading to death would be directly manifested as a leakage of these constituents from the cell. Two other approaches are indicative of thermally-induced membrane damage. Anilinonaphthalenesulfonic acid (ANS) penetrates into heat-shocked S. aureus (Allwood and Russell, 1968). This dye reacts with intracellular protein, but does not appear to have been used with other thermally-injured bacteria, an omission that should be rectified. The finding that the optical density (extinction) of SUSpensions of mycoplasma, bacterial protoplasts, spheroplasts (cf. with previous section), and L-forms falls when these are heated in an osmotic stabilizer at 80°C. (Razin and Argamon, 1963), indicates that heat clearly does influence the integrity of the cell membrane. The influence of temperature on the physicochemical structure of lipid membranes was studied by Byrne and Chapman (1964). It was suggested that the cytoplasmic membrane would be expected to be

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very sensitive to temperature changes since such membranes existed on the borderline of a temperature-sensitive phase transition. We are thus led to consider what sort of membrane damage could occur from which would ensue intracellular penetration or extracellular leakage. Two possibilities emerge, first, that there is a melting of the lipid component of lipoprotein membranes, and second, that there is physical damage of another kind, viz., that leading to small “holes” in the membrane (Marquis and Corner, 1967). From a biophysical point of view, both would seem feasible, with the latter more likely at temperatures below, say, 60°C. Membrane damage would lead to the loss of control, on the part of the cell, to concentrate small molecules intracellularly, which would, in turn, lead to a lowering of the internal osmotic pressure, which may itself affect the normal metabolic functioning of the cell. Cells not yet dead, but damaged, may be able to repair, at least to some extent, this membrane damage; however, membrane synthesis itself has been little studied, and it is not possible to predict how this may be achieved. There is little direct evidence indicating the role of membrane damage in the thermal injury of bacterial cells, although the leakage of material from cells may, in part, be a manifestation of the loss of ability of the membrane to control the passage of small molecules out of the cell. In view of the sensitivity of the membrane, it is reasonable to suggest that it may be of importance in the inability of vegetative bacteria to recover from thermal injury.

3. Ribosomes and RNA Degradation Califano (1952)concluded that the loss of 260 mp-absorbing material from heated suspensions of E . coli was a result of the breakdown of intracellular RNA. Hansen and Rieman (1963) also showed that heat treatment caused a loss of nucleic acids from gram-negative bacteria. A close study of the effects of thermal stress on viability and RNA breakdown in A. aerogenes was made by Strange and Shon (1964). At 47”C., RNA degradation was shown to occur, and preceded loss of viability, and it was concluded that the depletion of RNA was probably not the primary cause of death at this temperature. High temperatures have also been found to reduce the percentage of RNA in Streptococcus faecalis (Beuchat and Lechowich, 1968). As a result of RNA degradation, there is an increase in the nucleotide content in the soluble pool, followed by a leakage from the pool through the membrane (Allwood and Russell, 1968), which indicates

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that RNA breakdown precedes the leakage of 260 mp-absorbing material. Until recently, these experiments have been concerned with the degradation of cellular RNA, with no attempt being made to apportion reasons a s to what type of RNA is, in fact, especially thermolabile. There are three types of KNA in the cell: relatively small molecular weight transfer (t) RNA in the cytoplasm, which binds a specific amino acid, previously acitvated by combination with adenosine triphosphate (ATP); highly organized messenger (ni) RNA, which is transitory, and carries information from the DNA; and ribosomal (r) RNA, of high molecular weight, and found as ribonucleoprotein particles in the cytoplasm. These ribosomes occur as aggregates, the polyribosomes (polysomes), which comprises the functional unit in protein synthesis. The recovery of salt tolerance in S. uureus has been shown to parallel the recovery of the culture from sublethal heat treatment (Stiles and Whitter, 1965). Iaridolo and Ordal (1966) found that the recovery of salt tolerance after sublethal heat treatment could be inhibited b y actinomycin D which indicated that the synthesis of RNA was essential for recovery. This suggested that the degradation of RNA had occurred during exposure of suspensions stored at 55°C. This was later confirmed (Sogin and Ordal, 1967), and it was further shown that rRNA was degraded during heat treatment, and that its resynthesis was essential for recovery. It was postulated that, when heat-injured cells were transferred to a recovery medium, the extended lag period was in part due to the breakdown of ribosomes as a result of heat treatment, and that the cells were unable to reproduce uritil the ribosomes were regenerated. There has also been evidence for the degradation of ribosomal RNA in a ternperature-sensitive mutant of E . coli (Nozawa et ul., 1967). Studies on the thermophilic bacteria have shown that there is a correlation between ribosome stability and maximum growth temperature (Saunders and Campbell, 1965; Pace and Campbell, 1967); isolated ribosomes of thermophilic bacteria have been reported to be more heat-stable than ribosomes of mesophilic bacteria (Stenesh and Yong, 1967; Friedman et al., 1967). This need not necessarily imply, however, that the other types of RNA are not thermolabile. The extreme lability of mRNA has been well documented (Stacey, 1965) with the implication, therefore, that excessive mRNA degradation could occur resulting in the loss of an essential component of cellular synthetic processes.

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There is, then, evidence that rRNA degradation occurs in heattreated bacteria: however, there is no information available to suggest what happens to the protein part of ribosome particles. This is an important consideration, and the following scheme is tentatively put forward to explain the fate of ribosomal protein: (a) As a result of exposure of the bacteria to moist heat, there is a degradation of the RNA component of the ribosomes, with the protein fraction virtually unchanged. This would occur at lower temperatures, i.e., at temperatures that do not normally cause changes in protein structure leading to coagulation. (b) At higher temperatures, protein coagulation would prevent, to a considerable extent, the breakdown of the RNA component of the ribosomes; if, however, coagulation is secondary to some other effect in the cell, such as RNA degradation itself, then this degradation would proceed rapidly for a short period before protein coagulation intervened. The results of experiments conducted in our laboratory have supported these suggestions (Allwood and Russell, 1968). The changes induced in isolated nucleic acids during heat treatment at different temperatures in &To have recently been reported. Eigner et al. (1961) pointed out that RNA was degraded rapidly and was far less stable than single-stranded DNA at 50°C. Lindahl (1967) has shown that the heat inactivation of tRNA at 90°C. results in 63% loss of transfer function after 10-hour heating. Clearly, there is mounting evidence to suggest that there may be a correlation between the degradation of cellular RNA in vegetative bacteria and loss of viability during storage at high temperatures. The fact that resynthesis of RNA is essential for the recovery of heat damaged cells now adds force to this concept. We must wait patiently for more concise studies to show clearly at what stage during exposure to moist heat RNA degradation occurs, and what tolerance the vegetative cell shows to the loss of so vital a functional part of the cell unit.

4 . DNA and Mutation Wood (1956) suggested that nuclear damage could be the result of high temperature inactivation of bacteria and other unicellular organisms. The denaturation of native, double-stranded DNA is, presumably, a collapse of the hydrogen-bonded, double-helical structure, leading eventually to a dissociation of the complementary strands (Szybalski, 1967): as a result of this denaturation, there is an increase in extinction of DNA at 260 m p and a decrease in its viscosity. How-

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RUSSELL

ever, DNA must be heated to high temperatures (usually above 75°C.) before the structure collapses, and the cleavage of hydrogen bonds at high temperatures is often a gradual process. There is no endogenous degradation of DNA in starved cells of S. lutea at 37°C. (Burleigh and Dawes, 1967),and the DNA content of S. aureus cells stored in water at 60°C. remains constant (Allwood and Russell, 1968); conversely in a thermolabile, heat-treated psychrophile, DNA, of larger molecular weight than the protein, followed protein and RNA leakage out of the organism, an order which suggested that component release was under cellular control (Haight and Morita, 1966).In neither case was there evidence of DNA degradation. Eigner et al. (1961) showed that isolated double-stranded DNA was relatively heat stable. Baldwin (1964) has discussed the thermal stability of DNA and reported that the denaturation of most bacterial DNA preparations occurred between 70” and 90°C. However, Bridges et al. (1968) have recently shown that the sensitivity of various strains of E . coli to incubation at 52°C. paralleled their sensitivity to y-radiation, and concluded that these strains were inactivated, at least in part, b y damage to DNA of a similar type to that produced by ionizing radiations. There are also wider issues to be considered in terms of nuclear damage: are cells which survive a heating process to be regarded as mutants, since for their revival they may require nutrient substances not needed by control (unheated) cells? There is, however, no evidence available at present implicating the induction by heat of stable mutants of vegetative bacteria (Jordan et al., 1947; Jackson and Woodbine, 1963; Harries, 1968). Dry heat-induced mutations have been found to occur in bacterial spores, as already discussed, but these take place only at temperatures much higher than those with which we are concerned here.

5. Structural Protein Cougulution

The number of statements to the effect that death in heated bacteria is caused by protein coagulation or denaturation would lead one to expect that this contention had been subjected to adequate and critical experimentation. The reverse is the case, and only a few such studies appear to have been undertaken (Heden and Wyckoff, 1949; Allwood and Russell, 1968). The term “denaturation” is used here to denote the disruption of an original, natural, largely hydrogen-bonded and complex structure; presumably, heat induces a breakage of many of the hydrogen bonds and unfolding of the polypeptide chain, with, consequently, a collapse of the native protein structure.

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In general terms, protein denaturation may be completely reversible under some carefully controlled conditions, dependent on the degree to which denaturation has occurred. It is, then, to be wondered whether bacteria possess some sort of mechanism which enables them to “repair” this denaturation. However, the basis for the revival of heat-damaged cells on nutritionally complex media does not indicate that protein renaturation is involved (see later section). Moreover, loss of viability of heated bacteria would appear to occur before protein coagulation can be detected. Thus, we are of the opinion that gross denaturation of protein must occur after other changes within the cell which are responsible for death. A factor that may be of importance is the water activity ( a , ) of the environment in which the bacteria are heated; it is known that heat denaturation involves not just protein but the entire protein-water system (Brandt, 1967), for without water, protein cannot undergo denaturation. Work in this laboratory has clearly shown that the storage of cells in concentrated sucrose solution at 60°C. reduced the rate at which coagulation of the cellular protein occurs, without influencing the rate of loss of viability of the suspension, another indication that protein coagulation is not the primary cause of thermal injury in vegetative bacteria.

6. Functional Protein (Enzyme) Znactivation At first sight, enzyme inactivation in bacteria might be an obvious cause of thermally-induced death, but it is perhaps difficult to envisage how such a concept could result in exponential death. It is, of course, probable that some enzymes are inactivated on exposure of bacteria to supraoptimum temperatures, which may explain why bacteria which survive a sublethal heat exposure may, for a short period, require additional preformed nutrients which the cell cannot now build up from simple sources. Coenzymes, which are less likely to be destroyed by heat than apoenzymes, would be unlikely to be involved in the recovery process. Few experiments have, in fact, been carried out with a view to making a critical assessment of the role of enzyme inactivation in bacterial death. One study, by Rahn and Schroeder (1941) showed that whereas there was a 99% kill of BaciEZus cereus after 10 minutes at 46“C., the reductions in peroxidase and catalase activity were only 14 and 20%, respectively. However, one enzyme that is especially sensitive to heat is the malic dehydrogenase (MDH) of V. marinus MP-1 (Langridge and Morita, 1966); in cell-free extracts, MDH was inactivated at low temperatures, whereas in growing cells it was much

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less sensitive. The heat inactivation of formic dehydrogenase in whole cells of a facultatively psychrophilic strain of E . coli was far greater than in the mesophilic strain under the same conditions (Upadhyay and Stokes, 1963). It is to be wondered whether the thermolability of enzymes is responsible for such organisms being psychrophiles, and, indeed, Evison and Rose (1965) have suggested that the death of psychrophilic bacteria above their maximum growth temperature could be due to heat inactivation of their constituent enzymes. The thermal lability of a number of enzymes has been reported (Hagen and Rose, 1962). In nonsporing organisms, in general, some enzymes will be more thermolabile than others, and, consequently, the site of an enzyme in the cell may play at least a part in determining its inactivation by heat. It is also becoming apparent that the immediate physical environment may influence its stability together with its combination with other molecules in the cell matrix. To select one enzyme of abnormal thermosensitivity out of the potentially vast number possessed by the bacterial cell borders on the impossible. Perhaps some useful data might accrue by studying isolated cytoplasmic membranes, at which various enzymes are present (Hughes, 1962), and cell-free cytoplasmic contents. The difficulty here, in our opinion, is that reaction to heat treatment need not necessarily parallel what takes place in the heatexposed intact bacterial cell. It must also be stated that even if enzymes are destroyed, this need not prove fiatal to the bacterial cells, as the cells may still retain the ability to resynthesize the enzymes when the bacteria are transferred to a nutrient recovery medium and incubated. Our conclusion here must be that there is a lack of correlat'ion between enzyme inactivation and thermally induced bacterial death in mesophilic bacteria. V.

Repair of Thermal Injury

The observed synthesis of RNA during the recovery of salt tolerance of heat-treated S. uureus (Sogin and Ordal, 1967) has already been discussed. This finding clearly implied that RNA degradation had occurred during heat exposure of the cells. The synthesis of RNA and D N A in S . uureus during the heat-induced lag phase of growth has been studied in 'this laboratory, together with changes in the composition of the metabolic pool. It appeared that RNA synthesis was not impaired and D N A synthesis occurred normally. There was evidence

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to suggest that reconstitution of the metabolic pool was an essential stage in the process of recovery (Allwood and Russell, 1969b). Hershey (1939) investigated the recovery of heat-damaged cultures during the extended lag phase of growth following exposure. Evidence suggested that impairment of respiratory function occurred as a result of thermal stress. No further work has been reported on this problem, although we have been unable to confirm these findings. Correlation has been suggested between the maximum growth temperature of psychrophiles and the stability of their respiratory system (Robison and Morita, 1966). It remains to be seen if thermallyinduced breakdown of normal respiratory function, if it occurs, could lead to the death of vegetative bacteria. Much work has been carried out in studying the effects of different recovery media on the numbers of heat-treated bacteria able to grow and divide to produce visible colonies. We do not propose to review this work in any detail in this essay, since few of the results have given insight into the mechanism of thermal injury and death in vegatative bacteria. They have at least suggested that thermal injury does occur, implying that a proportion of a population, often variable in size, is more sensitive to their immediate environment. The reviews of Wills (1957) and Harris (1963) describe these results fully. Heater and Vanderzant (1957) reported that a number of amino acids were specifically responsible for the enhanced recovery of heatdamaged Pseudomonas jluorescens. This was believed to indicate that heat inactivation of an enzyme system had occurred, inducing the requirement for particular intermediates. Alternatively, factors present in the more complex medium contributed to particular synthetic processes capable of repairing certain cellular components inactivated by heat treatment. Hansen and Rieman (1963) surmised that the addition of metabolites to the recovery medium may stimulate enzyme resynthesis after heat treatment. Reporting the recovery of thermally injured S. aureus, Baird-Parker and Davenport (1965) suggested that the requirement for specific materials in the recovery medium indicated that catalase had been destroyed and therefore hydrogen peroxide produced during recovery was not destroyed and was toxic to cells. Clearly, such studies suggest possible causes for thermal injury in bacteria but do not provide information to show particular sites in the cell that are sensitive to high temperatures. The significance of results may often be difficult to assess, particularly since the viable counts of heat-treated suspensions are found to vary to a far greater degree than untreated suspensions. Also, many factors

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may be involved. This is well illustrated in Table I, which shows the viable counts of untreated suspensions and suspensions stored at 50°C. for 20 minutes. Cultures from three sources were compared: (1) A culture grown for 18 hours without aeration. (2) A culture grown for 18 hours with aeration. (3) A culture grown with aeration and harvested in the logarithmic phase of growth. Although the presence of yeast extract enhanced the recovery of tlie nonaerated culture, it appeared to have no influence on the recovery of the other cultures. It must be concluded, therefore, that the interpretation of the causes of injury from results obtained indicating specific nutrients enhancing the numbers of survivors able to grow and multiply on solid media, are very complex. A large number of factors are involved, many of which may not be apparent. It may be reasonable to suggest that recovery, as arranged in this manner, is a result of the need for nutrient materials lost to the cells during storage at high temperatures leading to starvation (the loss of materials from cells during storage has been discussed in an earlier section). It may therefore be a form of replacement therapy to overcome the starvation of small molecules, and may not imply that particular synthetic processes in the cell essential to normal growth are inactivated by heat treatment. TABLE 1 THE RECOVERY OF IIEAT-DAMAGED s. UUTeUS

FROM

DIFFERENT SOURCES"

Viable counts Source of culture

N.A.b

Nonaerated Aerated Logarithmic phase of growth

37.4 23.9 38.0

(X

10R/rnl.) N.A.

+ Y .E.C

100.9 29.3 37.8

Data of Allwood (1968). bN.A.,nutrient agar. "Y.E.,1% yeast extract (Difco).

VI.

Conclusions

There are various simultaneous or sequential changes which occur in heated bacteria. When the organisms are stored at very high temperatures (ca. lOOOC.) these changes take place at such a rapid rate that it is impossible to differentiate primary from secondary

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effects. Thus temperatures little above the maximum at which growth in a nutrient medium occurs must be used. Even so, it is still difficult to define the order in which these changes take place, and the picture is further complicated by the fact that in at least some bacteria, e.g., Sarcina lutea (Burleigh and Dawes, 1967), held under nongrowing conditions at ambient temperatures, there is membrane damage, degradation of RNA, and leakage of pool material into the environment. Therefore, in the case of an organism where the minimum temperature resulting in thermal death is, say, 50”C., one must differentiate between death due to heat at and above 50°C., and death below 50°C. caused by starvation accelerated by temperature. If such a premise is accepted, then this would be of use in explaining the slow death of bacteria under nongrowing conditions, such as when stored as washed suspensions. We have discussed the biochemical changes that have been found to occur during the exposure of vegetative bacteria to lethal temperatures and indicated the possible causes for the loss of ability of cells to grow arid reproduce. It is worth emphasizing that any analysis of the biochemical changes occurring in cells during thermally-induced death must be related to viability. The assay of viability is itself an arbitrarily definable character of cells and, apart from being a very variable and environmentally sensitive property of biological material, it is difficult to conclude what quantitative relationship it may have with thermally-induced damage and inactivation of bacteria. The complex interrelationships of changes occurring during thermally-induced damage to vegetative bacteria and their subsequent ability to recover from such treatment cannot be explained in simple terms. Thermal death appears to be due, not to such gross alterations in cellular organization as protein coagulation, but to far more subtle changes in intracellular labile molecules and organized systems which the cell finds difficult to reverse or overcome. As early as 1935, Belehradek (1935) believed that “with the advancing study of heat injury, it becomes more and more obvious that the effects of high temperature upon living systems are so complex that no theory could venture to reduce them all to a uniform basis.” In view of the universal degradation and disrupting influences of high temperatures on biologically active material, such a statement may well prove to be truer than we dare to consider. VII.

Summary

Because of the increasing use of heat sterilization and of freezing methods of preservation of biological materials during the last 50

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years, the microbiologist has been obliged to give considerable attention to the behavior of microorganisms at temperatures outside the limits of growth, and assessments of the efficiency of heat sterilization and pasteurization have been greatly studied. However, the causes of the thermal inactivation of vegetative bacteria have been neglected until recent years and much of the dogma on the subject is now proving to be at least a gross oversimplification of the problem. Thus, intracellular protein coagulation occurs above a particular temperature of exposure, but evidence is now mounting to show that it is not the cause of thermally-induced death of vegetative bacteria. More subtle changes are implicated, and studies of RNA degradation, changes in DNA, inactivation of specific bacterial enzymes, and changes in the cytoplasmic membrane are worthy of further consideration in the light of recent evidence. In view of the nonspecificity of heat as a physical agent causing inhibition of normal biological functioning, perhaps the causes of thermal death in vegetative bacteria may be a dislocation of the interrelated biochemical reactions and forces that go to make up a dynamic biological unit. REFERENCES Allen, M. B. (1953). Bucteriol. Reu. 17, 125-173. Allwood, M. C. (1968).P1i.D. Thesis, University ofWales. Allwood, M. C., and Russell, A. D. (1967a).Appl. Microhid. 15, 1266-1269. Allwood, M . C., and Russell, A. D. (1967b). Experientiu, 23, 878-879. Allwood, M.C , , and Russell, A. D. (1968).1. Bucteriol. 95, 345-349. A~~WIJIJL!, 14.C., a i r d Riissell, A. D. (1969n).]. Appl. n a c t e r i d . , 32, 68-78. Allwood, M. C., and Russell, A. D. (19Wb).].Appl. Bacterial., 32,79435. Ameliinxen, R. E. (1967).Biochinr. Biophys. Acttc 133,2442. Baird-Parker, A. C., and Davenport, E. (1965).J. Appt. BacterioE. 28, 390-402. Baldwin, H. L. (1964). In “The Bacteria” (I. C. Cunsalus and R. Y. Stanier, eds.), Vol. IV. Acadcmic Press, New York Beckett, A., Patki, G., and Robinson, A. (1959).J.Phurm. Pharnmcol. 11,360-373. Belehradek, J. (1935).“Temperature and Living Matter.” Borntrager, Berlin. Beuchat, I,. R., and Lechowich, R. V. (1968).A p p l . Microbiol. 16,772-776. Brandt, J. F. (1967). In “Thermobiology” (A. H. Hose, ed.). pp. 25-72. Academic Press, New York and London. Bridges, B. A,, Ashwood-Smith, M. J , , and Munson, R. J. (1968).53rd Gen. Meeting Soc. Gen. Microbid., Sept., 1968. Cambridge Univ. Press, London. Bmch, C. W. (1963).Symp. Life Sci. Spuce Res., pp. 357-371. Burge, R. E., and Draper, J. C . (1967).J . Mol. Biol. 28, 205-210. Burke, B. (1923)./. Infect. Diseases 33,274-284. Burleigh, I. G., and Dawes, E. A. (1967). Biochem. 1. 102,236-251. Byrne, P., and Chapman, D. (1964).Nature 202, 987-988. Califano, L. (1952). Bull. World Heulth Organ. 6, 19-34.

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Collection o f Microbial Cells’

DANIELI. c.WANG AND ANTHONY J. SINSKEY Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts I. Introduction.. ................................ 11. Centrifugation .............................. A. Principles ............................... B. Application, Equipment, and Pe .................... ......................................... 111. Filtration ...................... A. Principles .............................................. B. Application, Equipment, and Performance ............................. IV.

121 122 122 125 132 132 135 141 141 ............................. .................................. 142 143 V. Foam Fractionation ......................................................... A. Principles ....................................................... 143 B. Collection of Microbial Cells by Foam Fractionation ....... 144 146 VI. Miscellaneous Recovery Systems ....................................... 146 A. Ion Exchange ............................................................ 147 B. Biphasic Liquid Extraction .......................................... 148 C. Electrophoresis ....................... 150 VII. Summary ............... 150 References .............. ............................

I.

Introduction

The collection of microbial cells from suspending fluids is a task routinely encountered by individuals in laboratories, pilot plants, and production facilities. Advancing technology has placed new tools of collecting microbial cells either through improvements in existing methods or replacement of older techniques entirely. It is the intent of this paper to examine collection techniques which are presently available for the recovery of microbial cells. Simultaneously a brief review will also be presented on the classical and routine collection methods which have been employed in the past. The title of this review may be somewhat of a misnomer. Generally speaking, one refers to “microbial cells” when materials such as bacteria, algae, yeasts, and fungi are considered. Although this paper will discuss the collection of these substances, it is also our intent to present some of the more recent research studies on viruses, vaccines, ‘This is Contribution No. 1425 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts.

121

122

D. 1. C. WANG AND A . J . SINSKEY

bacteriophage, and mammalian cells. It should, however, be stated that the collection techniques which will be reviewed deal only with removal of materials from liquid suspension. The collection of cells from gases or vapors will not be discussed. The choice in selecting a method of collecting cells depends on many factors. Physical and chemical properties of the cells often dictate the exact method which can be employed. In the paragraphs to follow some of the pertinent properties and principles which are relevant to different collection techniques will be presented.

II.

Centrifugation

A. PRINCIPLES The most commonly used method for the removal of microbial cells from suspension is probably centrifugation. One of thc reasons why centrifugation is so often used lies in the overall simplicity of the process. Before presenting the details of this method of collection, the basic principles of centrifugal operation will first be summarized. 1. ~ ~ ~ e ~ eCentrifugution n t i ~ l When one considers the collection of microbial cells by centrifugation, the first question which is asked is the length of centrifugation time and what gravitational field must be employed. To answer this question let us first examine the theoretical principles underlying a centrifugal separator. The rate of settling of a solid particle through a liquid due to gravity can be expressed by Stokes’ law:

where

V

= settling

velocity of particles (cm./sec.)

pp = density of particle ( g n ~ / c r n . ~ ) pI, = density of liquid ( g m . / ~ m . ~ )

dp = diameter of particle (cm.) p = viscosity of liquid (gm./cm./sec.) g = gravitational constant (980 cIn./sec.2)

However, when the same particle is subjected to increased gravita-

COLLECTION OF MICROBIAL CELLS

123

tional field, such as that in a centrifuge, the rate of settling can be greatly enhanced. In this case the particle settling rate becomes

where w = angular

velocity of centrifuge (rad./sec.) r = distance of particle from axis of rotation (cm.)

Alternatively, if the particle being sedimented is accomplished by means of a continuous flow centrifuge, the flow rate through the machine can be estimated to be:

where

Q r, V, S,

= rate

of flow through centrifuge ( ~ m . ~ / s e c . ) radius of the centrifuge (cm.) = volume of liquid in the centrifuge = effective settling distance (cm.) = effective

From Eq. (2) it can be seen that the properties of the cells which govern the efficiency of a centrifugal collection device are: (1) the density difference between the cell and the suspending fluid; (2) the diameter (or size) of the particle; and (3) the viscosity of the suspending fluid. The index which typically characterizes the efficiency of a centrifugal collector is the particle size. When Eq. (2) is examined it can be seen that the settling rate of the cells in a centrifugal field is proportional to the square of the particle size. Thus, the variations in size of the different cells can exert a profound influence on the overall efficiency of a centrifuge. Table I illustrates the size range of some typical biological entities, as well as the type of centrifuge which can be employed for their removal. It should be emphasized at this time the types of centrifuge which can be used for collecting the cells shown in Table I are all based on the principle of differential centrifugation. This type of operation does not offer a high degree of resolution. For example, if a solution contains suspended particles having

124

D. I. C , WANG AND A . J. SINSKEY

TABLE I

Type of cell Virus and phage Bacteria

Size range (in microns) 0.01-0.10 0.3-3.0

Yeast

4.0-7.0

Mammalian tissue

5.0-20.0

Fungi

10.0-150

Type of operation Ultracentrifuge Large-scale commercial centrifuge Batch bottle laboratory centrifuge Large-scale commercial centrifuge Batch bottle laboratory centrifuge Large-scale commercial centrifuge Batch bottle laboratory centrifuge Large-scale commercial centrifuge Batch bottle laboratory centrifuge

Range of visibility Electron microscope Light microscope

Light microscope

Light microscope

Naked eye

different densities, as well as different particle sizes, at equilibrium, both types of particles may be located in the same sedimentation zone. For the recovery of large particulate materials such as bacterial, yeast, mammalian, and fungal suspensions, the degree of resolution achieved in differential centrifugation is generally adequate for practical purposes. This is due to the order of magnitude differences in the sedimentation rates between the solid particles to the contaminating components. However, if one examines the size region of virus and bacteriophage, the poor resolution achieved in differential centrifugation may not be adequate for recovery purposes. In view of this difficulty the principles of zonal centrifugation are often employed in the recovery of submicron materials.

2. Zonal Centrifugation The development of the zonal centrifuge owes much to N. G. Anderson and his associates of the Oak Ridge National Laboratory. Although much of their efforts have been devoted toward the separation of macromolecules, considerable strides have also been made in the recovery of submicron particles such as viruses and phages. Zonal centrifugation is based on two basic principles, as well as the combination of the two. The first of these is known as rate-zonal centrifugation where the heterogeneous particles are separated into

COLLECTION OF MICROBIAL CELLS

125

discrete zones due to the differences in the sedimentation rates. The second case, known as isopycnic-zonal centrifugation, particles are separated solely due to differences in buoyancy or banding density. The combination of rate- with isopycnic-zonal is probably more commonly employed in the recovery of submicron particles. This technique is unique in that when employed in virus concentration simultaneous purification can also be achieved. This is due to the higher degree of resolution which can be attained when these combinations are used together. To examine the principles which render the combination of zonal centrifugation more versatile than the conventional differential centrifugation, it would be necessary to examine again the sedimentation Eq. (2). From this equation it is evident that particles having different radii and densities could conceivably be sedimented at the same rate. This phenomenon wouId occur if the product of d; and (pp - p L ) as shown in Eq. 2 for the different particles is the same. However, if one isolates the particle size and particle density by a twostep procedure, it would then be possible to achieve a higher degree of resolution in the combined separation steps. This is precisely the principle which is applied in the combination rate- and isopycniczonal centrifugation. Thus one, for example, could use a rate-zonal centrifugation and isolate a distinct zone of particles due to rate of sedimentation. This zone can then be further resolved due to the differences in buoyancy.

B. APPLICATIONS, EQUIPMENT, AND

PERFORMANCE

1 . General The use of centrifuges to collect microbial cells, such as bacteria, yeasts, algae, and fungi, has been explored for many years. The types of machines available to the laboratory investigators, pilot-plant and production engineers include: (1)test tube centrifuges, (2) tubular bowl centrifuges for batch and continuous operation, and (3) disctype centrifuges for pilot-plant and production operations. Since the operational features of most of this equipment are well documented in the manufacturer’s technical bulletins, no further discussion along this line will be presented. Should the reader be interested in some of the production scale centrifuges, it is recommended that the recent article by Flood et al. (1966) be consulted. It is our intent in this portion of the paper to present some of the recent innovations in centrifugal developments.

126

D. I. C . WANC AND A. J. S1NSKF.Y

Some of the major advances which have been made in recent years are in the preparative-scale operation of viruses and vaccines recovery through centrifugation. The most commonly employed laboratory and preparative centrifuges for these purposes are the tubiilar bowl Sharples Laboratory Supercentrifuge and the zonal centrifuge. Results along these lines will be presented. 2. Virus Recovery

a. Diferentia 1 Centrifugation The theory of scaling-up differential centrifuges from laboratory data has been presented b y Ambler (1959). It was shown there that two parameters can be used to characterize a given centrifugal operation. The first of these is governed by the physical properties of the cell and of the suspending fluid. This is essentially the sedimentation velocity of the cell under unit gravitational field and this value can be calculated using Eq. (1). The second parameter characterizes the physical operational features of the centrifuge. Ambler (1959) presented the latter parameter as the sigma factor ( 2 )which is exprcssed a s Eq. (4).

It was also assumed that if one characterizes the cut-off point of the centrifuge corresponding to the 50% removal efficiency, then rearrangement of Eqs. (l),( 3 ) , and (4)yields the classical centrifuge scale-up equation shown as Eq. (5).

L

J L

J

Further examination of Eq. ( 5 ) shows that the ratio Q/I: for the removal of a given particle regardless of the scale of operation should be a constant. Stated in an alternate manner, when one compares different sizes of centrifuges for a given particle separation, it is possible to compare only the flow rates and the sigma factors in the following manner :

127

COLLECTION O F MICROBIAL CELLS

Using the Q / X factor as a means of characterizing the batch tubular Sharples No. 16 Supercentrifuge, Patrick and Freeman (1959) presented some laboratory data on the recovery of a 60 mp-diameter test virus. The viruses were propagated within the living tissues of chicken embryos. The results of their recovery studies are tabulated in Table 11. TABLE I1 VIRUS RECOVERYFROM BATCH TUBULAH B O ~ AS L DETERMINED BY BOWL FLUIDANALYSIS

Centrifiigation time (minutes)

15 30 60 240 ( I - ,

Virus removal efficiency (%) a t howl speed (r.13.m. x 1000)"

10 0 40 15

20

30

40

50

54 58 71 - I!

68

78

-

-

95 96

99 99

97 98 99

Not investigated.

It is evident from the results that by combining proper centrifugation time with the gravitational fields, quantitative recovery efficiencies of the desired material can be formulated. The investigators further analyzed the centrifugation data by comparing the Q/C ratios as a function of the recovery efficiencies. Data presented in this manner are extremely useful for scale-up purposes, as well as for extrapolating laboratory batch operation to continuous operation. The results of these calculations are graphically represented as the solid line shown in Fig. 1. The results show a distinct break occurring at approximately Q/X values between 0.5 x to 1.0 x cm./sec. At higher Q / C values 100%virus recovery will not be obtained. In order to examine the usefulness of their batch centrifugation data, Patrick and Freeman (1960) also performed continuous virus Centrifugation studies with the identical system previously used (Patrick and Freeman, 1959). Again the tubular bowl Sharples Supercentrifuge was used but continuously fed with the 60 mp-diameter test virus (Table 111).Results from these experiments were compared to the Q/C theory of Ambler (1959) for extrapolating batch processing data to continuous centrifugation operations. The results of this evalua-

128

D. I. C . WANG AND A. J. SINSKEY

VIRUS

RECOVERY IN

TABLE I11 FEED SHARPLES

CONTINUOUS

Virus removal efficiency (5%) at bowl speed (r.p.m. x 1000)”

Flow rate (ml./minute)

2.1 4.0 5.0 6.0 7.6 25.0 35.0 50.0

SUPERCENTRIFUGE

15

25

30

84.5

-

-

-

-

-

84.0 56.0

-

43.5

0.0

-

29.0

-

-

63.4

40

84.0 73.0

50

52

54

100.0

-

-

99.2

-

83.0

-

42.0

~~

‘I-,

Not investigated.

tion are shown as the dotted curve in Fig. 1. It can be seen that a similar “break” was also experienced corresponding to Q / X values between 0.5 to 1.5 X lo-” cm./sec. These findings substantiated that for geometrically similar centrifuges, the scale-up theory advanced by Ambler (1959)is reasonably sound for predicting centrifuge performance. These findings are extremely encouraging in that one has now on hand a tool for predicting the centrifugal performance of this type of a machine for other viruses or submicron particles.

b. Zonal Centrifugation The results presented thus far represent the recovery of submicron particles by means of differential centrifugation. The other main area of development of preparative scale centrifugation of submicron materials is the zonal centrifuge. A recent review on the status of virus, vaccine, and bacteriophage separation by means of zonal centrifugation was presented by Anderson (1966).Particular emphasis should be placed in terms of the commercially available preparative scale rotors and their capabilities in the concentration and purification of viruses and other submicron particles. The general performance characteristics of the latest zonal-rotor designs were presented by Anderson (1966) and shown in Table IV. Both the B-XIV and B-XV rotors can be operated as rate-zonal and isopycnic-zonal centrifuges. The general method of loading, unloading, and operating sequences of the zonal centrifuges was also presented by Anderson (1966). Isolation of various types of viruses, vaccines, and bacteriophages

129

COLLECTION OF MICROBIAL CELLS

20

0

40 60 Recovery efficiency ('10)

80

I00

FIG. 1. Batch and continuous centrifuge recovery efficiency of virus at various Q/Z values.

OPERATION

TABLE IV DATAFOR B-XIV AND B-XV ZONAL ROTORS ~~~~

Model B-XIV Aluminum B-XV Aluminum Titanium

Rotor weight empty (kg.)

Rotor volume (~rn.~)

Speed (r.p.m.)

Maximum centrifugal force (g)

3.75

649

30,000

60,000

7.44 12.7

1666

1666

21,000 26,000

45,000 60,000

has been performed using the B-series zonal-centrifuge rotors. Anderson et al. (1966) using the B-VIII rotor with various types of density gradient materials were able to isolate continuously various virus

130

D. 1. C. WANG AN13 A. J. SINSKEY

fractions. Some typical results of their studies are presented in Table V. Other performance characteristics of the B-VIII and B-IX rotors were also presented by Anderson et u1. (1966). TABLE V VIRUS ISOLATION WITH B-VlIl HOTOR"

Virus particle Adenovirus 2 Respiratory syncytial Moloney Moloriey

"Anderson et

Gradient material

Operating speed (r.p.1n.)

Average flow rate (liters/hnur)

Sample volume (liters)

Cesium chloridc

28,000

1.7

3

Cesium chloride Potass iu i n citrate C e s iuin tartrate

36,000 30,000 30,000

1.5 2.0

5 3 2

2.Y

d., 1966.

In another study by Reinier et al. (1966)continuous-flow centrifugation experiments were performed using the B-V zonal centrifuge rotor with live unfiltered polio virus (Sankett strain, type 3) grown in Maitland tissue cultures of rhesus monkey kidney. A total of 60 liters of polio-infected fluid was processcd continuously through the rotor over intermittent periods in 4 days. Typical performance 011 the percentage virus captured versus flow rate is illustrated in Fig. 2.

0

I

I

I

2

3

Flow rate ( L / hr )

Fig. 2. Efficiency of poliovirus capture at various flow rates with B-V rotor.

131

COLLECTION O F MICROBIAL CELLS

.The rc tor speed during separation was maintained at 40,000 r.p.m. It can be seen that an average flow rate between 2 to 3 l i t e d h o u r can be used for complete capture of polio virus using the B-V rotor. The performance using the B-V rotor in terms of concentration and purification are tabulated in Table VI. TABLE VI POLIOVIRUS CoNCENTRATION AND PURIFICATION WITH

Harvest fraction 110. Volume (ml.) Titer (log 10iml.) Concentration factor" Purification factor"

Original

1

60,000 150 6.5 8.6 l x 1X

B-V ROTOH

2

3

4

5

6

30 8.4

55 8.6

40 8.4

40 8.2

40 8.4

100x 8 9 x 120x 88x 5 6 x 73x 12X 36X 12X 16X 24X

7

8

40 8.6

100 8.2

126x 5 1 x 31X 1.4X

Based on infectivity. *Based on specific infectivity.

"

The results of the poliovirus centrifugation studies showed that the B-V rotor can b e operated at flow rates sufficiently high for practical purposes in virus recovery. Furthermore, this rotor appears to be capable of producing a highly concentrated and purified material. It thus appears that the present commercially available zonal centrifuge rotors offer the experimentalist an excellent tool for preparative scale operation in the recovery of submicron particles.

3. Other Centrifugal Operations Other developments in cell collection by centrifugation which are novel and worthy of mentioning include the study performed by Pretlow and Boone (1968).These authors showed theoretically and experimentally that higher resolution can be achieved in density gradient centrifugation by moving the centrifuge tube closer to the axis of rotation. Experiments with Ehrlich ascites tumor and HeLa cells mixture showed that the cells can be separated into different fractions when the location of the centrifuge tubes within a rotor is placed into their proper position. Calibration studies were also performed using divinylbenzene spheres with diameter ranges of 6 to 14 and 12 to 35 p which approximates the size range of many mammalian cells. The authors were able to show quantitatively the increase in resolution

132

D. I. C . WANG AND A. J. SINSKEY

through centrifugation in a Ficoll density gradient when the tubes were properly located. The effect of centrifugation on the viability of Burkitt lymphoma cells was studied by Wang et al. (1968). These investigators were interested to observe the degree of damage to the mammalian cells which may be caused by high-speed centrifugation. The importance in knowing this effect lies in the ability to predict the performance of higher-speed centrifugation for processing large volumes of mammalian cell tissue culture fluid. Experiments using Burkitt lymphoma cells were performed in a laboratory centrifuge at 0" and 25°C. and in centrifugal fields ranging from 25,000 to 42,200 g. The results show that no decrease in cell viability was encountered a t 0°C. and up to 42,200 g. However, reduction in cell viability was encountered when centrifugation was carried out at 25°C. and appeared to be influenced b y speed of centrifugation and time of centrifugation. These studies would indicate that when processing large volumes of mammalian cell culture fluid using high-speed centrifuges, proper judgment must be exercised if a high degrce of cell viability is to be retained. In the recovery of biological cell materials one often desires a method by which the components being separated can be collected coiitiriuously and aseptically. For example, it would be extremely desirable to be able to separate microbial or mammalian cells from the suspending fluid and to be able to recultivate the cells without the fear of the introduction of contaminating organisms. An approach to the development of this type of centrifuge was recently reported by Judson et al. (1968). These authors reported on a closed continuousflow centrifuge rotor which is able to fractionate blood into various cell components. Although asepsis of operation by this centrifuge rotor was not reported, it does appear that this approach may offer possible solutions in the area of aseptic collection of various cells.

Ill.

Filtration

A. PRINCIPLES Filtration as a means of collecting microbial cells is used in two different types of operation. The first of these is known as cake filtration. A typical laboratory example of this would be the removal of bacteria or fungi from a fermentation broth on coarse filter paper. On a production-scale operation, cake filtration is commonly employed to separate, for example, the mycelia from the fermentation broth in order to recover valuable metabolites in the filtrate. The

COLLECTION OF MICROBIAL CELLS

133

second type of liquid filtration is known as sterilization filtration. This is often used to remove completely microbial cells from suspension. When these two types of filtration are compared some distinct differences can be found. These differences will be presented in the following paragraphs.

1 . Cake Filtration Cake filtration is often used as an alternate method of clarifying microbial suspensions. It is, however, difficult to predict quantitatively the filtration rates of microbial suspensions. This is due to the drastic changes in the properties of filter cake which can be encountered due to differences in the fermentation medium, pH, aging, salt concentration, temperature of operation, size of the individual cells, filter cloth behavior, and many other variables. It is possible, however, to examine the general principles of cake filtration and observe the importance of the dependent variables which play a profound role in the rates of filtration. The most commonly and probably the most widely used model to describe the cake filtration phenomenon is shown mathematically as Eq. (7).

dV _ dt

e3

- ( 1 - E)'

AAP KS5 p L

(7)

where

V = volume of filtrate t = time or void volume of filter cake; volume of space filled by fluid/total volume of filter cake K = Kozeny constant So = specific surface area of filter cake, i.e., surface area per unit volume of solid A = filtration area AP = pressure drop across filter cake p = viscosity of fluid L = depth of filter cake E = porosity

When Eq. (7)is expressed in the familiar rate, resistance, and driving force concept, the following equation is obtained:

134

D. I. C . WANC, AND A. J. SlNSKEY

where a = specific resistance of the cake (1 - E)' -€3

KS8

However, the prediction of the specific resistance of a biological cake cannot be done readily. In addition, the specific resistance, a , of materials such as mold mycelia or bacterial cell paste behaves generally as a compressible cake. This means that the porosity changes throughout the filter cake. The changes in the cake porosity exert a profound influence on the rate of filtration. For example, a reduction of the cake porosity, E, from 0.80 to 0.50 which corresponds to only a 37.5% decrease would increase the specific resistance by 2600%. In biological materials, such as microbial cells, the ability to predict the specific cake resistance as influenced by variables such as p H , temperature, salt concentration, filtration pressure, and aging is extremely difficult, if not impossible. Therefore, the potential of using filtration results from one system to another is quite difficult. However, if one is scaling-up the same filtration process, careful data collection and intepretation can prove to be extremely useful. This approach, as will be seen later, renders large-scale cake filtration an extremely practical method for removing microbial cells.

2 . Sterilizution Filtration The other area where filtration is used, but quite different from cake filtration, is in liquid sterilization. Generally speaking, cake filtration involves the removal of large amounts of suspended solids. I n pharmaceutical processes, such as the antibiotic industry for example, mycelium concentrations ranging from 20 to 80 g. per liter are routinely filtered. In these instances, filtration removes the bulk of suspended cells and the filtrate still contains a small fraction of the suspended cells. On the other hand, there are many laboratory and production processes where filtration is employed to obtain a filtrate free of microbial contamination. Specifically, for example, the production of tissue culture media, bacteria-free intravenous solutions, pyrogen-free solutions, and various canned draft beers d l utilize filtration as a method for the removing of different types of contaminating materials. It must, however, be mentioned that in sterilization filtration, the initial

COLLECTION OF MICROBIAL CELLS

135

concentration of the contaminating organisms is usually of orders of magnitude lower than what is encountered in cake filtration. There are two types of filters which are used for liquid sterilization. The first of these is the depth filter where the removal of microbial cells is achieved throughout the thickness of the filter material. The particles being removed are trapped within the interstices of the filter as the liquid carrier stream moves through the filter medium, The other is the membrane filter where the cell removal is achieved through a sieving or screening action due to the distinct pore size of the membrane. From a theoretical point of view, the principle of the membrane filters for removing contaminating particular matters is relatively straightforward. These filters, effective only two dimensionally, are thin porous sheet structures produced from polymeric materials. The pore size during manufacture is carefully controlled to retain certain particle size suspensions. For example, there are commercially available 20 or more distinct pore-size grade membrane filters capable of retaining solid particles ranging from 0.01 to 15 ,u in diameter. It is, therefore, relatively simple in selecting a proper membrane filter to perform a desired task. The theory of depth filtration is much more complicated. Generally, this type of filter is manufactured in layers from inorganic fibers ranging from 0.03 to 8 p in diameter and bonded by an inert organic binder. When a liquid containing suspended solids flows through the filter medium, the particles are captured by the fibers due to adsorption, diffusion, inertial impingement, and impaction. These mechanisms are similar to those advanced in aerosol filtration by fibrous filters although experimental evidence in support of the theory in liquid filtration has been extremely sparse. It is beyond the scope of this review to examine from the mechanistic points of view on the modes of action of the depth filter. Instead, it is our intent to present some of the applications and results in utilization of these filters for liquid sterilization. The reader is encouraged to examine the works of Ives (1960, 1962, 1963, and 1965) for further discussion on the various mechanisms involved in depth filtration.

B.

APPLICATION, EQUIPMENT, AND PERFORMANCE

1 . Cuke Filtration There have been numerous publications dealing with various aspects of cake filtration. The more recent review on theoretical aspects of cake filtration was presented by Tiller (1966).In this review

136

D. I. C. WANG AND A. J. SINSKEY

Tiller showed the influences of various pertinent parameters on the rate of filtration. The author also presented methods of applying the known theory of cake filtration to monitoring and controlling plant oper1' t'ions. For laboratory filtration studies the choice of equipment is generally limited. Improvision by the individual investigator quite often is sufficient for the proper design of laboratory filtration units which are capable of obtaining nieaningful results. On the other hand, when the proper selection of a pilot or plant-scale filter is desired, good technical judgment using sound experimental data should be employed. To facilitate the proper selection of filtration and other solid separation equipment, Davies (1965) outlined a sequence of laboratory testing methods to aid in the final selection. Included in the work by Davies are the various types of filtration equipment and their general performance characteristics. In the same light as that presented by Davies, a much more descriptive arid comprehensive analysis was presented by Flood et al. (1966) on various types of commercial size filtration equipment. These authors described in great length the operational features and capabilities of various type of filters with special emphasis on the rotary drum filter. This discussion also encompassed various features of the multicompartment continuous operation rotary drum filters, continuous horizontal filter, scroll-discharge rotary horizontal filter, tilting-pan horizontal filter, belt filter, vacuum disk filter, horizontalplate batch filter, and cartridge filter. In addition, auxiliary equipment associated with filtration such as feed pumps, vacuum pumps, filtration feed tanks, and filter media were also presented. The reader is encouraged to examine this review for detailed information. The major use of cake-type of filtration for microbial cell removal is probably in the pharmaceutical industry. Generally, the filter is used to remove fungal mycelia from various fermentation broths in order to recover soluble metabolites. With the rising labor cost and keen competition in this industry, process optimization is continuously being applied. Thus, in these recovery operations the trend has been toward continuous filtration instead of batch filtration. The predominant type of filter which is used for removal of fungal niycelia is the vacuum rotary filter. The cake depdsited on the filter is usually slimy in nature and clogging of the filter medium and within the deposited cake is often encountered. To overcome the reduction in filtration rate due to binding, it is a conventional practice to employ a filter aid material. Filter aids include various grades of calcified

COLLECTION OF MICROBIAL CELLS

137

diatomaceous earth, calcined perlite, asbestos and cellulose fibers. Although the filter aids add to the operating cost, the gain in higher filtration rates far exceeds the additional material cost. Most recent approaches in filtration operation of mycelial fermentation broths call for a precoat of the filter aid onto the surface of the filter, as well as using the filter aid as a premix with the slurry. The increase in filtration and the reduction in cost when filter aid was incorporated have been reported by Dlouhy and Dahlstrom (1968). Dlouhy and Dahlstrom (1968) also presented some representative plant-scale filtration results for various fermentation broths. A summary of their results is shown in Table VII. It can be seen that operating under similar conditions such as precoating, admixing of filter aid, and filtration pressure (vacuum) a wide range of filtration rates can be anticipated. These results reflect the differences and unpredictable nature of the filter cake which is encountered with microbial cells. The results, however, are extremely useful in knowing the general performance of microbial cell recovery through cake filtration. Other studies on the clarification of microbial culture fluids by filtration include the recent study reported by Mahony (1968). In order to obtain a relatively clean liquid for membrane sterilization, culture fluid of Clostridium tetani was processed in completely enclosed tanks equipped with glass fiber tubes. Filtration rates for various types of glass fiber tube, as well as the effect of filter aids, were presented. Aside from the pharmaceutical industry which is routinely processing large volumes of culture fluid, there looms on the horizon another industry with the potential of operating in even larger scales. This is the production of single-cell protein on various types of substrates. In the overall economic analysis, cell recovery cost must be maintained as low as possible. This would be especially critical if small bacteria are to be considered as single-cell protein candidates. Existing processing methods for cell recovery were recently reviewed by Wang (1968). It was shown there that every attempt should be made to improve methods of cell collection. Work along this line is in progress as exemplified by the British patent specification (1967). Laboratory results showed that the filtrability of bacterial cells can be greatly enhanced through simple and mild physical and chemical treatments. Some typical results on the improvement in rate of filtration for Micrococcus cerificans cultivated on hydrocarbon are shown in Table VIII. It can be seen that through relatively mild and

Y

TABLE VII REPRESENTATIVE DESIGNAND OPERATING RESULTS FOR \IARIOUS FERMENTATION BROTHSO ~~~~~~

Fermentation broth: Filter type:

Filtration rate (gal./hr.-ft.2): Solid in slurry (%): Vacuum (in. Hg): Cake moisture (wt. %):

Albamycin

Bacitracin

Cortisone

Erythromycin

Knnamycin

Neomycin

Penicillin

Streptomycin

Vacuum precoat

Vacuum Vacuum precoat precoat

Vacuum or press drum

Vacuum drum

Vacuuni precoat

Vacuum precoat

Vacuum drum of precoat

Vacuum drum of precoat

6.25

50-60

4-8

40-80

10

2.1

3.2

35-45

3- 20

2-3

20

8

20

25

7

2-8

2-8

2-6

181.20

2

-

10-20

25

20

18-20

20

20

-

65

-

38-45

-

60-70

-

Woven glass

Precoat

Nylon

Nylon

Precoat

Precoat

Poly-propylene

Precoat

None

0.4-216

0.4-2.6

None

6

None

None

0-6

-

0.151b.i hr.-ft.'

Filter medium: Precoat Slurry admix, (wt. % of sluny): 1.5-3 Precoat consumption (1b.l 1000 gal. filtrate): 38.7 "

Ascorbic acid

-

Dloughy and Dahlstrom, 1968.

P

139

COLLECTION OF MICROBIAL CELLS

TABLE VIII IMPROVEMENTIN FILTRATION UTES OF 1% Micrococcus ceri.cans THROUGH HEATAND P H TREATMENT

PH treatment

Heat treatment

pH at filtration

No Yes Yes No

No No

7.0

Yes Yes

3.5 3.5 7.0

Temperature during filtration

(“(3 25 25 85 (15 min.) 85 (15 min.)

Filtration rate (ml./rninute) 0.53 (control) 2.22 10.00

0.16

simple treatment methods, a tremendous increase in the filtration rate can be obtained. The results presented on cake filtration have been on the successful operations. There have been probably many filtration processes where complete failures have been encountered. However, if careful analyses are made as to the reason for failure, often remedies may be found. Through improvements such as the use of flocculants (see Section IV), cake filtration will undoubtedly play a more important role in cell recovery in days to come.

2. Sterilization Filtration The use of membrane filters for routine laboratory liquid sterilization has probably been applied by most microbiologists. There are commercially available many types of sterilizing membranes in assorted pore diameters and sizes. In recent years membrane developments have progressed to the point where they can be used in production size installations. Typical applications, equipment description, and operation sequences for these large-scale membrane sterilization units were recently presented by Schaufus (1968). A more descriptive use of membrane filters for processing large volumes of draft beer and other beverages was also recently presented by Mulvany (1968). A single unit, containing stacks of membrane filters and capable of processing as much as 10,000 gallons per hour was reported to be widely used in several major beer installations. Typically, however, 20-plate units capable of processing at 3600 gallons per hour (Imperial) are used for routine sterilization of canned draft beer. The results from the plant filtration runs showed that by using a 1.2 p pore size membrane, complete removal of the yeast and significant reduction of the bacteria can be achieved. Shelf-

140

1).

I. C. WANG AND A. J. SINSKEY

life tests after 300 days storage at 75-80°F. of the aseptically canned beer showed the membrane sterilized product was far superior to the conventional heat pasteurized product. An economic analysis was also presented by Mulvany (1968) comparing the costs of membrane sterilized with flash pasteurized beer at various beer production levels. This analysis showed that the membrane method is competitive to the older beer pasteurization method. From these recent studies in membrane filtration, it appears that sterilization filtration is technically and economically feasible in production facilities. These developments offer methods of sterilizing heat-sensitive fluids, not only in routine laboratory operations, but also from the point of view of commercial operation. An older type of sterilization filtration than the membrane method is the depth filter. One inherent disadvantage associated with membrane filtration is the problem of membrane clogging. This is caused by colloidal materials or high microbial contamination which may be present in the fluid. This problem was emphasized by both authors, Schaufus (1968) and Mulvany (1968). One method in circumventing this problem in the membrane sterilization of beer is to have extremely low microbial contamination initially. For example, the total microbial counts including bacteria and yeast in the plant-scale beer filtration ranged only from 20 to 1000 orgganisms per 100 ml. of beer (Mulvany,

1968). The other approach in alleviating the clogging problem during sterile filtration is the the use of depth filters. Many types of filter material are commercially available. Wendland (1967) reported on the performances of five different types of asbestos filter pads in their ability to remove pyrogenic substances in salt solutions. It was shown that among the available filter material a wide range of results can be expected. The results, however, do show that filter pads are available which completely remove the added pyrogenic substance. In a series of reports Daniels and Hale (1960) and Hale and Daniels (1961) presented some theoretical aspects, as well as some experimerltal evidence on the behavior of asbestos pad depth filters. The authors were able to characterize the rate of depth filtration to the pertinent variables such as length of time during filtration; volume of filtrate passed per unit filter area, and the filtration pressure. Laboratory tissue culture medium sterilizations were performed and successful scale-up (400 times) to a small-scale pilot plant facility was also accomplished. Introducing a test organism (Sermtia marcescens) LIP to 108cellslml. in the feed solution yielded complete removal during depth filtration.

COLLECTION OF MICROBIAL CELLS

141

Last, Telling et al. (1966) presented,excellent studies on the use of membrane and depth filters for sterilizing large volumes (200 liters) of tissue culture medium. Detailed operational features for carrying out filtration sterilization were presented. It was concluded that membrane filters clogged readily with this type of fluid, whereas depth filters were less affected. The reader is encouraged to examine this publication for detailed discussion. IV.

Flocculation

A.

PRINCIPLES

Flocculation as a means of collecting cells from suspensions is an extremely useful and relatively simple method. If cells can b e induced to flocculate, this method can increase tremendously the efficiency in recovery of large quantities of microbial suspensions (Nakamura, 1961).Many chemical agents can be added as flocculating agents, but the proper choice in its selection must be made judiciously. For example, Nakamura (1961)pointed out that the many chemical agents used for coagulation and precipitation with ordinary contaminated water are not suitable for use in microbial suspensions. Flocculating agents suitable for microbial collection should meet at least some of the following requirements: 1. They must react rapidly with the cells. 2. They must be nontoxic. 3. They should not alter the chemical constituents of the cells. 4. Chemicals should have a minimum cohesive power in order to allow for effective subsequent water removal by filtration. 5. Neither high acidity nor high alkalinity should result upon the addition of chemicals. 6. The quantities of chemicals used must be small, highly effective, and low in cost. 7. The chemicals should be, under certain circumstances, capable of being removed by washing and preferably available for further use. Although many factors can influence flocculation of cells, the primary mechanism of flocculation appears to be due to charge neutralization. This ultimately results in coagulation and precipitation of the microbial cells. Bacteria and yeast in suspension at neutral pH generally possess negative charges due to the presence of one or more types of ionogenic groups on the cell surface. These groups may be amino, carboxyl, or phosphate (James, 1965; Chester, 1965). Therefore, flocculation depends upon the cell wall characteristics, the ionic environment, pH, and the flocculating additive used.

142

U. I. C. WANG AND A. J. SINSKEY

Many theories concerning the mechanisms of yeast flocculation have been summarized by Dunn (1955) and Jansen (1958). The reader is encouraged to review these publications for the detailed discussion.

B. FLOCCULATION OF MICROBIALCELLS The phenomenon of yeast flocculation has been studied primarily in the brewing industry. The studies were performed to explain the reason why yeasts have an inherent tendency to flocculate, the nature of the change from a nonflocculent to a potentially flocculent cell, and the interactions of potentially flocculent cells to form flocs (Mill, 1964). Thorne (1951, 1952) and Gilliland (1951) showed that yeast flocculence was an inherited characteristic which was dominant over nonflocculence. However, the nature of the change from a nonflocculent to a flocculent cell is not clearly understood. Jansen and Mendlik (1951) and Mill (1964) demonstrated that flocculation of a strain of brewers’ yeast was highly dependent upon the presence of calcium ions. Mill (1964) further showed that carboxyl groups of the cells were involved in the interaction with calcium. The flocs formed had a melting temperature” between 50” to 60°C. and were dispersed by urea, suggesting that hydrogen bonding is important in their formation and dissociation. These findings led Mill to suggest that flocculent yeast cells are linked by salt bridges formed by calcium atoms with carboxyl groups on the surfaces of different cells. The resulting structure is stabilized by hydrogen bonds formed between complementary patterns of carbohydrate hydrogens and hydroxyls on the cell surfaces. Nakamura (1961) working with yeast, bacteria, and algae found that calcium hydroxide and calcium chloride were the most effective inorganic flocculating agents. Cationic surface-active agents such as alkyl pyridinium salts, quarternary ammonium salts, and alkyl amines, gave effective reactions as chemical separating agents at concentrations of 0.01 to 0.1%. Busch and Stumm (1968) have demonstrated that bacteria can be flocculated with synthetic anionic and nonionic polyelectrolytes (Polyacrylamide, polystyrene sulfonate, polyglutamic acid, and dextran). Busch and Stumm’s (1968) results indicated that reduction of charge is not an absolute prerequisite for flocculation and agglomeration apparently results from specific adsorption of polymer segments and from bridging of polymers between cells. In the past ten years tremendous advances have been made in the production and manufacture of synthetic polyelectrolytes. The ability of these materials to induce flocculation in microbial cells has already “

COLLECTION OF MICROBIAL CELLS

143

been demonstrated by Busch and Stumm (1968).The avenue which has been opened by these synthetic flocculating agents will be an extremely important one in microbial cell recovery processes. Since the chemical structure and make-up of these synthetic flocculants can be defined to certain extents, it may be possible to select with confidence certain ones of these chemicals to aid in recovery of a given cell. It is the authors’ opinion that in the years to come the use of flocculating agents will contribute significantly in the reduction in recovery cost in various microbial systems. V.

Foam Fractionation

A. PRINCIPLES The basic principles of foam fractionation were recently reviewed by Lemich (1968). Extensive discussions on the type of substances that can be fractionated can be found in reviews b y Schoen (1966), Rubin and Gaden (1962), Cassidy (1957), and Shedlovsky (1947). Briefly, foam fractionation makes use of the principle that in a liquid foam system the chemical composition of a given substance in the bulk liquid is usually different from the chemical composition of some substance in the foam. The quantitative relationships for the equilibrium adsorption of the dissolved material at the gas-liquid interface is given by the simplified Gibbs equation:

r

1-

1 dY RT d In Ci

where y is the surface tension, R is the gas constant, T is the absolute temperature, r, is the surface concentration while Ci is the concentration of the adsorbed compound, i , in the bulk. In practice foam fractionation is accomplished by sparging an inert gas into the bulk liquid containing the substance to be fractionated. The gas is fed near the bottom of the liquid, and the bubbles rise to the top. For surface active systems a foam will be created and the overflow carries off selectively adsorbed solutes on the surface of the bubbles. For systems that do not foam, suitable surfactants may be added. The surfactants can then combine with the solute in question or simply adsorb it at the surface of the bubbles. The solute then can be carried off in the foam. A schematic diagram representing the operation is shown in Fig. 3. The operation and design of the foam column influence significantly

144

D. I. C. WANG AND A. J. SINSKEY

Overflow

Foam breaker

Collapsed

FIG.3. ~cheinaticflow diagram for foam fractionatinn.

the separation efficiency. There have been a number of studies which have concentrated on column design and operation (Brown, 1966; Grieves, 1968). Geometric variables are column diameter, height of liquid solution, height of the foam above the solution-foam interface, and bubble diameter. Operating variables are temperature, gas rate, foaming time, and dividing the surfactant feed in pulses instead of a siiigle dose. Other studies have been primarily concerned with methods of controlling the equilibrium characteristics between the microbial cells in the liquid and foam (Newson, 1966; Grieves, 1968). Important independent variables controlling the foam fractionation process from a solution equilibrium viewpoint include initial concentration of the species to b e collected, concentration of surfactmt, ionic strength, and pH.

H. COLLECTION

OF

MICROBIAL CELLS

BY

FOAMFRACTIONATION

Dognon (1941) found that tubercle bacilli were easily removed from suspension by foaniing while Escherichia coli,Staphylococcus albus, and Schizosacchuronryces sp. were concentrated with difficulty. However, the latter organism could be fractionated if Na2S04and CaC12were present. Boyles and Lincoln (1958) observed that masses of material collected above the liquid level in the head of foam when Bacillus anthracis was grown in aerated deep cultures were composed essentially of clean spores. This observation led to the conclusion that a collection process could be developed that would separate B. anthracis spores from vegetative cells and cellular debris in the

COLLECTION OF MICROBIAL CELLS

145

culture medium. In their study with spores of B. anthracis it was found that autolysis of cultures was essential but cultures with high spore counts were not required. Coarse spargers were more effective than fine or medium spargers. Other microbial cells such a s spores from autolyzed cultures of B . subtilis var. niger and cells of Serratia marcescens were also capable of fractionation by foaming. However, Pasteurella tularensis could not b e collected using their system. Last, the nature of the bacterial surface was found to affect the collection efficiency. For example, cells from a smooth strain of Brucella suis were not collected in foam under numerous conditions, whereas rough or mucoid type cells were effectively removed and collected. Gaudin et al. (1960a,b) found that B . subtilis var. niger spores could be separated from debris by conventional flotation techniques. Factors which influenced spore recovery were the age of the culture, the soluble materials present, the pH of the suspension, and whether or not fatty acids or amines were used as flotation agents. Fatty acid collectors were found to be selective for debris and vegetative cells, leaving the spores in suspension. Dioctylamine gave complete removal of spores. With hydrophobic organisms such as E . coli, Gaudin et 01. (1962a) found that the cells were rapidly concentrated from culture medium b y flotation in the presence of sodium chloride. Other salts were evaluated as flotation agents (Gaudin et al., 1962b). Phosphates were effective, but not as useful as NaC1. Carbonate gave good results but bicarbonate, sulfate, nitrate, bromide, and iodide did not promote flotation of the bacteria. Ammonium ions seemed to depress flotation. Grieves and Wang (1966) studied the foam separation of Escherichia coli with a cationic surfactant. Using ethylhexadecyldimethylammonium bromide at concentrations of 0.015 to 0.04 mg./ml., the cell enrichment ratio was found to vary from 10 to 1,000,000. The cell enrichment ratio was found to be an inverse power function of the initial surfactant concentration and an exponential function of foaming time. Levin et al. (1962) developed a froth flotation procedure for the removal of algae from dilute suspensions. No surfactants were needed. Cell concentration of the harvest was dependent upon pH, aeration rate, aerator porosity, feed concentration, and height of foam in the harvesting column. The studies on foam fractionation appear to show that specific conditions exist in order to achieve satisfactory foam fractionation. Although the predictability in quantitative terms of success is ex-

D. 1. C. WANC. AND A. J. SINSKEY

146

t r e n d y difficult, this method does seem to offer to investigators an unique tool for cell separation and recovery.

VI.

Miscellaneous Recovery Systems

A. ION EXCHANGE Ion exchange has received only limited attention as a method for recovery of cells from dilute suspensions. Various types of resin are available in granular bead forms with varying selectivities or affinities for various ions. Bacterial cells usually have charges on the surface; and it is, therefore, possible to adsorb the cells onto various type of ion exchange resins. It is beyond the scope of this review to examine the detailed principles and theories of ion exchange. The reader is encouraged to examine one of the classics on this subject by Helfferich (1962). There have been a few papers dealing with the application of ionexchange resin for the separation of microbial cells. Daniels and Kempe (1966) investigated the phenomenon of bacterial adsorption from aqueous suspensions onto synthetic anion and cation exchange resins. An interesting observation was seen when a suspension of Bacillus subtilis was mixed with an anionic exchange resin (Dowex 1 X 8, 200/400 mesh). Large flocculated particles were formed immediately due to ionic bridging. The authors concluded that the cells were removed from suspension by a true adsorptive process and not by filtration or sedimentation. The effect of pH during ion exchange operation was also examined. The bacterium is unique in that the surface charge can be altered through these pH variations. Thus, depending on whether the pII of the cells is above or below the isoelectric point, different types (anionic or cationic) of resins can be used for adsorption. This phenomenon was substantiated with their laboratory results. Other bacterial species investigated by Daniels and Kempe (1966) included Bacillus cereus, Escherichia coli, Proteus uulgaris, Pseudomonas ovalis, and Staphylococcus aureus. Various types of sorption occurred when these bacterial cells were exchanged with an anionic resin. For example, E . coli exhibited limited adsorption which became self-reversing while S. aureus, P . ovalis and B . cereus were shown to be strongly adsorbed. However, when the pH was lowered, desorption occurred presumably through a charge reversal. Proteus vulgaris exhibited strong adsorption with desorption promoted by the addition of salt while B . subtilis exhibited very strong adsorption with desorption promoted only by the combined action of low pH and the addition of salt.

COLLECTION OF MICROBIAL CELLS

147

Removal and collection of viruses by ion exchange resins has received more attention. LoGrippo (1950), using a strong basic exchange (Amberlite XE67), separated and purified Lansing and Thielen strains of polio virus from suspensions of mouse central nervous system tissues and from human feces. Muller and Rose (1952) using Amberlite XE64 (a carboxylate exchanger) were able to purify Type A influenza virus. Johnson et al. (1967) demonstrated that polycationic resins were very effective in removing tobacco mosaic virus and polio virus from aqueous suspensions. Further investigations are needed on determining optimal conditions for the use of ion exchange resins for the collection and removal of microbes and viruses from suspensions. These include: (1) the hydrophilic-hydrophobic ratio, ( 2 ) the type and extent of ionization, (3) charge distribution, and (4) the role of various ions and effects of suspension composition.

B. BIPHASICLIQUIDEXTRACTION Albertsson (1958) has reviewed the principles of extraction of microbial cells by using two-phase liquid systems. With low molecular weight substances in a two-phase system a finite partition coefficient (C,/C, = k ) is usually established (C, = concentration in phase 1, C:! = concentration in phase 2 ) . However, with higher molecular weight compounds

Cl/Cz = e exp [(Mh)/RT] where M is the molecular weight of the substance, h is a constant characteristic for a given phase system and the substance in question and R and T are the gas constant and temperature, respectively. With microbial cells M is usually replaced by the surface area of the particles. Complete separation of two kinds of particles can be achieved if the constant, A, has opposite sign. Therefore, high resolution can be obtained when the proper choice of the phase system is chosen. Albertsson (1958) evaluated many phase systems employing nonionic polymers for the isolation of various types of microbial cells. A phosphate and polyethylene glycol (285-315 M.W.) system proved to be very effective for extraction of cells. Using a variety of cell systems (Chlorella pyrenoidose, Seenedesmus oliquues, Seenedesmus quadricauda, and an Aerobacter strain) all could be selectively enriched in the polyethylene glycol phase. The concentrated and purified cells are then collected by centrifugation.

148

11. 1. C. W'ANC. AND A. J. SINSKEY

Sacks and Alderton (1961) studied the behavior of bacterial spores in a two-phase aqueous system consisting of polyethylene glycol

(4000 M .W.) and potassium phosphate. The authors demonstrated that several types of bacterial spores may be separated from vegetative cells using this solvent system. In addition, cellular debris can also be removed in this manner. Pendleton and Morrison (1966) studied thc separation of Bucillus thuringiensis spores from the protein crystals that are formed during sporulation with carbon tetrachloride. After extraction the aqueous phase was found to contain 98-99% crystals and only 1-2% spores. Extraction procedures have also been used to isolate, concentrate, and purify viruses from aqueous suspensions. Shuval et al. (1967) using a mixture containing 0.2% (w./w.) sodium dextran sulfate, 6.45% (w./w.)polyethylene glycol, and 0.3 M NaCl concentrated in a single step echovirus and poliovirus by factors of 52.5 to 200. The efficiency of recovery, however, ranged from 30 to 70%. Orlando et al. (1964) evaluated various methods for the removal of extraneous matter from vaccinia virus suspensions. After examining ten different procedures, it was found that differential centrifugation in combination with Freon extraction was the most successful method, The virus titers were found to be quantitatively retained with no stabilizing additives needed. Bachrach arid Polatanick (1968) developed a concentration and purification procedure for decigram quantities of foot-and-mouth disease virus from cell cultures that employs, in part, extraction with organic liquids.

C. ELECTROPIIOHESIS As discussed previously, microorganisms and viruses carry electrical charges and, consequently, can be induced to migrate in an electrical field. The mobilities of various bacteria and viruses in an electric field are shown in Table IX (Freeman, 1964). Some of the operating variables which affect the separation efficiency are field strength, ionic strength, time of run, and pII. Electrophoresis as a nieans of collecting and separating cells has been performed primarily on the laboratory scale (Polson, 1953, 1956; Largier, 1955, 1956).More recently Bier et al. (1967) evaluated a forced flow electrophoretic method for the concentration of bacteriophage. Using a dialysis membrane as part of the cell, the bacteriophage was induced to migrate electrophoretically onto the membrane surface. In this manner the membranes can be placed onto solid supports

TABLE IX ELECTROPHORETIC MOBILITIES OF CERTAINORGANISMS" Mobility Microorganism

Sbain

Pneumococcus Staphylococcus Pseudomonas Brucella arbortus E. coli bacteriophage

Type 1-46 Smooth F, Rough 80

Buffer

pH

p/sec./V./cm.

References 0

"

Freeman, 1964.

-

0.04 M Phosphate 0.013 M Phosphate 0.01 M Phosphate 0.08 M NaCl

7.3 7.4 6.9 7.5 5.78

4.2 1.8-2.0 2.0-2.1 3.72 4.36 x lo-,? (cm./sec./V./cm.)

Thompson (1932) Verwey and Frobesher (1940) Dyar and Ordal (1946) Stearns and Roephe (1941) Longsworth and MacInnes (1942)

n m

E

v)

150

U. I. C . WANG AND A . J. SINSKEY

for direct plaque assay. The results showed that bacteriophage can be separated by this method. Resnick et al. (1967)using stable-flow free-boundary electrophoresis showed successful separation of spores from diploid cells of Sacchammyces ceruisiue. Due to differences in electrical mobility, an aqueous suspension containing 99.04% of the spores was obtained. This separation allowed the investigators to prepare suspensions for other genetic analysis. It is the opinion of the authors that electrophoretic collection methods will still continue to have only limited practical applications. VII.

Summary

The recovery of microbial cells from suspending fluids is a task frequently encountered b y biologists, microbiologists, biochemists, and engineers. We have attempted in this review to examine some of the principles and applications of various recovery methods. These methods include: centrifugation, filtration, flocculation, foam fractionation, ion exchange, biphasic liquid extraction, and electrophoresis. In centrifugation, we have presented the principles of differential and zonal centrifugal operations. A review of the use of these types of centrifuges for preparative scale virus recovery was outlined. Filtration was presented on the large-scale recovery of cellular rnaterials from fermentation broths. In addition, laboratory- and plant-scale sterilization filtration by membrane and depth filters were also includcd. The use of flocculating agents was presented to show their influences in increasing the efficiency of microbial cell recovery. Foam fractionation was demonstrated to be of specific use in separating and fractionating microbial cells from their suspending fluids. Last, other miscellaneous methods such a s ion exchange, biphasic liquid extraction, and electrophoresis which have shown some degree of success in the laboratory for cell recovery were also examined. ACKNOWLEDGMENT

The authors wish to express their appreciation to the Natiorial Science Foundation, Grant Number GK-2860, for support in part iri preparation of the work for this review.

REFERENCES Albertsson, P. A. (1958). Biochim. Biophys. Actn 27, 378-395. Ambler, C. M . (1959).J.Biochern. M i c ~ o b i o lTechnol. . Eng. 1,185-205.

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Anderson, N. G . (1966). Science 154, 103-112. Anderson, N. G., Barringer, H. l’., Amburgey, J. W., Cline, G. B., Nunley, C. E., and Berman, A. S. (1966).Natl. Cancer Inst. Monogr. 21,199-216. Bachrach, H. L., and Polatatlick, J. (1968). Biotechnol. Bioeng. 10, 589-599. Bier, M., Bnicknew, G. C., Cooper, F. C., and Roy, H. E. (1967). In “Transmission of Viruses by the Water Route” (G. Berg, ed.), p. 57. Wiley, New York. Boyles, W. A,, arid Lincoln, R. E. (1958).A p p l . Microbiol. 6, 327-334. British Patent Specification (1967). No. 1,062,005 “Improved Process for Biosynthesis and Recovery of Microbial Cells,” March, 1967. Brown, D. J . (1966). Chem. Process Eng. 47, (5), 201-215. Bnsch, P. L., and Stumm, W. (1968).Enciron. Sci. Technol. 2(1),49-53. Cassidy, H. C. (1957). In “Technique of Organic Chemistry” (A. Weissberger, ed.), Vol. 10, Wiley (Interscience), New York. Chester, V. E. (1965). In “Surfixe Activity and the Microbial Cell,” Sci. Ind. Monogr. 19,pp. 59-66. Gordon & Breach, New York. Daniels, S. L., and Kempe, L. L. (1966).C h e m . E r g . Progr. Symp. Ser. 62 (69), 142. Daniels, W. F., and Hale, M. B. (1960).]. Biochem. Microbiol. Technol. Eng. 2,93-112. Davies, E. (1965).Trans. Inst. Chem. Engrs. 43, T256-T259. Dlonghy, P. E., and Dahlstrom, D. A. (1968).Chem. E n g . Progr. 64, 116-121. Dognon, A. (1941).Reu. Sci. 79, 613-619. Dunn, C. G. (1955).Am. Brewer 88 (l2), 42-46. Dyar, M. T., and Ordal, E. J. (1946).J. Bacteriol. 51, 149-167. Flood, J. E., Porter, H. F., andRennie, F. W. (1966).Chem. Eng. 16,163-181. Freeman, R. R. (1964). Biotechnol. Bioeng. 6, 87-125. Gaudin, A. M., Mular, A. L., and O’Connor, R. F. (1960a). A p p l . Microbiol. 8, 84-91. Gaudin, A. M . , Mular, A. L., and O’Connor, R. F. (1960b).A p p l . Microbiol. 8, 91-97. Gaudin, A. M., Davis, N. S., and Bangs, S. E. (1962a). Biotechnol. Bioeng. 4,211-222. Gaudin, A. M . , Davis, N. S., and Bangs, S . E. (196213).Biotechnol. Bioeng. 4,223-230. Gilliland, R. B. (1951). European Brewery Convention Congr., pp. 35-38, Elsevier, Arrrsterdarn. Grieves, R. B. (1968). British Chenc. Eng. 13 (l),77-82. (.r : ~ ~ c \ H. ’ eB., ~ ,and \Vang, S. (1966). Biotechiiol. Bioeiig:.8, 32.3-33fi. Hale, M. B.,and Daniels, W. F. (1961).J.Biochem. Microbiol. Technol. Eng. 3,139-150. Helfferich, F. (1962). “Ion Exchange.” McGraw-Hill, New York. Ives, K. J. (1960). Proc. Inst. Civil Eng. 16, 189-193. lves, K.J. (1962). Proc. S y m p . Interaction between Fluid and Particles, pp. 260-267, Institute of Chemical Engineers, London. Ives, K. J. (1963).Proc. Inst. Civil Eng. 25, 345-364. Ives, K. J. (1965). Trans. Inst.’Chem. EngrB. 43, T238-T247. James, A. M. (1965). “Surface Activity and the Microbial Cell,” Sci. Ind. Monogr. 19, pp. 3-22. Gordon & Breach, New York. Jansen, H. E. (1958).In “The Chemistry and Biology of Yeasts” (A. H. Cook, ed.), pp. 635-667. Academic Press, New York. Jansen, H. E., and Mendlik, F. (1951).European Brewery Convention Congr., pp. 5983. Elsevier, Amsterdam. Johnson, J. H., Fields, J. E., and Darlington, W. A. (1967). Nature 213, 665-6137, Judson, G., Jones, A., Kellogg, R., Buckner, D., Eisel, R., Perry, S., and Greenough, W. (1968).Nature 217, 816-818. Largier, J. F. (1955).Biochim. Biophys. Acta 16,291-292.

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Largier, J. F. (1956). Biochim. Biophys. Actu 21, 433-438. Lemich, R. (1968). In “Progress in Separation and Purification” (E. S. Perry, ed,) Vol. 1, p. 1. Wiley (Interscience), New York. Levin, G. V., Clendenning, J. R., Gihor, A., and Bogar, F. D. (1962). Appl. Mfcrobiol. 10, 169-175. LoGrippo, G . A. (1950). Proc. Soc. Exptl. Biol. Med. 74,208-211. Lonysworth, I,. G., and MacInnes, I). A. (1942).J . Gen. Physiol. 25, 507-516. Mahony, N. C. (1968). Process Biochem. 3, 19-22. Mill, P. J. (1964).J . Gen. Microhiol. 35, 53-60. Muller, R., and Rose, H. (1952). Proc. Soc. E z p t l . Biol. Med. 80, 27-29. Mulvany, J. (1968). Process Biochem. 1, 470-473. Nakamura, H. (1961).J. Biochem. Microbiol. Technol. Eng. 3,359-403. Newson, I. H. (1966).J. Appl. Chem. 16,43-49. Orlando, M. D., Riley, J. M., and Patrick, W. C., 111.(1964). Biotech. Bioeng. 6,321-328. Patrick, W. C., and Freeman, R. R. (1959).J. Biochem. Microbiol, Technol. E n g . 1,207215. Patrick, W. C., and Frccman, R. R. (1960).J . Biuchem. Microhiol. Technol. E n g . 2, 7180. Pendleton, I. R., and Morrison, R. R. (1966). Nature 212, 728-729. Polson, A. (1953).Biochim. Biophys. Actu 11,315-325. . 22,61-65. Polson, A. (1956). Biochim. B i o p h ~ sActu Pretlow, T. G . ,and Boone, C . W. (1968). Science 161,911-913. Reinier, C. R., Newlin, T. E., FIuvens, M . L., Baker. R. S., Anderson, N. C , ,Cline, C,. R., Barringer, H. P., and Nunley, C. E. (1966). Natl. Cancer Inst. Monogr. 21,375-388. Resnick, M. A,, Tippetto, R. D., and Mortimer, R. K. (1967). Science 158, 803-804. Rubin, E., and Gaden, E. L. (1962). In “New Chemical Engineering Separation Techniques” (H. M. Schoen, ed.), Chapt. 5. Wiley (Interscience), New York. Sacks, L. E., and Alderton, G. (1961).J. Bucteriol. 82, 331-341. Schaufus, C. P. (1968). Chem. Eng. Progr. 64,69-73. Schoen, H. M. (1966).Ann. N.Y. Acad. Sci. 137, 148-161. Shedlovsky, L. (1947). Ann. N.Y. Acad. Scl. 49,279-294. Shuval, H. I., Cymbalista, S., Fatal, B., and Goldblum, N. (1967). In “Transmission of Viruses by the Water Route” (G. Berg, ed.), p. 45. Wiley, New York. Stearns, T. W., and Roephe, M. H. (1941). J.Bucteriol. 42,411-430. TellinE, R. C., Stone, C. J., and Mnskell, M. A. (1966). Biotechnob Bioeng. 8, 153-165. Thompson, R. L. (1932). Am. J. H y g . 15, 712-725. Thorne, R. S. W. (1951). European Brewery Convention Congr., pp. 21-34. Elsevier, Amsterdam. Thorne, R. S. W. (1952).Wullerstein Lub. Comnun. 15,(50), 201-211. Tiller, F. M. (1966).Chem. Eng. 16,151-162. Verwey, W. F., and Frobesher, M. Jr., (1940).A m . J .Hug. 32,55-68. Wang, D. I. C. (1968). I n “Single-Cell Protein,” (R.I. Mateles and S. R. Tannenbaum, eds.), p. 217. M.I.T. Press, Cambridge, Massachusetts. Wang, I). 1. C., Sinskey, T. J., Gerner, R. E., deFillippi, R. P. (1968). Riotechnol. Bioeng. 10,641-649. Wendland, W. (1967).Filtrution Sepurution 2(5), 446-447.

Fermentor Design

R. STEEL AND T. L. MILLER The Upjohn Company, Kalamazoo, Michigan

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

IX. X. XI.

XII.

.

...................................... 153 ........................................... ~ . 154 . Fermentor Geometry ...... ....................... ...................... ... 155 Construction Materials ... ................................................ 157 Aeration-Agitation Systems ........ Agitator Shaft Seals ............................................... 159 Aseptic Operations .............................. ........................... 161 Air Filtration ... ... .................. ............, .......................... ... 164 A. Membrane Filter Media ........................... ... 164 B. Cox Filter Media .......... ................................ 166

I. Introduction 11.

C. Pall Trinity Filter Media ................................ 167 D. Biotech Filter Media ................................................ 168 E. Domnick-Hunter Filter Candles .....,._.... ..................... 169 169 F. Echo Air Filters ....................................................... ... 169 Mechanical Defoamers ............................... ....... 171 Antifoam or Nutrient Addition ............................. 172 ........................................... Instrumentation ....... A . pH Measurement and Control .... ....................... 172 B. Carbon Dioxide Measurement.. .. .......................... 174 C. Oxygen Measurement ............................................... 175 D. Temperature Measurement ....................................... 177 E. Pressure Measurement ....................................... 177 .......................... 178 F. Miscellaneous ... ........................ Continuous Fermentors .................... .......................... 178 A. Overflow Fermentors ................................................ 179 B. Packed Column (Tower) Fermentors ....... ... 180 C. Shaken Flask Fermentors ........... ...................... 181 D. Tube-Type Fermentors ............................................. 181 E. Cyclone Column Fermentors .................................... 182 F. Full Fermentors ...................................................... 183 G. Turbidostatic Fermentors .... References ............. .................... ...._.............._... 184 Appendix: Addresses of Equipme

.

I.

Introduction

171

The subject of fermentor design, depending on its scope, may cover a multitude of diverse types of equipment. The purpose of this paper is to bring together some of the alternate methods that various researchers have used to deal with fermentation problems and to provide information on available equipment. However this paper is not intended as an exhaustive literature survey. It is realized that each section of this report could be greatly expanded but space limitations 153

1S4

n.

STEEL AND T. L. MILLER

have required that we be selective in the choice of material for presentation. A previous review by Walker and Holdsworth (1) dealt with fermentor design, and Elsworth (2) and Chain et uZ. ( 3 ) have given detailed descriptions of their equipment. II.

Requirements

Those interested in conducting fermentations must first decide whether they need equipment designed for a special purpose or whether a general purpose design is preferable. The size of the fermentor will be determined from a number of considerations depending on whether the need is to obtain sufficient product for isolation, or whether collection of information (on the effect of process variables, strain or media screening, etc.) is most important. Small equipment (from 0.1 to 30 liters) is highly desirable where information gathering is the major objective. For indiistrial use a pilot plant fermentor should have flexibility for development of a variety of fernlentation processes which may have widely differing physical properties as well a s operating requirements. Although there is a certain amount of “ know-how” or art involved in fermentation research and development, it is to be hoped that improvements in fermentor design and fermentiition control will reduce the factor of equipment effects and enhance our scientific and technical understanding of process development and management. The availability of reliable commercial fernlentation equipment would be expected to offer greater uniformity and understanding of processes since results obtained in improvised “ homemade” equipment sometimes leave something to be desired. The selection of a portable or stationary fermentor installation is a matter of choice and circumstiince; each has certain advantages and disadvantages (4). Portable fermentors can be purchased in greater numbers per dollar outlay, hence more experimental data can be gained. When expense is no object a stationary fernientor installation has much in its favor: (1) Reduced handling and labor requirements. (2) Large samples can be taken or the complete fermentor harvested for purification studies. ( 3 ) Evaporation losses are not excessive. (4)Close control of media sterilization by a procedure (steam injection) that is used in production equipment. (5) Instrumentation and feed systems may be permanently attached, thus reducing contamination hazards. An excellent survey of comniercial laboratory and pilot-scale fermentors has been presented by Solonions (5, 6).

FERMENTOR DESIGN

111.

155

Fermentor G e o m e t r y

A conventional fermentor (Fig. 1) usually consists of an upright cylindrical tank fitted with four baffles, a jacket or coil for heating and cooling, an air sparger, a device for mechanical agitation, and an air filter. Depending on the requirements, other systems may be included for control of foaming and addition of nutrient. In addition to the basic instrumentation for indication and control of temperature, equipment may be added for recording and/or controlling of back pressure, pH, oxidation-reduction potential, dissolved oxygen, effluent oxygen and carbon dioxide, and other variables of interest. Continuous analysis of certain components of a fermentation beer can be accomplished with commercially available equipment.

+--T-----c( D / T = 0 . 3 0 - 0 50 B / D z 1.0-12

FIG.1. Some dimensional ratios of

Z/T=IO-Z.O W / T = 0.08 - 0.12 B

general purpose fermentor.

Illustrated in Fig. 1 are some of the more important dimensional ratios of a general-purpose fermentor. These ratios are not absolutely critical as indicated by the range of values given. The ratios may be deliberately changed for different fermentation processes. In particular, with production fermentations it is most economical to operate with the highest possible volume of beer to maximize the productivity per tank so that the value of Z is maximized. The maximum value of 2 may, in turn, be determined by the head space needed to control

156

R. STEEL AND T. L. MILLER

foaming. The actual dimensions of pilot and production fermentors will not be reviewed here but are available in the literature (7-11). The noncritical nature of dimensional ratios (within certain limits) is illustrated by the work of Oldshue (12). The five different geometric configurations shown in Fig. 2 provided a similar mass transfer

OTD

T

t

2.5D

P

OAD

FIG. 2. Virious geometric configurations; sparge ring diaIn./turbine diam., D/T superficial air velocity 0.1 ft./sec. (12).

= 0.33; Zl?’ is unyassed;

coefficient at equal power input/unit volume. This is convenient to know since we are assured that there is some leeway in selection of the number and spacing of impellers a t least for systems other than those where non-Newtonian behavior of the fermentation beer may control reaction rates. It would appear that the fermentation industry has not attempted to maintain geometric similarity of dimensional ratios in scale-up of equipment from pilot-plant to production scale. As far as the growth of unicellular organisms (e.g., yeast and bacteria) is concerned this does not pose a problem because the oxygen absorption rate correlates with power input/unit volume for fermentors of different sizes and shapes. As the scale of operation is increased the ratio DIT is maintained relatively constant whereas Z / D is usually increased. This keeps turbine size from getting too unwieldy and also improves the efficiency of oxygen transfer by providing a longer bubble path.

FERMENTOR DESIGN

157

Many alternative designs of fermentor are conceivable depending on the requirements and economics of the process. Cylindrical wooden tanks packed with beechwood shavings or coke have been used for many years for the production of acetic acid from ethanol. The Acetobacter spp. grow as a thin film on the solid support and substrate is percolated downward through the column and is recycled. The tube-type fermentor (13) and cyclone fermentor (14)offer other variations in geometry. While they could be used as batch fermentors they have been studied mainly as continuous fermentors, hence, their description is given in the section on continuous fermentors. A rotating drum fermentor (15) was used for gluconic acid production and a tower-type fermentor ( 1 6) with a large height/diameter ratio was used for the citric acid fermentation.

IV.

Construction Materials

A simple fermentor may consist of an open tank of wood or concrete construction provided that contamination does not pose a problem. Or even a hole in the ground lined with plastic sheeting may be all that is required. In these cases service life is not an important consideration because the replacement cost is relatively low. According to Irving (17),the brewing industry makes extensive use of carbon steel vessels with glass or phenolic-epoxy coatings. For fermentations with strict sterility requirements it is necessary to select materials that can withstand repeated steam sterilization cycles (120°C. for 30 minutes). For laboratory-scale equipment the fermentor body may be a Pyrex glass jar or a length of standard Pyrex pipe (18).The use of glass allows visual inspection of the fermentation beer whereas stainless steel construction offers zero breakage and better heat transfer. Pilot-plant fermentors are usually constructed of type 316 stainless steel, while production-scale vessels are stainless clad. In most cases stainless steel satisfies the requirements of chemical inertness, ease of cleaning, absence of toxic effects on the fermentation, and long life. V.

Aeration-Agitation Systems

The usual aeration system is composed of an open-pipe sparger or a ring or cross sparger with holes to break the air stream into small bubbles. If the holes are situated on the bottom of the sparge ring it is a simple task to clean the sparger internally by passing steam through it. Aeration may also be carried out with metallic or ceramic discs or candles but these are difficult to clean properly.

1%

R. STEEL AND T. L. MILLER

In addition to mixing ~irocluceclby aeration, mechanical mixing is achieved by one or more impellers located on the agitator shaft. The size and shape of the impellers may be important considerations for certain processes ( 1 9). Whilc a particular impeller design nay be satisfactory for a specific proccss but not another, certainly the most common impellers fur general purpose use are of thc flat-blade turbine design. Rushton and co-workers (20) found that the addition of baffles changed the fluid flow pattern from axial to raclial, improved mixing by elimination of the vortex, and also allowed more power to be imposed on the fluid. Compared to a marine propeller, the flat-blade turbine could be operated at constant speed in a baffled tank in fluids of widely different viscosities without increasing the power requirement. Herbert et d.(21) used a draft tube with a multiple baffle system to achieve high oxygen transfer rates in laboratory-scale fermentors. RoxLurgh, Spencer, and Salluns (22) reported that arrowhead hirbines were difficult to adjust to give uniform conditions in all ferinen tors. The power necessary for mixing clepends on the physical properties of the fermentation medium and the rate of aeration. For a generalpurpose laboratory-scale or pilot-plant fernieiitor, provision should be made for power inputs of 6 to 8 watts/liter. Keisman and Gore (23) instidled motors of sufficient capacity to supply 22-30 watts/liter to pilot-scale continuous fermentors. This allows for a high degree of flexibility for experirnentd work. In production-scalc equipment a power input up to 2 watts/liter is needed for mycclial-type beers (10). Various ~ifiitatiori/aerationsystems have been adapted for special piirposes. For example, the vortex system incorporates mechanical agitation i n an unbaffled tank with air introduced above the liquid to give a system with low-foaming properties. The Waldhof fermentor is an unbaffled txnk with a central draft tube and air is introduced at the tips of the impeller blncles via a hollow agitator shaft; this system gives ;I good vertical mixing. The Yeoman’s Cavitator and Fring’s Acetator (24) featlire a cavitation impeller and clraft tube to obtain a high rate of oxygen transfer per unit power input. Martin and Waters (16) designed a tower-type fermentor with no mechanical agitztion and sparged with pure oxygen to obtain the “pathological morphology” of Aapergillus riiger required for citric acid production. The pulsaerutiorl system of Heden (25) was used to control foaming. Chcmapec Inc. list several different agitation systems in their catalog; several of these are listed below along with certain of their features: Ultrumix: contains a draft tube-like insert for vertical mixing;

FERMENTOH DESIGN

159

oxygen transfer 100 mM 02/liter.hour; recommended for generalpurpose use. Emulgator with Draft Tube: holes or slits in that portion of the draft tube below the impeller for obtaining emulsions in multiphasic systems; recommended for hydrocarbon fermentations; oxygen transfer rates u p to 200 mM Or/liter.hour. Multistage Reactor: contains several impellers within a draft tube with small holes in the draft tube opposite the impeller (emulgator) blades to promote emulsification of immiscible phases, and larger holes in the draft tube between impellers to promote mixing; recommended for hydrocarbon fermentations; oxygen solution rates up to 400 mM 02/liter.hour.Another version contains a second draft tube concentric with first to promote vertical flow. While Chemapec Inc. offers a variety of agitation systems, there is still a serious lack of actual fermentation data that is needed to determine operating characteristics. Another agitation system offered by Chemapec Inc. is the VibroMixer agitator. This consists of a magnetic core that is activated by an external solenoid thus imparting a vibratory or reciprocating motion to the agitator shaft. The ampIitude of the vibration can be varied. Various designs of mixing element can be attached to the vibrating shaft to obtain different mixing patterns. Ulrich and Moore (26) used a flat disc with tapered holes (the direction of fluid flow is from the wide to the narrow direction of the taper) for mixing of 6-liter tissue culture suspensions. Moore et al. (27) also used a larger model of Vibro-Mixer for pilot-scale work. Miller (28) selected a Vibro-Mixer for used on a general-purpose fermentor. He noted that there was little information available on power requirements for mixing or oxygen transfer with this apparatus and he installed conductivity probes in the fermentor for use as a relative measure of mixing efficiency. VI.

Agitator Shaft Seals

It is essential for aseptic operation to effectively seal the agitator shaft as it enters the fermentor. This may be accomplished with a stuffing box or a mechanical seal. The stuffing box is usually packed with graphited-asbestos and lubricated with silicone, mineral oil, or antifoam oil (29). Since it is difficult to sterilize the packing there is an advantage to locating the stuffing box at least partially inside the fermentor to gain better heat transfer. When bench-scale fermentors are sterilized in an autoclave this is not as important.

160

H. STEEL AND T. L. MILLER

Friedland, Peterson, and Sylvester (30) described a lubricated mechanical seal composed of a stainless steel disc rotating on a stationary Magnolia bearing bronze seal. These fermentors were sterilized in an autoclave. Nelson, Maxon, and Elferdink (8) used a Garlock mechanical seal on 20-liter stationary fermentors. The seal was located on the underside of the fernientor head so that it was accessible to steam during sterilization. This seal requires no lubrication and the bearing surface is carbon against a stainless steel or ceramic insert. Kroll et al. ( 4 ) described a neoprene oil seal assembly with a Teflon steady bearing that was used on 50-liter fermentors. The useful life of the neoprene seal was about 6 months. Means et al. (13) used Durametallic carbon seals on a continuous fermentor. Reisman and Gore (23) reported minimal maintenance problems with a Teflon wedge-type double mechanical seal; this seal also had facility for maintaining dead-end lubrication with hot oil. An easily replaced cartridge seal is supplied by New Brunswick Scientific Co. Inc. (Fig. 3). In a more elaborate arrangement, steam may be passed through a lantern ring inserted between the packing in the stuffing box. How-

r

1 0

FIG. 3. Agitator shaft seal, cartridge type (courtesy of New Brunswick Scientific Co., Inc.).(1.) Teflon wedgelock ring; (2) tapered carbon hushing; (3)carbon-to-carbon sealing SUI.kce; (4) carbon insert; ( 5 ) Teflon packing; (6) compression spring; (7)retaining ring; (8) impeller shaft; (9)set screw; (10)shaft seal cartridge; (11)fermentor head plate; (12) “0”ring seal; (13)bearing housing; (14)ball bearing.

FERMENTOR DESIGN

161

ever, McCann et al. (11) reported that continuous steaming deteriorated the packing thus necessitating frequent maintenance. They changed to a gland packed with Plastalloy topped with a ring of Supeta packing. This system remained leakproof and could maintain sterility for at least 8 days. To maintain the seal in good condition it was pressure checked for leaks between fermentations. Detailed construction of a steam sealed system was given by Chain et al. (3); two stuffing boxes on the agitator shaft were separated by a steam chamber. The extra safety factor afforded by this design is essential for those working with pathogens since it is just as important to keep the culture in the fermentor as it is to prevent foreign organisms from gaining access to it. Commercial suppliers of fermentation equipment have their own designs combining both mechanical and steam seals. One method of countering the apparent difficulty of effectively sealing the agitator shaft is to use a Vibro-Mixer agitator. Since the shaft does not rotate it can be sealed with a diaphragm of neoprene or silicone rubber. This arrangement was used by Miller (28) for a generalpurpose fermentor and by Ulrich and Moore (26) for tissue culture. An agitator shaft driven by a magnetic coupling also eliminates the need for shaft sealing. The Virtis and New Brunswick Companies offer small-scale fermentors with magnetic drives. Cameron and Godfrey (31) described a magnetic drive for a 300-liter fermentor that was used successfully for the growth of various pathogens. VII.

Aseptic Operations

Portable fermentors usually are fitted with an abundance of flexible tubing connections to provide entry to the fermentor for air, sterile nutrient, and defoamer. Since certain of these connections are made after the fermentor is removed from the autoclave there are possibilities of contamination arising from this operation. Commercially available small-scale fermentors from Chemapec Inc. and Biotech Inc. utilize diaphragm sealed ports and tubing connections to the fermentor are made by piercing the diaphragm with a hypodermic needle. Biotech Inc. used a Stericonnector fitting to attach tubing to the fermentor. Stationary fermentors require permanent pipework to provide services. The basic philosophy of pipework installation for antibiotic plants was reviewed by Parker (32) and by Walker and Holdsworth ( I ) . Other descriptions of plant piping were given in detail by

162

n.

STEEL AND T. L. MILLEH

Chain and co-workers (3), Kroll et al. (41,and by Fuld and Dunn (33). Transfer of inoculum was described by Parker (32) and by Jackson (34). McCann et al. ( 1 1 ) described a system for inoculation of fermentors from a common seed tank that involved measurement of irioculurri volumc in a separate sterile vessel prior to its transfer to the fermentor. The complete transfer operation took only about 1 minute so that in most cases the character of the culture would not be drastically altered by adversc conditions that occurred during this manipulation. This arrangement is shown in Fig. 4. The transfer Seed

\ Sten m

Fe r m e ntor

I

Sterile nir

I

I

Common

--+

Sight gloss

Droib Horvesi line

FIG.4. Piping arrangement for irioculatioii of fermcntors from a

comiiioti

seed tank

(111.

vessel and adjoining pipework tire sterilized with steam under pressure and cooled under positive pressure of sterile air. Then seed culture, forced under pressure is measured into the transfer vessel, the drain valve on the seed tank is closed, and sterile air under pressure is put on the transfer vessel. The valves connecting the transfer vessel to the fermentor are opened to allow the introduction of inoculum into the fermentor. At least on pilot-scale equipment it is common practice to install a sampling point as a dip-leg from above the liquid level. This system must be maintained sterile during the course of the fermentation. The arrangcrnent used at Imperial Chemical Inclustries (11) is shown in Fig. 5. Between samplings, sterile air is kept between valves 1 and 2. To obtain a sample, valve 1is opened to clear the dip-leg of beer, the

FERMENTOR DESIGN

163

Formalin bucket is removed and the sterile air valve is closed. Valve

2 is opened and the sample is collected after the initial 200-300 ml. has run to the drain. Valve 1 is closed and the pipework is resterilized with steam, bleeding through valve 2. When sterilization is complete the steam supply valve and valve 2 are closed and sterile air is admitted. The Formalin bucket is replaced. Steam

Sterile ir

FIG. 5. Piping for a sampling port ( 1 1 ) .

H e d h (25) has described a Stericonnector fitting used to attach tubing securely to a fermentor port. Commercially available equipment still necessitates the attachment of tubing to a rigid pipe under pseudoaseptic conditions. An improvement might be a small threeway valve attached to the tubing when autoclaved, that is closed when the tubing and nutrient reservoir is removed from the autoclave, and is turned to the steam bleed position when the fermentor is sterilized. While these arrangements may be satisfactory for pilot equipment, other arrangements should be made for larger scales of operation. Flexible hoses are unreliable even though an operator can “walk the hose” to drain condensate from the low points; there is still the hazard of contamination lurking in minute cracks in the internal wall. Rigid piping is the preferred arrangement. The subject of valve selection is still controversial. The most commonly used valves for lines in contact with fermentation beer (e.g., the drain line and sample line valves) are of the diaphragm type. The valve body may be welded to the fermentor to eliminate the use of threaded pipe and to reduce the volume of beer holdup between the valve and the tank (8). The useful life of a diaphragm valve de-

164

H. STEEL AND T. L . MILLEH

pends on the diaphragm material, the type of service it is subjected to, and the “touch” of the fermentor operator that uses it. Teflon diaphragms give longer life than other materials when used on intermittent steam service. If they are abused by screwing them down very tightly every time they are used, the “follower” (which is partially imbedded in the Teflon diaphragm) will sooner or later abrade, and eventually break the diaphragm. Reisnian and Gore (23) used hightemperature butyl rubber diaphragms. A leak-proof on-off valve with a relatively long life was described by workers at the Ajinomoto Co. (35) as being suitable for steam service. It is common practice to provide steam entry as close to possible to the valve seat by tapping into the valve boss. Chemapec Inc. supply a steam-flap valve for continuous sterilization of either side of a valve. VIII.

Air Filtration

The behavior of fibrous bed depth filters is well documented in the literature (36-40). Although they have served the fermentation industry relatively well in the past, techniques for absolute filtration of air are desirable. Several of these are in the formative stages of commercial development and certain of them (Millitubes, Ultipor cartridges, Cox M-780) are currently recommended by manufacturers for pilotplant and production-scale operations. In addition, disc and cartridge configurations are available in other materials (Echo filter, Domnick Hunter filter, and Biotech filter) which, although they are not considered as absolute filters, nevertheless have a high efficiency for removal of microorganisms from air supplies. A. MEMBRANEFILTERMEDIA Cellulose nitrate membranes have been used for the sterilization of liquids for a number of years. They also effectively remove microorganisms from air to a predetermined size level (depending on the pore size) with the assurance that any organism larger than this size will be removed. Millipore membranes of cellulose nitrate are autoclavable at 250°F. for 30 minutes. The rate of air passage through a cellulose nitrate membrane (Millipore) is a function of the pressure drop across the membrane (Fig. 6). Membrane discs and holders range in size from 47 to 293 mm. diameter. Membrane cartridges (Millitubes) are 2% inches in diameter and are available in 22- and 31-inch lengths. PR/MF Millitube cartridges combine microfiber glass pre-

165

FERMENTOR DESIGN

t

Gelman

I

0.05

U l t ipor

10

.I2

D

Pressure drop, PSlG

FIG.6 . Pressure drop-flow rate relationship for various filter media. Cox AA-20, pore size 0.20 p; Acropor AN-200, pore size 0.20 p; Millipore GS, pore size 0.22 p; Ultipor .12, removes particles 0.35 p and larger from aqueous solution. (Data from manufacturers’ literature.) SCFM = standard fb3/minute.

filtration and Millipore membrane filtration in a single unit. The membrane material is held between two sheets of Dacron mesh and applied around a plastic core. A layer of prefilter material is then applied and the entire cartridge is covered by an outer polypropylene sleeve which protects against back-pressure surges and rough handling. Multitube filter holders (Fig. 7 ) adapted with large-diameter connections for the filtration of air, accommodate up to 20 Millitube cartridges of either a 22- or 31-inch length by means of leak-proof 0ring seals. The assembled units are ethylene oxide sterilized before use with a convenient portable device especially designed for the multitube system. According to the manufacturer Millitube cartridges have proved effective in removing organisms not only from dry air but from moist air as well. The Gelman Instrument Co. supplies membranes in a variety of materials, certain of which are autoclavable. Although the AcroporAN membranes are listed as autoclavable in a holder in the Gelman 1968 catalog, they were listed as not autoclavable in the 1967 catalog.

166

R. STEEL AND

'r.

L. MILLER

FIG. 7. Multiple filter cartridge assembly for air filtration. (Courtesy of Millipnre Corp.)

The relationship between pressure drop and air flow rate for 0.20 F Acroyor-AN membrane is shown in Fig. 6. Gelman recommends a 0.45 p Acropor-AN Inernbrane for air sterilization.

B.

Cox FILTERMEDIA

One shortcoming of cellulose nitrate membranes is their fragility. This objectionable feature has been overcome in the filter medium formulated b y Cox Instrument Co. The filter medium (designated M-780) is composed of glass and asbestos microfibers bonded in epoxy resin to a thickness of 780 F. This filter medium is available in a

FERMENTOR DESIGN

167

variety of pore sizes. The rated pore size (0.20 p is the smallest) is controlled by adjustment of ingredients and packing density. For a given filter medium the density is lightest at the upstream surface, in effect, forming a prefilter to retain larger particles without clogging the inner pore structure. Air is routed in the inner structure through an ever narrowing maze of verticaI and horizontal flow paths. Whereas film or membrane filters act as a screen to trap particles on their upstream surface, the M-780 retains contaminants not only on the surface but also throughout the entire filter matrix. The openings are progressively reduced in size until at the last 10% or so of filter thickness the rated pore size is reached (Fig. 8). The filter medium is rugged compared to membrane filters and can withstand sterilization temperatures to 200°C. This firm also markets filter housing in aluminum or stainless steel to accommodate single elements for low flow rates, or multiple-stacked elements for higher air flow rates. While the 0.45 p pore size may be satisfactory for air sterilization, the 0.20 p pore size offers a higher degree of assurance provided that the higher pressure drop required across the filter can be provided in your equipment. Pressure drop-flow rate data are given in Fig. 6. Equipment can be supplied for production-scale requirements.

C. PALL TRINITYFILTERMEDIA Pall Trinity Micro Corporation supplies Ultipor filter medium which is an epoxy impregnated fiber backing to which extremely fine inorganic fibers are epoxy resin bonded. This material withstands autoclaving at 250°F. for 30 minutes. The Ultipor .12 cartridges are pleated so as to have a large surface area for filtration. A cartridge with 5.5 ft.2 of effective filter surface.is contained within physical dimensions of 2 % inches in diameter and 9% inches in length. The manufacturer claims that Ultipor .9 will provide sterile air but certainly the Ultipor .12 will provide a greater degree of assurance because of its smaller pore size. The pressureflow rate relationship for Ultipor .12 is shown in Fig. 6. The pleated cartridge configuration is necessary to provide the high surface area required to obtain reasonable air flow rates. For sterilization, steam is passed to both sides of the cartridge. Hardware is available from this firm for pilot-scale fermentors and multiple units can be assembled for production-scale equipment with no apparent limit on capacity. A paper by Singer and Hacker ( 4 1 ) described their filter test method using filtration of Serrutia marcescens from aqueous buffer soIution to characterize pore size and

168

R. STEEL AND T. L. MILLER

FIG.8. Photomicrograph of downstream side of Cox AA-20 filter medium. (Courtesy o f Cox Iristrument Co., x 10,000.)

filter integrity. The procedure is used to guarantee absolute retention of bacteria 0.35 p and larger.

D. BIOTECH FILTERMEDIA Biotech Inc. supplies a glass fiber paper disc for air filtration. The filter has a removal efficiency of ~ ~ 9 9 8for%the removal of particles of

FERhlENTOH DESIGN

169

dioctylphthalate of 0.3 p mean diameter in an air stream with a linear velocity of 14.2 cm./second. The filter is autoclaved in the holder and is replaced for each fermentation. The maximum air flow rate is 12 liter/minute for the unit described.

E. DOMNICK-HUNTER FILTERCANDLES Domnick-Hunter manufactures filter elements composed of borosilicate glass microfibers (mean fiber size, 0.5 p) which are tightly packed and reinforced to minimize fiber migration. Manufacturers' tests with dioctylphthalate and methylene blue (0.4 p mean diameter) show penetration less than 0.001% and no penetration of bacterial spores. The filter candles withstand repeated steam sterilization and steam can be passed directly through the filter element. The useful life of the filter ends when the resistance to air flow becomes too high or the maximum permissible pressure differential of 10 13.s.i.g. is reached. To extend the life of the filter, prefilters on air and steam are recommended. Hardware is available to handle flow rates u p to 4000 standard ft."/minute with air to 30 p.s.i.g. and a 2 p.s.i.g. pressure drop.

F. ECHOAIR FILTERS The Echo air filter, manufactured b y Eikoh Kasei Co. Ltd. (Tokyo, Japan), is claimed to have a collection efficiency exceeding 99.999% when the superficial air velocity is 80 cm./second. It consists of a disc of polyvinyl alcohol 3 to 5 mm. thick which acts as a depth filter. In use the disc is held rigid between the flanges of a holder. For sterilization steam is passed into chambers on each side of the filter medium for 30 minutes at 120°C. Deterioration of the filter medium with use is claimed to be slight; even with weekly sterilization the filters have an expected life in excess of 2 years. The filter element is replaced with a new one when the pressure drop is twice that of its initial value. Filter life is extended by use of a prefilter and the manufacturer recommends a bed of urethan foam for removal of dust and oil. This equipment is available for all scales of operation.

IX.

Mechanical Defoamers

Foam generation by agitated-aerated fermentations is usually combatted b y addition of various chemical defoaming agents. The presence of certain chemical defoamers may reduce the oxygen solution rate thus decreasing the rate of growth and/or product formation. They may also interfere with purification processes. Another undesirable result of foam is that one sometimes has to allow sufficient head

170

R. STEEL AND T. L. MILLER

space for it, thus reducing the effective tank capacity. However, all chemical defoamers do not necessarily have adverse effects. Phillips (42) described the application of water-soluble antifoam agents of the Pluronic series (Wyandotte Chemicals Corp.) which did not reduce the oxygen solution rate appreciably. There are occasions when foam destruction by chemical antifoams brings about an increase in oxygen solution rate (43). Nevertheless a reliable mechanical defoaming system would eliminate the problems which sometimes arise from the use of chemical antifoam agents. Martin and Waters (16) described a foam breaker they used with the citric acid fermentation. Foam was ejected from a jet onto the surface of an umbrella-shaped rotating disc. The emulsion was broken by centrifugal force when it struck the walls of the receiver. The gas phase continued upward out of the receiver while the liquid phase drained by gravity-feed back into the fermentor. It was necessary to aid the mechanical system by making periodic additions of octadecanol in lard oil. Phillips et al. (44) were able to grow Torulopsis utilis in a conventional agitated-aerated fernientor without addition of chemical antifoam. The effluent gas and foam were passed from the fermentor through a foam breaker nozzle at velocities of 100-300 ft./sec. Interchangeable nozzles of varying internal diameter were used to impart sufficient velocity to the foam, dependiiig on the rate of air flow through the fermentor. The sudden acceleration through the nozzle resulted in almost complete destruction of the foam. A deflector baffle directed the liquid to the bottom of the receiver from where it was pumped back into the fermcntor. The effluent gas passed out the top of the receiver and was vented to the atmosphere. This is a relatively simple apparatus that worked effectively for a yeast fermentation; it would probably also perform satisfactorily for bacterial fermentations. However, inycelial fermentation beers may offer some problems because of accumulation of growth in the receiver and adjoining pipework. Chemapec Inc. has designed a foam separator consisting of several cones mounted on a rotating hollow shaft. The cones open downward into the fermentor. Foam and effluent gas enter the spaces between the rotating cones and the liquid phase is separated by centrifugal force. The effluent gas passes out of the fermentor through the hollow shaft. To our knowledge there have been no reports in the literature dealing with the evaluation of this device. The power requirements for the foam separator will be subject to a number of variables such as

FERMENTOR DESIGN

171

foam density, gas flow rate, cell concentration, and type, so that it is difficult to estimate power costs. Ebner, Pohl, and Enenkel(24) described a mechanical foam breaker originally designed for use with the Frings Acetator. It consists essentially of a rotor with radial blades which turn within a spiral housing at 1000-1450 r.p.m. Centrifugal action separates the phases the liquid returns to the fermentor and the gas passes to atmosphere. The power consumption is said to vary with the type and quantity of foam from 0.30-1.2 kw-hr./lOOOft.3of gas in foam. The Teknika Sonic Defoamer operates at a transducer frequency of 12,000 C.P.S. The pressure variation approaches that of a vacuum during part of each cycle so foam bubbles burst because their internal pressure is higher than the surrounding partial vacuum caused by the sonic energy. The unit requires 8.0-11.6 standard ft.3/minute of air (sterile) at 20 to 40 p.s.i.g. pressure for operation. Miller (28) reported that a 10 kc. Sonifier could cope with 50-60% of fermentation foams (150-liter fermentor) that he encountered. Hence it was the primary foam control system. However with difficult foaming problems, with which the Sonifier could not cope, a backup system was activated which injected an antifoam agent onto the foam surface. The Kearfott Sonijet generates sound intensity of 150 db. in the frequency range of 8 to 10 kc. and requires an air flow rate of 14.5 standard ft.3/minute at 20 i1.s.i.g. pressure. Defoaming systems are custom-made; the nature of a particular foaming problem will determine the installation capacity and configuration. Whether foaming is controlled by chemical or mechanical defoamers, it is necessary to detect the foam level in order to activate the defoaming system. This is accomplished with resistance and/or conductance probes (8, 1 1 , 28, 30,33). X.

Antifoam or Nutrient Addition

Sterile antifoam or nutrients may be added to the fermentor by gravity feed, forced by sterile air under pressure or pumped. A simple arrangement for intermittent manual batch addition was used by Kroll et al. (4). A sterile calibrated vessel was filled from a sterile reservoir and additions of measured amounts were made by the shift operator. Bartholomew and Kozlow (45) considered the proper selection of a solenoid to be an important feature of their defoamer system. The seats should be resistant to repeated steam sterilization in the presence

172

11. 5TEEl.

AND T. L. MILLER

of antifoain oil and the valve body should be easily cleaned. McCann et al. (11) eliminated the solenoid and used air pressure to feed antifoam to bench-wale fermentors. In production-scale operations a central defoamer tank may be manifolded to a series of fermentors with an additional metering tank located at each fermentor. The proper operation of a continuous fermentor requires the addition of nutrient medium in a continuous arid constant manner. In small-scale (laboratory) kinetic studies a dependable feed rate is absolutely essential in order to achieve meaningful results. Regardless of the feed device used it is desirable to maintain an unvarying hydrostatic head pressure of medium in the storage reservoir that supplies the feed device; a Mariotte bottle is often used for this purpose (46). Peristaltic pumps are popular for delivering nutrient medium to fermentors. With these devices the liquid is driven through a length of flexible tubing by metal fingers, a cam, or roller action. The Sigmamotor pump is often used (21, 47, 48). These pumps have the advantages of simplicity and that the mediiiin does not contact the moving parts of the pump. The disadvantiiges are that the feed is delivered in a pulsating fuhion and that the feed rate changes as the tubing stretches or slips in the tubing holder. Piston-type positive displacement punips may give a more constant feed rate and are often used when the medium does not contain suspended solids. Two pumps that have been used are the D.C.L. Micropump (21) and the Milton Roy pump (49). Motor-driven syringes also offer a means of continuous addition of nutrients (50). A liquid metering device may be used in place of a pump to deliver the feed medium at a constant rate (13, 51). In this case a precisely controlled volume of medium is isolated in a chamber between two valves. The medium contained in the chamber is injected into the fermentor by the automatic sequential opening of the proper valves. Under controlled conditions such an arrangement gives an unvarying semicontinuous feed. A device for use in small-scale continuous fermentations has been described in detail (51). XI.

Instrumentation

A. p11 MEASUREMENT AN11

CONTHOL

The importance of control of pH in many feimentations is well established. In some fernlentations pH control is achieved by natural buffers present in the complex ingredients that make u p the medium. In addition, compounds such its CaCO:$are sometimes added to the

FERMENTOR DESIGN

173

medium for the purpose of maintaining a desired pH range. However, such methods are usually only partially successful and the control of pH within narrow limits requires the periodic addition of acid or base, or both, to the fermentation beer. The handiest way to accomplish this is with pH-sensing electrodes in constant contact with the fermentation beer; then pH control may be achieved by the automatic addition of acid and/or base. The pH electrodes may project directly into the fermentor (8,28,52) or they may be inserted into a recirculating loop outside of the fermentor (53). It is desirable in either case to have electrodes that withstand steam sterilization so that they can be sterilized in place. Such an electrode has been described by Fiechter, Ingold and Baerfuss (54). This electrode, assembled in a concentric configuration that incorporates both the glass and reference electrodes in a single unit, is available commercially from Chemapec Inc. The unit also has provision for the application of pressure to the reference electrode. It is necessary to apply pressure to the reference electrode when it is mounted in the side of a fermentor or in a recirculating loop. A pressure-equalizing hole above the electrolyte level is used with electrodes mounted inside of a pressurized fermentor. This insures that fermentation beer will not pass into the electrode. Steam sterilizable pH electrodes are also available from New Brunswick Scientific Co., Electronic Instruments Ltd. (ELL.), and Beckman Instruments, Inc. An electrode-holder assembly that allows the removal or insertion of the glass or reference electrode during the fermentation is manufactured by E.I.L. However, the glass electrode sold by this company is not recommended for sterilization above 100°C. Both Chemapec Inc. and E.I.L. market a flow-through pH electrode assembly that makes provision for cleaning the electrode without removing it from the unit. Such an arrangement is useful in fermentations where the eIectrodes tend to become fouled by material depositing on the electrode surfaces. i n other arrangements the electrodes are sterilized with ethylene oxide or ultraviolet radiation and then placed in the fermentor or in a closed circulating loop (55, 56). These methods of sterilization introduce the risk of contamination when the electrodes are placed in contact with the fermentation medium. For operation in fermentations, glass and reference electrodes are connected to a pH meter which is in turn connected to a pH controller unit or a recorder-controller. An electrical signal from the p H controller initiates the addition of acid or base to the fermentor as needed to maintain pH control. i t is desirable to add acid or base in

174

R. STEEL AND T. L. MILLER

small amounts, with a time delay between additions, to allow for mixing and thereby avoid overshooting the pH set point. The above mentioned companies and others that supply pH electrodes also sell pH controllers.

B. CARBONI~IOXIDE MEASUREMENT Carbon dioxide and water are the most common products of aerobic microbial metabolism. The COz produced along with the oxygen consumed gives a measure of the aerobic metabolism of fermenting cultures. In addition, a knowledge of the amount of COZ produced is essential for obtaining a carbon balance for a fermentation. Probably the most widely used instrument for measuring COZ in fermentation effluent gases is the infrared gas analyzer. The operation of such an instrument relies on the infrared-absorbing properties of COZ. The operation of the instrument depends on a detector which is sensitive to the energy produced when infrared radiation is absorbed by C 0 2 in the sample chamber of the instrument. The percentage of COz can be read directly from a meter on the front of the amplifier section of the instrument or the signal from the analyzer may be printed out on a strip-chart recorder. Infrared gas analyzers are available from a number of instrument suppliers including Beckman Instrument Co. and Mine Safety Appliances Co. Maxon and Johnson (57) described an interesting method for the continuous measurement of COe. It consists of bubbling the COecontaining gas from a fermentor through a dilute solution of NaOH that contains phenol red indicator. The bicarbonate ion concentration of the solution, and thus hydrogen ion concentration, is directly proportional to the pCOe of the gas passing through the solution. Therefore, the C 0 2 concentration can be measured indirectly by the photometric determination of the absorbance of the phenol red indicator. The electrical output from the photocell may be plotted on a stripchart recorder, and COZ concentration is then obtained by use of a suitable standard curve. This is a simple and inexpensive method for determining COe, but the instrument required periodic attention for continuous operation. The Harvard Apparatus Co. has available an instrument for determining C 0 2 that utilizes the measurement of changes in thermal conductivity of gases with changes in composition. There are other methods and variations of the above methods for determining gaseous COZ. In addition, dissolved C 0 2 may be measured b y the use of a Teflon coil, such as described by Phillips and Johnson (58) for dis-

FERMENTOR DESIGN

175

solved oxygen measurement, in the fermentation beer. The carrier gas stream (NP) from the coil is then passed through a CO2 gas analyzer such as one of those described above. C. OXYGENMEASUREMENT Oxygen is an essential nutrient in all aerobic fermentations. An insufficient dissolved oxygen level in the fermentation beer may limit the rate of microbial growth and product formation. Indeed, the dissolved oxygen level is sometimes the key in scale-up of fermentations. In the design of equipment for aerobic fermentation processes the oxygen transfer rate of the fermentor is a most important consideration. Traditionally, the measurement of dissolved oxygen has relied on the electrolytic reduction of oxygen at an electrode surface. Wet titration (Winkler) methods have proved to be awkward and often inaccurate for measuring dissolved oxygen in fermentation beers. Polarographic methods using dropping mercury or rotating platinum electrodes offered a somewhat better means of measuring oxygen concentration. The introduction of the membrane-covered electrode by Clark (59) made measurement of dissolved oxygen in the fermentor much more practical. Initially the membrane electrodes used in fermentors were also of the polarographic type (60-62). Some of these electrodes had the added advantage of being steam sterilizable (61,62). Phillips and Johnson (58) devised an oxygen diffusion method for measuring dissolved oxygen. With this procedure nitrogen carrier gas is passed through a long coiled oxygen permeable-plastic tube in contact with the fermentation beer. Oxygen dissolved in the beer diffuses through the tubing at a rate proportional to the oxygen tension. The oxygen content of the carrier gas emerging from the tube is measured by passing it through an oxygen analyzer. The dissolved oxygen concentration can then be calculated. While this method effectively measured dissolved oxygen it required a large “membrane” surface area for oxygen diffusion and also the use of an oxygen gas analyzer. The most recent and perhaps the best method for measurement of dissolved oxygen is the galvanic cell oxygen probe (63, 64). These probes generate their own electrical current which is proportional to the oxygen being reduced at the cathode. The anode and cathode of the cell are separated from the medium by an oxygen-permeable membrane. Improvements of these probes have been introduced (65,66). Probes constructed according to the method of Johnson et al.

176

1%. STEEL

AND T. L. MILLER

(63, 66) are inexpensive and easy to fabricate; in addition, they are steam sterilizable, long-lived arid fkiirly rugged. Some suppliers of commercially available oxygen probes are given in Table I. TABLE I SUPPLIERS OXYGENP ~ o i i e s

Beckinan

777

Polarographic (membrane)

yes

Lee Scientific

100

Pnlarographic (memhrane )

yes

Honeywell

55145-01 and 02 Polarographic (membrane)

Tcchnnlogy Inc.

P016OL and B

Polarographic

no

0-116 mm. IIg

Electronic Instr. Ltd.

A15A

Galvaiiic (64)

nu

0-40% 0,

Union Carbide

1101

(incmhrarie) Polarographic

no

0-15 p.p.m.

E. H. Sxrgvut & Co.

S-38640-10R

Calvanic (membrane)

no

0-50 mg. OJliter

Delta Scientific

75

Galvanic (membrane)

?

0-100%0

O-lOO% Or

?

110,

2

In aerobic processes it m a y be dcsirable to control the dissolved oxygen concentration at a given level or it may only be necessary to insure that it docs not fall helow the critical level (67). Control of the oxygen tension at a given level may be achieved by altering ferrnentation variables such as agitation rate (68), aeration rate (69), and partial pressure of oxygen (pOe) in the influent gas (70, 71)or the fermentor head pressure a s indicated by the following equation (72): N,4= K g . a (Pg

~

Pe)

where

N , = rate of 0 2 transfer by the fermentor K g = overall 0 2 transfer coefficient a =total interfacial area of gas bubbles in the fermentor ( K g and a are generally expressed together) Pg = 1 1 0 2 in the influent gas Pe = dissolved O2 tension in the medium

FERMENTOR DESIGN

177

Thus, it is clear that increasing K g , a, or Pg on the right hand side of the equation results in increased oxygen transfer. The aeration and agitation rates control the interfacial area ( a ) and transfer coefficient ( K g ) , while the pO2 of the influent gas determines Pg. Therefore, to achieve control of dissolved oxygen in the fermentor it is necessary to measure the dissolved oxygen concentration with one of the probes described above. The signal from the oxygen probe in the fermentor is then made to regulate, by suitable electrical arrangements, one or more of the above-mentioned fermentation variables. The measurement of gaseous oxygen in fermentor influent and effluent gas streams is essential for determining the rate of oxygen consumption b y the respiring microorganisms. Measurement of gaseous oxygen in the fermentor gas streams may be accomplished by use of a membrane-type oxygen probe (73) or a paramagnetic oxygen analyzer (68, 70). Paramagnetic type gas analyzers are available commercially from Mine Safety Appliances Co., Beckman Instruments, Inc., Leeds and Northup, and several other companies.

D. TEMPERATURE MEASUREMENT The temperature within the fermentor is measured by one of the conventional sensing devices such as a thermister, thermocouple, bimetallic strip, mercury-filled column, or liquid-filled bulb. The temperature control mechanism may be operated by an electrically transduced signal from the sensing device. On large-scale fermentors, temperature control is achieved by circulating cold water through coils inside the fermentor. With laboratory fermentors cooling may be obtained with a cold-finger within the fermentor, while heat may be supplied b y an immersion heater or by radiation with an infrared lamp. Alternatively, water of a controlled temperature may be circulated through a jacket or bath surrounding the fermentor. Instrumentation for temperature control is described by Reisman and Gore (23) and others (8,9,11).

E. PRESSUREMEASUREMENT It is often desirable to conduct fermentations under positive head pressure. This helps to suppress foam, effectively increases the p 0 2 within the fermentation beer, and is a preventive measure against contaminants gaining access to the fermentor via leaky valves or seals. The head pressure is usually measured by diaphragm, manometer, Bourdon tube, or bellows-type detector. Pressure control is achieved by manual or automatic manipulation of a valve on the effluent air line.

178

R. STEEI. AND T. L. MILLER

F. MISCELLANEOUS Fuld and Dunn attempted to measure and control sugar concentration in a yeast fermentation (74). They contemplated measuring specific gravity or optical rotation of the fermentation beer, but concluded that continuous measurement of refractive index (R.I.) was the best method. The method required that the beer be free of cells and debris. A closed recirculating loop cycled the cell-free sample through the refractometer; a sugar solution was added to the fermentor on a signal from a recorder-controller. Ethanol produced by the culture interfered with the R.I. measurement (33). The measurement of the redox potential as an indication of 0 2 tension has been suggested. The relationship between redox potential and oxidizing capacity was studied by Squires and Hosler (75).The correlation between microbial growth and redox potential level is not well understood. However, the demonstration of the reproducibility of redox electrodes (76) should stimulate further investigations of this relationship. Generally equipment for the measurement of redox potentials is available from the firms manufacturing pH measuring equipment. In order to calculate the power input it is necessary to measure the torque applied to the fermentor agitator shaft. Reisman and Gore (23) measured torque with a strain gauge torquemeter, while Nelson et al. (8) used a spring-loaded torque indicator. The input horsepower may be calculated if the agitator speed (r,p,m,) and torque (in.-lb.) are known. LKB Instruments (Biotech Inc.) markets a continuous flow microcalorimeter for monitoring the growth pattern of microorganisms. While there have been relatively few papers on the subject, automation is becoming more common in the fermentation industry. Ajinomoto Co. workers (35)have described their computer-controlled glutamic acid plant and Dista Products (77)published briefly on their installation at Speke, England.

XII.

Continuous Fermentors

A continuous fermentor is a device for maintaining a steady state population of microorganisms. This is accomplished by continuous introduction of feed medium and simultaneous withdrawal of fermentation broth at the same rate. The theoretical aspects of such systems have been covered in many excellent reviews (78-80). In general, a well-designed continuous fermentor conforms to the same standards as outlined in the preceding sections on batch fermentors. I n fact, a

179

FERMENTOR DESIGN

continuous fermentor is often simply a batch fermentor with provision for maintaining constant volume (chemostat) or cell density (turbidostat) while nutrient medium is being continuously added. However, a wide variety of unique continuous fermentors have been designed mainly for laboratory use; the purpose for which the fermentor is to by used usually dictates the design. Many of the continuous fermentors described in the literature can supply oxygen to growing organisms only at a very limited rate. Such fermentors can only support growth of high concentrations of microorganisms at low growth rates. Unfortunately, data on the rate of oxygen transfer of a fermentor, such as those discussed by Johnson (a]), usually are not included with fermentor descriptions. This section will mainly describe methods for maintaining constant volume or cell density in the fermentation vessel. The discussion will deal principally with single-stage stirred fermentors, although the application of these fermentors may be extended to more sophisticated multistage and cell recycle systems. These fermentors may be equipped for measurement and/or control of pH, dissolved and gaseous O x ,C 0 2 in effluent gas, etc., as described in other sections of this report.

A. OVERFLOW FERMENTORS Regardless of the overall fermentor design, the most popular means for controlling volume in continuous fermentations is by the overflow method. The overflow outlet may be in the side (21, 79, 82) or the bottom (83,84) of the fermentor. In addition, the withdrawal from the top surface of liquid in the fermentor may be achieved by entrainment of the liquid in the effluentair stream (21,85), or by use of a standpipe (68) or a side arm. Figure 9 illustrates several generalized types of apparatus for maintaining constant volume by the overflow method.

a

/J=J

\ X q Q

(61 Standpipe

(A)Side

( C 1 Gooseneck

h

Effluent

md: :

f=Q

( D l Gas entrainment

FIG.

L

p

( E l Teapot

9. Methods for maintaining constant volume.

180

R. STEEL AND T. L. MILLER

Each of these designs has certain advantages and disadvantages. The withdrawal of culture fluid from the liquid surface (Fig. BA,B,D) may result in the removal of a disproportionate concentration of cells if foaming occurs. Furthermore, if the substrate is immiscible with arid less dense than water (such as in hydrocarbon fermentations), then a disproportionate amount of substrate may be withdrawn. Withdrawal of culture fluid by gas entrainment requires a positive pressure within the fermentor; in addition, a gas-liquid separator must b e incorporated into the withdrawal line (85). On the other hand, withdrawal of culture fluid by these methods (Fig. 9A,B,D) is probably the simplest and most direct way of achieving volume control. Withdrawul of culture fluid from below the liquid surface by methods (C) and ( E ) helps to eliminate the problems related to foaming and low density water-immiscible substrates. However, other problems may be encountered. For example, the goose-neck arrangement introduces an area without trirbulence (poor mixing) which is subject to anaerobic conditions and clogging with cells. The teapot apparatus is subject to relatively large volume fluctuations resulting from the turbulence caused by agitation; this method also introduces a more or less stagnant area in the “spout.” A general method that can be used for volume control by withdrawal of culture fluid from any part of the fermentor uses a manometric sensing device that actuates a valve allowing some beer to escape. This set-up is described later in the discussion of the cyclone column fermentor.

B. PACKEDCOLUMN(TOWER) FERMENTORS An interesting, although seldom used, variation of the continuous fermentor is the packed column or tower fermentor. In this case a cylindrical column is placed in a vertical position and packed with particles of some relatively inert material, e.g., wood, polyethylene, or concrete chips. A solution of medium and microorganisms is fed into the top of the column. The microorganisms adhere to and grow as a thin film un the solid support. After good growth is obtained on the supporting material, a solution of the desired substrate is percolated through the column. The broth containing the product flows from the bottom of the column. This is by no means a new concept; indeed, the vinegar generator (86) which is an example of a packed column fermentor, is a relatively old concept. In this example the substrate is ethanol, the organism is Acetobacter, the solid support is beechwood shavings, and the product is acetic acid. A recent patent describes the conversion of sulfite waste liquor to ethanol in such a

FERMENTOR DESIGN

181

fermentor (87). The application of this fermentor to other oxidation and reduction bioconversions appears reasonable.

C. SHAKENFLASKFERMENTORS One of the earliest and simplest batch fermentors consisted simply of a shaken flask containing the culture fluid. On a rotary shaking machine the bulk of the liquid moves around the sides of the flask; oxygen is introduced at the liquid film-air interface and adequate oxygen transfer is obtained (81). Therefore, it is not surprising that a shake flask was converted into a continuous fermentor (88). Volume control was accomplished by overflow through a side arm on the flask. With the device described, an oxygen transfer rate of 0.3 mM 02/literminute (sulfite method) was reported (88). Such a device offers a simple and convenient method for continuous fermentation since no elaborate equipment is required. The method is, of course, limited to small-scale studies. A variation of the above method is one in which the flask itself rotates about its longitudinal axis. Devices have been described where the rotating flask is in a horizontal position (89) or inclined at an angle (90). In either case, the culture liquid is moving with respect to the walls of the flask in much the same manner that it moves in a shaken flask on a rotary shaker. Vessels with working volumes of 100 (90) to 800 ml. (89) have been described. Volume control was obtained by overflow, siphoning, or suction of the culture liquid. Such fermentors are claimed to give good gas transfer and no foaming when operated at speeds of less than 400 r.p.m. (90). However, they cannot be scaledup without decreasing the aeration capacity.

D. TUBE-TYPEFERMENTORS An interesting multistage continuous fermentor used for the cultivation of filamentous organisms was described by Means et al. (13). It consisted of a horizontal tube 8 inches in diameter and 18-ft. long separated into nine compartments each 2-ft. long. The compartments (Fig. 10) were separated by metal plates each bearing an overflow hole 1 inch in diameter which established the liquid volume within the compartment. The agitator shaft was composed of two sections each 9%-ft. long to which were attached, at right angles, stainless steel blades. The blades were spaced N inches apart and when rotating they passed through a comb-shaped baffle plate mounted vertically to the bottom of each compartment. Volume control was effected by overflow from one compartment to the next; the hold-up volume of

182

R. STEEL AND T. L. MLLLER S t i r r i n g blades

Overflow hole

Shaft

-

I///,

,n

I / /

1

W////////A

&I

13 -

I

I

,

I

Shaft

FIG. 10. Cross-sectional view along the longitudinal axis of a compartment of a tube-type fermentor (13).

each compartment could be varied independently b y adjusting the position of the overflow hole. Air was introduced at the bottom of each compartment. The advantages claimed for this type of fermentor are uniform oxygen availability throughout the bulk of the medium and minimal hang-up of mycelium on the fermentor walls.

E. CYCLONECOLUMNFERMENTORS A unique fermentor designed especially for the growth of filamentous cultures was described by Dawson (14). This apparatus consists of a vertically positioned cyclone column (Fig. 11). Culture fluid is pumped from the bottom of the column through a closed loop (reAir

Flowmeter

I

Chiller

Air

\

Nutrient reservoir

__

I1

receiver

-_

. _.

. -~

Cyclone column

Pump

FIG. 11. Simplified diagram of cyclone column fermcntor; all details are not shown (14).

183

FERMENTOR DESIGN

circulating arm) and reenters at the top of the column. The entering fluid runs down the wall as a relatively thin film. Nutrients and air are fed in near the bottom of the column while the effluent gases pass out the top. The advantages claimed for this type of fermentor include decreased wall growth, good gas exchange and no foam. The liquid volume is controlled manometrically as shown schematically in Fig. 12. One arm of the manometer “U” tube is attached to an inlet Connection to fermentor

A

Air (slow

\

Manometer

Constriction (damps sudden fluctuations)

FIG. 12. Manometric control of the volume in a continuous fermentor (14).

air stream and the other to the top of the fermentor. The manometer is filled with an electrolyte solution, the level of which responds to the hydrostatic head in the fermentor, i.e., h = h’. When the electrolyte in the manometer makes contact with the electrode contact wire, the relay is actuated through appropriate circuitry. The relay causes a solenoid valve to be energized thereby releasing a small amount of culture fluid from the fermentor and reducing the hydrostatic head pressure. This type of volume control device can be used in conjunction with almost any kind of continuous fermentor. The method was employed in a continuous fermentor designed for hydrocarbon fermentations (73) in which case culture withdrawal from the bottom of the fermentor under full hydrostatic head pressure was achieved. The cyclone fermentor has been used to obtain so-called “continuous phased growth” (91, 92). Synchronous cell growth (Candidu utilis) was initiated by the addition of a relatively large amount of medium (50% of the operating volume) to the fermentor resulting in the subsequent displacement of an equal volume of fermentation

184

11.

STEEL AND T. L. MlLLEH

broth. This fermentor was then used to furnish inoculum for a second cyclone fermentor, operated in series, where the synchronous growth was perpetuated. By this technique it is possible to obtain a high proportion of cells in a similar physiologic state.

F. FULL FERMENTORS The working volume of a fermentor is usually about 50-75% of its total volume. However, in some instances it is possible to operate with a completely full fermentation vessel. In this case the fermentor volume may be changed only by substitution of a vessel of different size. With aerobic fermentations the cell density is limited by the amount of dissolved oxygen in the nutrient feed medium; obviously with anaerobic fermentations other factors will be limiting. Completely full fermentors have been used for laboratory studies of oxygen transport and utilization by microbial cells (49, 53, 94). In these studies the fennentors were simply glass round bottom flasks ranging in volume from 167 to 500 ml. Agitation was achieved by a magnetic stirring bar in the fermentor. With aerobic systems such fermentors are most useful for studying the kinetics of substrate utilization. Higher cell populations may be obtained in anaerobic systems, but provision must be made for venting the gases produced.

G. TURRIDOSTATIC FERMENTORS A turbidostatic fermentor is an apparatus designed for maintaining a constant optical density (O.D.) in the fermenbtion vessel. In such systems constant volume is achieved by one of the methods previously described e.g., overflow device. However, in contrast to the chemostat, the turbidostat maintains constant optical density by varying the nutrient feed rate. With some of the earliest devices described, cell propagation and optical density measurement took place in the same vessel (55-97). In such systems the fermentor consisted of a small vessel within a colorimeter, These designs were necessarily of small volume and required special provisions for keeping the walls of the growth chamber free of adhering cells (96,57). Other devices have been described where the colorimeter photocell is external to the fermentor (21) in which case the culture fluid is circulated through the colorirneter sampling chamber by a pump. REFERENCES

1. Walker, J. A. H., and Holdsworth, H. (1958). In “Biochemical Engineering” (R. Steel, ed.),p. 225. Heywood, London.

FERMENTOR DESIGN

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2. Elsworth, R. (1960). Zn “Progress in Industrial Microbiology” (D. J. D. Hockenhull, ed.), p. 103. Heywood, London. 3. Chain, E. B., Paladino, S., Ugolini, F., and Callow, D. S. (1954). Rend. Zst. Super. Sanita 17, 87. 4. Kroll, C. L., Formanek, S., Covert, A. S., West, J. M., and Brown, W. E. (1956). Znd. Eng. Chem. 48,2190. 5. Solomons, G. L. (1967).Process Biochem., March. 6. Solomons, G. L. (1968). Process Biochem., August. 7. Pfeifer, V. F., Vojnovich, C., and Heger, E. N. (1952). Ind. Eng. Chem. 44,2975. 8. Nelson, H. A., Maxon, W. D., and Elferdink, T. H. (1956). Ind. Eng. Chem. 48,2183. 9. Anon. (1960). Chemist 36,377. 10. Steel, H., and Maxon, W. D. (1961). Ind. Eng. Chem. 53, 739. 11. McCann, E. P., Parker, A,, Pickles, D., and Wright, D. G. (1961). Truns. Inst. Chem. Eng. 39,461. 12. Oldshue, J. Y. (1966). Biotechnol, Bioeng. 8, 3. 13. Means, C. W., Savage, G . M., Reusser, F., and Koepsell, H. J. (1962). Biotechnol. Bioeng. 4, 5. 14. Dawson, P. S. S. (1963). Can. J . Microbiol. 9, 671. 15. Herrick, H. T., Hellbach, R., and May, 0. E. (1935). Ind. Eng. Chem. 27, 682. 16. Martin, S. M., and Waters, W. R. (1952). Ind. Etlg. Chem. 44, 2229. 17. Irving, G. M. (1968). Chem. Eng. 75, 100. 18. Lumb, M., and Fawcett, R. (1951). J . Appl. Chem. Suppl. 2, 594. 19. Maxon, W. D. (1959). J . Biochem. Microbiol. Technol. Eng. 1,311. 20. Rushton, J. H., Costich, E. W., and Everett, H. J. (1950). Chem. Eng. Progr. 46,467. 21. Herbert, D., Phipps, P. J., andTempest, D. W. (1965).Lab. Pract. 14,1150. 22. Roxburgh, J. M., Spencer, J. F. T., and Sallans, H. R. (1956). Can. J . Technol. 34, 389. 23. Reisman, H. B., and Gore, J. H. (1966). 59th Ann. Meeting Am. Znst. Chern. Eng., Detroit, Michigan, Dee. 24. Ebner, H., Pohl, K., and Enenkel, A. (1967). Biotechnol. Bioeng. 9,357. 25. Heden, C. G . (1958). Nord. Med. 60, 1090. 26. Ulrich, K., and Moore, G . E. (1965). Biotechnol. Bioeng. 7 , 507. 27. Moore, G . E., Hasenpusch, P., Gerner, R. E., and Barns, A. A. (1968). Biotechnol. Bioeng. 10, 625. 28. Miller, 0. C. 17th Ann. Chem. Eng. Con$, Niagara Falls, Ontario, Canada, Oct., 1967. 29. Dworschak, R. G., Lagoda, H. A., and Jackson, R. W. (1954).Appl. Microbiol. 2,190. 30. Friedland, W. C., Peterson, M. H., and Sylvester, J. C . (1956). Znd. Eng. Cheni. 48, 2180. 31. Cameron, J., and Godfrey, E. I. Paper presented 3rd Intern. Fermentution Symp., New Brunswick, New Jersey, Sept., 1968. 32. Parker, A. (1958). I n “Biochemical Engineering” (R. Steel, ed.), p. 95. Heywood, London. 33. Fuld, G. J., and Dunn, C. G . (1957). Znd. Eng. Chem. 49, 1215. 34. Jackson, T. (1958). In “Biochemical Engineering” (R. Steel, ed.), p. 183. Heywood, London. 35. Mori, M., and Yamashita, S. (July, 1967). ControZ Eng., p. 66. 36. Gaden, E. L., and Humphrey, A. E. (1956). Znd. Eng. Chem. 48,2172. 37. Humphrey, A. E., and Gaden, E. L. (1955). Znd. Eng. Chem. 47, 924. 38. Sadoff, H. L., and Almoff, J. W. (1956). Znd. Eng. Chem. 48,2199.

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39. Humphrey, A. E., and Deindoerfer, F. H. (1961). Folia Microbiol. 6 , 1. 40. Dorman, R. G . (1967). Chem. rl+ Znd., p. 1946. 41. Singer, H., arid Hacker, C. H. (1968). Chem. Eng. Progr. 64, 75. 42. Phillips, D. H. U.S. Patent 3,142,682. 43. Chain, E. R . , and Gualandi, G. (1954). Rend. 1st Super, Sanita. 17,5. 44. Phillips, K. L., Spencer, J. F. T., Sallans, H. R., and Roxburgh, J . M. (1960). J . Biochein. Microhiol. Technol. Eng. 2, 81. 45. Bartholomew, W. H., and Kozlow, D. (1957).Ind.E n g . Chem. 49,1221. 46. Ricica, J. (1966). In “Theoretical and Methodological Basis of Continuous Culture of Microorganisms” (I. Malek arid F. Zdenek, eds.), p. 157. Academic Press, New York. 47. Hosenberger, R. F., and Elsden, S. R. (1960).J.Gen. Microbiol. 22,726. 48. Finn, R., and Wilson, R. E. (1954). Agr. Food Chem. 2,66. 49. Button, D. K., and Carver, J. C. (1966).J. Gen. Microbiol. 45,195. 50. Moss, F., and Saeed, M. (1967). In “Progress in Industrial Microbiology” (D. J. D. Hockenhull, cd.), p. 209. Heywood, London. 51. Johnson, M. J. (1967). Biotechnol. Bioeng. 9, 630. 52. Hosler, P., and Johnson, M. J. (1953). Ind. Eng. Chem. 45, 871. 53. Denison, F. W., West, I. C., Peterson, M. H., and Sylvester, J. C. (1958). Ind.E n g . Chem. 50, 1260. 54. Fiechter, A., Ingold, W., and Baerfuss, A. (1964). Chew&.Ing. Tech. 36,1000. 55. Callow, D. S., a i d Pirt, S. J . (1956).]. Gen. Microbiol. 14,661. 56. Deindoerfer, F. H., and Wilker, E. L. (1953). Ind. Eng. Chem. 49, 1223. 57. Maxon, W. D., and Johnson, M. J. (1952). Anal. Chem. 24, 1451. 58. Phillips, D. H., and Johnson, M. J. (1961). J. Biochem. Microbiol. Technol. Eng. 3,261. 59. Clark, L. C. (1956). Tram. Am. Soc. Artijicial Internal Organs 2, 41. 60. Bandyopadhyay, B., Humphrey, A. E., and Taguchi, H. (1967). Biotechnob Bioeng. 9, 533. 61. Carritt, D. E., and Kanwisher, J. W. (1959). Anal. Chem. 31, 5 . 62. Phillips, D. H., and Johnson, M. J. (1961). Sci. Reyt. Ist. Super. Sanita 1,190. 63. Johnson, M. J., Borkowski, J., and Engblom, C. (1964). Biotechnol. Bioeng. 6,457. 64. Mackereth, F. J. H. (1964).j . Sci. Instr. 41, 38. 65. Flynn, D. J., Kilborn, D. G., Lilly, M. D., and Webb, F. C. (1967). Biotechnol. Bioeng. 9,623. 66. Borkowski, J., and Johnson, M. J. (1967). Biotechnol. Bioeng. 9, 635. 67. Phillips, D. €I., and Johnson, M. J. (1961). J . Biochem. Microbiol. Technol. Eng. 3, 277. 68. Moss, F. J,, and Bush, F. (1967). Biotechnol. Bioeng. 9, 585. 69. Lengyrel, Z. L,, and Nyiri, L. (1965). Biotechnol Bioeng. 7, 91. 70. Harrison, D. E. F., and Pirt, S. J. (1967).J . Gen. Microbiol. 46, 193. 71. Maclennan, D. G., and Pirt, S. J. (1966).J . Gen. Microhiol. 45, 289. 72. Arnold, B. H., and Steel, R . (1958).In “Biochenlical Engineering” (R. Steel, ed.), p. 149. Heywood, London. 73. Miller, T. L. (1966). Ph.D. Thesis, University of Wisconsin, Madison, Wisconsin. 74. Fuld, G. J., and Dunn, C. G. (1956). 16th Ann. Meeting Inst. Food Techttol., S t . Louis, MissouriJune. 75. Squires, R. W., and Hosler, P. (1958). Ind. Eng. Chem. 50, 1263. 76. Garcia, L. H., Daniels, W. F., and Rosensteel, J. F. (1967). Biotechnol. Bioeng. 9, 626.

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Anon., (1968). Manuf. Chem. Aerosol News, June. Powell, E. 0.(1965).Lab. Pract. 14,1145. Herbert, D., Elsworth, R., and Telling, R. C. (1956). J . Gen. Microbiol. 14, 601. Maxon, W. D. (1955). Appl. Microbiol. 3, 110. Johnson, M. J. (1958).7 t h Intern. Congr. Microbiol., Stockholm, Sweden. Sikyta, B., and Stezak, J. (1964).Arch. Microbiol. 49, 341. DeHaan, P. G., and Winkle, K. C. (1955). Antonie uan Leeuwenhoek]. Microbiol. Serot. 21, 33. 84. Rotman, B. (1955).J . Bacteriol. 70, 485. 85. Maxon, W. D., and Johnson, M. J. (1953). Ind. Eng. Chem. 45,2554. 86. Vaughn, R. H. (1954). In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.), Chapt. 17. Chemical Publ., New York. 87. U.S. Patent 3,402,103. 88. Owen, S. P., and Johnson, M. J. (1955).Agr. Food Chem. 3,606. 89. Heden, C. G., Holme, T., and Malmgren, B. (1955).Acta Pathol. Microbial. Scand. 37,42. 90. Perrett, C. J. (1957).J . Gen. Microbid. 16, 250. 91. Dawson, P. S. S. (1965). Can. J . Microbiol. 11, 893. 92. Dawson, P. S. S. (1966). Nature 210, 375. 93. Johnson, M. J. (1967). J . Bacteriol. 94, 101. 94. Borkowski, J. D., and Johnson, M. J. (1967).Appl. Microbiol. 15, 1483. 95. Bryson, V. (1952). Science 116,48. 96. Northrop, J. (1954).J . Gen. Physiol. 38, 105. 97. Anderson, P. A. (1956). Rev. Sci. Instr. 27,48.

77. 78. 79. 80. 81. 82. 83.

Appendix: Addresses of Equipment Suppliers Beckman Instruments Inc., 2500 Harbor Boulevard, Fullerton, California. Biotech Inc. (Sweden). LKB Instruments Inc., 12221 Parklawn Drive, Rockville, Maryland, 20852. Chemapec Inc., 1 Newark Street, Hoboken, New Jersey, 07030 Cox Instrument Division, Lynch Corp., 15300 Fullerton Avenue, Detroit, Michigan 48227 Delta Scientific Corp., Box 493, Hicksville, New York Domnick-Hunter (Engineers) Ltd., Washington Steel Works, Washington, County Durham, England Durametallic Corp., 2104 Factory Street, Kalamazoo, Michigan Eikoh Kasei Co. Ltd., C.P.O. Box 2064, #3,2-chome, Kanda-Misakicho, Chiyodoku, Tokyo, Japan Electronic Instruments Ltd., through Cambridge Instrument Co. Inc., 73 Spring Street, Ossining, New York 10562 E. H. Sargent and,Co., 4647 West Foster Avenue, Chicago, Illinois Fermentation Design Inc., P.O. Box 205, Durham, Pennsylvania Garlock Inc., Palmyra, New York Gelman Instruments Co., P.O. Box 1448, Ann Arbor, Michigan 48106 Harvard Apparatus Co., Inc., 150 Dover Road, Millis, Massachusetts Honeywell, Industrial Div., Fort Washington, Pennsylvania 19034

188

R . STEEL AND T. L. MILLER

Kearfott Div., General Precision Inc., Little Falls, New Jersey Lee Scientific Corp., 545 Technology Square, Cambridge, Massachusetts 02139 Leeds and Northrup, 4907 Stenton A v ~ I ~Philadelphia, u~, Pennsylvania 19144 T h e London Co., 811 Sharon Drive, Westlake, Ohio 44145 Marton Equipment Co., ,50 Federal Street, Beverly, Massachusetts 01915 (D.C.L. Micropump) Millipore Filter Corp., Bedford, Massachusetts 01730 Milton Roy Co., 1300 East Mermaid Lane, Philadelphia, Pennsylvania Mine Safety Appliances Co., 201 N. Braddock Avenue, Pittsburgh, Pennsylvania Mixing Equipment Co., Inc., 147 Mt. Head Boulevard, Rochester, New York New Bmnswick Scientific Go., Inc., 1130 Somerset Street, New Bninswick, New Jersey 08903 Pall Trinity Micro Corp., P.O. Box 1172, Cortland, New York 13045 Sage Instruments, Inc., 2 Spring Street, White Plains, New York 10601 (syringe pump) SigIliaiiiotor Inc., 3 North Main Street, Middleport, New York 14105 Technology, Inc., 7400 Coloncl Glenn Highway, Dayton, Ohio 45431 Teknika, Iiic., 634 Asylum Avenue, Hartford, Connecticut Union Carbide Corp., Electronics Division, 5 New Street, White Plains, New York 10601 T h e Virtis Co., Inc., Gardiner, New York Yeomans Brothers Co., 1999 North Ruby Street, Melrose Park, Illinois

The Occurrence, Chemistry, and .Toxicology of the Microbia I Peptide-Lactonesl

A. TAYLOR Atlantic Regional Laboratory, National Research Council of Canada, Halifax, Nova Scotia

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

189 190 192 111. Chemistry of Peptide A. Isolation of Peptide-Lactones from Cultures ............ 192 202 B. Determination of Structure of P 224 C. Synthesis of Peptide-Lactones ... 239 IV. Toxicology of Peptide-Lactones ..................... 239 A. Association Phenomena of Pept B. Antibacterial Properties of Peptide-Lactones ............ 253 C. Toxicity of Peptide-Lactones to Other Micro258 258 D. Antitumor Activity of Peptide-Lactones .............. E. Toxicity of Peptide-Lactones to Animals .................. 259 ................................. 263 263

I. Introduction

11. Production o

I.

Introduction

Macrocyclic compounds are among the most common known metabolic products of microorganisms, perhaps because of their growthinhibiting properties. In general they are heterocyclic compounds, that range from the carbocyclic macrolides, on the one hand, to the cyclic polypeptides, on the other. Between these two groups are a large number of metabolites that combine the structural features of both, that is, they are cyclic polypeptides that are also lactones. These compounds are the subject of this review. The term “depsipeptide” coined by Shemyakin (1960) can be applied to the group, but it also includes macrocyclic lactones such as antimycin (Yonehara and Takeuchi, 1958) and esters like bottromycin (Waisvisz and Van der Hoeven, 1958; Nakamura et al., 1965) which are omitted from this report. The microorganisms producing the compounds described here are common and are ubiquitously distributed on this planet (Table I, Brazhnikova et al., 1956; Jung-Sheng Tsai et al., 1958). The metabolites have therefore been isolated by many workers and their ’Issued as NRCC No. 11102.

189

190

A. TAYLOR

chemical complexity has made comparisons difficult. The result has been an extraordinary proliferation of names for the same compound, for example, actinomycin D has at least 13 names in the literature and ostregrycin B has about 12. An object of this review is, therefore, to attempt to coalesce this information in an exhaustive manner in the hope that the lists of organisms, compounds, and their physical properties will be helpful to other workers. It is highly probable that there are many important omissions, especially as it has become the habit to refrain from mentioning new compounds in abstracts. I hope, therefore, that colleagues will not deny themselves the pleasure of pointing out these omissions. It has also become the habit to publish the same work several times, and this is often helpful when multiple publication is in different languages. For the purposes of this review I have tried to quote papers giving experimental details and have, in some cases, collected like publications in a group. Various aspects of the subject matter have been reviewed previously. The chemistry of depsipeptides has been described by Shemyakin (1960, 1965), Kupryszewski (1962), Schroder and Lubke (1963), Losse and Bachmann (1964), Russell (1966), and Shemyakin and Ovchinnikov (1967). The mode of action of the ostreogrycins was briefly described by Vazquez (1964,1967), and the actinomycins have been the subject of many reviews, e.g., Brockmann (1960) and Katz (1967). The mechanism of the binding of actinomycin to deoxyribonucleic acid (DNA) was reviewed by Reich (1966), and excellent accounts are available of the history, production, and chemistry of the ergot alkaloids (Stoll, 1952; Vining and Taber, 1963).The mass spectroscopy of depsipeptides has been surveyed by Lederer and Das (1967).

II.

Production of Peptide-Lactones

The basic biologically active compounds present in ergot were the first depsipeptides to be studied in detail. A crystalline compound was isolated by Tanret (1875)and 40 years later Stoll(1918,1945) was successful in the isolation of the first pure peptide-lactone- ergotamine [(I),R = lysergyl, R’ = Me, H” = CHzCaH5].The properties of the producing Cluviceps sp. illustrate a number of general points. They have been isolated from grasslands throughout the world, only certain heterokaryotic strains produce the alkaloids (Spalla et al., 1969),and those that do synthesize the metabolites, produce a complex mixture of compounds differing only in the nature of the amino acid residues. Few figures are available for the percentage of isolates of a

MICROBIAL PEPTIDE-LACTONES

191

species that produce peptide-lactones. In an obviously large screening program Tonolo (Arcamone et al., 1961) states that "few" isolates produced ergot alkaloids. Waksman et al. (1946) reported that about three isolates out of about 10,000Actinomyces produced actinomycins (II), and Brockmann and Grone (1954) found that 21 isolates from a collection of 2140 produced pigments having similar chemical and biological properties to the actinomycins. Katagiri has stated (Katagiri and Sugiura, 1961) that 2% of the Streptomyces species he examined produced quinoxaline antibiotics. On the other hand, w e (Dingley et al., 1962) obtained sporidesmolides (111) from all isolates of Pithomyces chartarum that produced conidia in laboratory culture. Previous work by Perrin (1959) strongly suggests that the same is true for organisms growing in the field (Russell et al., 1962). Thus, there are many gaps in our knowledge of the ecology of these microorganisms, especially with respect to their distribution according to soil type, season, and weather conditions. Organisms that produce peptidelactones in culture are listed in Table 1, In most cases the isolates listed in the table were obtained from soil samples; where other sources are reported by the authors they are given. In all cases a considerable proportion of the peptide-lactones produced were found in the tissues. This varies from about 25% in the case of pyridomycin [(IV), Maeda, 19571 to 100%in the case of the sporidesmolides (111);most of the examples given in Table I lie at the upper end of this range. T h e majority of the organisms given in Table I produce their characteristic peptide-lactones in surface and submerged culture. The Pithomyces sp. are exceptions, for they do not sporulate in the shake flask culture conditions used (Dingley et al., 1962). In general, the antibiotics have been produced on complex media, at 25°C. by fungi and at 30°C. by Actinomyces. An interesting point is the great increase in yield of valinomycin obtained by growing Streptomyces fulvissimus at 37" instead of 27°C. (Brockmann and Schmidt-Kastner, 1955; Brockmann and Geeren, 1957). Sporidesmolides (Butler et at., 1962) and actinomycins (Brockmann and Pfennig, 1953; Goss and Katz, 1960) are biosynthesized on chemically defined media. There is some evidence that the depsipeptides produced by Fusaria sp. are degraded when added to cultures of Fusaria other than the producing isolate (Lacey, 1950). Katz and Pienta (1957) have shown that an Achromobacter sp. isolated from a barnyard soil degrades actinomycins. It was found that young growing cultures did not degrade actinomycins while ca. 170 pg./ml./hour were degraded by cells 32 hours old. More recently Perlman and his colleagues (1966) have shown that actinomycins (11) are converted into actinomycin

192

A. TAYLOR

monolactone and then into actinomycinic acid by an Actinoplanes sp. (IMRU 824). When this species was incubated with echinomycin (V), etaniycin (VI), or vernamycin (VII) the culture filtrates showed no biological activity. This work has been extended and it has been shown (Perlrnan and Capek, 1968; Perlnian and Hou, 1969) that the enzyme that hydrolyzes the 16-membered lactone ring of actinomycins is inducible and thus differcnt from the enzyme(s) which hydrolyzes depsipeptides of greater ring size, e.g., etamycin. The use of these enzymes is, therefore, of great diagnostic value. Ill.

A.

Chemistry of Peptide-Lactones

ISOLATION O F

PEPTIDE-LACTONES FROM CULTURES

The isolation of peptide-lactones from cultures free of other chemical species is usually easy, though the ergot alkaloids are notoriously sensitive to heat, light, and oxygen. A major difficulty in the field, however, is the isolation of a single metabolite, free of its closely related companions. Apart from the difficulties of separation it is hard to define criteria of purity. For example, sporidesmolide I [(111), R = Me, R’ = R” = CHMe2)ldoes not depress the melting point of its D-do-isoleucine analog [(III), R = Me, R’ = CHMeEt, R” = CHMez)] and their infrared spectra are identical. The best criteria available at the present time are: quantitative amino acid analysis of an acid hydrolyzate and high resolution mass spectroscopy of the peptidelactone. Neither method is foolproof: the former because of the frequent occurrence of unknown amino acids (Table 11)and the instability of some of these and of known amino acids in the hydrolysis reaction mixture. In the latter case some isomeric depsipeptides have identical mass spectra (Bertaud et al., 1965) and it is often difficult to decide whether homologous compounds of lower molecular weight are present. The ergot alkaloids were separated by fractional crystallization. In the ergotoxine group Stoll and Hofmann’s (1943) use of the di-(ptoluy1)-L-tartaric acid salts for the separation of ergocristine [(I), R = lysergyl, R’ = CHMez, R” = CHrCsH5], ergocorriirie [(I), R = lysergyl, R’ = R” = CHMe21, and ergokryptine [(I),R = lysergyl, R’ = CHMe2, R” = CH2CHMer],is a magnificent example of the classic approach.‘ More recently, Russell (1962) achieved the separation of sporidesmolide I and sporidesmolide 111by fractional crystallization using amino acid analysis of hydrolyzates of fractions as a criterion of purity. Such ‘Numbers beneath struchires are correlated to the material as it appears in Table IV.

193

MICROBIAL PEPTIDE-LACTONES

ORGANISMSKNOWN

Organism and place of isolation

Actinomyces daghes tanicus flavescens kurssanovii

TABLE I PRODUCE PEPTIDE-LACTONES

TO

Antibiotic produced and yield (gg./ml.)

6613 ? Etamycin

Brazhnikov et al. (1959)

Actinomycin-Xz 1-4725

Actinomycins

Sokolova et al. (1965) Gauze et al. (1964), Brazhnikova et al. (1965) Preobrazhenskaya et al. (1958) Belova and Stolpnik (1966) SevEik et al. (1956)

Actinomycins Beauvericin

Dalgliesh et d . (1950) Hamill et al. (1969)

Ergot alkaloids

Arcamone et al. (1961)

Enniatin B

Plattner and Nager (194%); Tirunarayanan and Sirsi (1957a); Farmer (1947) Farmer (1947) Farmer (1947); Guhrillot-Vinet et al. (1950) Plattner and Nager (1 9 4 8 ~ ) Plattner and Nager (1947, 1948c) Plattner and Nager (1 948c) Plattner and Nager (1 9 4 8 ~ ); Farmer (1947); Cook et al. (1948) Plattner and Nager (1 9 4 8 ~ )

flavochzomogenes

Echinomycins

Actinomyces sp.

Neotelomycin

Actinomyces (BU 306) (Czechoslovakia) Actinomyces (X-45) Beauveria bassigna (NRRL 3352) Claviceps paspali Stevens and Hall (Puspulum,Rome, Italy) Fusarium avenaceum

fructigenum lateritium

References

Fmctigenin (33) Enniatins A, B (40)

Enniatin B (470) oxysporum Schlecht var. aurantiacum (Lk), Wr oxysporum Schlecht Enniatin A oxysporum Schlecht

Enniatins A, B (900)

sambucinum Fuck.

Lateritiin I Sambucinin

scirpi

?Enniatins A and B (1000)

(continued)

194

A. TAYLOR

TABLE I (Continued)

Organism and place of isolation

Antibiotic produced and yield (pg./ml.)

References

Gibberella baccata Isaria cretacea cretacea strain B Isaria sp. Micromonospora sp. (608)

Sambucinin Ennidtin B Enniatins A, B Isariin (45)

Tirunarayan and Sirsi (1961) Gaumann et al. (1960) Vining and Taber (1962)

PCsNHoNdh Isarolides Actinomycins

Nocardd asteroides

Peptideolipin-NA

Taber and Vining (1963) Briggs et al. (1966) Fisher etal. (1951) Guinand et al. (1958); Guinand and Michel (1963)

Fusarium s p .

(ATCC 9969)

Oospora destructor Pi thomuces chartarum ( I M I 74473) (Rye grass, Hamilton, New Zealand) cynodontis maydicus (IMI 98084, 46232) sacchari (IMI 102686, 120724,120725 Serratia marceScens (9-3-€3) Strep tomyces antihioticus aureus (S-2-210) candidus canus (ATCC 12646 and 7) (Florida) chrysomueus (Gottingen) echinatvs (ETH 8331) (Angola) fimicarius (007) (Taiwan) fradiae (Horsham, U.K.)

Destruxins A, B Sporidesrnolides I, 11,111 (100) Pithomycolide (0.05)

Tamura et ol. (1963) Russell (1962); Rriggs et 01. (1964)

Angolide (90) Sporidesmolide IV (77 2 30) Angolide (20-240)

Ellis (1965) Bishop et al. (1965)

Serratamolide (12.5) Actinomycins A (50) Triostins (25) LL-A0341A (lo), LL-A0341B (100) Telomycin (200-300) Actinomycins C (19) Echinomycin (12) Actinomycins Actinornycins Z (96)

Riches et al. (1967) Wasserman et al. (1962) Waksman and Woodruff, (1940,1941) Katagiri (1959); Kuroya et al. (1961) Whaley et al. (1966) Hooper et al. (1962) Brockmann and Pfennig (1953) Corbaz et al. (1957) PB-WGn Liu and Te-Ch'un Ch'iu (1960) Bossi et al. (1958)

195

MICROBIAL PEPTIDE-LACTONES

TABLE I (Continued)

Organism and place of isolation

Antibiotic produced and yield (FgJmlJ

fuloissimus

Valinomycin (29)

graminofaciens (Texas) griseus (NRRL 2426) griseus (P-D 04799,04955) jamnicensis bidensis (ATCC 11415) oliuaceus (ATCC 12019) ostreogriseus (Jordan Valley, Israel) p yridomyceticus

tsusimaensis u m brosus

Brockmann and Schmidt-Kastner (1955) Streptogramin Charney et al. (1953) Etamycin (100) Heinemann et al. (1954) Griseoviridin, etamycin (170) Bartz et al. (1954,1956) Monamycins Hassall et al. (1969) Vernamycins Donovick et al. (1955) PA-114 Sobin et al. (1957) Ostreogrycins Whitfield et al. (1958) Pyridom ycin Valinom ycin Actinomycins U

Streptomyces s p . (Illinois isolate 65-24) Streptomyces sp.

Levomycin

Streptomyces sp.

Actinomycins F

Staphylom ycins

Viridiogrisein S trep tomyces sp Streptomyces sp. (PRL 1642) Valinomycin (50) Streptomyces sp. Echinomycin Streptomyces sp. Oncostatin C (INA 39/59) Streptomyces sp. Amidomycin Streptomyces sp. (SV 1784) Actinomycins H (South Africa)

Streptomyces sp. (Sotenich 3 ) (Eifel) Streptomyces sp. (5901) (Blood agar, New York) Streptomyces sp. (New Mexico)

Streptomyces sp. [732 (175211

References

(0.33) Actinomycins X Valinomycin Thiostrepton

Quinomycins (5)

Okami et al. (1953) Nishimura et al. (1964) Schmidt-Kastner et al. (1960) Carter et al. (1954) DeSomer and Van Dijck (1955) Farbenfabriken-Bay er (1960) Horvath et al. (1959) MacDonald (1960) Maksimova et al. (1965) Ptociennik et al. (1961) Taber and Vining (1957) Brockmann et al. (1959a) Brockmann and Pfennig ( 1953) Brown et al. (1962) Vandeputte and Dutcher (1955);Perlman and Hou (1969) Yoshida and Katagiri (1967)

AMINO ACIDS

Amino acid GH7N02 P-Alanine D-Alanine

ISOLATED

FROM

Source

Destruxin B Griseoviridin Peptidolipiii-NA Vernamycin B,Bs

C3H7N03 D-Serine

C4HYN02 L-N-methylalanine

D(-)-2-Aminobutyric acid

CsHgN02 D( ?)-Proline

TABLE I1 PEPTIDE-L.4CTOXES NOT h-ORhIALLY FOUND IN PROTELU HYDROLYZATES"

Melting point

b I D

- 13.4"

q41.2, N HCI

PK,

Derivatives

References

Tamura et al. (1963) Guinand and Michel (1966) Bodanszky and Ondetti (1963) ? 4

Echinomycins Triostins

Destruxin B Pithomycolide Actinomycins Z E-l29B, doricin

N-2,4-dintrophenyl, m.p. Keller-Schierlein and 175", [a];*19" (ql.1, Prelog (1957) AcMe)

Tamura et al. (1963)

-6.7"

Eashvood et al.

c32.7,H20

(1960) Charles-Sigler and Cil-Av (1966)

Ostreogrycin G 205-207" 26.6" Hydroostreogrycin A ~,1.6,H20

Delpierre et a2. (1966)

5 IF;

CsHsNOs allo-~-3-Hydroxyproline trans-3-H ydroxyproline

Etamycin Telomycin

LL-A0341-B

cis-3-H ydroxyproline

Telomycin

248-250" 56.7" c,2,Hz0 - 15.3" c,l,HzO 17.4". c.0.5 N HC1 - 18" ~,0.55,Hz0 -91.5" c,0.61,HzO

Sporidesmolide I

CJLNO, ~(-)-4-Oxopiperidine- Ostreogrycin B 2-carboxylic acid CsH13N02 D-Leucine

D-alloisoleucine

Actinomycin CS Peptidolipin-NA Sporidesmolide 11 Angolide

247" 276"

9.3

Haskell et al. (1954) 0-Me, m.p. 217-219" [a]$-25.3" (c,l,HzO)

Irreverre et al. (1963); Morita et al. (1963);Sheehan and Whitney (1963) Whaley et al. (1966)

0-Me, m.p. 212-214" la]$- 110" (c,l,HpO)

Irreverre etal. (1963); Morita et al. (1963)

-28.3" c,2,6 N HCl

N-2,4-dinitrophenyl m.p. 129-130"

Russell (1962)

- 14" c,l,HzO

HClide, m.p. 175-180" [a];' 3.8" (~,2,H20)

Eastwood et al. (1960)

- 15.5" c,2.6N HCI

Etamycin Sporidesmolide I Isariin

1.7

- 15.2" HzO

2.2

9.6 Naphthalene-2-sulfonate, m.p. 185-187" [aID- 10" (c,3,90% EtOH) 3,5-Dinitrobenzoate m.p. 178"

Haskell et al. (1954); Russell (1962); Vining and Taber (1962) Brockmann et al. 1951); Russell (1965) c

(continued)

$

TABLE I1 (Continued)

w

cc

00

Amino acid

Source

D-ISOleuCine

Monamycin

L-N-mefhylvaline

Enniatin B, lateritiin-I, destruxin-B Echinomycins Actinom ycins

Melting point

[a],

A,,,

-31.7 N HCI 285"

35" c,1.44,6 iV HCI

Loge

Derivatives

References

N-2,4-dinitrophenyl Bevan e t a / . (1969) [a]65-87" N NaOH N-2,4-dinitrophenyl m.p. Cook et 01. (1949); 181" [a]?482' Brockmann et al. (1951) Tamura et al. (1963) ?

CsHnN03 erythro-~-3-Hydroxy- Telomycin leucine Neotelomycin LL-AO341-bB C;Hi,NO, L-N-methylleucine

L-N-methylisoleucine

218-222" 35" c,O.41 N HCI

Spordesmolides I, 11, IV Enniatin A

N-2,4-dinitrophenyl m.p. 173-174"

Sheehan e t al. (1962) Whaley et al. (1966)

21" c,1.5,H?O

A7-2,4-dinitrophenyl m.p. 152-153"

Russell (1962)

44.8"

N-2,4-dinitrophenyl 1n.p. 150" [a]?499" (c,0.79,CHC&)

Plattner and Nager (1948h)

o-nitrophenylsulfenyl m.p. 146" [a]iO 135" ( c .1,AcOEt)

Eashvood et al. (1960);Koenig et ~ l(1967) .

c,1.16,5 N HCI

CBHsN02 L(+)-Phenylglycine

Ostreogrycin B Doricin

239"

2

67.5" c,0.6 3' HCI 150"(s) q0.6 N HCI

4

r

$

CBHI~NPO~SZ L(+)-N-methylcystine

Triostin C

175-182" 34" 217Ys) c,1.02 N HCI 787s) c,0.78 N HCI

C~HITNOP 3,4-Dimethyl-2methylaminovaleric acid

Etamycin ?Quinomycin C Triostin C .

315-316" 41.9" (dec.) c,1.05,5N HCl

CSHIINOP ~(+)-2-Methylamino- Etamycin phenylacetic acid C II H 14N202 2-Methyl-3-hydroxy-4- Pyridomycin amino-5-(pyridyl-3')valeric acid

245-246" 118" q4.8 N HCI

(sub.)

Otsuka and Sh6ji (1965); KellerSchierlein et al. (1959)

Sheehan e t a / .(1957), 1958); Sheehan and Howell (1963); Sh6jietal. (1965)

257

Sheehan et al. (1957,1958)

2.42

177- 180" (dec.)

CI~HI~NZOZ P-M ethyltryptophan

Telomycin

250"

CizHwNz02 4-Dimethylaminophenylalanine

Ostreogrycin B Doricin

208"

Ogawara et al. (1968)

22.6" c,O.5 N HCI

289

3.70

249

4.60

"Abbreviations: c, concentration; dec., decomposition; s, synthetic; sub., sublimation.

Sheehan et al. (1968)

251, DiHClide A,, 257,262,266 mp, (198,232,190,120)

Eashvood e t a / . (1960); Jolles et al. (1965)

200

A. TAYLOR

methods did not succeed with other groups of peptide-lactones. Brockmann and Crone (1564) showed that actinomycins had different partition coefficients in diphasic systems that contained sodium naphthalene-2-sulfonate in solution in the aqueous phase, thus permitting countercurrent distribution studies of the Craig type, and also partition chromatography on papcr. In this way they were able to show that all the actinomycins (11) produced b y Streptornyces sp.

I

RHN

P

MICROBIAL PEPTIDE-LACTONES

201

were mixtures differing only in the nature of the amino acids present in the peptide-lactone moieties. The method has been used extensively, particularly analytically, and it is interesting that the crude, rapid technique of circular chromatography on paper discs was originally, and remains, the preferred procedure. The partition coefficients of the actinomycins in these systems differ significantly only between homologs, e.g., actinomycins C1, Cz, and Cs and separation of isomers have been achieved by further chromatography on standardized alumina. Similar techniques have been used for the separation of the ostreogrycin group of antibiotics based on the observation of Smith (1958) that adequate distribution of the antibiotics could be achieved in diphasic systems by using urea, acetamide, or nicotinamide and propylene glycol to increase their solubility in the aqueous phase. The system was exploited in a series of patents (Eastwood et al., 1958; Fantes and Boothroyd, 1959; Mervyn, 1962) to separate the ostreogrycin mixture into some of its components. Bodanszky and Ondetti (1963) have also studied this problem and have achieved the separation of the ostreogrycin group into the vernamycins B,-s and doricin (Bodanszky and Sheehan, 1963), by countercurrent distribution of the mixture in the system toluene-methanol-water (4 : 3 : 1) followed by 1500 transfers of the doricin containing fractions in the system toluene-chloroform-methanol-water (5 : 5 : 8 : 1). In the former solvents the partition coefficients of vernamycins Bu-s were 2.1, 0.7, 1.3, and 0.5, respectively. Such methods of separation are not always successful. Taber and Vining (1963) obtained three antibiotic zones after countercurrent distribution and silicic acid chromatography of the antibiotics from Isaria cretacea strain B but concluded that all were mixtures. Similarly, Bertaud et al. (1965) obtained only partial resolution of the sporidesmolide mixture remaining after removal of some of the sporidesmolide I, by countercurrent distribution in the system formic ac id-b en zen e. Despite the elegance, and partial success of these chemical approaches to the problem of separation of depsipeptide mixtures it has been found that biological procedures are often the methods of choice. One method involves screening a large number of isolates to find one which produces one metabolite in much greater quantity than its companions. The analyses of ergot from different sources quoted by Stoll (1952) illustrate this point; the isolation of actinomycin D by Manaker et al. (1954) is another example. However, a more common approach is to add to the culture medium an amino acid known to be present in one of the components of the mixture. Schmidt-Kastner

202

A. TAYLOR

(1956) showed that when DL-valine was added to the culture medium of Streptomyces chrysomallus the proportion of actinomycin D present in the actinomycins synthesized increased from 10 to 83%. Bertaud et al. (1963) showed a similar effect of D L - d i n e on sporidesrnolide I production by Pithomyces chartururn and it is known that L-isoleucine increases the production of quinomycin B by Streptornyces sp. 732 (Yoshida, 1961; Yoshida and Katagiri, 1962, 1967). In all of these examples enhanced production of one metabolite in the mixture is also accompanied by an increased yield of the mixed peptide-lactones. The alternative possibility, i.e., suppression of the synthesis of all components of the mixture except one is also known. Thus the effect of sarcosine on actinomycin production by Streptornyces antibioticus 3720 depressed antibiotic production by about 70% but resulted in almost exclusive production of actinomycins FS and Fg. Similarly Bertaud et al. (1963) reported that DL-isoleucine suppressed production of sporidesmolide I and this allowed the isolation of a new isoleucine containin6 sporidesmolide (Bertaud et al., 1965).The biosynthetic implications of work of this type are discussed below, but one has the impression that the practical possibilities have not always been appreciated nor exploited. In summary, the isolation of peptide-lactones still presents considerable problems, and one of the most educational results of the work done so far is to expose the inadequacy of the methods of separation currently known. OF STRUCTURE OF B. DETERMINATION

PEPTIDE-LACTONES

Assuming that the isolation of a single component has been achieved, elementary analysis, or mass spectroscopy reveals the presence of C , H, N, and 0,an infrared spectrum shows the presence ofamide and ester functions, and finally vigorous acid hydrolysis results in the discovery of amino acids and sometimes hydroxy acid residues,.a peptide-lactone may be present. These are the criteria that have been used in assembling Table 111, a list of natural products whose full structure has not been reported. In Table IV a list of peptide-lactones whose structure appears to be firmly based is given. This latter table is regarded as the core of this review since it presents the physical properties of the known peptide-lactones, a few of their chemical derivatives, and the names that have been given to these compounds. Table IV is compiled strictly in conformity with increasing molecular formula; this could not be done in Table 111 for obvious reasons, hence the list is assembled alphabetically. A number of depsipeptides in the echinomycin and triostin series have not been included as

MICROBIAL PEPTIDE-LACTONES

203

I have failed to find any of their physical properties. These compounds are given in Table V I together with some of their toxicology. The gross structure of the stendomycin group of antibiotics (XLI) is now known but separation into single components has not yet been reported. 1 . Chemical Degradation of Peptide-Lactones Having the above evidence that a peptide-lactone was under investigation, most workers have proceeded to determine the structure and proportions of the various amino acids present in acid hydrolyzates. In general it has been necessary to isolate the various amino acids, and not rely on their partition coefficients, to identify them. Althrough their stereochemistry can often be determined enzymically, this is not always possible, e.g., in the case of N-methylamino acids. Some evidence is available, however, (Charles-Sigler and Gil-Av, 1966) that gas-liquid chromatography is useful in assigning the configurations of amino acid fragments. A list of amino acids found in peptide-lactones is given in Table 11. Amino acids have only been included in Table I1 if they have been isolated in crystalline form and their structures determined. This restriction results in the omission of several amino acids for which there is good evidence for their being constituents of peptide-lactones, e.g., a,&dehydrotryptophan present in the telomycin group (Sheehan et al., 1963, 1968). The amino acids have normally been obtained after extraction of the hydrolysis reaction mixture with an organic solvent. In the extract there is often a mixture of acids. The analysis of the mixture then reveals, usually, the presence of a-hydroxy acids and/or a,P-unsaturated acids, the latter arising from the elimination of a P-hydroxy function. Such phydroxy acids have therefore been isolated by careful alkaline hydrolysis of the depsipeptide. A list of hydroxy acids that have been isolated (the same restriction as in Table 11) is given in Table V. No enzymic methods are known for the determination of the stereochemistry of hydroxy acids. Once the structure and proportion of the amino acids (and hydroxy acids) has been determined the next step is to find how they are assembled in the natural product. Smith and Timmis (1937)were able to isolate small quantities of the ketopiperazine (XFI) by pyrolysis of ergosine [(I), R = lysergyl, R’ = Me, R” = CHMe2] and S-hydroxypicolinamide has been obtained by pyrolysis of etamycin (Arnold et al., 1958)and ostreogrycin B (Eastwood et al., 1960). Little work has been done on selective fission of peptide bonds. Eastwood et al. (1960)

NATURALPRODUCTS

TABLE I11

OF UNKNOW7S STRUCTUHE WHOSE Kh'OWZr PROPERTIES SUGGESTTHE PRESESCEOF A PEPTXDE-LAC TONE^

Compound 362

Producing organism

Melting point

1415

Streptomyces sp. ATCC 13694 Streptomyces 1415

11072 RP

Streptomyces caelicus 222" Streptomyces gongeroti

2 10" (dec.) 90-95"

200-215"

Actinoidin Actinomycin EL

Nocardia actinoides Streptomyces chrysornalus

Actinomycin H b

Streptomyces SV 1784 255-257"

A4ctinoniycinU

Streptomyces umbrosus

Actinomycin Z,

Streptomyces fradiae Streptomyces sp. SV 1784

Actinoinycin Z,

StTeptomyces fradiae

Amidoinycin

S treptomyces

[a],

-

-34.5" c,l,MeOH

1760 1700

- 100" c,l,MeOH 117" c,l,HrO

256-260" -372" 251-252" c,O. 19,CHCls -712" c,0.2,AcMe -269" ~,0.26,CHCl, 192' 19.2" c,l.2,EtOH

Hydrolysis products

gly ala pro val phe leu + 1 other

gly-proleu Ileu

41.I.C.(Fg./ml.) Salmonella < 10 Klebsiella < 10 E. coZi > 200 B . subtilis 0.1 S.aureus 5 !vl ycobacteria

Reference 1

2

3 4 ?

1745 1640 1580

NRRL 2791

PRL 1642

vmdxan.-'

thr sar pro Ileu SMeval NMeIleu thr sar val NMeval ? K M e ala ? oxopro thr sar Val B. subtilis pro SXfeval + 1 other thr sar val 2-hi e-3-trxopro \i Xleala

5 6

7 1.3

8 9 10 9

1740 1660 1525

D-val

Cundidu albicans

0.6

11 12

Nocurdia asteroides

or-Arninobutyrylpeptidolipin-NA cf. XVIII

Aspartocin

300" (dec.)

Streptomyces griseus var. spiralis S . violaceus

58" HClide c,l,H,O

Fusarium avenaceum

Beauvericin C4JLN309 Card(c)inophyllin

Beauvaria bassiana

93"

Streptomyces sahachiroi M-14 from soil from Date City Japan Claviceps purpurea (Spain)

220" (dec.) 197" (dec.)

Bacillus mesentericus

238"

139"

- 101" c,I,EtOH 65.8" c,l,MeOH

Ileu L-a-aminobutyric acid 3 hydroxy acids asp glu phe S. aureus val pro gly B . subtilis arg cysteine E . coli M . phlei ~ I asp Y pro S. aureus val 6-Measp B . subtilis D-or-pipecolic E . coli acid a$-diamino-butyric M . raiiae acid S. aureus L-NMeval B. subtilis M . phlei L-NMephe

129"

1740 1670 1735 1630

100

-24"

-103"

c,l,EtOH

14

0.004 100 1.25

15.5 4.0 250 62

15

1 0.2 6.3

18

16

17

41 19

20

Candida albictins

c,0.66,MeOH

Fusarium fruc tigenum

1740 1665

26.4" c,2.l,MeOH

Avenacein

Fructigenin

13

ala D-ala D - ~ Z ~ O -

+

Strep tomyces arsitensis

Datemycin

thr val pro

220"

1740

val

21

1735 1720 1695 1639

DL-leu asp Val glu 3-hydroxy tridecanoic acid

22

L-NMeval

S . aureus B . subtilis M.phlei

0.75 0.5 5.0

18

-

Kl

(continued)

0

to

3 5,

TABLE 111 (Continued)

Compound

Producing organism

Melting point

[&ID

vmax cm.-'

Hydrolysis products

M.I.C. (pg.im1.)

1.0 0.5 100

Glumam ycin

Streptomyces ?S. momyceticus

230" (dec.)

8" c,2,EtOH

1755 1670

asp Val pro S.uureus D-pipecolic acid B. subtilis aB-diaminoE. coli butyric acid 4(?)-Proteus isodecenoic acid uulgaris

Griseococcin i'C2iHxN4012

Streptomyces griseus 448N

230-240"

227" c,l,AcMe

1735

B . subtilis

0.8 -1.6

Isarolides Lateritiin (I)

Isaria sp. Fusarrum lateritiurn

Lateritin (11)

Fusarium la teritium

125"

-92" c.l.2.EtOH

S.aureus B . subtilis M.phlei S.aureus B . suhtilis

2 1 5 1 1 5

Leucinam ycin ?12M-88-A3

Streptomyces cinnamoneus

235-237"

-82" c,0.5,0.1 N HCl

phe val L-NMeval

121-122" -95.6" c,l,EtOH

L-KMeval

M.-phlei-1650

Levomycin Streptomyces ?echinomycin (Illinois isolate C ~ H ~ J S O I O 65-24)

222-224" -290" c,2,AcMe -323" c,l,CHCl,

1750 1700 1650

LL-A0341-A & B Streptomyces C ~ ~ H T P N I Z O I I ( candidus IS)

225-230" -104" c,l.l,MeOHHLO,1: 1

1740 1655

Monamycin

Streptomyces jamaicensis

126"

(HClide)-6T c.O.9.EtOH

asp gly pro eIu val leu arg *he+ 3 others 4 unknown 1volatile acid

thr allo-thr

ser pro gly ala P-oxyleu trans-3-oxyproline

23

100 24

25 18

100 6.25 100 1 10 80 <1

S.uureus B. subtilis E . coli M . smegmatis

12 2 100 6

; i 4 r

18

~

S.aureus B. subtilis E. coli S.aureus B . subtilis

E. coli M. phlei

Reference

26

27

28

42

3

a

Matamycin ?althiomycin

S treptomyces matensis

Melanosporin

S treptomyces

Neotelomycin

Actinomyces sp.

Radicicolin

C ylindrocorpan

Sambucinin

radicicola Fusarium sambucinium

Saramycetin X 5079C %2-7758

S treptomyces saraceticus NRRL 2831

Stendomycins A, B

Streptomyces sp. probably S. endus

melanos po rus (Pesaro, Italy4

173" (dec.)

132- 134" 30" c,l.G,MeOH

86-87"

Fuck.

Thiostrepton (siomycin)

Streptomyces sp.

Unnamed

Pithomyces maydicus IMI 98084,46232

36.6" c,O.l,MeOH

270-280" (dec.)

1730 1670 1725 1650

-99" c,l,H?O - 193" c,l.4,MeOH -83" c,l,EtOH 40.2" c,l,HeO -32" c,2,8 M urea A-92.4" B-83.4" c,l,EtOH

246-256" -98.5" c,l,HOAc

ply ser arg cystine 2 others 3 unknown amino acids

+

S. uureus 5 B. subtilis 5 E . coli 50 Corididu albicans 2.5 E . coli > 100 MYCObacterium ATCC607 10

29 30

31 32 L-NMeval 1689

1755 1660 1610

1740 1680 1640

asp gly thr pro cystine ser gly ala pro val leu d o - t h r alloIleu %amino2-butyric acid 11-Melauric acid or other fatty acid D-cystine, Ileu Val, ala thr, pro lys gly asp glu val NMeval a-oxyisocaproic acid

S.aureus 1 B . subtilis 0.25 M.phlei 5.0 Paecilomyces oarioti

S. aureus B. subtilis Botrytis

12.5 15.5 1.56

cinerea Epidenno- 0.78 phyton fEoccosum ATC 10227 B. subtilis 0.03 M . tubercu- 3.0 losis B.C.G. E . coli > 50

18

33

34 35 36

43

37 ._

(continued)

I9 0

-.I

TABLE I11 (Continued) ~

~

Compound Ussamycin

Producing organism

Me1ting point

[fflo

Strep tomyces lacendulae 7KI

Val6-peptidolipin- Nocardh NA asteroides Wildfire toxin

Pseudomonas tabaci

221-223' 70" (CHCI,)

Hydrolysis products

vmaU c n c '

gly ala leu ser asp glu lys pro Val Phe D-ala pro val thr D-allo-Ileu 3 oxy acids lactic acid tab toxinine

+

~

~

hl.1.C. (pg./ml.) antitumor

~~

Reference 38

39 40

"Abbreviations: standard contractions are used for L-amino acids; YMe, N-methyl; c , concentration 0x0, keto; oxy, hydroxy; S . aureus, Staphylcoccus aureus; B., Bacillus; E . , Escherichia; M . , Mycobacterium. "Actinomycins J Actinomycins X (Helc. Chim. Acta 41, 1646; Nishibori, (1956).Actinomycins L and M have also been reported, but definitive properties have not been given. 'Infrared spectrum given, but there appears to be an error in scale. References: 1, Philippe (1962); 2, Sgarzi et a / . (1961); 3, Rhone-Poulenc (1962); 4 , Nakazawa et al. (1961); 5, Shorin etal. (1957); 6, Schmidt-Kastner (1956); 7, Brockmann et al. (1959a); 8, Schmidt-Kastner et al. (1960, 1962); 9, Bossi et ul. (1958); 10, Brockmann and Manegold (1965);11, Vining and Taber (1957); 12, Schulz (1966); 13, Guinand et al. (1966a); 14, Ceriotti (1960); 15, Shay et al. (1959); 16, Martin and Hausman (1960); 17, Kirsch et a / . (1959); 18, Cook et a / .(1949); 19, Hata and Sano (1956); 20, Kuroya and Koyama (1959);21, Abe et al. (1959); 22, Ito and Ogawa (19.59);23, Shibata et al. (1961); 24, Takeuchi et al. (1962); 25, Briggs et al. (1966); 26, Mizuno eta!. (1967); 27, Carter et al. (1954); 28, Whaley et al. (1966); 29, Semi et al. (1958); 30, Arcamone et ul. (1959); 31, Belova and Stolpnik (1966); 32, Evans and White (1966); 33, Baudet and Cherbuliez (1964); 34, Thompson and Hughes (1963);35, Bodanszky et al. (1967,1968); 36, Muramatsu and Bodanszky (1968); 37, Bishoo and Russell (1967); 38, Goncalves d e Lima et al. (1963);39, Guinand et a / . (196613);40, Woolley e t al. (1952); 41, Hamill et a / . (1969); 42, Hassall et al. (1969); 43, Vanderputte and Dutcher (1955); Perlman and Hou (1969); Anderson et al. (1970).

209

MICROBIAL PEPTIDE-LACTONES

TABLE IV PROPERTIESOF NATURALLYOCCURRINGPEPTIDE-LACTONES OF ESTABLISHEDSTRUCTURE XXVI, Griseoviridin m,p. 228-230" (from pyridine), 160" (from MeOH), [a];' -237" (c,0.5,MeOH), A,, 220.5 m y (E 44,000), v,, (tetrahydrofuran) 3425, 1739, (Nujol) 3300, 1748, 1684,1645, 1600, 1515,1412, 1374, 1317, 1276, 1188, 1105, 1044, 1029, 991, 957, 893, 845, 770, 759 em.-', unit cell dimensions: (a) 10.72, (b)9.58, (c) 11.705 A, p = 93" 33' 2 3'. Diacetate, m.p. 137-140" (decomp.), [a];' -230" (c,0.44, MeOH). [Ames et al. (1955);Fallona e t a l . (19644.1 IX, Angolide m.p. 261-262", [a]k2-83" (c,l,CHCl,), ,,v (KBr) 3330, 1750, 1670, 1575, ern.-', mle 426 (d2),382 (d2),367 (d2),297 (dl),282 (dl), 197 (d,), 182 (dl),169, 167,154(dl),141,112 (d,),86 (d,), 69 (4).[Russell (1965); Macdonald and Shannon, (1964);Kiryushkin et al. (1964).1 (XVII, R = H), Serratamolide m.p. 159-160" [a];54.8" (c,2.5,EtOH), 7.9" (c,2.3,CHC13), Ditrityl ether m.p. 186-1873, ditetrahydropyranyl ether m.p. 146-148". [Wasserman et al. (1962); Shemyakin et al. (1964); Castro et at. (1959).] IV, Pyridomycin m.p. 231-233" (decomp.), [a]k1-62" (c,l,dioxan-H20, 2: l), p& 4.17, (EtOH) 264, 270, 305 m p (E: 92, 80, 177),A, (0.1 N 9.17, A,, HCl) 228,265 (sh.) 304 m p (E: 2.446,170,197),A,, (0.01N NaOH) 333 m p (E: 2,168),,,v (KBr) 3450, 1730, 1670,1650cm.-', d 1.477 gin. unit cell dimensions of dihydrolironiide; (a) 19.23, (h) 8.02, (c) 12.20 A, p = 103.6". Dihydrobromide n1.p. 218-220" (decamp.), monoacetate m.p. 187-188" (decomp.), pyridomycin acid m.p. 187-188" (decomp.). [Maeda (1957); Ogawara et a1. (1968); Koyama et al. (1967).1 XXVII, Ostreogrycin A, Staphylomycin MI, Pristinamycin II,, Virginiamycin MI,, PA-114-A, Vernamycin Al,Antibiotic 899, Streptogramin-? m.p. 203-205", -218" (c,0.34,EtOH), A,, (95% EtOH) 228, 272 mp (log E 4.51,4.00),A, (6 N HCl) 303 mp (log E 4.20), A,, (0.2 N ethanolic NaOH) 293 mp (log E 4.34), v,, (CHCI,) 1725, 1670, 1636 (sh.), 1619 (Nujol) 3390, 3290, 1736, 1725 (sh.), 1673, 1646, 1618, 1583, 1541, 1420, 1330, 1157, 1116, 1034,973,869,746 cm.-' [Delpierre et al. (1966).] (D-prolyl derivative of XXVII), Ostreogrycin G m.p. 122-127" (decomp.), [a]? 78" (c,1.36,EtOH), vmX 3290, 2970, 1736, 1669, 1624, 1582, 1537, 1437, 1394, 1370, 1346; 1279, 1213, 1186,1110,1047,980,931,894,869,806,752 cm.-', mle 527 (2% of base peak), 509 (57% of base peak). [Kingston et al. (1966a).] XLII, Destruxin A m.p. (125") 188", [a]::-224.8" (c,2.25,MeOH),v,, 1735, 1689, 1625

2.

-

(continued)

2 10

A. TAYLOR

TABLE IV (Continued) cm.-', T (C[PH]C13)4.75 (multiplet) p.p.ni.,catalytic hydrogenation gives CzJ~48N,0,m.p. 190". [a]? -240" (c,O.&,MeOH),methyl 0acetyldihydrodestruxinate A, - 191", (c,l,MeOH). [Suzuki et al. (1966); Kodaira (1962).] XLIV, Pithomycolide (EtOH) 220,275 mp m.p. 242-244", [aID-60" (c,0.095,CHCl3),,,A ( E 13,000, 835) u,, (KBr) 3356, 3077, 1739, 1718, 1669, 1642, 1534, (CCl,) 3322, 1757, 1738, 1689, 1658 cm.-', T 2.06, (H), 2.66 (5H), 2.70 (5H), 3.72 (2H), 5.26 (3H), 7.02 (4H), 7.18 (3H), 7.81 (H), 8.53 (6H),8.89 (3H), 9.01 (3H) p.p.m., mle 552. [Briggs et al.(1964).] (I, R = lysergyl, R' = Me, R" = CHMe,), Ergosine m.p. 228" (decomp.), [a]b-179" (CHC13),hydrochloride m.p. 235" (decomp.), ergosinine m.p. 228", dihydro derivative m.p. 212". [Smith and Timmis (1937).] (XLII, R = CH,CHMe,), Destnixin B m.p. 234" (decomp.), -228" (c,O.S,MeOH), umlx 1732, 1690, 1660, 1635 cm.-' Destruxinic acid R cyclohexylammonium salt m.p. 176", [a19 -126" (c,O.S,MeOH). [Tamrira et al. (1963); Kuyama and Tamura (1965); Kodaira (1962).] (I, R = lysergyl, R' = R" = CHMe,), Ergocornine m.p. 182-184", [a]iO-188" (c,l,CHC13), - 105" (c,l,pyridinc) Isoergocornine m.p. 228", [a]: 409" (c,l,CHCl,), dihydroergocornine m.p. 18T,[a]gl-48'. [Stoll and Hofinmn (1943).] (I, R = lysergyl, H' = CHMe,, R" = CH,CHMe,), Ergokryptine ~ ~ (c,l,CHCl,), -112" (c,l,pyridine) Isom.p. 212-214", [ C Y ] -187" ergokryptine m.p. 240-242", [a]? 408" (c,l,CHC13), dihydroergokryptinc m.1). 235", [(ul$-4l0. [Stoll and Hofmann (1943).] (111, R = H, R' = R" = CHMe,), Sporidesmolide 111 m.p. 277-278", 294-295", [a]A8-79.2" (c.0.7,HOAc). [Russell (1962); Hussell et nl. (1964); Ovchinnikov et ul. (1965a, 1966b).] (I, R = lysergyl, R' = Me, R" = CH,C6H5),Ergotamine m.p. 212-214" (decomp.), [a];0-160" (CHCI,), tartrate m.p. 203" (decomp.). Ergotaminine [a12 385" (CHCl,), dihydroergotamine m.p. 239", [ ( u ] E O-64" (pyridine).[Stoll(1918, 1945).1 (X, 4,10,16-N = M e , L-3,9,15-R=PPr, D-R=PPr), Enniatiii B m.p. 175", 177-179" (synthetic), -107.9" (c,0.631,CHCI3). [Plattner and Nager (1948a); Plattiier et al. (1963); Shemyakin et al. (1963a, 1965); Losse and Raue (1968).1 (111, R- Me, R' = R" =CHMe,), Sporidesmolide I m.p. 261-263", [aID-217" (c,l.S,CHCl,), -98" (c,l.J,HOAc), u, (KBr) 3350, 2968, 2930, 2869, 1753, 1679, 1646, 1529, 1464, 1410, 1381, 1363 em.-', m/e 638 (dR).596 (da), 582 (d:J, 289 (d2),285 (&I), 100 (d,), principal spacings (A, CuK, radiation) d, 12.10,7.02, 6.08, 4.60, 4.05, 3.53,3.37, 3.04, 2.82, 2.66, 2.31, 2.18, 2.01, 5.22, 4.87,

MICROBIAL PEPTIDE-LACTONES

211

TABLE IV (Continued)

4.23, 3.97, 3.45,3.19,2.96,2.80. [Done et al. (1961);Russell (1962); Bertaud et al. (1963);Shemyakin et al. (l962,1963a).] XIX, Isariin m.p. 250", mle 637.4421, v,, (KBr) 3300, 3060, 2950, 2920, 2855, Isariic acid m.p. 1735, 1650, 1533, 1470, 1450, 1380, 1190 194-200". [Vining and Taber (1962); Wolstenholme and Vining, (1966).] (111, R = Me, R' =CHMeEt, R" = CHMe,) m.p. 228", [a]:: -228" (c,0.25,CHCI3),195" (synthetic), umax (KBr) 1755, 1675, 1520, 1460, 1390, 1380 cm.-', principal spacings (A, CuK, radiation) d, 12.25, 7.11, 6.14, 5.20, 4.87, 4.65, 4.26, 4.10, 3.49, 2.83. [Bertaud et al. (1965); Shemyakin et al. (196313, 1965).] (111, R = Me, R'=CHMe,, R" =CH,CHMe,), Sporidesmolide IV n1.p. 232-233", [alD-215" (synthetic)-212" (c,2,CHC13),umax(KBr) 1745, 1685, 1640 em.-' Alkaline hydrolysis gives L-a-oxyisocaproylL-valyl-L-N-methylleucine m.p. 167-169", [aID- 100". [Bishop and Russell (1964, 1967); Ovchinnikov et al. (1966); Kiryushkin et al. 1965).] (I, R = lysergyl, R' = CHMe,, R" = CH,C6H,), Ergocristine m.p. 165-170" (decornp.), [a]? -183" (c,l,CHCl,), -93" (c,l,pyridine). Isoergocristine m.p. 226", [a]:" 366" (c,l,CHC13), dihydroergocristine m.p. 180", [a]? -56" (pyridine). [Stoll and Burckhardt (1937).1 (X, 4,10,16-N = Me, L-3,9,15-R = EtMeCH, D-R = CHMe,), Enniatin A, Baccatin A (mixture of enniatins A and B) m.p. 122", la], -91.9" (c,0.926,CHCI3),-94.5" (c,0.74,CHCI3,synthetic), umax 1740, 1660, 1165 em.-', principal spacings (A, CuK, radiation), 12.60, 11.8, 11.2, 10.7, 9.30, 9.11, 8.50, 7.13, 6.23, 5.27, 4.98, 4.86, sublimes unchanged at 127-128"/10-4 mm. [Plattner and and Nager (194813); Plattner et al. (1948);Quitt et al. (1963);Shemyakin et al. (1963b, 1965);Hall (1960).] [VII,R=CH~C~H5,Rf=Et,X+Y=-(CH~)~-l m.p. 163-165", -61.2" (c,l,EtOH), Amax (EtOH) 304 m p (e 75601, A,, (0.1N NaOH) 332 mp (e 8350),umax 1755 em.-' [Ondetti and Thomas (1965).] (VII, R = CH2C6H,,R' = Et, X + Y = -CHzCH2COCH2- ), Staphylomycin S, Antibiotic 899, Virginiamycin S m.p. 240-242", [aID-28" (c,l,EtOH), A, 207, 304 mp (E) 590, 86), pK, 9.0, umax (CHCl,) 1735, 1725 em.-' [Vanderhaeghe et al. (1957);Kiryushkin e t a l . (19671.1 (VII, R = p-Me,NC,H,CH,, R' = Et, X = CH,CO,H, Y = H), Doricin m.p. 170-190", [a]? -92" (c,l,MeOH). [Bodanszky and Sheehan (1963).1 VI, Etamycin, Viridogrisein, 6613 -

(continued)

212

A. TAYLOR

TABLE IV (Continued) 1n.p. 168-170" (decomp.), [a@ 62" (c,5,CHCI,), 31" (c,S,EtOH), A,, (EtOH) 304.5 m p (log e 3.91), v,, 3290, 1755, 1640, 1520 cm.-', diacetate A,, 270 m p (log E 3.55), etamycinic acid 1n.p. 140-143" (decornp.), [a]?-7.9" (c,S,EtOH). [Sheehan et al. (1957); Arnold et al. (1958); Brazhnikova et al. (1959).] (VII, R = p-MeZNC,H,CHz, R' Et, X + Y =-CCH2COCHZCH, -), <:4a€I,4N,O,o, E129B, PA-114l3, Ostreogrycin B, Mikamycin B,, Vernamycin B,, Pristinamycin I,, 1253.5 RP, (1-4725. Synergistin, Pyostacin, Streptogramin, Virgimycin, are names given to mixtures of closely related antibiotics of this group). m.p. 266-268" (when tenaciously held solvent of crystallization has been removed), [a12 -66.8" (c,0.5,MeOH), -57.5" (c,0.25,EtOH), A,,, (EtOH) 259, 305, 365 m p (c 18,280, 8580, 985), A,,, (0.1 N ethanolic NaOH) 246, 334 m p ( E 21,600, 9150), v,, (Nujol) 3390, 3330, 3280, 1737, 1718, 1677, 1649, 1624, 1523, 1302, 1249, 1195, 1167, 1132, 1060, 1003, 904, 809, 762, 696 cm.-', pristinomycinic acid I, -44", -42" (synthetic, c,l,MeOH). [Eastwood et nl. (1960); Watanabe (1961); Gauze et al. (1964); Hobbs and Celmer (1960); Preiid'homme et ul. (1965, 1968); Pevzner et ul. (1964); Celmer and Sobin (1955).] C50HBON,2012Sz, (V, R=quirioxalyl-2, R'=R"=Me,CH,X-,Y=-SCH,-,Y+X= - SCH2-), Echinomycin, Quinoniycin A, Actinoleakin, X-948, X-53, 6270,.?Levomycin I T I . ~ . 217-218"; 236-238" (decomp.), [a]? -310" (c,0.86,CHC13), A,, (EtOH) 245, 320, 330 Inp (log ~4.51,3.75,3.75), echiriornycinic acid, amorphous, m.p. 158-160", [a], -178". [Cnrbaz et al. (1957); Berger et n l . (1957); Brazhnikova et al. (1959); Otsuka and Sh6ji (1967); Keller-Schierlein et al. (1959).1 (V, R = quinoxalyl-2, R' = R" = Me,CH, X = Y = H, X + Y = CSOIIB2Nlz0,2S2, -CI12SSCH2-)), l'riostin A in.]). 245-248" (decomp.), [a]h4- 157" (c,O.97,CHCl3),A,, (MeOH) 243,320 mp (log E 4.81,4.07). [Otsuka and Sh6ji (1967).] C,oH8eN70,,, XVIII, Peptidolipine-NA m.p. 232-233", [ a ] ,42" (CHCI,), vmax 3250, 3080, 1740, 1640, 1530, 1250, 1160 cm.-', m/e 963.6599, peptidolytic acid m.p. 107-110", [aID-9.8" (c,2.34,CHC13). [Barber et ul. (1965); Guinand et ~ l . (1964);Guinand and Michel(1966).] (V, R = quinoxalyl-2, R' = R" = CHMeCHMe,, X = Y = H, X + Y = C,,H,,N,,O,,S,, -%H,SSCH, - ), Triostin C [nlF -143.9" (c,l.121,CHC13), ,,A (MeOH) 243, 320 mp (log c 4.87,4.13). [Otsuka and Sh6ji (1965).1 (XI, L-6,18,30-H = Me, L-9,21,33-R = Me,CH, U-R = Me,CH), C,,H,N,O,,, Valinomycin, N-329B m.p. 190°, 187" (synthetic), [a];' 31" (c,l.6,C6H,), 32.8" (c,1.25,C6H6),

MICROBIAL PEPTIDE-LACTONES

2 13

TABLE IV (Continued) 17.4" (c,0.97,MeOH), u,,, 3235, 1660, 1530 em-', unit cell dimensions (a) 10.14, (b) 14.47, (c) 22.2 A, ty = 105.0",f3 = 86.9", y = 90.4", (d)= 1.15.6 [(C[2H]3)2S0, 200 M Hz], 0.9 (CH,, Val. 2-oxyisolvaleryl), 1.25 (CHI, lactyl), 2.2 (P-CH, Val, 2-oxyisovaleryl), 4.25 [a-CH, (D)L-val], 4.45 [a-C€-I, (L)D-Val, J N H - - C H = 8 Hz], 4.80 (d, a-CH, 2-oxyisovaleryl), 5.10 (q, a-CH, lactyl), 7.86 (d, NU, (D)L-Val), 8.34 [d, NH, (L)D-Val] p.p.m. Cell dimensions of potassium aurichloride complex: (a) 11.04, (b) 16.06, ( c ) 13.61 A a = 124.0", P = 97.4". [Brockmann and Schmidt-Kastner (1955); Brockmann and Geeren (1957); Brown et al. (1962); Mathieson (1959); Shemyakin et al. (1966b); Ohnishi and Urry (1969); Pinkerton et al. (1969).] (11, R = R' = R" = R"' = CHMe,, X = X' = Me, Y = Y' = H), Actinomycin F,,, Actinomycin 11, Actinomycin A,, m.p. 215-216", [a];' -157" (c,0.24,CHC13),A,, 237, 429, 447 mp (log E 4.55, 4.33, 4.37), u,,, 3390, 2960, 2930, 2351, 2330, 1750, 1660, 1587, 1517, 1489, 1401, 1319, 1230, 1200, 1131, 1079 cm.? [Johnson and Mauger (1959).1 VIII, Telomycin [a]:* -133" (c,l,MeOH-H,O,Q:l), A,, (EtOH-H20, 2: 1)221, 277, 290, 339 mp ( E 63,732, 13,746, 11,890, 22,058), ,,v 1745, 1640, 1525 em.-', p& 2.1, 8.7, telomycinic acid [a];*-32" (c,l,MeOHH,0,2: l), pK, 3.6. [Misiek et al. (1957); Sheehan et al. (1968).] (11, R = R' = R" = R"' = CHMe,, X' + Y' (X --f Y) =- CH,COCH, or-CH,CH,CO--, X(X')= Me, Y(Y')= H),ActinomycinX,, m.p. 246-247", [a]D -418" (c,O.l,AcMe), -340" (c,0.05,AcMe), -200" (c,O.O25,AcMe), A,, (MeOH) 443 m p ( E 24,900). Reduction with Al(OpPr), gives actinomycin &,, m.p. 251-252" [a]? -468" (MeOH) 443 m p ( E 25,300). [Brockmann and (c,O.e,AcMe), A,, Manegold (1962).] (11, R = R = R ' = R " = CHMe,, X' -+ Y' (X + Y) = - (CH,), -, X(X') = Me, Y(Y') = H), Actinomycin F,, Actinomycin 111, Actinomycin A,,,, ?Actinomycin XOy m.p. 237-238", [a];' -205" (c,0.22,CHC13), A,, (CHC1, - EtOH, 1: 10) 240,430,450 m p (log ~4.54,4.38,4.41), u,,, 3470,2965,1758, 1664, 1642, 1588, 1515, 1489, 1410, 1380, 1362, 1322, 1303, 1272, 1230, 1202, 1131, 1100, 1080, 953, 781 cm.-' [Johnson and Mauger (1959).] (11, R = R ' = R " = R " ' = C H M e , , X + Y (X'+Y')=-(CH,),--,X'+ Y' (X Y) = -CHzCHzCO-), Actinomycin &, Actinomycin V, Actinomycin A,, Actinomycin By m.p. 249.5-2503, -359" (c,O.e,MeOH), -288" (c,O.e,AcMe), A,, (MeOH) 443 mp ( E 24,700). Reduction with hydrogen and platinum in acetic acid gives actinomycin D and actinomycin X,,p [Roussos and Vining (1956); Brockmann and Manegold (1960).] --f

(continued)

2 14

A. TAYLOR

TABLE IV (Continued)

(11, R = R' R" = R"' = CHMeZ, X Y X' + Y' = -(CHZ)3-), Actinomycin D, Actinomycin lV, Actinomycin I, Actinomycin C, , Actinomycin X,, Actinomycin D,,, Actinomycin A,,, Actinomycin B,", Actinomycin J (probably a mixbire), Dactinomycin, Oncostatin C, Oncostatin K, 5-67 (possibly a mixture) m.p. 236" (R.&V.), 242" (B.&C.), [a]F -268" (c,0.25, 95% EtOH), h,,, (05%EtOH) 242,444 nip,,,A (cyclohexilne)446 ilrp. [Kuussos and Vining (1956); Brockmann and Crone (1954); Brockmann and Lackner (1968);Victor et (11. (l%M).] (11, R = R' = R" = R"' = CHMe,, X + Y (X' + Y') = -(CH2)$-, C,,H,,N,,O,,, X + Y' (X + Y) = - CI12CII,CHOH-)), Actinomyein &p, Actinomycin A,, Actinomyein B, m.p. 245-24T, 2 3 7 3 , [ C X ] -260" ~ ~ (c,O.e,AcMe), A,, 242, 441 mp, monoacetate m.p. 240-242", [a]: -283" (c,O.e,AcMe),Amax 443 rnp (c 24,300), rnonopalniitate m.p. 194-196", [a]iO -219", A,,, 442 I I I ~ (c 25,000). [Brockmann and Pampus (1955); Roussos arid Vining (1956).] (11, epimer of actinomyciri A,), Actinomycin &p C,2H,,Nl,0,,, m.p. 245-246", A,,, (MeOH) 443 inp (c 23,400). [Brockmann and Manegold (1962).] C63H88N12016, (11, R = R"' = R"(H') = CHMe,, R'(R") =CHMeEt, X+ Y = X ' + Y' = - (CH,), - ), Actinomycin C,, Actinomycin VI m.p. 238", [a],-325" (MeOH), A,,, 446 m p (e 30,000). Hydrolysis with barium hyrli-oxide gives depeptidoactinomycin m.p. 2.30" (decomp.), diacetate m.p. 1W (decomp.), NMe derivative m.p. 217-224", [a]:' -180" (c,0.08,AcMc). [Brockmann and Crone (1954); Brockmann and Lackner (1968);Brockmam et al. (1959b).] CB4HwN120,,, (11, R' = H" = CHMeEt, R = R"' = CHMe,, X + Y = X' + Y' = - (CH,), -), Actinomycin C3, Actinomycin VII, Aurantin A,, Cactinomycin m.p. 232-235", [aID -321" (MeOH),,,A 446 m p ( E 28,300), N-CH,CH,OH derivative m.p. 216-224", [a& m P -1 10" (c,0.3, MeOH). Hydrolysis with Ba(OH)2gives depcptidoactinomycin 1n.p. 230" (decomp.). [Brockmann and Grone (1954); Brockmann and Lackner (1968).]

C,,HOBN,,O1,,

were able to obtain peptide fragments by treating ostreogrycin B with acetic and hydrochloric acids at 37°C. for 14 days. They were thus able to determine the amino acid sequence by classic peptide chemistry methods. Partial acid hydrolysis was successfully used by Otuska and Sh6ji (1965) who obtained quinoxalylserine, quinoxalylserylalanine, and an ester, which was not characterized but gave yuinoxaline-2-carboxylicacid, serine, alanine, and Me2CH,CHMe,CH(NHMe)C02H, on hydrolysis of triostin C. The isolation of

MICROBIAL PEPTIDE-LACTONES

215

peptides leading to the sequence serylalanyl-N-methylalany1-Nmethylvaline was reported from desthioechinomycin by KellerSchierlein et al. (1959). Such methods were not applicable to etamycin [(VI), Sheehan et al., 1957)] where Edmann,degradation was applied to etamycinic acid to determine the amino acid sequence, or to telomycin [(VIII), Sheehan et al., 1968)l where peptide fragments leading to the sequence aspartylserylthreonylallothreonylalanine were obtained by partial alkaline hydrolysis; an observation possibly prompted by the report of the production of leucylhydroxyproline by hydrolysis of etamycin (VI) with baryta (Arnold et al., 1958). The most important use of base treatment in peptidelactone chemistry has, however, been its use to selectively cleave the lactone group(s). Numerous examples of this reaction will b e found in Tables I11 and IV. The product is a hydroxy acid whose equivalent weight may be determined by titration. The C-terminal amino acid can be found by a Dakin-West or Edmann procedure and the remainder of the peptide sequence is often deducible from its mass spectrum (see below). It is sometimes preferable to use hydrazinolysis (Stoll et ul., 1950) rather than hydrolysis as the resulting hydrazides are commonly easier to crystallize. The method was also used by Russell (1965) who showed that hydrazinolysis of angolide (IX) gave the hydrazides of a-hydroxyisovaleryl-D-doisoleucine and a-hydroxyisovaleryl-L-leucinehaving m.p. 222"-223°C. and 188"-189"C., respectively, and easily separable by chromatography. The selective reactivity of the lactone group(s) has been exploited in two other ways. Thus Sheehan et at. (1958)and Eastwood et at. (1960) showed that a threonine residue in etamycin (VI) and in ostreogrycin B (VII) was not susceptible to attack by chromic anhydride in acetic acid in the presence of pyridine. As this threonine residue was oxidized by this reagent in etamycinic acid, for example, it was deduced that this hydroxyl was involved in lactone formation. Stoll et al. (1951) showed that the cyclol group in the depsipeptide fragment of the ergot alkaloids was selectively cleaved by reduction of the alkaloids with lithium aluminium hydride. However lithium borohydride is a more useful reagent since it does not reduce amide links but does cleave ester groups in peptide-lactones. Thus KellerSchierlein et al. (1959) demonstrated that the hydroxyl groups of the D-serine and the carbonyl groups of the N-methylvaline residues (production of N-methylvalinol on hydrolysis) were involved in the lactones of echinomycin (V). The experimental procedures described above usually serve to define the gross structure of peptide-lactones. However, when two or

TABLE V HYDROXY ACIDS FOUND N HYDROLYZATES OF PEPTIDE-LACTONES"

Acid C,HsOz L-Lactic acid

C~HIOO, 2-Oxyvaleric acid

Source

Melting point

[a]*

References

Valinomycin Wildfire toxin

Phenylphenacyl ester, m.p. 151", Brockniann and Geeren (1957) [ ~ ] ; ~ - 5 . 8(~,4.1,HzO) ~

Dihydrodestruxin B

Acetate, B.p. 103"/0.8mm. [a];5-33" (c,S,MeOH) Cyclohexylamine salt, m.p.

L-2-Oxyisovaleric acid Sporidesmolides I, 111, IV, angolide, pithomycolide D-2-Oxyisovderic acid Enniatins A, B Val inomyc in Beauvericin

67-68'

20"

c,4,CHC13 67-69'

Sporidesmolide I V 80-81"

2,4-Dinitrophenylhydrazone, m.p. 167-168"

-12.3" c,l,HpO h'a salt -21.9"

Tamurd et al. (1963) Russell (1962)

?

*4

140-142", [u]D-~' (~,5,H20)

-1.92" Phenylphenacyl ester. m.p. c,12.3,H~O 108-lOY, 112" (B. & G . ) &

CBHLOOB 3-Methyl-2-oxocaproic Pyridomycin acid C~~H~UO, ~-2-Oxy-4-methyl valeric acid

Derivatives

1

Fischer and Scheibler (1908); Plattner and Nager (1948a,b); Brockniann and Geeren (1957)

Ogawara et al. (1968)

Acetate, b.p. 150-152°/0.5 mm. Bishop and Russell (1967) [a];* -40.5" (~,3.6,CsHfi) t-butyl ester, h.p. 105-107"/ 15 mm., [(~1~~-39.5" (c76.6,CsH6)

r 0

9

C9HioO3 ~-2-Oxy-2-phenylpropionic acid C~Hi602 5-Methyl-6-isopropyl2-oxopyran

Pithomycolide

114-1 16"

Briggs et al. (1964)

Hydroostreogrycin A

(b.p.) 96" 120-130"/ c,2,CHC13 15mm.

Delpierre et ul. (1966)

CioH~o03 3-Oxycapric acid Serratamolide (3-oxydecanoic acid)

CiiHi3NO3 2-Methyl-3-oxo-5(pyridyl-3)-valeric acid CI~HJ-IO~ 3-Oxylauric acid

CzoHmO3 D(-)-3-Oxyeicosonic acid

47"

- 19"

Cinchonidine salt m.p. 119Cartwright (1957) ~,2.1,CHC13 120", [a]$-82.7" (c,2.5, CHCl,) methyl ester, m.p. 13"

Griseoviridin

189-191"

Ames and Bowman (1956)

Pyridom ycin

160-161"

Ogawara et al. (1968)

Isariin

63"

-15.2" c,3.4,CHC13

Vining and Taber (1962)

Peptidolipin-NA

58"

-8" c,3.52,CHC13

Cuinand and Michel(l966)

"Abbreviations used are found in previous tables. bInitials used as in Beilstein to refer to different values for the same compound by different workers.

218

A. TAYLOH

more ester functions are present in the macrocyclic ring(s) ‘*II 1 accurate molecular weight is also required to determine the ring size. The determination of molecular weight of peptide-lactones by measuring their colligative properties in solution has led to misleadingly low results, e.g., actinomycins [(11),Waksnian and Tishler, 1942)1, enniatins [(X), Plattner and Nager, l948a,b)] and valinomycin [(XI), Brockmann and Geeren, 1957)l. However, Brockmann et al. (1963) obtained a value 1149 SO for valinomycin by equilibrium ultracentrifugation, and though a lower value was assumed by Mathieson (1959) the former seems to be correct since the synthetic product is apparently identical to the metabolite (Shemyakin et al., 1965). A thermoelectric method of determining molecular weights in trifluoroacetic acid has been developed by Feigiria (Kiryushkin et al,, 1964) and successfully used for the determination of the molecular weight of angolide (IX). The best method of determining molecular weight is by mass spectroscopy.

*

2. Mass Spectroscopy of Peptide-Lactones The first mass spectroscopy of a peptide-lactone was carried out by Reid who denionstrated that the molecular weight of sporidesmolide I was 638 (Russell, 1962). This enabled a structure to be advanced with confidence arid it is significant that sporidesmolide I was the first peptide-lactone whose proposed structure was substantiated synthetically (Shemyakin et al., 1963a). Mass spectroscopy of sporidesmolides I and I11 and the analogous depsipeptide (111, R = Me, R’ = CHMeEt, R” = CHMe2)was studied by Shannon and his co-workers (Macdonald et al., 1964; Russell et al., 1964). The most abundant ions in these mass spectra were due to fission of the side chains b y a mechanism such as XI11 + XIV. They pointed out that side chain fission might also proceed by mechanism XV and that both of these processes might be followed by homolytic fission of the ring. The mass spectra suggested that the ions generated by these processes proceeded to fragment sequentially at each amide and ester group as indicated in XVI, a supposition supported by the arialogous spectra of the deuteriated molecules. These results have been generally confirmed by other workers. Thus Hassall and Thomas (1966) found that the main ion reactions of a series of depsipeptides related to serratamolide (XVII) were side chain fission and homolysis of the ring. Barber et al. (1965) wcre able to determine the sequence of amino acids in peptidolipin-NA (XVIII), an important study since it demonstrated the use of a characteristic end group (C19H:&0 - ) to

219

MICROBIAL PEPTIDE-LACTONES

All arymmetrlc center% are probably L

nm

xm:

(Flpurer In brackets

XIP

r e f e r to increases in moss In the

xn

d e u t e r i a t e d molOCule)

220

A. TAYLOR

R and R ' = amino a c i d s i d e chains

xxm X :H, OJN3*0R

M.

R'

Me

R , R', R " . M e 2 C H , E t M s C H , M I Z C H C H p

a:

facilitate the identification of the series of ions indicating the amino acid sequence. Similar studies by Wolstenholme and Vining (1966) and Kiryushkin et al. (1967)applied the method to isariin (XIX) and staphylomycin S (VII, R = CHZCsH5, R' = Et, X + Y =

22 1

MICROBIAL PEPTIDE-LACTONES

-CH2CH2COCH2-), respectively, and the work of Kingston et al. (1966a) showed that this type of fragmentation also occurred in macrocyclic compounds where the peptide and lactone moieties were separated by chains of up to ten saturated carbon atoms. The formula assigned to isariin mass spectroscopically was confirmed independently by Biemann et al. (1966). These workers adopted a different approach. Isariin was converted into methyl isarate and the mass spectrum of the latter scanned for ions having the elementary composition ClaH2:lN0 (also C1aH2sN02) 1.007 and subsequently for all other possible ions, i.e., CI3Hz3NO Me, C13H23NO CHMez, and ClsH,3N0 CHzCHMez(the program can obviously be extended to include any amino acid and/or a-hydroxy acid residues). The method depends on an IBM 7094 computer, calculating the elementary composition of all the ions in the mass spectrum having an abundance greater than an arbitrarily chosen value, and hence the selection of ions having the correct elementary fit for a possible amino acid residue and rejection of all others. The computer output suggested two sequences of amino acids for isariin were the most probable on the basis of the sum of their ion intensities. These were: glycylvalylleucylalanylvaline and glycylvalylleucylalanylglycine.These two are, of course, easily distinguishable on the basis of other chemistry of isariin; they could also be distinguished mass spectroscopically since elimination of the side chains, which gives rise to this type of ambiguity, occurs only in the sequence involving aminoacyl ions. The method used for determining N-Me residues reported by Macdonald et al. (1964) does not work in all cases, e.g., no deuteriation was observed in the case of isariin. Recently Das et al. (1968)reported that peptides could b e N-methylated with methyl iodide and silver oxide. Examination of the spectra of the N-Me and N-C[2H]3derivatives and comparison of their spectra with that of the parent compound showed those residues which were unaffected by the reaction and hence were N-methylated. The above fragmentations apply to peptide-lactones where the heterocyclic ring is greater than 14 membered. The fragmentation of 12-membered rings is somewhat different. Macdonald and Shannon (1964) reported that the molecular ion of angolide first lost CO, and subsequently fragmented in an analogous manner to XVI. These results were confirmed by Bochkarev et at. (1965) who studied eight depsipeptides of the type XX (Table VI, Shemyakin et al., 1961) and showed that the following reaction sequence ocM - CO: M - COr - XNCHR' curred: M+ M--0,-XNCHR-OCHRCO+, etc. The mass spectra of 9- and 10-membered depsipeptide rings have been studied by Wul'fson et

+ +

+

-

-

+

-

222

A. TAYLOH

al. (1963) who showed that processes similar to the cyclol-depsipeptide isomerization (see below) also occur in the mass spectrometer. Thus the cyclol XXI givcs fragment ions at m/e 99 arid m/e 70 also produced by the depsipeptide XXII. A further technique, introduced b y workers at the Institute for Natural Products in Moscow (Wul’fson et ul., 1964, 1965a,b; Puchkov et al., 1965) consists of subliming the peptide-lactone into the source instead of inserting it directly as a solid. Under these conditions three types of fragmen-tation were reported to occur: loss of C o t , common only in 12-membered rings; formation of morpholines of the type XXIII; and more rarely, the formation of ketenes. Unfortunately, the course of these reactions depends not only on the temperature of sublimation but also on the metal from which the source of the instrument is manufactured; dctailed studies on these parameters have not been reported. The value of mass spectroscopy in determining molecular formulas of peptide-lactones is well established; its utility as a criterion of purity and also as a means of providing information concerning the composition of mixtures is likewise respectable. Its use in the determination of sequences of amino acids is not proved since, with one exception, all the compounds studied have been examples whose structures were more or less known prior to analysis. The report of Briggs et al. (1966) on the structures of the isarolides offers an opportunity for an enthusiast to prove its validity in sequence determination, since these workers have suggested the composition and the structures of a complex mixture of depsipeptides almost entirely on the basis of mass spectroscopy. The above degradative methods serve to define the gross structure of most peptide-lactones. Other conformational and associative problems are discussed below following the section on synthesis of the metabolites. However, the above methods were not sufficient to define the structure of the actinomycins since they did not resolve the two possibilities, i.e., two separate 16-membered lactone rings or a single 32-membered macrocyclic system (Palmer et al., 1964). This problem has recently been solved by Brockmann and his colleagues (Brockmann and Boldt, 1968; Brockmann and Lackner, 1968a). Thus 2-deainino-2-hydroxyactinomycin Cs [11, R’ = R” = CHMeEt, R = R ” ‘ = CHMe2,X + Y = X’ + Y’ =- (CH&- Brockmann and Franck, 19541 on treatment with hydrogen peroxide in acetic acid, followed by catalytic reduction gave the lactone (XXIV, H = COC02Me) in 36% yield. Finally actinomycin CBwas synthesized by oxidative condensation of the lactone (XXIV, R = XXV; see also Meienhofer, 1968).

MICROBIAL PEPTIDE-LACTONES

223

A further group of peptide-lactones - griseoviridin (XXVI) and osterogrycins A and G (XXVII)-have had their structures determined by methods other than those described above since they did not provide a number of amino and/or hydroxy acids on acid hydrolysis (see however, Okabe, 1961). Ostreogrycin G gave proline of undetermined configuration on hydrolysis and both ostreogrycin A and G gave traces of glycine. Greater insight into their structures came from hydrogenation studies. In all cases hydrogenation led to mixtures. Ames and Bowman (1955, 1356) showed that griseoviridin, after exhaustive hydrogenation, gave perhydrogriseoviridin which gave D-alanine and a hydroxy-10-aminodecanoic acid after hydrolysis. This reaction was studied in greater detail by de Mayo and his co-workers (Fallona et al., 1964a) who were also able to isolate alanylalanine and 5hydroxycaproic acid. Partial hydrogenation gave an hexahydrogriseoviridin whose diacetate, Fallona et al. were able to show, gave a mercaptolactone on alkaline hydrolysis which was assigned the structure (XXVIII). Oxidation of griseoviridin with manganese dioxide gave a ketone whose ultraviolet spectrum showed the presence of -(CH =CH)&O -, and this compound still possessed 3 double bonds. These degradation studies led to the suggestion of structure XXVI for griseoviridin which was alleged to uniquely explain the ability of the antibiotic to form “salts” (Fallona et al., 196413). However ostreogrycin A is also known to form salts (Donovick et al., 1955). A similar experimental approach was used by Todd and his coworkers in their studies of the degradation of ostreogrycins A and G (Delpierre et al., 1966; Kingston et al., 1966b). They used the technique of ozonolysis followed by reduction of the ozonides to carbonyl compounds with hydrogen and a 5% pallidized strontium carbonate catalyst. This provided characteristic degradation products of the unsaturated hydrocarbon fragments of the molecules. The posit.ion of the lactone group was determined by reduction of a partially reduced ostreogrycin A (Hydro-A) with lithium borohydride; the product on hydrolysis giving prolinol, and the y-lactone (XXIX), the latter arising by rearrangement of the &lactone (XXX) in acid solution (Delpierre et al., 1966). The appearance of traces of glycine in hydrolyzates of ostreogrycin A was shown ingeniously, to be due to the decomposition of an oxazole moiety, thus providing an instructive exception to the use of amino acid analysis as a criterion of purity of peptide-lactones. Their degradative evidence &as supported by a complete analysis of the proton magnetic resonance (p.m.r.) spectrum of ostreogrycin A (Kingston et al., 1966a) in which chemical shifts

224

A. TAYLOR

and coupling constants were assigned to all 35 protons; an effort unique in peptide-lactone chemistry.

c.

SYNTHESIS OF

PEPTIUE-LACTONES

The methods of constructing peptides from their constituent amino acids are numerous arid it will be assumed that the reader is acquainted with them. Here the important differences between the synthesis of peptides commonly found in proteins and those in peptide-lactones will be of prime concern. This section is divided into three parts, the first dealing with nonenzymic synthesis, the second dealing with conformational problems (since the cyclization of the linear peptidelactones provides much information on this subject), and the last section with biosynthesis. 1 . Nunenzymic Synthesis of Peptide-Lactones An obvious difference between the synthesis of peptides found in proteins and of peptide-lactones lies in the availability of the constituent amino acids. These are often molecules of considerable complexity and the synthesis of the correct stereoisomer commonly presents formidable difficulties. Thus, although DL-u-aminophenylacetic acid has been available by a Strecker reaction since 1906 (Zelinsky and Stanikoff, 1906), its resolution, except by enzymic means, was reported only recently (Koenig et al., 1967).Similarly the four stereoisomers of 3-hydroxyproline have been known since 1919 but only comparatively recently (Morita et al., 1963) has a simple synthesis been available and the separation of the isomers is still a lengthy procedure (Sheehan and Whitney, 1963).Even in the case of the N-methylamino acids, a simple preparative procedure was described only a few years ago (Quilt et al., 1963a), the products obtained by the old Fischer method usually containing the starting material. 3,4-Dimethyl-2-methylaminobutyric acid is present in triostin C (V, R = quinoxalyl-2-, R’ = R” = CHMeCHMe2, X = Y = H, X -+Y = -CHz- S - S -CH2- ), etamycin (VI) and probably quinomycins B, and C. A synthesis of the diastereoisomeric mixture was reported b y Sheehan and Howell (1963), and later Japanese workers (Sh6ji et al., 1965) were able to separate the mixture and assign the configuration of the isomers by p.m.r. spectroscopy. Two other amino acids present in peptide-lactones, but not so far found in proteins, erythroP-hydroxy-L-leucine, present in telomycin (VIII, Sheehan et al., 1962) and 4-oxopipecolic acid, present in ostreogrycin B [VII, R = -CHeCsH4NMez, R’ = Et, X + Y = - CH2COCH2CH2-(Jolles et aZ., 1965)], have been synthesized during the last few years.

MICROBIAL PEPTIDE-LACTONES

225

Enniatin B has been synthesized by three groups of workers. On March 6, 1963, Plattner and his co-workers (1963) submitted their paper on the synthesis of this antibiotic which was published with the alacrity for which the Swiss are justly famed. Two days later, the Russian group (Shemyakin et al., 1963c) submitted their synthesis starting from an intermediate they had prepared (Shemyakin et al., 1962) for the synthesis of the structure proposed for enniatin B 15 years earlier by Plattner and Nager (194th). The former synthesis, with a few improvements has just been reported (Losse and Raue, 1968) again. The syntheses are given in detail in Fig. 1 as they illustrate some general differences in the procedures used in depsipeptide synthesis as compared to those in use in the synthesis of peptides present in proteins. Thus in general the lactone bonds are formed first, principally because the weak nucleophilic nature of the hydroxyl groups requires the use of mixed anhydrides to form the ester link. However, Stewart (1968) has demonstrated that imidazole also catalyzes the reaction of amino acid esters, e.g., cyanomethyl, p-nitrophenyl, with hydroxy compounds and this observation may introduce greater flexibility in the synthesis of peptide-lactones. Of course the use of acetylimidazole for the formation of the lactone bonds in the actinomycin series has been known for some years (Brockmann and Manegold, 1967) and Ondetti and Thomas (1965) cyclized the linear peptide of a biologically active vernamycin analog using cyclohexyl-3-(2-morpholinoethyl)carbodiimide. This selfimposed limitation of primary formation of ester bonds has inevitably meant that the protecting groups used at each end of the depsipeptide chain have had to be removable in conditions where the ester link was stable. Thus the groups shown in Fig. 1 have been used almost exclusively for the protection of the amino end of the chain, apart from the use of nitroso (NO), which was reported to resist hydrogenation and which was removed with hydrogen chloride in benzene (Quitt et al., 1964). The acidic end of the chain has almost always been protected by esterification with t-butanol since these esters are readily solvolyzed under anhydrous conditions. The greater flexibility attained by formation of the Iactone as the final step in the synthesis is well illustrated by Brockmann and Lackner’s (1968b) synthesis of actinomycins where the intermediate actinomycinic acids were constructed by starting with formylthreonine and using the stable benzyl esters to protect the acidic end of the growing peptide chain. Since N-alkylamino acids are commonly present in peptidelactones the belief has grown that successful formation of peptides such as those shown in Fig. 1 required the activation of the acidic

226

A. TAYLOR

CH Mez I C,H,CH,OCONCHCOOSO,

be

I

CH M e z

CHMe2 I C6 H5 CHzO CON CH C 0 0 CH C OOC Me

7 .%I CHMe,

CHMe2 I

I C~HJCH~OCONCHCOOCHCOOH

I

I

Me

CHMe2

CHMe, I

X = C,H,CH,OCONCHCOOCHCOCl I Me

CHMe2

+

CHMe2 I N H M e CHCOOCHCOOCMe3 I CHMe2

CHMe2 CHMe2 I C 6 H 5 C H 2 0 C O N h H COOCHCO N C H C O O C H C O O C Me3 I I / 1 I Me CHMe2Me CHMe,

\

Hydroqsnalyrls

CHMe2 C H Me2 I I NHCHCOOCH CONCHCOOCHCOOCMeJ I I I CHMe, M e CHMe,

+

CHMe2 CHMe, CHMe2 I I I C6H,CH20CONCHCOOCH C O N C H C O O C H C O N CHCOOCH COOC Me, I I I I I I Me CHMe, M e CHMe2 Me CHMee

J

0 @

Hydrogsnolysla

@

Et3N

Htl

0PCI~

Enniatin-8

( X ,4,10,16-N L- 3, 9,15-R D-R

= Me, = OPr, BPr; 31%)

MICROBIAL PEPTIDE-LACTONES

227

CH M e 2 CHMe2 I I N 0 2 ~ C H 2 0 C O : C H C O 0 CI H C O NI C H C O O CI H C O O C M e 3

-

CHMez M e Me

NO2

0 -

C H 2 OCO

CHMe2

/Et,O

CHMe2 CHMe2 I I C H COO C H CO N C H COOCH COOH I I I Me CHMe2 M e CH M e 2

SOCl,

t N02QCH20COti4

CHMe2 C H Me2 1 I CHCON CHCOOCHCOCI

:HCOO

I

Me CHMe2

I

+

Me C H M e 2

CH M e 2 I N H CHCOOCHCOOCMe3 I I Me CHMe;!

1

0 nct @ HBr/AcOH

CHMe2 CHMe2 I I I (HBr) H N CH C O O C H C O N C H C O O C H C O N C H C O O C H C O O H I I I I I I Me CHMe, M e CHMe2 M e CHMe2 CHMe,

I

@ SOCI, Q Et,N

(BJ

Enniotin

-6

,

20°, 3Ohr.

(60% )

FIG.1. Syntheses of enniatin B. (A) Plattner et al. (1963). (B) Shemyakin et al. (1963).

function as a mixed anhydride and most syntheses reported have used exclusively acid chlorides or benzene sulfonic anhydrides. It is surprising that the use of such methods has led to so little racemization, but a careful consideration of the data given in Table X reveals that it is likely that this has sometimes occurred. Further, it is unlikely that such methods are necessary since dicyclohexylcarbodiimide and triethylamine were used for the formation of all the peptide bonds in actinomycins where at least three of the four amide functions involve imino acids. Last, the cyclization of the linear peptide (depsipeptide) is a step not normally required in the synthesis of protein-like peptides, but since this has important connotations with the confonnation of the macro-ring its discussion is deferred. The above discussion has been deliberately critical and has, therefore, bordered on the churlish, since many natural products have been successfully

228

A. TAYLOR

synthesized by these means. Apart from enniatin B (Fig. I), angolide (Kiryushkin et aZ., 1964), enniatin A (Quitt et ul., 1963b; Shemyakin et al., 1963c), enniatin C (Ovchinnikov et ul., 1964), which had little biological activity (but cf. [MI,, Table X), destruxin B (XLII Kuyama and Tamura, 1965), sporidesmolide I (Shemyakin et al., 1963a), its D-isoleucyl, and D-UlhiSOleUcyl analogs (Shemyakin et ul., 1963a), sporidesmolide I11 (Ovchinnikov et al., 1965), sporidesmolide IV (Kiryushkin et al., 1965), and valinomycin (Shemyakin et al., 1966) have all been synthesized. In addition numerous analogs of the enniatins (Shemyakin et aE., 1962; Studer et d.,1965; Losse and Raue, 1968) and valinomycins (Shemyakin et aZ., 1966), some of which are given in Table X, have been reported. The synthesis of the structures proposed originally for the enniatins, for esperinic acid (Ovchinnikov et al., 1966a), and valinomycin have been reported, a n d a synthesis of pristinomycinic acid I, was described by Jolles et al. (1965). Finally the interesting reaction: +

0 11

+

ONCCH2C0,CMe,

CHZ

described several years ago by Ugi and Fetzer (1961) does not appear to have been exploited. 2. Conformational Problems

A 9-membered depsipeptide ring (XXXI) was suggested by Jacobs and Craig as an adequate expression for the peptide portion of the ergot alkaloids. However certain reactions described above as diagnostic for structure determination in the peptide-lactone field did not give the expected products. Thus the amino acid moieties, when treated with alkali or hydrazine, were not isolated as linear peptides or hydrazides but as dioxopiperazines or their derivatives. Second, reduction of, e.g., ergokryptine with lithium aluminium hydride did not give a linear amino alcohol but the piperazine derivative XXXII. On the other hand, thermal degradation at 200"C/0.001 mm. produced a compound having the formula C17H18N204 for which Stoll proposed XXXIII (though this is not possible on the basis of its infrared spectrum which suggest the formula XXXIV). These considerations led Stoll to propose the cyclol expression I for the depsipeptide moiety

229

MICROBIAL PEPTIDE-LACTONES

of the ergot alkaloids. The first evidence of transannular isomerization involving a peptide group was obtained by Cohen and Witkop (1955) who showed that the cyclic peptide ketone (XXXV) obtained by oxidation of octahydroquinoline, rearranged to the cyclol XXXVI. In the early 1960's several groups of workers (Shemyakin et al., 1965; Sheppard, 1963; Stich and Leemam, 1963; Griot and Frey, 1963; Glover et al., 1965) realized that 9- and 10-membered cyclodepsipeptides existed in solution as a mixture of the isomeric forms

CH20Ac

-*

(

m ,R =

COCH,

)

230

A. TAYLOR

XXXVIIa, b, and c. The chemical and spectroscopic data that support this result are given in the papers quoted. In general, if the macroring is 9- or 10-membered and especially in the former case the cyclol is the most stable isomer. The reservation is inserted here because Kazmierczak and Kupryszewski (1967) have obtained evidence that in /3-hydroxypropionyl-6-methyl-2,5-dioxomorpholines the cyclodepsipeptide is the most stable isomer. In rings greater than 12-membered the ester isomer is the one present in greatest abundance. Hence a reaction leading to a hydroxyacylpolypeptide results in the formation of a depsipeptide, e.g., XXXIX + (XVII, R = COCH,). These results have been exploited in three ways. Hofmann, Frey, and their collaborators were able to synthesize the depsipeptide ergot alkaloids and in a brilliant application of the method Shemyakin and co-workers (Shemyakin et al., 1964; Antonov et al., 1965) synthesized serratamolide (XVII, R = H) uia its diacetate. Although the Russian group has shown that racemization occurs with cyclol formation (Shemyakin et al., 1965), it is not always complete and they have suggested that acylation of peptides, possibly uia the intermediacy of thioesters is a biosynthetic mechanism for peptide synthesis (see below). They have also drawn attention to the point made by Arnold et al. (1958) that a reaction similar to hydroxyacyl incorporation would offer a biosynthetic mechanism for the synthesis of cyclodepsipeptides from their linear analogs. This has been shown to be true in the case of actinomycin monolactone which is lactonized by Streptomyces antibioticus (3720) and by cell-free extracts (Perlman and Capek, 1968). The phenomenon of cyclol formation shows that 9- and 10-membered cyclodepsipeptides in solution take up a conformation conducive to transannular reaction. The question then arises: do cyclodepsipeptides of ring size 12 or greater also adopt a most stable conformation and, if so, what structural parameters are involved? This question is closely connected with the toxicity of these materials and will be discussed in greater detail below. Here the physical and chemical properties that have been measured, which are pertinent to the question, will be presented. While there is no certainty that the conformation of the ring in a crystal is retained when it dissolves, this possibility may be true. A complete X-ray crystallographic analysis of pyridomycin (IV) has been reported (Koyama et al., 1967) and the arrangement of the ring atoms are shown in Fig. 2. It is to be noted that the amide group in this 12-membered depsipeptide is trans. Of great interest are recent reports of optical rotatory dispersion (ORD) studies on peptide-lactones. The compounds that have been investigated; enniatins (X) (Shemyakin et al., 1967b), stendomycins (Bodans-

FIG.2. Model of the ring atoms of pyridomycin (a few hydrogen atoms have been added to improve definition) showing the conformation of the ring in the crystal.

232

A. TAYLOR

al., 1968), show Cotton effects in the ultraviolet at ca. 230, 263, and (213) 269 nip, respectively. Unfortunately too few compounds have been examined so far for precise conclusions to be drawn. However it is clear that these Cotton effects are not due to the presence of known asymmetric chromophores active in the 200-350 mF region of the spectrum. It has therefore been assumed that these phenomena are manifestations of the conformations of the macro-rings. Shemyakin’s group (Ovchinnikov et al., 1965) have also pointed out that the chemical shift of the N-C& signal in cyclodepsiDeBtides is solvent dezky a n d Bodanszky, 1968),and actinomycin Cs (Crothers et

ENN IATIN-A ---

20,000 -

S T E N DOM YC I N ***..*.**ACTINOMYCIN-C3 O o q u e o u s solution pH 6-95 @methono1

-

-

10,000

CQIA -

0

@ ........1..

..... , .-............... *,..,...

.......;

; 250

.......

.?-.=.--b--

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

300

1

I

350mp X

-

Q............. .... 10,000 -

t

20,000

FIG.3. Optical rotqtory dispersion curves of peptide-lactones.

MICROBIAL PEPTIDE-LACTONES

233

pendent and that in diketomorpholine, for example, where the ester and amide bonds are cis, a diamagnetic shift was observed when solutions in chloroform were compared to those in pyridine. By contrast the chemical shift of the N- M e signal in enniatin B (X) was displaced downfield when the same comparison was made, thus supporting the conclusion that the amide bonds in Wmembered cyclodepsipeptides are trans. Even in the conformationally rigid 12membered depsipeptides the configuration of the amide groups is trans (cf. pyridomycin, IV), and hence the multiplicity of the signals assigned to the asymmetric CH proton in certain of the diastereoisomers was interpreted as an indication of the presence of several conformers of equivalent thermodynamic stability. The limited conformational possibilities of 12-membered depsipeptide rings is illustrated also by the behavior of the corresponding linear depsipeptides when cyclized by the acid chloride method in dilute solution. It has been pointed out, in the case of peptides, by Schwyzer et al. (1956) that the cyclization of dipeptide cyanomethyl esters gives the same cyclic product as that obtained from tetrapeptide cyanomethyl esters. Later (Schwyzer et d.,1964) they showed that glycolylglycine and its dimer behaved similarly. In the meantime a considerable amount of work had been done in Moscow on the cyclization of linear depsipeptides. Thus it was shown that dipeptidyla-hydroxy acids gave 18-membered cyclodepsipeptides in yields varying from 21-41%. On the other hand, linear N-methylated tetradepsipeptides gave the cyclic compounds in much greater yield (Table VI). It is noticeable that considerably lower yields were obtained for the stereoisomer with an all-D configuration which was also attended by the production of dimer, i.e., cyclooctadepsipeptide. Similar results were obtained when the N -Me group was replaced b y N-H. Ovchinnikov and his co-workers argued (1963a, 1963b) plausibly, that these results reflected the conformations of the linear depsipeptides in dilute solution; models of the stereoisomer of the L,D,L,D configuration having all the peptide bonds trans, was in a particularly favorable conformation with respect to intramolecular reaction and a recent study (Konnert and Karle, 1969) of the structure of this compound by X-ray crystallography has confirmed this conformational analysis. Conversely the possibilities of hydrogen bond formation in the N - H compounds was greater, thus there was an increased chance of association and hence intermolecular reaction. It is interesting that the stereoisomer cyclized in greatest yield (L,D,D,D) clearly exists as two thermodynamically stable conformers according

234

A. TAYLOR

TABLE VT CYCLIZATI~N OF STEREOISOMEIIS OF VALYL-CY-HYDROXYISOVALERYLVALYI,-~UHYDROXYISOVALERIC ACIDS (XL) CHMe:! CHMe2

McLCH

I

0- CHCONXCHCO

XNHCHCO,C€ICONCHCO,CHC02H

I

CHMe2

I

I

X

I

I

CHMe2

I

A

1

I

[COCIINXCOC~I-OI,t

I

CHMCL

CHMep

XLIII

XL

Configuration at asymmetric center (right tu left in XI,)

Yield (%)

L,D,L,D

H

Me

L,D,D,L, L,L(?),D,D,

Me Me Me

VAW-

Me

D,D,D,D

I1 Me

D I D, D I D

Physical properties of the productXLII1, n = 1

X=

L,D,LP

L,D,D,D

I

CHMev

n=l

n=2

m.p.

tali?

6 70 75 70 40 45 18 8

19

319-320" 228" 144" 229" 229"(227") 129"

-51" 4.8" 218" 0" 0" 66"

8

13

156"

6 1"

to its p.1n.r. spectrum. One awaits with great interest similar studies on the large number of valinomycin analogs synthesized by the Russian school (Table X) because of the pertinence of this information to membrane function (see below). In summary, the data supports the idea that peptide-lactones are not a randomly distributed mixture of all possible conformations in solution, but that relatively few of the possibilities are preferred. However, even in the rigid 12-membered depsipeptides, one conformation is not exclusively present and an important factor governing the number of conformers is the absolute configuration of the asymmetric centers of the molecule.

3. Biosynthesis of Peptide-Lactones Apart from its intrinsic interest, this subject has some theoretical importance. Peptide-lactones unlike many "secondary metabolites" are synthesized in quantities that account for an appreciable proportion of the dry weight of the cell. Clearly the mechanism of the biosynthesis of the peptide bonds in peptide-lactones differs from that of the synthesis of, say, an enzyme, since the end product is different.

MICROBIAL PEPTIDE-LACTONES

235

In recent years, our increasing knowledge of the mechanism of protein synthesis has allowed speculation on the manner in which it differs from the biosynthesis of peptide-lactones. Very little experimental evidence, however, is available, and this survey is largely devoted-to exposure of the gaps in our knowledge. It was clear from the work of Schmidt-Kastner (1956) that addition of DL-amino acids to cultures of Streptomyces chrysornal~usprofoundly altered the yield and composition of the actinomycins produced. This work was extended and elaborated by Katz and his colleagues but the first demonstration that the amino acid added was incorporated intact was given by MacDonald (1960,1967) who showed that ~ - v a l i n e - l - ' ~ C was incorporated into the D-valyl, L-valyl, and D-a-hydroxyisovaleryl residues of valinomycin. Further, because the radioactivity was located, almost entirely, in the carbonyl groups of these hydrolytic fragments it is permissible to assume that the carbon skeleton of ~ - v a l i n e - l - l ~is C incorporated into the antibiotic intact. Similar results were obtained in studies on the biosynthesis of sporidesmolide I (Butler et al., 1962) where it was shown that 20% of added ~-valine-U-l~C and 16% of ~-leucine-U-'~C were incorporated into the depsipeptide. Approximately 20% of D L - p r ~ l i n e - U - ~and ~C ~ ~ - p h e n y l a l a n i n e - U - ' ~added C to Claviceps purpurea Fr. (Tul) cultures were found in the ergotamine produced (Majer et al., 1967) and Basmadjian et al. (1969) have shown that lysergylalanine is incorporated as a unit into this alkaloid. Finally Salzman et al. (1964) added ~ - v a l i n e - ' ~ C -of ~ ~known N isotopic proportion to cultures of Streptomyces antibioticus and found that the valine isolated, subsequently, from the cell protein and from the actinomycin produced had similar radioactivity and isotopic distribution. It can be concluded that all the carbon and nitrogen atoms of the amino acid are incorporated into the antibiotic. It is a plausible assumption that this holds for the incorporation of amino acids into other peptide-lactones because this is also known to b e true in the case of penicillin and other microbial polypeptides. A difficulty arises at this point, because of the differing asymmetry of the valine residues in the protein of S. antibioticus and in the actinomycin produced; both arise from L-valine added to the culture and not from D-valine. Though most of the organisms producing peptide-lactones are able to utilize D-valine and other amino acids of the D-series, these compounds, in all cases that have been examined, are not utilized in the formation of the corresponding residues in the peptide-lactones synthesized. Indeed they are often inhibitory (Katz, 1960; Katz and Weissbach, 1963). Bertaud et al. (1963) showed that

236

A. TAYLOH

the L-isomers of DL-valine and DL-isoleucine were preferentially utilized b y Pithomyces churtarum and were able to isolate almost pure D-valine from culture filtrates. The effect of adding isoleucirie is complex and experimentally it is still difficult to obtain all four stereoisomers in an optically pure form. Russell (1967) has studied this problem and has shown that degsipeptide production by P . churturum is depressed by all four stereoisomers of isoleucine. However, very little decrease was observed in the cases of the erythro-D and threo-u isomers (92 and 96% of the controls, respectively). The isoleucine, normally present in proteins (erythro-L) and threo-Lisoleucine had a more marked effect, sporidesmolide production being about 65% of the controls, but the former resulted in the production of sporidesmolides of the type (111, R = Me, R’ = CHMeEt, R” = CHMep) while the latter resulted in the formation of a very complex mixture of depsipeptides, which were not separated. Katz and his colleagues (Katz, 1960; Katz et al., 1961) have obtained similar results in their studies of isoleucine precursors of actinomycin biosynthesis b y S. antihioticus and S . chrysomullus, but they were curiously uncritical of the optical purity of the isoleucines used. Similar studies on the effect of isoleucine on quinomycin biosynthesis are summarized in Table V I I (Yoshida and Katagiri, 1967). These results strongly TABLE V I I EFFECT OF STEREOISOMERIC ISOLEUCINES ON QUINOMYCINS PRODUCED BY Streptomyces Sp. 732

Amino acid

None L-IsoIaucine

D-Isohcine D,L-Isoleucine

Concentration of Quinomycins produced amino acid added Yield ofquinomycins imw bg./ml.) A B C D 0 0.3 0.3 0.6

5 4 5 4

55 56 51 5

15

44 41 5

20

85

0

10

1 3

0 0

(W) E 0 0 9 0

suggest that the L-isomers, at any rate, of valine arid isoleucine are epimerized enzymically before incorporation into the depsipeptide and this epimerization occurs without rupture of C -C or C -N bonds. Russell (1967) has obtained some circumstantial evidence for such epimerization enzymes, but these await investigation. They are clearly of considerable theoretical interest and the answers to such questions as “By what means does the cell segregate such enzymes to

MICROBIAL PEPTIDE-LACTONES

237

enable protein synthesis to continue?” may well extend our knowledge of the chemistry of these procedures. A further difficulty will have been observed, i.e., in most cases the effect of adding amino acids to the culture medium on the composition of peptide-lactones produced has been detected only by crude paper-chromatographic analysis. There are very few examples, even of the isolation of a single pure component from the depsipeptide mixture produced. Despite this caution, there appears to be evidence that when an amino acid is added to the culture media it does not replace, indiscriminately, any amino acid residue in the peptide-lactone. Thus Ciferri and his colleagues (Ciferri et al., 1964, 1965)demonstrated that sarcosine added to cultures of Streptomyces V-187 was not incorporated into the sarcosine residues of the actinomycins synthesized (which were derived from glycine) but replaced the proline residues. This work was an elegant demonstration of the vulnerability of the proline position in the actinomycin molecule to bioreplacement by a large number of imino acids. These include L-hydroxyproline, which incidentally showed that in Streptomyces this amino acid can be incorporated into the actinomycin directly, or as in the case of the biosynthesis of collagen, by oxidation, with 0 2 , not H 2 0 (Diegelmann et al., 1969), of a proline residue already present in the peptide (Katz et al., 1962), ~-thiazolidine-2-carboxylic acid (Nishimura and Bowers, 1967), 4-fluoroproline, 4-chloroproline, 4-bromoproline, piperidine2-carboxylic acid, azetidine-2-carboxylic acid (Yoshida et al., 1968), cis- and trans-4-methylproline, and cis- and trans-5-methylproline (Yoshida et al., 1966a). 3-Methylproline (cis and trans) in concentrations of 3.3 x 10-~and 4.5 x lopti M , respectively, were not incorporated into actinomycins but inhibited actinomycin synthesis by 50% (Yoshida et al., 1966b). Similarly a number of substitution products of piperidine-2-carboxylic acid were also inhibitory (Katz, 1967). This work reveals two similarities between peptide-lactone biosynthesis and protein synthesis: the utilization of L-amino acids, and the specificity of their position in the peptide chain. It also reveals the assembly of the peptide is not carried out by mechanistically identical processes since some of the residues can be varied by altering the concentration ratios of the amino acids presented to the organism. Whether a similar phenomenon occurs in protein biosynthesis has not to my knowledge been investigated. One other similarity between actinomycin and protein synthesis was observed by Weissbach et al. (1965), who showed that washed cells of S. antibioticus synthesized actinomycins at a greater rate when all the constituent amino acids:

238

A. TAYLOR

valine, glycine, methionine, proline, and tryptophan were presentD-tryl)tophan inhibits actinomycin synthesis (Albertini et a,!., 1966). Jt was inevitablc that chloramphenicol would be used a s a tool to substantiate the ovcrall difference between actinornycin synthesis and protein synthesis. Katz et al. (1965) showed that 30 pg./inl. of cliloraiiiphei.lico1 inhibited protein synthesis by about 90%, while the production of actinomycins was doubled. Conversely, addition of 0.1 pg./nil. of a stereoisomeric mixture of 3-methylprolines increased protein synthesis by 25% and decreased actinomycin synthesis by 70%. Chloramphenicol is known to react with ribosomes and this has led to the dognia that it specifically inhibits ribosomal synthesis of peptides. It follows that the peptides found i n actinomycins (and other microbial polypeptides) are not biosynthesized by a ribosomal mechanism. There are, however, certain difficulties with this theory. Actinorriyciri is proclucecl when growth hns ceased, in the presence or absence of chlorarriuheIiico1, arid it follows that the enzymes required for the synthesis of the antibiotic are synthesized before the addition of the cliloratiiphenicol (Marshall et ul., 1968), but despite this no actinomycin is produced. This may be because the actinoniycin produced is degraded at the same rate, which requires the assumption that the degradative enzyrnc is particularly labile or that a final oxidative step is repressed by ii metabolic degradation product of protein, e.g., anthranilic: acid ( S a h i a n et nl., 1969). These difficulties are important and suggest that the epiinerizing enzymes discovered by Russell (1967) should be closely investigated since they are uniquely c.oncernec1 with peptide-lnctone synthesis. Another avenue which has not been investigated is the source and mechunisni of incorporation of hydroxy acids into peptide-lactones. It is known that L-valine is incorporated (though less efficiently) into the 1.-a-hydroxyisovaleric acid residues in sporidesmolide I (Butler et d.,1962) and into the D-a-hydroxyisovaleric acid residues of valinomycin (MacDonald, 1960). However L-a-hydroxyisovaleric acid is not incorporated into valinomycin but its D-stereoisomer is (MacDonalcl and Slater, 1968) and it is tempting to assume that the L-valine precursor is first isomerized into u-valine and then converted to the hydroxy acid of correct stereochemistry for incorporation. The way these rcsidues are assembled may illuminate some of the problems p s c d above, but t h e y also have an additional interest. It is now known that a-amino acids can be substituted into valinomycin and enniatin B (Losse and R u e , 1969) with retention of biological activity and, conversely, that a-hydroxy acids can be substituted for tu-amino acids in, for example, bradykiriin often with en-

MICROBIAL PEPTIDE-LACTONES

239

hanced activity (Shemyakin et al., 1966a). The question therefore arises: how many amino acid residues in an enzyme, for example, could be replaced by a-hydroxy acids having the same configuration and side chain? The biosynthesis of peptide-lactones shares another feature in common with protein synthesis since no intermediate between the precursor amino acid and the depsipeptide has been found. Perlman and Capek (1968) have shown that actinomycin monolactone is a precursor of actinomycin, but here all the peptide bonds have been formed. Recently Slater and Spencer (1968) have synthesized pure ~-lactyl-~-valine-l-~~C, and MacDonald and Slater (1968) showed that this compound was almost certainly hydrolyzed before the valine fragment was incorporated. Thus the activation, the mechanism of formation of, and the way in which the order of the residues is controlled, in other words all the important features of peptide-lactone biosynthesis, remain unknown.

IV.

Toxicology of Peptide-Lactones

A. ASSOCIATIONPHENOMENA

OF

PEPTIDE-LACTONES

At first sight it may seem unusual to commence a description of the biological activity of a group of natural products with an excursion into thermodynamics. However to anticipate the findings to be presented below, it has been found that most of the biological properties of the peptide-lactones can be attributed, at least in part, to their ability to modify the surface properties of biological polymers and aggregates thereof. Three main groups of this type of phenomenon have been studied: the association of actinomycins and echinomycins (Sato et al., 1967a,b) with deoxynucleic acids (DNA), the effects on the permeability of membranes by cyclodepsipeptides of the valinomycin and enniatin types, and finally the surface properties given to the fruiting bodies of Pithomyces species by the occurrence of depsipeptides on their surfaces. Each of these topics will be discussed in turn but as indicated above it is likely that the phenomena are all associated with the stable conformations adopted by peptide-lactones in solution.

1. Association Phenomena between Peptide-Lactones and DNA In Table IV, there are a few examples of the many discrepancies in the literature for values of melting points and optical rotations of the various actinomycins. A possible explanation of these results was given by Brockmann and Manegold (1962) who showed that the

240

A. TAYLOR

specific optical rotation of actinomycin X,, was greatly dependent on concentration, solvent, and temperature. It has been known for many years that cyclic decapeptides, e.g., tyrocidines, existed in solution as aggregates (Pedersen and Synge, 1948) and that such association was not dependent on, e.g., intermolecular 7~ bonding between tyrosine residues, but on the conformation of the peptide ring (Ruttenberg et al., 1966; Schwyzer and Ludescher, 1968). Further, biological activity is only observed in those tyrocidines having the capacity to associate. The nature of the association between molecules of actinomycins was suggested by Palmer et a,!. (1964) from X-ray diffraction measurements of a crystalline mixture of actinoinycins B. These workers showed that to accommodate the datz it was necessary to assume that two molecules of the antibiotic were associated. In solution, Berg (196s)determined the molecular weight of actinomycin C1 polarographically and obtained a value of 5100 in water. Miiller arid his co-workers (Muller and Emme, 1965; Crothers et al., 1968) determined the molecular weight of actinomycin C:, in aqueous solutions of differing concentrations b y sedimentation equilibrium. They concluded that at concentrations of about 13pg./ml. the antibiotic was mostly dimerized and they obtained thermodynamic values for the equilibrium 2 monomer dimer Crothers et at. (1968) also gave ORD data on actinomycin Cs some of which has been algebraically transformed and incorporated for comparison in Fig. 3. It is not clear to me how this data supports, except in the general terms of Brockmann and Manegold (1962), a dimeric structure for actinomycin CJat these concentrations. Apart from the formation of aggregates of like molecules, actinomycins also have the property of association with other herterocyclic molecules, notably nucleosides, and particularly with deoxyguanosine. Thus Kersten (1961) showed that the absorption band at 450 m p in the spectrum of the actinomycins was shifted on addition of deoxyguanosine and a new band appeared at about 465 mp. His data suggested that 1 mole of actinomycin was associated with 1 mole of deoxyguanosine. Miiller and Spatz (1965) estimated the molecular weight of the actinomycin C3-deoxyguanosine complex and found that Kersten’s figure had to be revised upward by a factor of 2. Since both binding sites of the actinomycins were equivalent but independent it was suggested that the actinomycin dimer was the reactant. The stability constants of the complexes of deoxyguanosine with a large number of actinomycins were determined by Muller and Spatz (1965)

+

241

MICROBIAL PEPTIDE-LACTONES

and some of their values are given in Tables VIII and IX. The values given for AH" and AS" in Table VIII have been compiled from the literature and they reveal that the reaction is energetically very similar to the association of two molecules of actinomycin (AH"= 15 kcal./mole; ASo = -38 e.u./mole). Crothers and Ratner (1968) showed that the stability of the actinomycin-deoxyguanosine complex was decreased when small quantities of methanol were added, the change in solvent resulting in greater negative entropy of complex formation, this result is also in accord with solvent effects in the dimerization of the actinomycins. I have listed a number of actinomycins in Table XI which TABLE VIII THEHMODYNAMIC CONSTANTS OF THE REACTION BETWEEN ACTINOMYCINS AND DEOXYGUANOSINE Actinomycin

k

CI C,

2.6 x 103 2.6 x 103

Serylactinomycin"

AH" kcalimole

AGO

ASo e.u./mole - 15.1 -19.8

9.1 -10.3

-

-4.44 (-4.26,10% MeOH)

7.4 x lo2

"11.

BIOLOGICAL ACTIVITY OF

A

TABLE IX NUMBEROF RELATEDACTINOMYCINS

Actinomycin (11)

X+Y Xa -(CHr):,-(CHx)zeC1- (CHx):+Xop - (CH2)SXos -(CHI),XI, X = M e , Y = H &, X = M e , Y = H Xo, X = M e , Y = H Fe X = M e , Y = H C1

X' -+ Y'

M.I.C." (pg.im1.)

- CHZCH~CO- (CHA-( C H A -CHZCHzCHOH -CH~CHZCHOH -C H I C H ~ C O -( C H A -CH,CH,CHOH X'=Me,Y'=H

1.5 0.7 > 100 0.025 0.4 0.7 0.3 0.01 0.15

LDsob

k"

(rng./kg.) (X l O F }

0.4 1.1

1.1 2.6

>8

1.7 2.3

Fi i . i x 107 2.3 X lo6 < 102 1.3X lo6 3.8 x lo6

1.5-3 6-8

1.6

"M.I.C., minimum inhibitory concentration against Bacillus subtilis. *LD,o in mice, dosed subcutaneously. 'k, Stability constant of actinomycin-deoxyguanosine complex. d K , Stability constant of actinomycin-calf thymus DNA complex eC1, etc. Trivial names given to actinomycins; eC, = enantiomer of actinomycin C,, X, is epimeric with Xoa at OH group of hydroxyproline residue.

242

A. TAYLOR

differ only in the nature of the amino acid present in the position occupied by proline in actino~nycinD and the values obtained by Muller and Spatz (1965) for the stability constant of their complexes with deoxyguanosine. For comparison the antibacterial activity (Brockmann and Manegold, 1962) and also their toxicity to mice (Pugh et al., 1956) are included. It is clear that the only relationship to be found is that all the actinomycins form complexes and all are biologically active! All of this work was originally stimulated by the observations of Kirk (1960) and Rauen et u1. (1960) who showed that actinomycins complex with DNA. They showed this in three ways. (1) The antibacterial activity of actinomycin against Staphlococcus aureus and other bacteria was lost in the presence of exogenous DNA; DNA, but not ribonucleic acid (RNA), reversed the inhibition of protein synthesis by actinomycins. (2) The actinomycin-DNA complex absorbed at 465 m p and not at 445 m p , and (3) the sedimentation coefficient of the actinomycin-DNA complex was different from that of DNA; the complex could be seen to migrate as a distinct boundary giving a colorless supernatant and a yellow subnatant. These observations released a torrent of research on the nature of the binding of the DNA-actinomycin complex and scant justice can be done to this here. The treatment is divided into two parts, the former dealing with the physical chemistry of the association and the latter with the kinetic use of actinomycins as toxicological agents. It has been shown that several types of complex of DNA with actinornycins are formed, but obligatory requirements are that the nucleic acid should contain deoxyguanosine residues (but see Wells, 1969) and that the actinomycin lactone groups and amino group should be intact (Grabowska, 1966). Mekler and Zhadriov (1966) have shown that the DNA from T2 phage formed several complexes with actinomycin D which could be separated on cross-linked dextran gels. The complexes are dissociated b y dilution (Kirk, 1960)and by the addition of urea or dodecyl sulfate (Liersch and Hartmann, 1965). Cellert et al. (1965) found that the stability constant of the “tightly bound” actinomycin with calf thymus (and other) DNA’s was about 5 X lo6, or at least three orders of magnitude greater than that of the actinomycin-guanosine complex (see also Table IX). The reaction was associated with a large increase in entropy (ASo = 31 e.u./mole actinomycin bound) and they concluded that this was due to dehydration of actinomycin since the antibiotic was not dimerized under their reaction conditions. They found that few of the guanosine residues in the DNA’s examined were

MICROBIAL PEPTIDE-LACTONES

243

complexed to tightly bound actinomycin and concluded that the theoretical stoichiometry of binding with a DNA consisting entirely of guanosine-cytosine (G-C) residues would be 1 actinomycin/l2 residues, while a DNA with 25% G-C residues would theoretically bind 1 actinomycin/l9 residues. Gellert et ul. (1965) also studied the dichroism of actinomycin-DNA complexes of ratio 1:40 and 1 : 14 because a lower dichroism (E 1/ e 11 ) at 440 m p than at 260 m p implies that the actinomycin chromophore is not perpendicular to the helical axis. In both actinomycin complexes the transition moment was calculated to be 23” off perpendicular to the axis of the helix, and hence the actinomycin chromophore was unlikely to be intercalated between the DNA bases. A similar conclusion, based on X-ray diffraction fiber diagrams (Hamilton et al., 1963) had been suggested earlier. The conclusion has, however, been challenged (Miiller and Crothers, 1968). These workers have interpreted the results of equilibrium measurements, viscosity, and sedimentation studies of a number of DNA’s of different molecular weights and rates of dissociation of actinomycin C3-DNA complexes in terms of intercalation of the actinomycin chromophore between base pairs. The precise structure of the complexes, therefore, remains unknown but the increase in temperature at which thermal dissociation of the complex occurs, when compared to the DNA alone (Haselkorn, 1964),and the decrease in viscosity and increase in sedimentation coefficient of the C ytoplauga johnsonii DNA (35% G-C) with < 8 mM actinomycin (Kersten et al., 1966) infers that the DNA helix is more tightly coiled. It seems that further progress in this field awaits the synthesis of actinomycin analogs and polydeoxynucleotides though these will have to be of high molecular weight. Some work has been done on the former. Thus Brockmann and Lackner (1964) synthesized the analog of actinomycin C1 with L-serine in place of L-threonine and showed that its antibacterial activity and stability constant with deoxyguanosine were reduced (Miiller and Spatz, 1965, Table VIII). Brockmann and Schramm (1966) prepared the optical antipod of actinomycin C1 and found that it had no antibacterial activity (cf. enniatin B), and was unable to complex with DNA (Miiller and Crothers, 1968), which appears to invalidate the intercalation theory. Finally Mauger and Wade (1966) have synthesized a derivative of gramicidin S in which the ornithine residues are linked amidically to the carboxyl functions of the actinomycin chromophore. This compound was of interest because it was once thought possible (see above) that the actinomycins were a single 32-membered peptide-lactone. Mauger and Wade’s compound did not complex with

244

A. TAYLOR

DNA at neutral pH, a r i d it was 200 times less toxic to mice than actinomycin D. Kirk’s report (1960) that actinomycin D inhibited RNA synthesis as well as protein synthesis has received much subsequent attention and support. Perry (1962) found that 3.3 x lo-* M actinomycin D inhibited the incorporation of [3H]-cytidine into the nucleic acids of the nucleolus and cytoplasm but had less effect on its incorporation into the nucleic acids of the nucleus. Experiments using labeled actinomyciris revealed the presence of firmly bound isotope in the nucleus (Dingman and Spoon, 1965). Goldberg et al. (1962) showed that RNA synthesis primed by synthetic deoxyadenine-thymidine polynucleotide (polydAT) or the DNA from Cancer borealis (a polydAT containing about 2.5% G-C residues), were not affected by actinomycin though other workers have obtained somewhat different results (Widholm and Bonner, 1966). Kahan et al. (1963) also found that the inhibitory effect of actinomycin on RNA synthesis primed by DNA was dependent on the primer containing deoxyguanosine residues. Curiously, the RNA synthesized in the presence of actinomycin with the crab DNA as primer was still of the same composition as that synthesized in the absence of the antibiotic. The effect of actinomycin on the functions of DNA as a “template” is, in part, reversible since Becker et al. (1966) found that the DNA recovered from the liver of rats treated with actinomycin D had impaired priming activity in a bacterial RNA polymerize system, while the DNA, isolated from the same source and extracted with isopropanol, had greater activity. This effect of actinomycin is only observed with a native, double-stranded DNA. Denatured DNA (Widholm and Bonner, 1966) and single-stranded DNA (Kawamata, 1963) are not affected in their priming properties by actinomycin. This work has led to the simple belief that the effect of actinomycin on cells is highly specific, i.e., it is complexed to certain guanosine residues of the DNA of cells and hence can be used as a sensitive tool to inhibit certain manifestations of the genome. Experimental results conflicting with this belief, e.g., Eisenstein et al. (1966) have a distinct air of heresy. Much information has been achieved, however, b y the application of this dogma and some of the most useful (though the choice is probably invidious) are given below. The examples are given merely by way of illustration. Thus Widholm and Bonner (1966) found that actinomycin D inhibition of RNA synthesis was a sensitive means of demonstrating the irreversibility of the melting phenomenon of native DNA’s. Burdon (1966) showed that methylation of sRNA by

MICROBIAL PEPTIDE-LACTONES

245

Krebs I1 ascites tumor cells was inhibited by actinomycin D and deduced that this was consistent with the idea that only freshly synthesized RNA was methylated. Elkhom and Laland (and others, 1965) showed that while actinomycin D inhibited protein synthesis by Bacillus brevis the drug had no effect on the biosynthesis of gramicidin S, and hence the mechanism of the synthesis of the former was different from that of the latter! Wilt (1965)and Wainwright and Wainwright (1966) showed that 2 pg./ml. of actinomycin D inhibited the incorporation of radioactive uridine into the HNA of high molecular weight but did not inhibit hemoglobin synthesis by chick blastodiscs. The differentiation of Pleurodeles waltlii (newt) is affected by actinomycin D and Duprat et al. (1966) obtained evidence to show that embryos at the myoblast stage were equipped with messenger RNA’s sufficient to allow normal development. Levy et d.(1965) studied the production of interferon by chick embryo cells infected with ungunya virus. No interferon was produced when actinomycin was added 0-1.5 hours after infection, but addition of the antibiotic 2 hours after infection did not prevent interferon production - the inference being that 2 hours were required to mobilize the interferonsynthesizing mechanism. In summary, the formation of complexes between native double helix DNA with actinomycins is established. Some of the structural parameters required are known as are the hndamental thermodynamics of the reaction. Study of the effect of this reaction when it occurs within the cell have thrown much light on the nature of some replication processes, but there has been a tendency to believe that because other reactions of actinomycins with cell constituents have not yet been observed, they do not exist. 2. Effects of Peptide-Lactones o n the Properties of Membranes There is a good deal of general evidence that peptide-lactones modify the properties of membranes. Thus Warner (1961) pointed out that a model of etamycin could b e constructed in such a manner that one side of the macrocycle was lipophilic and the opposite side lipophobic. Amino acid transport by Streptococcus faecalis is inhibited by actinomycins (Holden and Utech, 1967) and ostreogrycin A is irreversibly adsorbed by rabbit (Tanaka et al., 1962) and human (Maillard and Pellerat, 1965) erythrocytes. Resistance in Bacillus subtilis and Escherichia coli to ostreogrycin A is also thought to be due to modifications of the transport properties of the cell wall which prevents the drug from entering the cell (Ennis, 1967). The antibac-

246

A TAYLOR

terial activity of glurriamycin is enhanced by increasing the concentration of Ca” (Matsui et al., 1963). Much work has been done on the binding of geptide-lactones to lipoproteins (Andreoli et ul., 1967; Lev and Buzhinsky, 1967; Tosteson et nl., 1968; Johnson arid Barigham, 1YG9), Pache and Ziihner (1969) showed that the inhibition of growth of Bacillus suhtilis by valinomyciri and enniatins A and B was reversed by addition of lipoprotein from hen’s egg yolk to the cultures. The lipoprotein had no effect on the antibacterial activity of actinomycin. Effects of valinomycin on the permeability of erythrocytes (Tosteson et al., 1967) and adipose cells from rat epididymal fat pads (Kuo and Dill, 1968) have been reported. Recently, stimulated by the work of Pressman and hib colleagues (see below) the effect of the peptide-lactones on the transport of cations by synthetic membranes has been examined in detail. Mueller and Rudin (1967) studied the effect of enniatin B and valinomycin on “bimolecular” lipid menibranes. Thcy showed that when the antibiotic was added to a system consisting of n monovalent salt solution (e.g., NaCI) of different concentration on either side of the membrane, simple diffiision did not occur but a negative membrane potential was set up on the side of the menibrane exposed to the higher salt concentration. In other words, the cations d i f i s e d through the membrane preferentially. If two different monovalent salt solutions, e.g., NaCl or KC1 of the same concentration were placed on either side of the membrane and then valinornyciri added (1 pg./rnl.), it was found that one cation diffused through the membrane selectively, thus setting up a membrane potential. This system allowed the comparison of the potentials set up between pairs of alkali metal cations; the values obtained for Li+, Na+, K+, Hb’ ,and Cs being 0, 8,151,172, and 135 mV, respectively, in the 0, 21, and 90 mV, recase of valinomycin, and for Na+, K+, and CS+, spectively, in the case of enniatin B (10 pg./Inl.). Clearly this phenomenon is dependent either on the reaction of the depsipeptide with the membrane, thus modifying its properties, or on a reaction of the depsipeptide with the cation, the product having a different stability constant with each alkali metal, or on a combination of both. A good deal of progress has been made on this problem by the Russian school (Shemyakin et al., 1967b) who have been able to isolate the potassium thiocyanate complex of enniatin B. They have X-ray diffraction data on the complex (Shemyakin, private communication; Wipf et al., 1968) and have shown analytically that it had the composition K eniliatin B+ CNS-. The formation of these complexes was followed by studying the change in the ORD curve (Fig. 3) of enniatin B (and other related

MICROBIAL PEPTIDE-LACTONES

247

compounds) on addition of K+. The Cotton effect shifted hypsochromically during the addition of 1 mole of potassium thiocyanate. Second, they found that the addition of enniatin B or its enantiomer (Shemyakin et al., 1967a) to KCNS solutions resulted in a decrease in conductivity due to lowered mobility of the complexed ion. This experimental data led directly to the value 3.7 x lop3for the stability constant of the enniatin B-K+complex; -4.90 kcal./mole for its free energy of formation and 5.25 A for its effective Stokes' radius. The ester C =0 stretching frequency in the complex was shifted bathochromically with respect to the frequency observed in enniatin B, thus demonstrating that the ester carbonyl groups were involved in complex formation. The data obtained for other alkali metal cations closely fitted the results described above for the membrane potentials generated by these cations in the presence of peptide-lactones. However the values for the stability constants of enniatins of differing structures were not proportional to the minimum growth inhibitory concentrations of the antibiotics and Shemyakin et al. (1967b) proposed that this required a third factor-a membrane receptor site which accepted the depsipeptide, which subsequently reacted with the cation. They noted, in support of this, that the stability constants were solvent dependent; no complexes were formed in aqueous media. Pressman reported, at the 3rd International Fermentation Symposium, that he had obtained essentially similar results. The difficulty at the moment lies in deciding how far these results are applicable to transport problems in living systems. Certainly, they do not have the required specificityLippe (1968) has shown that the resistance of a lecithin membrane decreases about 1000 times when solutions of thiourea and enniatin B are added to the system. The experiments described in the preceding paragraph were of course, stimulated by work which stemmed from the observations of McMurray and Begg (1959) that 10-7-10-8 M valinomycin uncoupled oxidative phosphorylation by rat liver mitochondria, and that of Gaumann and Obrist (1960) that enniatin A at M lowered the water permeability of protoplasts of Rhoeo discolor. Further progress was not made for several years until Pressman (1965, 1967) applied the use of ion-sensitive electrodes to the problem of studying active transport of ions by mitochondria. Generally the addition of valinomycin to rat liver mitochondria resulted in an increase in the intramitochondria1 [K+]. It was found that ions such as acetate at pH 6.35 increase the net K' uptake but the rate at which it entered the mitochondrion was lower than that normally observed in the absence of

248

A. ’IAYLC~H

valinomycin. Phosphate also promoted net K+ uptake (Pressman, 1964; Moore and Pressman, 1964; Ogata and Rasmussen, 1966; Azzi and Azzone, 1965, 1966). The increased uptake of K+ was usually dependent on the presence of an oxidizable substrate and its extent depended on the substrate used (Harris et al., 1966). The accumulation of K+ is accompanied by mitochondrial swelling, decreased fluorescence, and stimulation of respiration. The pH of the suspending medium decreased and conditions can be found where the loss of H+ from the mitochondria is balanced b y their K+ gain. However, this ratio is variable, e.g., at pH 6.35 the value of K+/H+may be 10. Considerable attention has been directed at the energetics of this valinomycin-induced transport of K+ against the concentration gradient. Thus when C1- is the major anion in the medium the oxygen consumed is higher than when acetate is the major anion. This is consistent with the idea that the energy demand for movement of K+ depends on the availability of a freely permeable anion since C1- is known to enter slowly (the possibility of acetate being a substrate does not seem to have been considered).When respiration inhibitors, e.g., rotenone are added to mitochondrial suspensions, it is still possible to observe valinomycin-induced K+ uptake provided adenosine triphosphate (ATP) is also added. Cockrell et al. (1966)showed that a maximum value of 7 wdS obtained for the stoichiornetry of K+ transport per equivalent of ATP at pH 6.7, 25”, 2.5 mM K’, arid 20 m M acetate, corresponding to a “thermodynamic efficiency” of about 80%. Since both reactions are temperature dependent (Azzi and Azzone, 1966), Cockrell et al. (1966) were able to obtain the activation energy of K+ transport in the presence of an oxidizable substrate (9.8 kcal.) and in the presence of ATP (10.8 kcal.). This result suggests that the rate-limiting step in both processes is similar. However, the response of the mitochondria to valinomycin is not merely to accumulate K+. Using 4zK+ Harris et al. (1967) were able to show that 40% more K+ was taken u p by the mitochondria than estimated by determining the decrease in [K+] in the suspending medium. Using this technique it was possible to show that when the [K+] of the medium was constant continued. At this point the expulsion of K+ must be uptake of 12K+ equal to its intake and clearly the rate of turnover can be calculated. It was found that this turnover rate was a function of the valinomycin concentration. It is clear from this work of Pressman and his colleagues that valinomycin affects the permeability of the mitochondria1 membrane and that this effect is a function of the structure of the antibiotic (Pressman, 1965; Table X). It is strange that no reports of the fate of

MICROBIAL PEPTIDE-LACTONES

249

radioactive valinomycin (MacDonald, 1960) in mitochondrial systems has been reported and that some of the more subtle changes in structure (e.g., cyclostereoisomers, Table X) synthesized by the Russian school, have not been tested for their ability to induce the rate of turnover of K+ in these particles. It is probable that valinomycin affects other mitochondrial processes apart from the transportation properties of the membrane (though whether these are dependent on this effect is not known). Hofer and Pressman (1966) found experimental conditions where adenosine 5'-phosphate was phosphorylated when rat liver mitochondria were treated with valinomycin and Bygrave and Lehninger (1966) showed that 5 p M valinomycin decreased ADP-ATP exchange by about 70%. Harold and Baarda (1967) have shown that valinomycin at lop6M inhibited the growth of a strain of Streptococcus faecalis, an organism that derives its energy, at least in part, from glycolysis, and not at all by oxidative phosphorylation. In this case inhibition of growth was attended by loss of K+ from the cells and this (i.e., growth) could be reversed by increasing the [K+]. When the cells were grown in media containing rubidium instead of potassium and then transferred to medium containing valinomycin and K+ (or H+, s6Rb+,or Cs+) the intracellular Rb+ was displaced, the exchange being temperature dependent. It appears that valinomycin merely increases the permeability of these cells to K+ (since normal media do not contain Rb+ or C s + ) .Similar claims have been made in the case of rat liver mitochondria by Lynn and Brown (1965, 1966) who pointed out that valinomycin did not stimulate the production of L3Hzl0 from (HOOC*CH[3Hl)z. Baltscheffsky (1960) and his co-workers (Baltschefkky and Arwidsson, 1962; Baltscheffsky and von Stedingk, 1966) have studied the effect of valinomcyin on photophosphorylation phenomena in chromatoplasts from Rhodospirillum rubrum. It is known that photophosphorylation in this organism is stopped by the addition of 2heptyl-4-hydroxyquinoline-l-oxide. When phenazine methosulfate is added to this blocked system, phosphorylation starts and is not inhibited by valinomycin. It was, therefore, argued that there are two phosphorylation sites, only one of which is blocked by valinomycin. When the organism was incubated in the dark with 3 X lop6M valinomycin for 4 minutes and then illuminated with about 30,000 lux. the uptake of inorganic phosphate was not affected for illumination periods less than 5 minutes, provided ADP had not been added to the system. Sat0 et al. (1966) have obtained closely similar results working with the same system, and Plengvidhya and Burris (1965)showed that

OPTICAL ROTATION

.4ND

KJ

TABLE X TOXICOLOGY OF SYYTHETIC CYCLODEPSIPEPTIDES

u1 0

P

M.I.C. (pLg./rnl.)* Compound" C~HSN~OY, CJOHSINSOY,

X, L-R = ePr X, L-3,9,15-R=@Pr,D-R=pPr CxHi3N309, X,4-N= Me, L-3,9,15-R=@Pr,D-R=fiPr C~LHS~N@Y, X,4,1O-N=Me, L-3,9,15-R=PPr, D-R=PPr CJJHS~N~OY, X, 4,10,16-N= Me,L-3,9,15-R=V r , I)-R = @Pr

C~JLN~OY, CiiHsiN,0~, CdLdL09, C~fiHJ'J,Oy, CIBH7RN~OlBr C~tlH7,NsOla,

X, 4,10,16-N=Me, D-3,9,15-R=pPr, L-R=@Pr X, 4,10,16-N= Me, L-3-R=PPr, L-9,15-R= EtMeCH, D-R=oPr X, 4,10,16-N= M e , L-3,9,15-R= EtMeCH, D-R=@Pr X, 4,10,16-N= Me, L-3,9,15-R= MerCHCHL,D-R =@Pr XI, D-3,15,27-R=@Pr,L-9.21,33-R=PP~, L-R = M e XI, D-3,15,27-R=ePr, L-9,21,33-R="r, L-6,1&,30-R=Me, D-R= Me

[MI;''

- 134" -629" -681" -582"

(-684") 5820

-533" -540"

S. aureus Botrytis cinerea

30 180 96 73 9 9 2 1.5

-137"

0"

10

K-'

1.3

9 4.5

25 50 50

-1

0.46 0.61 0.15

3s

XI, D-3-R= Me, L-6,9,18,30-R= Me, L-21,33-R=PPr, D-R=PPr XI, D-3-R= Me, L-6,18,30-R= Me, L-9,21,33-R=@Pr,D-R=PPr XI, L-6,18,30-R= Me, L-9,21,33-R = PPr, D-R = @Pr XI, L-6,18,30-R= Me, D-3,15,27-R=PPr, L-R=oPr XI, L-6,18,30-R= Me, D-R=PPr XI, L-6,18,30-R = Me, D-12,24,36-R = BPr, L-R = SPr XI, L-6,18,30-R= Me, L-3,9,21,33-R=@Pr,D-R=@Pr XI, L-6,18,30-R= Me, L-21,33-R =PPr, D-R= PPr XI, L-6,18,30-R= Me, L-9,12,21,33-R=PPr, D-R=PPr XI, D-6-R = Me, L-18,30-R = Me, L-9,21,33-R = PPr, D-R = PPr XI, L-6,18,30-R=Me, L-9-R= EtMeCH, L-21,33-R=BPr, D-R=PPr XI, L-6,18,30-R= Me, D-3-R= EtMeCH, L-9,21,33-R=pPr, D-R = PPr XI, L-f3,18,30-R = Me, D-3-R = Me2CHCHi, L-9,21,33-R = PPr, D-R=PPr XI, L-6,18,30-R= Me, D-3-R= MeKHCHr, L-9-R= Me2CHCHz, L-21,33-R=PPr D-R=PPr XI, L-18,30-R= Me, L-6,9,21,33-R=PPr, D-R=PPr XI, L-6,18,30-R= Me, L-9,21,33-R=0Pr, D-3,15,27-R= EtMeCH, D-R=@Pr XI, D-3,12,15,24,27,36-R=@Pr, L-R=PPr XI, 7-0= NH, L-6,18,30-R= Me, L-9,21,33-R=PPr, D-R=@Pr XI, 13-O= NH, L-6,18,30-R= Me, L-9,21,33-R=@Pr, D-R=oPr XI, 7-0=NH, L-18,30-R= Me, L-6,9,21,33-R=@Pr,D-R=PPr

372" 540"

8 0.7 0.8 50 50 50 8 6 SO 50 0.6 0.8

450"

0.7

513"

8

284" 322"

50 50

0" 830"

50 1 1 0.7

358" 389" 364"

0.7 0.7

50 9 9 50

100

13

2.2

0.7 0.7" 0.7

SO 2

"Nomenclature: To avoid repetition, the enniatin type nucleus is given as X and in the table the substituents are located by position number. The cipher "4-N = Me" means that the nitrogen atom at position 4 bears a methyl substituent, not H as shown in X; similarly the cipher "70 = NH" means that the oxygen at postion 7 in XI is replaced by NH. The ciphers D-R and L-R mean that all the groups not previously specified have this configuration. All numerals refer to ring positions. "M.I.C., minimum inhibitory concentration. rK+, induced transport of potassium ions by rat liver mitochondria as described by Moore and Pressman (1964). "This compound was assayed against Botrytis allii and the M.I.C. obtained is quoted. The molecular rotation in this case was calculated from other data for comparative purposes.

252

A . TAYLOR

2.2 x M valinomycin inhibited, by 25%, photophosphorylation by spinach leaf chloroplasts catalyzed b y phenazine monosulfate. On the other hand, Avron and Shavit (1965) using chloroplasts from “Swiss-chard” leaves obtained only weak uncoupling of photophosphorylation by valinomycin at concentrations greater than lop6M . It therefore appears that the results obtained depend on the precise biological conditions (including the experimenter) used, particularly with respect to the methods used for isolating, e.g., mitochondria. The situation is thus confused at the moment, though the measurement of the stability constants of the potassium enniatin complexes, is probably a start on its resolution. The reaction of the enniatins and valinoniycin with synthetic lecithin membranes is one of great physicochemical interest, and its extension to biological membranes might be expected in the future (McCarty, 1969).

3. Occurence of Peptide-Lactones on the Surfuce of Conidiu of Pithomyces S p . In their report of the production of sporidesmolides by Pithomyces charturum, Done et al. (1961) showed that there was a correlation between the number of spores produced by a surface culture of the organism arid the weight of sporidesmolides isolated therefrom. Later Dingley et ul. (1962) found that this correlation applied to different isolates ofthe fungus which differed in their ability to produce conidia. In the meantime Bertaud had been examining spores of the fungus electron microscopically and had demonstrated that their surface was coated with crystalline structures since they gave an electron diffraction pattern in the electron microscope and an X-ray powder diagram in the X-ray diffraction camera. The angle of the reflections found in this Debye-Scherrer diagram had the same value as those of the mixed sporidesmolides that had been isolated by extraction. Two other independent lines of experimental evidence were used to identify the crystalline structures on the spores with sporidesmolides. First it was found that sporidesrnolides were slowly extracted from spores by brief contact with benzene, and since an analysis of the lipid extracted was different from that of the total lipid of the spores it was concluded that only spore surface material was being removed. Second, the number of crystalline structures on the spore could be sharply reduced by growing the organism on media containing isoleucine. This (see above) resulted in a marked reduction in the quantity of sporidesmolide extracted from the cultures but a slight increase in the number of spores produced (Bertaud et al., 1963); these spores were not

MICROBIAL PEPTIDE-LACTONES

253

covered with spicules. These results have been greatly extended by Russell and his colleagues (Riches et al., 1967; Bishop et al., 1965) and all Pithomyces sp. examined so far (P. maydicus, P. sacchari, P. cynodontis) have crystalline coats of cyclodepsipeptides on their spores. The composition of the depsipeptides present in the coat may well b e of taxonomic importance, however, the physical properties conferred on the spores by these materials probably play an important role in their dispersion and in the control of water movement into and out of the spore. This modification of the surface properties of the biopolymers of the conidia of Pithomyces, is to my knowledge unique, but it suggests that modification of other biological macromolecules b y peptide-lactones may be important. T h e sporidesmolides are characteristic of the sporulation process in Pithomyces and there is thus an analogy here with the production of bacitracin by Bacillus sp. a process also associated with sporulation.

B. ANTIBACTERIAL PROPERTIES

OF

PEPTIDE-LACTONES

Many of the peptide-lactones were discovered when fermentation liquors were tested for bacterial growth-inhibiting properties. When such activity was obtained the fermentation broths were assayed for their ability to inhibit the growth of a wide range of species. In Table XI the minimum inhibitory concentrations of peptide-lactones of known structure against a number of bacteria in vitro are given. The bacteria have been selected from a large number of species investigated because they represent those most commonly tested. The data are, of course, not strictly comparable but despite the wide geographical dispersion of laboratory location and the different techniques of analysis used, the results obtained are remarkably consistent. I n Table I11 some of the antibacterial properties of antibiotics of unknown structure (and/or purity) are given. In general the same organisms have been selected for the same reasons. Most of the peptide-lactones inhibit the growth of gram-positive bacteria and have little effect on gram-negative organisms. There are some exceptions, notably pyridomycin (IV) and griseoviridin (XXVI). Etamycin, the B group of ostreogrycins, and telomycins (but, curiously, not the related LL-A0341) do not inhibit the growth of acidfast bacteria. Valinomycin and ostreogrycin A have little activity against Bacillus subtilis. Garrod and Waterworth (1956) found that the mixed ostreogrycins inhibited the growth of more than 50 strains of Staphlococcus aureus including those resistant to penicillin. About 75% of the strains ex-

TABLE XI TOXICITSOF PEPTIDE-LACTONES OF

m 0 W N STRUCTURE"

LDs0(mice) (mg./kg.)

Minimum inhibitory concentration (pg./ml.)

B. Compound Griseovirin

E. coli

> 100

Serratamolide Pyridomycin Ostreogrycin A

subtilis

25 100

S. aureus

M.plzlei

1-4

H37R\, 100-300 avian 25 1.5-5 M.607 50

50

50

200 100

200 0.78

Candida albicans >200

i.p.

i.v.

75

oral

> 100

Ehrlich et al. (1954); Bartz e t a l . (1954) Wasserman et al. (1962)

300

> 5000

1100

Destruxin A

References

(silkworms) 1.35 0.34 16.9 (Anguillula acetil 100 pg./ml.

Maeda (1957) Englishetal. (1955) Kodaira (1962)

0.28

Destruxin B Enniatin B

Sporidesmolide I Enniatin A Staphylomycin S +M

>lo0

37-50

9

3.3

37

> 100 > 100 > 1000 > 100

> 100

> 100 1.5

> 100 1.25 3.3

> 100

6 3.3 1

6.3 0.2

9

> 100(snzegmatis) 7

Kodaira (1962) Shemyakin et al. (196313); Bacikova et al. (1965); Plattner and Nager (1948~) Shemyakin et al. (1963b) Shemyakin et al. (1963b); Gaumann e t al. (1947) DeSonierand van Dijck (1955)

?

Etamycin

, , 200 ’

Ostreogrycin B

100

Echinomycin

1000

2.5

0.31

H37Rv 0.78-1.6

>200

3.12

6.25

M. 607

> 100

0.1

100

10 (mega-

552

470 > 1000 > 2500

Ehrlich et a1. (1954);Dickison et a/.(1954);Heinemann et al. (1954) English et al. (1955)

100 (tuberculosis)

Corbazet ol. (1957)(see Table VI)

3.13 0.75 (synthetic)

Shemyakin et a / . (1966a); Brockmann and SchmidtKastner (1955) Goss and Katz (1960) Gourevitch e t a / . (1957);Tisch et al. (1957) Goss and Katz (1960) Brockinann and Manegold (1962) Brockmann and Manegold (1962) Pugh et al. (1956) Pugh et nl. (1956) Brockmann et al. (ISSYb) Brockmann and Lackner (1968) Brockmann and Lackner (1968)

therium) Valinomycin

50

50

0.8

Actinomycin F, Telomycin

1000

0.15 1.6

0.32 8

0.2 0.7 1.5 0.068 0.48 0.2 1 1

0.39

Actinomycin F g Actinomycin X,, Actinomycin Xz Actinomycin D Actinomycin A, Actinomycin C2 Actinomycin C3

0.13 0.4

1.o

4.0 0.3

1000

1000

6-8 1000 1.5-3

0.7

0.3 ( s . c . ) 1.1(s.c.) 8 (s.c.)

1000

“Abbreviations are found in previous tables. When the organism at the head of a column in the table was not used, the results for a related species are given; this is indicated in the column. The next figure in the column refers to the organism given at its head, except for Kodaira’s results. When two results are given for the same compound against the same organism, these are results from difierent laboratories, and are given for comparative purposes.

256 A. TAYLOR amined were inhibited at 0.25 pg./ml. and the remainder at 0.5 pg./ml. These workers found that this mixture of antibiotics was bactericidal at 10 pghnl., but English et uZ. (1955) found that 16 serial subcultures of S. uureus in tan unstated concentration of mixed ostreogrycins resulted in a 20-fold increase ill the coricentration required to inhibit growth. Similar results were obtained by Ehrlich et al. (1954) in the case of etamycin. It may be concluded that the peptide-lactones are bacteriostzts, even though in an antibacterial assay the end point is insensitive to the number of bacteria used in the inoculum (Garrod and Waterworth, 1956). It appears that the exploitation of the phenomenon of resistance of bacteria to compounds such as valinomycin or actinom! cin has not been used to help the biochemical elucidation of the mode of action of these materials. Many such strains are known. Katagiri and Sugiura (1961) have shown that strains of Surcina Zutea resistant to actiriomycin are also resistant to echinomycin. Similarly strains of S. u w e w resistant to actiriornycin D are resistant to other actinomycins (Pug11 et al., 1956), and finally strains of this species resistant to ostreogrycins are also resistant to the macrolides erythromycin and spiramycin (Cybulska and Jeljaszewicz, 1967). A property common to inany peptide-lactones is the synergism observed between chemically related and unrelated metabolites in their effects on the growth of microorganisms. The phenomenon was first recognized in the case of ostreogrycin. English et aZ. (1955; Tanaka et d., 1962) showed that a 50% mixture of ostreogrycins A arid B inhibited the growth of Bacillus subtilis at 0.8 pg./ml. while the former alone was inhibitory at 100 pg./ml. and the latter at 3 pg./ml. It may be possible to deduce from the data given by Ehrlich et al. (1954) that a similar synergism occurs with mixtures of griseoviridin and etamycin, but this is not mentioned by these workers. Enniatins A and B are also known to behave synergistically (Giiumann et al., 1960). Thus 19 pg./ml. of 50% mixture was found to be the ‘‘LD50’’in Gaumann’s algal growth test, enniatin A having a similar effect at 30 pg.lm1. and enniatin B at 45 pg./ml. This synergistic behavior is not restricted to peptide-lactones. There is some evidence that macrolide antibiotics behave synergistically with peptide-lactones (English et uZ., 1955; Garrod and Waterworth, 1953; Vazquez, 1964) though this view has been challenged (DeSomer and van Dijck, 1955; Meyers et uZ., 1965). The problem has been studied in detail by Chabbert and Acar (1964) who showed that the modes of action of ostreogrycins A and B were different. Thus Staphylococci which were resistant to ostreogrycin A, retained their sensitivity to ostreogrycin B and also retained the bacteriostatic synergistic response to both antibiotics. Although the

MICROBIAL PEPTIDE-LACTONES

257

ostreogrycin A group is chemically the most closely related to the macrolides, Staphylococci resistant to, e.g., erythromycin, were less sensitive to the polypeptide ostreogrycin B. The French workers also showed that the antibacterial effect of penicillin was antagonized by the ostreogrycins and vice versa. Biochemically, Laskin and Chan (1964, 1965)confirmed the work of Vazquez (1964) that ostreogrycin A affected the binding of tRNA’s to ribosomes but were unable to show that ostreogrycin B enhanced the effect. A synergistic effect of ostreogrycin A and staphylomycin S on protein synthesis by Bacillus subtilis has, however, been reported by Cocito (1969). It was shown that the turnover of sRNA’s was decreased and protein synthesis, as measured by incorporation of radioactive amino acids, or phage (2C) replication, ceased. Finally, although Meyers et al. (1965) were unable to demonstrate antibacterial synergism between ostreogrycins and other antibiotics, combinations of ostreogrycin A at 0.25 pg./ml. with 10 pg./ml. ostreogrycin B, 2 mpg./ml. actinomycins B, and 1 pg./ml. carbomycin resulted in a 50% inhibition of growth of Earle’s L cells (NCTC 929) whereas 45 pg./ml., 6 mpg./ml., and 3 pg./ml. were required for the same effect by these antibiotics alone. It is usually considered that the enniatins and valinomycin have no therapeutic effect on bacterial infections in experimental animals, though Tirunarayanan and Sirsi (1957b) reported that 100 mg./kg. of enniatin B administered intraperitoneally, affected the course of experimental tubercuIosis in mice. The same workers (1957a) reported that the antibacterial activity of enniatin B was competitively inhibited by inositol and choline. The peptide-lactones of the ostreogrycin B type (e.g., telomycin, etamycin) all have therapeutic effects in experimental bacterial infections in mice. Thus English et al. (1955) reported significant protection of mice infected with Streptococcus pyogenes, Diplococcus pneumoniae, and Staphylococcus aureus at a dose of 100 mg./kg. of mixed ostreogrycins. The synergism observed in vitro was also observed i n vivo and the effect was greater: 400 mg./kg. of either ostreogrycin A or B did not have a therapeutic effect when dosed subcutaneously to mice infected with S. pyogenes. Benazet and Cosar (1965) also found that a 50% mixture of ostreogrycins A and B was the proportion of the two antibiotics most effective in therapeutic tests and that the mixture was ten times more active when dosed orally than subcutaneously. They provided evidence that ostreogrycin A was more effective when dosed orally, but ostreogrycin B was most effective when administered subcutaneously; the toxicity of ostreogrycin B was also dependent on its route of administration. Like many other workers Benazet and Cosar (1965)

258

A. TAYLOR

found that the somewhat cruder commercial product was more active than the 50% mixture of pure ostreogrycins A and B. Ehrlich et al. (1954) demonstrated that etamycin and griseoviridin were also therapeutically effective. The latter protected mice from experimental ~ e m ~ ~ ~pertussis i i ~ uinfections s and the former was effective against experimental intestinal amebiasis (Endunzoeba histolytica) and bovine mastitis.

c.

TOXICITYOF PEPTIDE-LACTONES TO OTHER MICROORGANISMS

The peptide-lactones inhibit the growth of a wide range of fungi, actinomycetes, protozoa, etc. Some of their activities are given in Tables I11 and XI. Since Candida alhicans is the fungus most commonly used for assay purposes, this has been chosen as representative in Table XI. When other fungi have been tested and no data has been found for the activity of a particular compound against Candida the organism tested is written in the table; following entries in the table refer to Candida unless otherwise stated. Pugh et al. (1956) showed that the growth of Aspergillus niger, Penicillium notatum, Candida albicans, and Trichoderma koningii were not affected by 100 pg./ml. of actinomycins but that 30 pg./ml. inhibited sporulation O f A. niger and T. koningii. Jirovec (1951) found that the enniatins exhibited toxicological properties against certain protozoa and these compounds have also been reported to have anthelminthic activity (Bicikova e t al., 1965). Actinomycin also showed anthelminthic activity but telomycin arid etamycin did not.

D. ANTITUMOR ACTIVITY OF PEPTIDE-LACTONES In 1942 Robinqon and Waksman reported that actinomycins had a cytotoxic action on the spleens of experimental mice, however it was 10 years before Hackmann (1952) obtained evidence that actinomycin C was more toxic to transplanted Walker carcinoma cells in rats than to normal cells, and another 2 years before this news crossed the Atlantic (Reilly e t ul., 1953)and the selective toxicity of actinomycins to ascites tumors and the like was explored with American thoroughness (Gregory et al., 1956; Sugiura, 1956). Since that time an enormous amount of work has been done, various courageous patients with, for example, Hodgkin’s disease have been treated by intrepid physicians without effect. This work has been ably reviewed by Maddock et al. (1960) and other authors in this issue of the Annals of the New York Academy of Science. It might perhaps be summarized by the dictum that a therapeutic effect can be demonstrated only if the tumor is growing at a rate faster than the turnover rate of cells in tissues like

MICROBIAL PEPTIDE-LACTONES

259

spleen, bone-marrow, etc. (Schwartz et ul., 1966). In short the therapeutic index is usually too small to permit satisfactory therapy. Most of the work described above has been carried out using actinomycin D or a mixture of the depsipeptides. Pugh et ul. (1956) examined the effect of several pure actinomycins against the Gardner lymphosarcoma (6C3HED) in the ascites form and against an adenocarcinoma (C3HBA) in mice. The actinomycin C group of antibiotics was found to be the most effective therapeutically while actinomycin X2 was inactive. It is interesting that the introduction of a single carbonyl group should have this effect, and in view of the large number of actinomycins, claimed to have been made with variations in this part of the molecule (see above), that more details of their antitumor properties do not appear to have been published. The actinomycins, however are not the only group of peptidelactones having carcinostatic activity. Shorin et aZ. (1959) and Rossolimo et al. (1959) showed that the echinomycin group of antibiotics, when administered at the maximum tolerated dose (given in parenthesis) inhibited the growth by 70430% of the Crocker sarcoma (and lymphosarcoma) in mice (450 pg./kg., subcutaneously) and by 50%, the Ehrlich adenocarcinoma in rats (140 pg./kg., orally). They concluded that the antibiotics principally affected lymphoid tissue. Zaretskii et aZ. (1961) obtained similar results working with leukemia in mice and in addition obtained some evidence that etamycin at its maximum tolerated dose had an effect against myeloid leukemia (see also Shorin et al., 1961).Further work on the antitumor effect of the echinomycin group has been reported by Katagiri and his colleagues (Katagiri and Sugiura, 1961; Sato et ul., 1967a; Matsuura, 1965)who worked with the various components of the echinomycin complex. They found marked differences in the response of the Ehrlich carcinoma (ascites) to the three echinomycins examined: quinomycin C being far the most effective (Table XII). Another group of quinoxalinecontaining antibiotics, the triostins, also show antitumor activity and their therapeutic index (in mice) is considerably greater than the echinomycins (Matsuura, 1965, Table XII), principally because they are much less toxic. However Katagiri and Sugiura (1961)were unable to demonstrate antitumor properties of the triostins against tumors in rats, hamsters, or chickens. E. TOXICITYOF PEPTIDE-LACTONES TO ANIMALS Several peptide-lactones are toxic to insects, and this subject has been reviewed by Huang (1969).The destruxins (Table IV), metabolic products of Oosporu destructor and Aspergillus ochraceus have been

STRUCTURE .4ND

TABLE XI1 TOXICITY OF ECHINOMYCIK (v)ANTIBIOTICS" Minimum inhibitory concentration (pg./ml.) LD50

R'

Antibiotic ~~

~~

R"

S. E. B. Sarcina Clos- HuLa aureus co2i subtilis tutea tridiurn cells

Antitumor dose

Ackvity

mice4.p. (mg./kg.)

(pg./kg./day)

tumor indexb

0.4

25

0.77

~

~~

0.02

Echinomycin (V) (X- Y = - S C H r Y + X=- S C H r ) Quinomycin B

-CHMel

-CHMe2

0.1

100

0.02

- CHMeEt

0.02

100

0.01

0.005

0.001 0.001

0.04

Quinomycin C m.p. 218" [alo-250" Triostin C (V)

-CHMeCHMel

-CHMeEt @lo) -CHMeCHMe*

0.02

100

0.01

0.005

0.001 0.001

0.04 0.08[

0.01

0.01

? (allo)

(do) -CHMeCHMez

(allo)

(do) -CHMeCHMer (allo)

0.1

100

0.05

0.05

0.001 0.01

100

(X=Y=H, X+Y=-CHSSCWr-) "Katagiri and Sugiura (1961); Matsuura (1965);Gtsukaand Sh6ji (1967);Shorin etal. (1959). T u m o r Index [Packed cell volume x amount of ascites fluid (treated)]/[Packed cell volume Shorin etal. (1959).

X

amount of ascites fluid (contro1s)l.

2 20

1.25 1250 313

toxic 0.69 0.07 0.52

'

MICROBIAL PEPTIDE-LACTONES

26 1

shown to be toxic to silk-worms by Kodaira (1962). However the longsuffering mouse has received most attention b y toxicologists investigating the unsavory properties of the peptide-lactones. The results that have been obtained on the toxicity of well-characterized peptidelactones are summarized in Table XI. It is clear from the table that the ostreogrycins, etamycin, and telomycins are conspicuously less toxic than the other depsipeptides that have been investigated. This results in a favorable therapeutic index and, hence, these compounds are the ones that have been examined in a pharmacodynamic sense. In addition, because of the hope aroused by Hackmann’s discovery of the therapeutic effect of actinomycin against tumors, the pharmacology of these materials has also been examined in detail. Each of these four groups will, therefore be discussed briefly, in turn. Tisch et al. (1957) examined the acute and subacute toxicity of telomycin in mice, rats, rabbits, and dogs. It was nonirritant to the three latter species and blood levels of about 1 pg./ml. were obtained for 6 hours for each mg./kg. dosed. No telomycin was detected in blood after oral doses of less than 100 mg./kg. Dogs given 200 mg./day intramuscularly did not lose weight, but were anemic, the change being reversible. When 5-100 mg./kg. of telomycin were given intravenously to anesthetized dogs a histamine-like drop in blood pressure was observed, which was also reversible-the rate of recovery depending on the dose. The toxicology of etamycin has been studied by Dickison et al. (1954), Ehrlich et al. (1954), and Zaretskii et al. (1961).The animals used b y these investigators included mice, hamsters, rats, cats, guinea pigs, rabbits, and dogs. Mice tolerated oral administration of 1gm./kg./ day and showed only small kidney lesions on post-mortem examination. Dogs tolerated single oral doses of etamycin of 0.8 gm./kg. but at higher levels serious leukopenia was observed. When dogs were dosed with 0.5 gm./kg./day, they survived about 7 days and postmortem examination revealed lung and kidney lesions which were not very comprehensively described. Leukopenia was also observed in cats treated with 1 gm./kg./day but attempts to induce this phenomenon in rabbits and mice failed. Detectable blood levels of etamycin were observed in rabbits 6 hours after dosing orally with 0.8 gm./kg. and 2 hours following a dose of 0.25 gm./kg. Essentially similar results were obtained with dogs. A maximum of 20% of the dose can be recovered in the urine of animals dosed orally or intravenously, while about 12% was found in the feces. It is interesting that the etamycin disappeared rapidly from feces that were kept, possibly by microbial action (cf. Katz and Pienta, 1957).

262

A. TAYLOR

The acute toxicity of thc ostreogrycins is low. Benazet and Cosar (1965) found that the LU,,, in mice of ostreogrycin B was 2 gm./kg. when dosed orally, while the LDSoof ostreogrycin A was > 5 gm./kg. Watanabe (1961) showed that dogs given 100 mg./kg:./dayof mixed mikamycin A and ostreogrycin B for 3 months suffered no clinically obvious toxicological symptoms. Tanaka et al. (196213) showed that tritiated ostreogrycin B and iriikarriycin A were absorbed from the gastrointestinal tract, and radioactivity was found in most tissues after slaughter of the animals. Maximum blood levels were obtained about 1 hour after oral doses. Dubost and Pascal (1965) found that humans dosed with 2 gm. of mixed ostreogrycins had blood levels of 0.4 pg./ml. after 1 hour, 0.9 pg./ml. after 2 hours, and 0.5 pg./ml. after 4 hours. These workers also showed that after the same dose 3 mg. were excreted in the urine in the first 2 hours after dosing and about 1 mg. in the following 2 hours. Scott and Waterworth (1958) treated 55 Ineu and women of age ranging 16-67 years with crude mixed ostreogrycins, orally, at a dose rate of 3 gm./day. No adverse effects were reported, and the patients, who had severe local staphylococcal infections progressed at about the same rate as a similar group dosed with 1 gm./day of erythromycin. It is possible that the ostreogrycins would have found a place in clinical practice but for the advent of better agents, and the difficulty of obtaining therapeutic blood levels, possibly due to selective adsorption of mikamycin A and ostreogrycin A on erythrocytes (Tanaka et nl., 1962a; Maillard and Pellerat, 1965). Shorin et n2. (1959) found that the maximum tolerated doses of echinomycin in rats, rabbits, and dogs were 140, 25, and 20 pg./kg., respectively. Thus the echinomycins are considerably more toxic than the actinomycins. Robinson and Waksman (1942) showed that mice tolerated a dose of mixed actinomycins of 150-250 pg./kg., intravenously, intraperitoneally, or subcutaneously, and 5-10 mg./kg. orally. In chronic toxicity tests 75% of the mice treated survived 30 days treatment with 25 pg./kg., though no rats survived this regimen. No rabbits survived a single dose of 1 mg./kg. These findings have been confirmed repeatedly in the last 27 years, for example, Hackmann (1954) working with rats and mixed actinomycins C found that 6 daily doses of 5 pg./kg. resulted in reduction in size of the spleen, thymus, and axillary lymph nodes, while the adrenals were enlarged. Study of the toxicity of three pure actinomycins (Pugh et al., 1956) did not invalidate the results given above. The figures obtained are given in Table XII; the 4-oxoproline derivative, actinomycin Xz, is the most toxic, its LDSobeing about 0.4 mg./kg. when dosed to mice subcutane-

MICROBIAL PEPTIDE-LACTONES

263

ously, while actinomycin B1 seems to be much less toxic -the spleen weight of mice receiving 0.5 mg./kg. being reduced by only 5%. V.

Conclusions

The chemistry of the peptide-lactones finds an honorable place in the edifice of modern organic chemistry, although there are still many difficult problems associated with the separation of closely related metabolites. Currently, depsipeptide chemists are concerned with the subtleties of conformation that govern the ability of the metabolites to form complexes with biopolymers and with smaller ionic species; the object of the research being an understanding of the function of these molecules in living systems. The techniques that are being used are, intellectually, of high stature and there is no doubt that much interesting chemistry will emerge. From a biological point of view, it is perhaps permissible, to question the relevance of this chemical approach. This scepticism may b e illustrated by the fact that, despite the work done on the chemistry of the reaction of actinomycin with DNA, no useful antitumor therapy has emerged. Other examples will readily occur to readers of these pages. To me, the inference is that the biological approach has been inadequate or nonexistent. There is no detailed knowledge of the distribution of the microorganisms producing peptide-lactones, with respect to climate, season, soil type, etc. It is not known (with the probable exception of the sporidesmolides) whether these metabolites are produced by the organisms growing in their natural habitat, and if so whether this production is a function of climate, etc. We are therefore ignorant of the role of these microorganisms and their metabolites in soil fertility and animal health. Has not the time arrived, when attention to such mundane problems is overdue? ACKNOWLEDGMENTS

I wish to thank my colleagues Dr. D. W. Russell and Dr. L. C. Vining who read and corrected the manuscript. Many colleagues in different parts of the world have been most helpful by sending me reprints of papers I had missed and typescripts of papers in the process of publication.

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Microbial Metabolites as Potentially Useful Pharmacologically Active Agents

D.

PERLMAN AND

G. P.

PERUZZOTTI

School of Pharmacy, The University of Wisconsin, Madison, Wisconsin I. Introduction .... .............. ............... 11. Types of Pharm A. Insulin-like Activity .............................................. B. Estrogenic Activity ............................................... C. ACTH-like Activity ............................................... D. Diabetogenic Activity ... E. Antispasmodic Activity. F. Antiinflammatory Activi G. Cardiotonic Activity .............................................. H. Salivation Induction .............................................. I. Emetic Activity .................................................... J. Epinepherine-like Activity. ........... K. Sedative .............................................................. L. Ergot, Muscarine, Serotonin, and Psilocybin Alkaloids ........................................................... M. Miscellaneous Activities ............ N. Toxicity ........ ...... ......................... 111. Summary ................................................................... References .................................................................

I.

277 278 278 279 280 280 280 28 1 282 283 284 284 285 285 287 287 288 288

Introduction

The continuing successes of microbiologists and chemists in finding among microbial metabolites clinically useful antibiotics has stimulated a renewed interest in screening cultures for antibiotic production. These promising research programs together with the complete chemical characterization of over 500 antibiotics has convinced many chemists that microbial metabolites constitute an inexhaustable untapped reservoir of new types of interesting chemical structures. Despite the brilliant chemical syntheses proposed in some-laboratories for preparation of a number of the important antibiotics, large-scale production of most antibiotics is still most economic by the timetested fermentation processes. However, the demonstrated advantages of the semisynthetic penicillins, the cephalosporins, the tetracyclines, the lincomycins, and the rifamycins have encouraged many chemists to try to prepare, by chemical modifications of the natural product, significantly improved chemotherapeutic agents. These studies have led, in turn, to a better understanding of the chemical structure/biological activity relationships in certain antibiotics, e.g., 277

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the penicillins, and eventuully to much better design of research programs for preparation of antibiotics. Although most of the interest in microbial metabolites has been channeled toward observations of their antibiotic properties, there have been several studies of certain mebhol ites which have other ~~linriiiacological/biolo~ic~~l properties. Indeed, considering the small number of nietabolities examined, a relatively wide variety of pharrnacologicnl activities has been reported. Among those mentioned are: hypoglycemic activity, estrogenic activity, ACTH-like activity, chibetogenic activity, antiinflamrnatory activity, cardiotonic activity, emetic activity, sedative activity, and antispasmodic activity. In many instances the observations are not well documented, and most instances as rclativcly impure inaterials were tested, it is dif€icult to judge the value of many of the reports. However, this literature does show that rnicrobial metabolites should be corisidered in searching for new pharm~icologicallyinteresting compounds, and it is the purpose of this review to summarize some of the more interesting observations. II.

Types of Pharmacological Activity Reported

Since niany of the reports are fragmentary, we have elected to try to mention the highlights of all without too careful evaluation of the claims inade for the compounds and the procedures used to demonstrate the activity reported. This iiiay not b e entirely justified since some of the observations are at least 40 years old but, on the other hand, does present the whole literature and encourages the reader to examine the original reports.

A.

INSULIN-LIKE ACTIVITY

The early reports of the success of insulin-containing preparations in the treatment of diabetes, eiicouraged Wintcr m c l Smith (19234 to examine yeast and other products for this activity. They rioted that a preparation from yeast caused lowering of blood sugar level in rabbits upon injection and convulsions in rats. This was confirmed by Collip (1923a,b) and Funk and Corhitt (1923). ‘The onset for the yeast extract was slow, liowcver, and the duration of action was longer than that of insulin. Winter arid Srnith (1923b) tested an alcoholic extract of the yeast on 7 cliabeteq afflicted patients, k i d 5 showed a marked improvement. Further study b y Hutchinson et al. (i923a,b) showed that extracts from different sainples of commercial yeast differed widely in their

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279

activity. Working on the hypothesis that a contaminating microorganism in the yeast might be the “active” material, Hutchinson et al. (1923b) isolated a coliform bacillus which produced “insulin-like” activity. This hypothesis (that yeast per se was not directly involved) was substantiated when Winter and Smith (1925) reported that they had rejuvinated a yeast that had lost its ability to produce the insulinlike activity by growing the culture in a lactose-containing medium. Other accounts of the insulin-like activity of yeast include those of Simola (1927), Kaufmann (1928), and Shizume (1937). More recently K ~ i oet al. (1966a,b) noted that a protease from S treptomyces griseus displayed insulin-like effects on the metabolism of isolated fat cells from rat epididynial tissue. These effects included enhanced conversion of glucose to CO, and lipid, and repression of the lipolysis stimulated by corticotropin and norepinepherine. More than 95% of the proteolytic activity could be essentially eliminated by acid treatment without loss of the insulin-like properties. The authors suggest that the effect noted is on the cell membrane in a manner unrelated to the conventional enzymatic function. Although Hayashi et al. (1964) noted activity in vivo (rabbits) with the S. griseus preparations, Kuo et al. did not observe this.

B. ESTHOGENIC ACTIVITY In 1928 McNutt noted that sows consuming fungus-infected feed developed enlarged, tense, and elevated vulvae, enlarged mammary glands, and, in severe cases, prolapse of the vagina and rectum. Similar conditions were noted in Ireland (McErlean, 1952) when Fusarium infected barley was fed to sows. In another study the observations of vulvovaginitis in animals fed molded grain led Stob et al. (1962) to isolate a crystalline anabolic and uterotrophic compound from the Gibberella zeae infected corn by solvent extraction followed by column chromatography on magnesium silicate. In a similar experimental program Christiansen et al. (1965) examined the metabolic products from 97 fungal cultures from spoiled grain and noted that 12 Fusaria isolates produced materials which caused weight increases of the uterus in female rats. Two crystalline factors were isolated, one of which was shown to be ergosterol. The structure of an active material from Gibberella zeae was shown by Urry et al. (1966) to be an enantiomorph of 6( 10-hydroxy-6-oxo-transl-undeceny1)-P-resorcyclic acid lactone. This structure was confirmed by Taub et a1. (1967) who reported the total synthesis of the estrogenic material. An estrogenic response in female rats could be elicited

280

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by injecting intramuscularly as little as 20 mcg. of the material (named zearalenone by Urry et al., or designated as F-2 by Mirocha et al., 1967).In a mouse uterus assay this material was found to be 6.3 x times as active as estrone (Mirocha et al., 196th).T h e importance of zearalenone as a useful estrogenic agents has still to be determined; it or similar factor may play some role in the infertility of dairy cattle fed infected hay (Mirocha et al., 1968b). C. ACTH-LIKE ACTIVITY Nelson et al. (1954) noted a drop in adrenal ascorbic acid in hypophysectomized rats injected with residues from a bacitracin-containing feed supplement. Further study showed activity in concentrates of cells of bacitracin-producing organisms including Bacillus licheniformis and Bacillus subtilis, as well as Serratia marcescens and Lactobacillus leichmannii. Similar results were obtained by Chedid and Boyer (1958a,b,c) in their studies with Salmonella typhi and Salmonella enteritidis, who also noted the elevation of hepatic glycogen in the treated rats. In a somewhat related study, Danowski et al. (1962) noted development of Cushing’s syndrome in conjunction with Nocardia asteroides infection and suggested that the organism might produce an ACTHrelease, an ACTH-like polypeptide, or an adrenocortical steroid. D. DIABETOGENIC ACTIVITY Streptozotocin is an antibiotic complex which was isolated from Streptomyces uchromogenes (Vavra et al., 1960; Herr et al., 1960; Herr et al., 1967). Rakieten et al. (1963) noted that 100% of dogs and rats receiving a single intravenous dose of 50 mg./kg. body weight of streptozotocin developed a sustained and prolonged hyperglycemia. Histological examination of the islets of Langerhans in streptozotocintreated rats and dogs showed disruption of the islets and marked decrease or complete absence of granules in the beta cells (Junod et al., 1967; Arison et al., 1967).Animals given nicotinamide injections did not develop the diabetes (Dulin and Wyse, 1969; Schein et al., 1967).

E. ANTISPASMODIC ACTIVITY Colisan, a peptide antibiotic produced by a strain of Bacillus brevis (Leon and Bergmann 1965; Leon and Bergmann, 1968) isolated from a mesenteric lymph gland of a patient who succumbed to paralytic ileus (Reitler and Boxer, 1946), has high antiprotozoal activity and exerts a pronounced antispasmodic effect on the smooth muscle of the

METABOLITES AS PHARMACOLOGICAL AGENTS

28 1

intestine and other organs (Rudlich, 1957). It produces a contraction of the smooth muscle followed by a prolonged relaxation (Bergmann et al., 1960) and exerts its effect against any one of the following smooth muscle stimulants: acetylcholine, histamine, 5-hydroxytryptamine, nicotine, serotonin, and barium chloride; Bergmann et al. (1961; Leon and Bergmann, 1968) suggested that colisan effects the permeability of the cell membrane. Patulin, an antibiotic from Penicillium patulum (Birkinshaw et al., 1943), was found by Ambache (1958)to block contraction of isolated hamster colon induced by irin. It also inhibited contraction of isolated guinea pig ileum induced by nicotine, by prostaglandin, by pilocarpine, by histamine, by 5-hydroxytryptamine, and by acetylcholine (Eliasson, 1958). In a related study Ohashi et al. (1963) found patulincontracted tracheal muscle preparations could not be relaxed with atropine, with antihistaminies or antiserotonin agents. The polypeptide antibiotic, colistin, in high dosage can entirely antagonize the facilitative effect of neostigmine on contraction of striated muscle, and it can block the cumulative effects of neostigmine (Naranjo and d e Naranjo, 1966). They also concluded that the colistin acted directly on the striated muscle, and also at the level of the neuromuscular junction partly blocking the motor end receptors, and that it blocks cholinesterase.

F. ANTIINFLAMMATORYACTIVITY Griseofulvin, a metabolite from several species of Penicillium (Brian et al., 1955), was first isolated in 1939 from P . griseofulvum Dierckx (Oxford et al., 1939) and its structure determined some years later (Grove et al., 1951). The antifungal potency in vitro (Brian, 1949) against dermatophytes led to in vivo experiments in guinea pigs infected with Microsporum canis (Gentles, 1958).A highly inflammatory reaction which developed in all the control animals was prevented by the griseofulvin treatment (Gentles, 1958). Cochrane and Tullet (1959) investigated the active inflammatory cattle ringworm in man and found the rapid disappearance of inflammatory signs when treated with griseofulvin. Further examination of the antiinflammatory action of griseofulvin showed potency in the cotton pellet method in rats and the tuberculin skin sensitivity method in guinea pigs (D’Arcy et al., 1960). This potency was not associated with a cortisone-like action since griseofulvin had no activity in the coldstress tests or the liver glycogen deposition tests in adrenalectomized mice.

282

n.

PERLMAN AND G. P. PERUZZOTTI

G. CARDIOTONIC ACTIVITY Ozaki et al. (1954) first reported the cardiotonic activity of the polyene antifunga1 agent trichomycin on the p e r h s e d toad heart. A few years later Umeda et al. (1957) in a screen for cardiotonic agents using embryonic heart of O~yziaslatipes (Japanese killifish) found several metabolites from streptomycetes had activity. A number of antibiotics have been examined and the results summarized in Table I obtained. TABLE I CARDIOTONIC EFFECTS OF SOME POLYENE ANTIBIOTICS Antibiotic IIamycin

Trichomycin

Nystatin

Amount used

Obsclvtd cflcct

Test system

Heference

0.05 mcg./ml. Frog heart

Reduced force of contraction Dave et al., myocardial stimulation 1962 0.2 nicg./ml. Frog heart Increased cardiac output; in- Arora, creased diastolic tone; sys- 1962a tonic arrcst of ventricles 10 mcg./ml. Rat heart Systolic arrest; reduced Arora, water content; reduced K+; 1962b increased Na+ 0.05 mcg./inl. Amphihian and Increased myocardial con- Arora and mammalian tractility; increased Sinka, hearts cardiac output 1963 30 nicg./kg. Dogs (i.v.) Reduced amplitude of P Arora and wave; increased arterial Arora, pressiire; ventricular fibril- 1966 lation 0.05 nicg./nil. Frog heart Reduced force of contraction; Dave rt uZ,, in yocardial stimulation; in- 1962 creased cardiac output 0.2 mcg./ml. Frog heart Increased ccirdinc output; Arora, 1962a increased diastolic tone 0.5 incg./ml. Frog heart Systolic arrest of ventricles; Arora and incrcased K i tissue conArora, tent 1963 0.5 mcg./ml. Habbit heart Systolic arrest of ventricles Arora, 1963a I mcg./ml. Rat heart Systolic arrest; reduced K+; Arora and increased Nn+ Arora, 1964 0.5 mcg./ml. Guinea pig Systolic arrest; rcduced K+; Arora, heart increased Na+ 1966a 20 nicg./ml. Frog heart Systolic arrest and reduced Arora, amplitude of contraction l963b --

~~

~

(continued)

METABOLITES AS PHARMACOLOGICAL AGENTS

283

TABLE I (Continued) Antibiotic

Pimaricin

Amount used

Test system

30 mcg./ml.

Rat heart

40 nicg./ml.

Rabbit heart

40 mcg./ml.

Guinea pig heart Guinea pig heart

35 i n c d m l .

35 mcg./ml.

Rabbit heart

Pentam ycin

20 mcg./ml.

Guinea pig heart

Lagosin

2 mcg./ml. 6 mcg./ml.

Guinea pig heart Frog heart

6 mcg./ml.

Rat heart

Fungichromin 10 mcg./ml. Eurocidine

20 mcg./ml.

Endomycin

20 mcg./ml.

Candidin

20 mcg./ml.

Ainphotericin 15 mcg./ml.

Guinea pig heart Guinea pig heart Guinea pig heart Guinea pig heart Rabbit heart

Observed effect

Reference

Systolic arrest and reduced amplitude of contraction Systolic arrest and reduced amplitude of contraction Systolic arrest; increased Na+;reduced K+ Systolic arrest of ventricles; increased Na+; decreased Ki Increased amplitude of contractions; systolic arrest; increased coronary flow Diastolic arrest of ventricles; decreased K+; decreased coronary flow Decreased K+; H 2 0content increase Increased amplitude of contractions; increased cardiac output; systolic arrest of ventricles Increased amplitude ofcontractions; increased cardiac output Decreased K+; increased H 2 0content Decreased K+; increased H 2 0content Decreased K+; increased H 2 0content Decreased K+; increased HzO content Decreased amplitude of contractions; diastolic arrest of ventricles; decreased coronary flow

Arora, 1964a Arora, 1964a,h Arora, 1966a Arora, 1965a Arora, 1966b Arora, 1965a Arora, 1965b Arora, 1965c

Arora, 1965c Arora, 196513 Arora, 1965b Arora, 1965b Arora, 1965b Arora, 196613

H. SALIVATIONINDUCTION

Numerous reports of excessive salivation in dairy cattle and horses after feeding on second cutting red clover were received by Byers and Broquist (1960, 1961). Preliminary study of the factor involved (given the trivial name “slobber factor”) showed alkaloid properties including a positive Dragendorf test. Smalley et al. (1962; Crump et al., 1963) studying the same problem determined that a culture of Rhixoc-

284

D. PERLMAN AND G. P. PERUZZOTTI

tonia leguminicola was involved in the spoilage and produced the active material. The pure material was isolated by Rainey et al. (1965) and by Aust and Broquist (1965) from R. leguminicola fermentations. Aust et al. (1966) and Whitlock et al. (1966) characterized the material as l-acetoxy-8-amino-octahydroindolizidineand gave it the trivial name of “slaframine.” This was later revised to (lS,GS,8aS)-l-acetoxy6-amino-octahydroindolizine(Gardiner et al., 1968). Pharmacological studies by Aust et al. (1968) showed that slaframine is not the active compound but is converted to the active metabolite by liver microsoma1 enzymes. It is a potent stimulator of exocrine glands, and stirnulates pancreatic activity.

I. EMETICACTIVITY Dounin (1926) observed the widespread occurrence of Fusaria on the cereal crops in Russia and suggested that the illnesses (vertigo, headache, nausea, and emesis) resulting from consumption of bread made from the Fusarium-spoiled grain might be related to the metabolism of the fungus. Sometime later Mundkin and Cochrane (1930), Roche et al. (1930),and Dickson et al. (1930) all described feeding experiments in which scabbed barley or an extract of the spoiled grain induced nausea and emesis in pigs, horses, and dogs. The emetic principle in stored barley was found to remain active after 56 months storage in a grainery (Shands, 1937). In 1941 Prentice et al. noted that a number of species of Fusarium including F. moniliforme, F. gramineurum, F. avenaceum, F. poae, F. sporotrichoides, F . equestri, F. culmorum, F. nivale, F. roseum, and F. scirpi var. acuninatum (Prentice and Dickson, 1968) produced emetic materials when grown in laboratory media. The active substance(s) was extractable from scabbed grain, too, by water (Dickson et ul., 1930) and by methanol or ethyl ether (Hoyman, 1941), and appeared to be the same material isolated from Fusarium moniliforme grown in submerged culture (Prentice, 1962). A polypeptide nature was suspected (Yrentice and Dickson, 1968) though not proved.

J.

EPINEPHERINE-LIKE ACTIVITY

Williams et aZ. (1967) presented evidence that crude filtrates from Bacillus anthracis cultures contain a substance that mimics the vascular effects of epinepherine when injected intravenously into pithed rats. An initial transient drop in blood pressure was noted followed by marked elevation in both systolic and diastolic pressure.

METABOLITIES AS PHARMACOLOGICAL AGENTS

285

This was in turn followed by a diastolic pressure drop leaving a widened pulse pressure. The conditions for production of the active principle differ from production of the anthrax toxin.

K. SEDATIVE Monorden, an antibiotic from Monosporium bonorden (Delmotte and Delmotte-Plaquee, 1953) also known as radicol (Mirrington et al., 1964; McCapra et al., 1964) was reported to have remarkably low toxicity, while acting as a potent sedative without other obvious side effects on the nervous system. In a systematic screen of fungus products for inhibitors of dopamineP-hydroxylase, Hidaka et al. (1969) noted that fusaric acid (5-butylpicolinic acid) was especially effective. Intraperitoneal injection of fusaric acid at a dose of 20 mg./kg. into rabbits, rats, cats, or dogs caused significant decreases in blood pressure from about 30 minutes to 6 hours after the injection. When the dose was increased to 50 mg./kg., the blood pressure decrease was greater and the lowered pressure was maintained for more than 24 hours. After an injection of 100 mg./kg. there was a marked decrease of norepinepherine in the heart and almost complete recovery after 12 hours. The decrease of norepinephrine in the brain was slight in both brain and spleen. The decrease of norepinephrine in the heart and adrenal is thought to be due to inhibition of dopamine-P-hydroxylase by the fusaric acid and the decrease of norepinephrine in angiovascular system is thought to be the cause of the hypotensive effect.

L. ERGOT,MUSCARINE,SEROTONIN, AND PSILOCYBIN ALKALOIDS Two hundred years ago Munchhausen (1765) showed that ergot is a fungus and not a deformed rye seed. A century later following Tulasne’s taxonomic studies and Brefeld’s pure culture isolations (1881), research on ergot alkaloid production by Claviceps was in progress in a number of laboratories. Today there is an extensive literature on the chemistry (Stoll and Hofmann, 1965; Hofmann, 1965), the microbiological production (Abe and Yamatodani, 1964; Vining and Taber, 1963), and the pharmacology (Hoffer, 1965; Rothlin, 1957) of both naturally occurring and semisynthetic compounds. The ergot alkaloids include the clavine group and the lysergic acid and isolysergic acid alkaloid group. With only one exception, these compounds all contain the tetracyclic nucleus ergoline with a substituent at C-8 and a methyl group at N-6. The claving alkaloids are only nonergoline ergot alkaloids known are the chanoclavines. The

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D. PERLMAN AND G . P. PEHUZZOTTI

nonclavine alkaloids occur in nature in epimeric pairs differing in configuration around C-8. These compounds are amide derivatives of lysergic acid and isolysergic acid. Ergometrine and ergometrinine are examples of compounds which are simple arnide derivatives, while ergotanline and ergotaminine are examples of the series with complex polycyclic peptide side chains. The biologically active ergot compounds belong to the lysergic acid series. Abe was the first to demonstrate ergot alkaloid production by Claviceps species in saprophytic culture (Abe and Yamatodani, 1964). Continued screening of Cluviceps strains showed that a strain of C.litoralis Kawatani produced peptide alkaloids. Stoll e t al. (1965; Vining and Taber, 1963) confirmed the production of agroclavine and elymoclavine. In 1961 Arcamone et al. described a strain of C. paspali which produced large quantities of a-hydroxyethyllysergamide together with small quantities of lyserganiide. More recently Tonolo (1966) described a strain of C. purpurea which produced a large amount of ergotamine in submerged culture. Muscarine was one of the first substances discovered which reproduced some of the responses to stimulation of the parasympathetic nervous system. Although muscarine is usually associated with Amanita muscaria it has also been found in several other fungi including: Arnanita pantherina, Boletus luridus, Boletus satanas,

Clitocybe rivulosa, Clitocybe dealbata, lnocybe asterospora, Inocybe horigardi, Znocybe cookei, Inocybe lateracia, Znocybe fostigiata, Znocybe napipes, Inocyhe patouillardi, lnocybe umhrina, Inocybe rimosa, and Russula emeticu. The history, chemistry, and pharmacology of muscarine has been reviewed b y Bowden and Mogey (1958), Waser (1961,1965),and Wilkinson (1962). Serotonin and 5-hydroxytryptophari have been found in the following Panaeolus species (Benedict and Brady, 1969): P. campanulatus, P. foenesecii, P. acuminatus, P. fontinalis, P. semiovatus, P. subbalteatus, and P. texensis. Both were absent in P. solidipes. Psilocybin and psilocin have been isolated from Psilocybe mexicana (Hofmann et al., 1958) and other fungi (Benedict and Brady, 1969):

Conocybe cyanopus, Conocybe smithii, Panaeolus subhalteatus, Psilocybe aztecorum, Psilocybe haeocystis, Psilocybe caerulescenes, Psilocybe cubensis, Psilocybe cyanescens, Psilocybe fimeruria, Psilocybe pelliculosa, Psilocybe quebecensis, Psilocyhe semilaneata, Psilocybe semperviva, Psilocybe wassonii, Psilocybe yungensis, and Psilocybe zapotecorum. These mushrooms are used in induce intoxi-

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287

cation and hallucinations (Hofmann, 1960). When grown in laboratory media the carpophores, sclerotia, or mycelia formed contain psilocybin and psilocin (Catalfomo and Tyler, 1964; Hofmann et al., 1958; Heim and Hofmann, 1958; Leung et al., 1965; Singer, 1958).

M.

MISCELLANEOUSACTIVITIES

Griseofulvin, an antifungal agent frequently used to control ringworm of the scalp and nail bed infections, was found (DePasquale et al., 1963) to give relief to patients suffering from angina pectoris. Schaffner and Gordon (1968) noted that heptene macrolide antifungal agents including candicidin B and amphotericin B, as well as the pentene filipin reduced serum cholesterol levels when the antifungal agents were orally administered to dogs for 3 weeks. This treatment produced a reduction in the volume of the prostate gland in young dogs, and marked gland volume reductions in old dogs with established natural prostatic glandular hyperplasia (Gordon and Schaffner, 1968). A microorganism identified as Oospora astringenes (Yamamoto and Yamamoto, 1960) isolated from air of an asthmatic patient’s room produced, in liquid culture media, 3 isocoumarin derivatives named oospolactone, oosponal, and oosponglycol (Yamamoto, 1961; Yamamot0 et al., 1961; Yamamoto et al., 1962; Nitta et al., 1963a,b,c).The pharmacological effects of the group were determined using guinea pig trachea where 10 mcg./ml. of oosponol resulted in tracheal muscle contraction in the in vitro experiments (Ohashi et al., 1962).This contraction was not antagonized by atropine, by antihistaminics, or by antiserotonics. The oospoglycol showed weak relaxing action in these tracheal muscle preparations.

N. TOXICITY The majority of observations of the pharmacological effects of antibiotics have been tests of the toxicity of the materials. These have been usually carried out on semipure or purified materials in studies coordinated with efficacy tests. If the therapeutic indexes are less than 2, the substances are usually considered too toxic to be worthy of further study. Umezawa (1967) includes toxicity information in his survey of antibiotics produced by streptomycetes, and additional information is found in various handbooks (Altman and Dittmer, 1964; Spector, 1956). The mycotoxins and other fungal products have recently been of considerable interest as the economic effects of these substances have

288

D. PERLMAN AND G . P. PERUZZOTTI

TABLE I1 TOXIC FUNGALMETABOLITES ~~

Type of toxicity

Name of compound

Microbial source

Reference

Ruhratoxins Aand B Penicillium rubrum Burnside et al., 1957 Moss et al., 1968 Townsend et al., 1966 Wilson and Wilson, 1962 Amanitins and Phal- Amanita phalloides Wieland, 1963 loidins Hepatotoxicity and Aflatoxins AspergihsfEauus Wogan, 1966 Schoental, 1967 hepatocarcinogenic Citreoviridin Penicillium citreo- Sakahe et al., 1964 uiride Pithomyces chart- Hodges etal., 1963 Hepatotoxicity and Sporidesmins facial eczema arum Hemorrhagic Unnamed scirpene Fusarium tricinctum Bamhurg et al., 1968 Gilgan et al., 1966 Hemorrhagic and Crotocin Cephalosporium Glaz et ul.. 1966 ' CNS crotocinigenium Mirocha et al., 1968a Penicillium species Mirocha et al., 1968a Inflammatory Verrucarins A to J Myrothecium Bamburg et ul., 1968 verrucaria Roridins A to E Myrothecium Harriet al., 1862 roridum Muconomycins A Myrothecium Guarino et al., 1968 uerrucaria and B Trichothecin Trichothecium Freeman et al., 1959 roseum Freernan and Gill, 1950 Freeman and Morrison, 1949 Freeman, 1955 Godtfredsen and Vangedal, 1964 Irritant Teleocidin Streptomyces Nakata et al., 1966 species Takashima and Sakai, 1960 Scirpene Fusarium scirpi Brian et al., 1961 Gangrenous Butenolide Fusurium piuale Yates et al., 1967a,b Aspergillus Van der Merwe et al., Fatty infiltration of Ochratoxin A liver ochraceus 1965 Nephrotoxic Penicill ium Sakai, 1955 Citrinin citrinum Phototoxic Psoralens Sclerotinium Perone et al., 1964 sclerotiorum Scheel et al., 1963

H epatotoxicity

METABOLITES AS PHARMACOLOGICAL AGENTS

289

been considered. Many have been examined for their mechanisms of toxicity, whiIe the study of others has been concerned with the relationship of structure to activity (Brook and White, 1966; Ciegler and Lillehoj, 1968; Borker et al., 1966; Townsend, 1967). Some of the effects of selected compounds are listed in Table 11. Ill.

Summary

The studies of microbial metabolites for pharmacological activity mentioned above have often been by-products of programs studying these compounds for other therapeutic effects, and since the interest has usually involved negative attributes, e.g., toxicity, it is not surprising that most of the observations have not been encouraging as far as discovering useful pharmacological agents. Perhaps if the surveys of the compounds were oriented toward finding positive, useful compounds for certain syndromes, a number of leads worthy of exploitation would result. Bohonos and Piersma (1966) pointed out that many of the compounds discovered in screening microbial metabolites for various pharmacologic activities were new types of chemical structures differing from those cansidered to have these pharmacologic activities. It is likely that exploitation of these leads will eventually result in better understanding of both the pharmacologic activity and the chemical structures needed for these activities. REFERENCES Abe, M., and Yamatodani, S. (1964).Progr. Ind. Microbiol. 5,205-229. Altman, P. L., and Dither, D. S. (1964).“Biology Data Book.” Federation of American Societies of Experimental Biology, Washington, D.C. Ambache, N. (1958).J.Physiol. 140,24P. Arcamone, F., Chain, E. B., Ferretti, A., Minghetti, A., Pennella, P., Tonolo, A., and Vero, L. (1961).Proc. Roy. Soc. (London)B155,26-54. Arison, R. N., Ciaccio, E. I., Glitzer, M. S., Cassaro, J. A., and Pruss, M. P. (1967). Diabetes 6,51-56. Arora, H. R. K. (1962a).J.Pharm. Pharmacol. 14,320. Arora, H. R. K. (196213).Med. Exptl. 7,280-286. Arora, H. R. K. (1963a).Med. Exptl. 11,88-94. Arora, H. R. K. (1963b).J.Pharm. Pharmacol. 15,562. Arora, H. R. K. (1964a).J.Pharm. Pharmacol. 16,356-358. Arora, H. R. K. (1964b).Med. Exptl. 10,239-244. Arora, H. R. K. (1965a).Med. Pharmacol. Exptl. 12,239-244. Arora, H. R. K. (1965b).Med. Pharmacol. Exptl. 13,57-62. Arora, H. R. K. (1965~). Med. Pharmacol. Exptl. 13,155-160. Arora, H. R. K. (1966a).Med. Pharmacol. Exptl. 14,571-575. Arora, H. R. K. (1966b).Med. Pharmacol. Exptl. 14,98-103.

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Arora, H. R. K., and Arora, V. (1963).Med. E x p t l . 9:l-4. Arora, H. R. K., and Arora, V. (1964).Arch. Intern. Pharmacodyn. 150,451. Arora, H. R. K., and Arora, V. (1966).Med. Pharmacol. E x p t l . 15,473. Arora, H. R. K., and Sinka, Y. K. (1963). Ind.J. Med. Res. 51,453-463. Aust, S.D., and Broquist, H. P. (1965).Nature 205,204. Aust, S. D., Broquist, H. P., and Rinehart, K. L. (1966). J . Am. Chem. Soc. 88, 28792880. Aust, S. D., Broquist, H. P., and Hinehart, K. L. (1968). Biotech. Rioeng. 10,403-412. Bamburg, J. R., Marasas, W. F., Riggs, N. V., Smalley, E. B., and Strong, F. M. (1968). Biotech. Bioeng. 10,445-455. Benedict, R. G., and Brady, L. H. (1969). In “Fermentation Advances” (D. Perlman, ed.). Academic Press, New York. Bergniann, F., Reitler, R., Chaimovitz, M., and Bergmann, D. (1960).B r i t . / . Phurmucol. 15,313-318. Bergmann, F., Chaimovitz, M., Leon, S. A., and Preiss, B. (1961). Brit. J . Phamnacol. 18,302-310. Birkinshaw, J. H., Michael, S. E., Bracken, A., and Raistrick, H. (1943). Lancet 245, 625-630. Bohonos, N., and Piersma, H. D. (1966).Bioscience 16,706-714. Borker, E., Insalata, N. F., Levy, C. P., and Witzman, J. S. (1966).Aduan. Appl. Microb i d . 8,315-351. Bowden, K., and Mogey, G. A. (1958).J.Pharm. Pharmacol. 10,145-156. Brefeld, 0 . (1881). “Botanische Untersuchingen Ubcr Schimmelpilze.” H. Schonigh, Munster. Brian, P. W. (1949).Ann. Botany (London)13,59-77. Brian, P. W., Curtis, P. J., and Hemming, H. G. (1955). Trans. Brit. Mycol. Soc. 38, 305-308. Brian, P. W., Dawkins, A. W., Grove, J. F., Hemming, H. G., Lowe, D., and Norris, C. L. F. (1961).J.Exptl. Botany 12,l-12. Brook, P. J., and White, E. P. (1966).Ann.Reu. Phytopathol. 4,171-194. Burnside, J. F., Suppel, W. L., Forgacs, J., Carill, W. T., Atwood, M. B., and Doll, E. R. (1957).Am.J.Vet. Res. 18,817-824. Byers, J. H., and Broquist H. P. (lY6O)./.Dairy Sci. 43,873. Byers, J. H., and Broqnist, H. P. (1961).J.Dailrj Sci. 44,1179. Catalfomo, P., and Tyler, V. E. (1964).Lloydia 27,53-63. Chedid, L., and Boyer, F. (1958a).Compt. Rend. Acad. Sci. 246,2664-2667. Chedid, L., and Boyer, F. (195813).Compt. Rend. Acad. Sci.246,2801-2804. Chedid, L., and Boyer, F. (1958~). Compt. Rend. Acud. Sci. 246,2937-2940. Christensen, C. M., Nelson, G. H., and Mirocha, C. J. (1965). Appl. Microbiol. 13, 653-659. Ciegler, A., and Lillehoj, E. B. (1968).Aduan. Appl. Microbiol. 10,155-219. Cochrane, T., andTullet, A. (1959). Brit. Med.]. 2,286-287. Collip, J. B. (1923a)J. Biol. Chem. 56 513. Collip, J. B. (1923b).Proc. Soc. E x p t l . Biol. Med. 20,321-323. Crurnp, M . H., Smalley, E. B., Henning, J. N., arid Nichols, R. E. (1963)./.Am. Vet. Med. ASSOC. 143,996-997. Danowski, T. S., Cooper, W. M., and Braude, A. (1962). Metub. Clin. E x p t l . 11, 265272. D’Arcy, P. F., Howard, E. M., Muggleton, P. W., and Townsend, S. B. (1960).J . Pharm. Pharmacol. 12,659-665.

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AUTHOR INDEX Numbers in parentheses are reference numbers and indicate that an author’s work is referred to althoueh his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed. v

A

Abe, M., 208,263,285,286,289 Acar, J. F., 256, 266 Adam, J., 208, 273 Adelburg, E. A,, 93, 118 Aida, H., 22(129), 31 Aida, K., 15(64, 80, 85, 86, 88), 27(183), 30,31,33 Alas, G., 60, 61, 62, 85 Albertini, A., 237, 238, 263,266 Albertsson, P. A,, 147, 150 Albuquerque, F., 208,267 Albury, M. N., 69,83,86,87 Aldanova, N. A,, 213, 221, 222, 228, 264, 273, 274, 276 Alderton, G., 148, 152 Alieva, R. M., 20(123), 21(123), 31 Allen, M. B., 116 Allwood, M. C., 101, 105, 106, 107, 109, 110, 113, 114,116 Almoff, J. W., 164(38), 185 Altman, P. L., 287,289 Amano, K., 72,83 Ambache, N., 281,289 Ambler, C. M., 126, 127, 128, 150 Amburgey, J. W., 129, 130,151 Amelunxen, R. E., 94, 116 Amerine, M. A., 36, 37, 38, 40, 53, 5 4 5 6 , 57, 58, 59, 60, 61, 62, 72, 83, 86 Ames, D. E., 209,217,223,264 Ameyama, M. 19(116), 27(116, 176, 177), 31,33 Amici, A. M., 190, 274 Amoore, J. E., 39, 40, 83 Anderson, B., 208,264 Anderson, N. G., 128,129,130,151,152 Anderson, P. A. 184(97), 187 Andreoli, T. E., 246,264,275 Andrews, F. N., 279,293 Anslow, W. K., 195,276 Antoniani, C. E., 48, 83 Antonov, V. K., 209, 210, 211, 218, 222, 229, 230, 246,247,264,273,274,276

Araki, T., 208,246,270,274 Arcamone, F., 191, 193,208, 264,286,289 Archer, G. T., 60,83 Argainon, M., 104, 106, 118 Argoudelis, A. D., 280, 291 Arima, K., 282, 293 Arison, R. N., 280, 288 Arkima, V., 46, 83 Arnold, B. H., 176(72), 186 Arnold, R. B., 203, 212,215, 230,264 Arora, H. R. K., 282, 283, 288, 290 Arora, V., 282, 290 Arsenault, G. P., 221,264 Arwidsson, B., 249, 264 Asai, I. 14(44), 25(146), 26, 27(165, 183), 29,32,33 Asano, K., 25(148), 32 Asao, Y., 73, 83,88 Ashwood-Smith, M. J., 110, 116 Atwood, M. B., 289,290 Augustin, J. A. L., 96, 118 Ault, R. G., 11(5),28 Aung Tun-kyi, 233,272 Aust, S. D., 284, 290 Averyanova, V. V., 14(38), 29 Avigad, G., 15(81, 82, 83, 84), 30 Avron, M., 252, 264 Axel, R., 94, 108, 117 Axelrod, D., 245, 269 Axelrod, E., 244, 266 Ayapaa, T., 46, 83 Azzi, A., 248, 264 Azzone, G. F., 248, 264 B

Baarda, J . R., 249, 268 Babel, F. J., 64, 84 Bachmann, G., 190,197 Bachrach, H. L., 148,151 BHcikovi, D., 254, 258, 264 Badings, H. T., 67, 83 Baerfuss, A., 173, 186 Barwald, G., 46, 47, 84

295

296

AUTHOR INDEX

Baird, D. K., 11(5), 28 Baird-Parker,A. C., 113,116 Baker, R. S., 130,152 Baldwin, R. L., 110, 116 Baldwin, R. S., 279,293 Ball, C. L., 214,275 Ball, S., 195,276 Ballotta, R., 208, 272 Baltscheffsky, H.,249, 264 Barnburg, J. R., 289, 290 Bandyopadhyay, B., 175(60), 186 Bangharn, A. D., 246, 268 Bangs, S . E., 145, 151 Bantz, A. C., 14(31, 34), 29 Barber, M., 212, 218, 264 Barns, A. A,, 159(27), 185 Baron, S., 245, 269 Barringer, H. P., 129, 130, 151, 152 Bartholomew, W. H., 171, 186 Bartz, Q. R., 195, 197, 254, 264, 268 Basmadjian, G., 235, 264 Bates, F. J., 15(53), 30 Battifora, H., 244, 266 Baudet, P., 208, 264 Bailer, E., 54, 85 Bavisotto, V. S., 46, 48, 83 Beaven, V., 237,275 Becker, B,, 215,229 Bccker, F. F., 244, 264 Becker, K., 47,48,88 Beckett, A., 105, 116 Beech, F. W., 62, 63, 86 Beetschen, J. C., 245, 266 Behal, V., 13(30),29 Beidler, L. M.,36, 37, 39, 83 Beilby, A. L., 209, 266 Belehradek, J., 115, 116 Belloc, A., 212,271 Belova, I. P., 212, 271 Belova, Z. N., 193, 208, 264 Belyakova, M . S., 15(58), 30 Bknazet, F., 257, 262, 264 Benedict, R. G., 286,290 Benvegnin, L., 59, 83 Berg, C., 38, 83 Berg, H., 240, 264 Berg, H. W., 57, 58, 84 Berg, R. W., 247, 269 Berger, J., 212, 264 Bergmann, D., 281, 290

Bergmann, F., 280, 281, 290, 292 Bergy, M. E., 280, 291 Berman, A. S., 129, 130, 151 Bernhauer, K., 12(16), 13(25), 15(52), 17(98, RQ),19,27(99,104),29,30,31 Bernsee, G., 15(70,71),30 Berridge, N. J., 65,66,85 Bertaud, W. S., 192, 201, 202, 211, 235, 252,264 Bertazzoli, C., 208, 264 Bertrand, G., 12, 29 Bcssell, C. J., 201, 266 Betina, V., 254,258, 264 Beuchat, L. R., 107, 116 Bevan, K., 198, 264 Bezzubov, A. A., 62, 87 Bhacca, N. S., 285, 292 Biemann, K.,221, 264 Bier, M., 148, 151 Birkinshaw, J. H., 281, 290 Bishop, E., 194, 208, 211, 216, 253, 264 Bittera, V., 195,268 Blick, S. R., 48,83 Boaz, H. E., 193,208,267 Bobadilla, G. F., 60,83 Bochkarev,V. N.,221,222,264,276 Bodanszky, A,, 208,232,265 Bodanszky, M., 196, 201, 208, 211, 232, 265,270 Boehni, E., 228,274 Boeseken, J., 12(17), 29 Boezardt, A. G. J., 19(107), 31 Bogar, F. D., 145, I52 Bohonos, N., 194, 197,198,208,273,276, 279,288,290,291 Bohstedt, G. H., 284, 292 Boldt, P., 222, 265 Bolhafer, W. A., 194, 267 Boller, A., 211, 271 Bone, D. H., 27(179), 33 Bonner, J., 244,276 Boone, C. W., 131,152 Boothroyd, B.,201, 267 Borisova, V. N., 193, 265 Borker, E., 288, 290 Borkowski, J. D., 175(63, 66), 176(63), 184(94),186,187 Bossi, R., 194,208,265 Bowden, K., 286,290 Bowers, W. F., 237,270

297

AUTHOR INDEX

Bowman, R. E., 209, 217, 223,264 Boxer, J., 280, 292 Boyd, W. C., 38,83 Boyer, F., 280,290 Boyles, W. A., 144, 151 Bracken, A., 281, 290 Brady, L. R., 286, 290 Brandt, J. F., 111, 116 Braude, A., 280,290 Braun, A . C., 208,276 Brazhnikova, M. G., 189, 193, 208, 212, 265, 274 Brefeld, O., 285, 290 Brennan, J.. 195, 213, 265 Brenner, M. W., 48, 83 Brian, P. W., 281,288,290 Bridges, B. A., 110, 116 Bridtker, N., 94, 109, 110, 117 Briggs, L. H., 194,208,210,217,222,265 Brock, A., 286, 287, 291 Brockmann, H., 190, 191, 194, 195, 197, 198,200,208,213,214,216,218,222, 225,239,240,242,243,255,265 Brook, P. J., 288, 290 Broquist, H. P., 283, 284, 290, 291 Brown, D. J., 144, 151 Brown E. V., 17(100), 20,21(100), 22(100), 31 Brown, R., 195, 213, 265 Brown, R. H., 249, 269 Brown, W. E., 154(4), 160(4), 162(4), 171(4), 185 Bmch, C. W., 96,116 Brucknew, G. C., 148,151 Brumsted, D. D., 49, 83 Bryson, V., 184, 187 Bucek, W., 19(113), 20(113), 31 Buckley, S. M., 258, 272 Buckner, D., 132,151 Bucks, E. J., 208,269 BudkSinsky, Z., 12(Y), 13(9), 28 Bujas, Z., 38, 83 Burch, G. E., 287,291 Burckhardt, E., 211, 274 Burdon, R. H., 244, 265 Burge, R. E., 103, 104, 116 Burikov, V. M., 220, 269 Burke, B., 90, 116 Burkhardt, H. J., 288,294 Burks, J. W., 287, 291

Burleigh, I. G., 110, 115, 116 Burnside, J. F., 288,290 Burris, R. H., 249, 271 Busch, P. L., 142, 143, 151 Bush, F., 176(68), 177(68), 179(68), 186 Butler, G. W., 191,235, 238,265 Button, D. K., 172(49), 184(49), 186 Butts, R. M., 69, 70, 86 Buzhinsky, 246,269 Byers, J. H., 283, 290 Bygrave, F. L., 249,265 Byrne, P., 106, 116 C

Califano, L., 105, 107, 116 Callow, D. S., 154(3), 161(3), 162(3), 173 (55), 185,186 Calvin, A. D., 38, 87 Cameron, J., 161, 185 Campbell, L. L., 94, 108, 118 Capek, A., 192,230, 239, 271 Capelleri, G., 58, 84 Cappucci, C., 53,83 Capt, E., 59,83 Carill, W. T., 288,290 Carlin, G . T., 42, 44, 83 Carr, J. G., 15(172), 27(172), 32 Carrington, H. C., 11(5), 28 Carrion, J. P., 233, 272 Carritt, D. E., 175(61), 186 Carson, K. J., 104, 117 Carter, H. E., 195, 208, 266 Cartwright, N. J., 217, 266 Cassani, G., 237, 266 Cassaro, J. A., 280,289 Cassidy, H. G., 143, 151 Castor, J. G. B., 60, 83 Castro, A. J., 209, 266 Catalfomo, P., 287, 290 Catlin, G . , 248, 268 Caul, J. F., 39, 83 Cavalla, J. F., 209, 264 Celmer, W. D., 212,266, 268 Ceriotti, G . , 208, 266 Chabbert, Y-A., 256, 266 Chaimovitz, M., 281, 290 Chain, E. B., 154, 161, 162, 170(43), 18.5 186,191,193,264,286,289 Chambers, C. W., 91, 117

298

AUTHOH INDEX

Champagnat, A,, 35, 83 Chan, W. M., 257,269 Chao, K.-C., 284, 293 Chapman, D., 106,116 Chapman, H. R., 65,66,85,86 Charles-Sigler, R., 196, 203, 266 Charney, J., 194, 195, 266, 267 Charpentie, Y., 212, 271 Chedid, L., 280,290 Cheldelin, V. H., 14(35), 15(65, 66, 67, 68), 29, 30 Chen-Chu Pao, 189,268 Cherbuliez, E., 208, 264 Chester, V. E., 141, 151 Chiasson, L. P., 96, 117 Chick, H., 90, 99, 117 Chikaike, T., 189, 270 Christensen, C. M., 279, 280, 289, 290, 392 Christensen, M. D., 69,83 Christopherson, J,, 94, 103, 117 Ciaccio, E. I., 280,289 Ciegler, A., 288, 290 Ciferri, O., 237, 238, 263, 266 Clark, L. C., 175, 186 Clarke, D. A., 258, 272 Clarke, R. T. J., 191, 235, 238, 265 Clendenning, J. R., 145,152 Cline,G. B., 129,130,151,152 Cochrane, R. L., 284,292 Cochrane, T., 281,290 Cocito, C., 257, 266 Cockrell, R., 248, 268 Cockrell, R. S., 248, 266 Coffey, G. L., 254,255,256,258,261,266 Coffinan, J. R., 43, 44, 87 Cogati, C. V., 282, 291 Coggins, R. A., 15(90, 172), 27(172), 31, 32, 62, 88 Cohen, L. A., 229,266 Cole, E. W., 43, 83 Colebrook, L. D., 194, 210, 217, 265 Collip, J. B., 278,290 Cone, C., 221,264 Cook, A. H., 193, 198,208,266 Cook, P., 246,275 Cooke, G. M., 55, 85 Cooney, D., 280,293 Cooper, F. C., 148,151 Cooper, W. M., 280,290

Corbaz, R., 194, 212, 255, 266 Corbitt, H. B., 278, 291 Corner, T. R., 107, 117 Convin, A. H., 209,266 Cosar, C., 257, 262,264 Costich, E. W., 158(20),185 Coulon, T., 19(115),27(170),31,32 Cousin, D., 95, 117 Covert, A. S., 154(4), 160(4), 162(4), 171(4), 185 Cox, S. F., 193, 198, 208, 266 Craig, L. C., 240, 272 Crothers, D. M., 232, 240, 241, 243, 266, 270 Crowell, E. A,, 47, 52, 58, 61, 83,84, 85 Cruess, W. V., 53,54,56,57,58,59,83,87 Crump, M. W., 283,284,290,292,293 Csangi, E., 289,291 Cull, K. M., 255, 261, 266 Cummins, J . T., 15(65, 66), 30 Curda, D., 69,84 Curnutte, B., 43, 44, 85 Curran, C., 195, 266 Currie, J. F., 106, 11 7 Curtis, P. J., 281, 290 Cybulska, J., 256, 266 Cymbalista, S., 148, 152 Czarnecki, H. T., 48, 83 Czulak, J., 66,83 D

Dahlstrom, D. A,, 137, 138,151 Dalgliesh, C. E., 193, 266 D’Angio, G. J., 258, 270 Daniels, S. L., 146, 151 Daniels, W. F., 140, 151, 178(76),186 Danyluk, S. S., 214,275 Dann, M., 194, 197, 198, 208, 276 Danowski, T. S., 280, 290 D’Arcy, P. F., 281, 290 Darlington, W. A., 147, 151 Das, B. C., 190, 208, 212, 218, 221, 264, 266,267,269 Datta, A. G., 27(169, 174, 175), 32 Dave, C. U., 282,291 Davenport, E., 113,116 Davies, E., 136, 151 Davies, H., 99, 110, 117 Davies, J. G., 65, 83

299

AUTHOR INDEX

Davies, J. S., 198, 264 Davies, J. T., 39, 83 Davis, B. R., 194, 210, 217, 265 Davis, N. S., 145, 151 Dawes, E. A., 110, 115,116 Dawkins, A. W., 288,290 Dawkins, P., 213, 271 Dawson, P. S. S., 15(59),30, 157(14), 182, 183(14, 91, 92), 185, 187 Day, E. A., 64, 65,85 de Barros Coelho, J. S., 208, 267 DeBoer, C., 280,293 de Bmyn, C. A. L., 20,31 DeClerk, J., 45, 84 DeFeo, J. J., 288,291 deFillippi, R. P., 132, 152 DeHaan, P. G., 179(83), 187 Deibel, R. H., 71, 84 Deindoerfer, F. H., 164(39), 173(56), 186 de Leeuw, F. J., 12(13), 29 Delmotte, P., 285, 291, 292 Delmotte-Plaquee, J., 285, 291, 292 Delmouzos, J. G., 70, 84 Delpierre, G. R., 196, 209, 217, 223, 266 Delvaux, E., 12(21),29 de Mayo, P., 209,223,267 de Naranjo, E., 281,288,292 Denison, F. W., 173(53),186 Denisov, Yu. V., 222,272,276 DePasquale, N. P., 287, 291 DeSomer, P., 395, 211, 254,256,266,287 Dettori, R., 101,118 Devreux, A., 47,85 Dickerson, R. E., 222,240,271 Dickison, H. L., 255,261,266,275 Dickson, A. D., 284,291,292 Dickson, J. G., 284,291,292 Diegelmann, R. F., 237,266 Dietz, A., 280,293 Dill, I. K., 246, 269, 279, 291 Dimmock, N. J., 102,117 Dingley, J. M., 191,252,266 Dingman, W., 244,266 D i t h e r , D. S., 287,289 Dloughy, P. E., 137, 138, 151 Doczi, B., 195,268 Dodd, E. A., 38,87 Doetch, R. N., 64,84 Dognon, A., 144,151 Doi, M., 16(188),33

Doll, E. R., 289,290 Dollar, A. M., 72,87 Domodaran, M., 14(48),29 Done, J., 191,211,252,266 Donovick, R., 195,223,266 Dorman, R. G., 164(40),186 Dornbush, A. C., 208,269 Doubler, M. R., 208,265 Doudoroff, M., 93,118 Dounin, M., 284,291 Draper, J. C., 103,104,116 Drews, B., 46,47,84 Dmmmond, P. E., 203,273 Dubost, M., 262,266 Dulin, W. E., 280,291 Duncan, C. J., 37,84 Dunn, C. G., 142, 151, 162, 171(33), 178, 185,186 Dunn, R., 5 3 , 8 5 Dunning, J. W., 12(18), 14(18), 29 Duprat, A. M., 245, 266 Dutcher, J. D., 195, 208, 223, 266, 275 Dworschak, R. G., 159(29), 185 Dyar, M. T., 149,151 Dyer, B. M., 195,276

E Eagon, R. J., 104, 117 East, D. N., 15(56),30 Eastwood, F. W., 196, 198, 199,201, 203, 209, 212, 215, 217, 223, 266 Eble, T. E., 280, 291 Ebner, H., 158(24), 171, 185 Ehrlich, J., 195, 254, 255, 256, 258, 261, 264,266 Eigner, J., 94, 109, 110, 117 Eijkman, C., 90,117 Eisel, R., 132, 151 Eisenstein, R., 244, 266 Elferdink, T. H., 156(8), 160, 163(8), 171(8), 173(8), 177(8), 178(8), 185 Eliasson, R., 281, 291 Elkhom, T. S., 245,267 Elliker, P. R., 65, 67, 86, 87 Elliott, J. A., 66, 84 Ellis, J. J., 288,294 Ellis, M. B., 194, 267 Elsaesser, T., 15(51),30 Elsasser, T., 13(29),29 Elsden, S . R., 172(47), 186

300

AUTHOR INDEX

Elsworth, R., 15(56), 30, 154, 178(79), 179(79),185, 187 Emme, I., 240, 270 Emmons, 1).B., 66,84 Enebo, L., 47, 48, 49, 50, 84,87 Enenkel, A., 158(24),171, 185 Engbloni, C., 175(63),176(63),186 Englard, S., 15(81,82, 83, 84), 30 English, A. K., 195, 254, 255, 256, 257, 267,274 Ennis, H. L., 245,267 Epshtein, R. B., 14(47),29 Erge, D., 235,264 Ettel, V., 23(136),26(136),32 Etthger, L., 194, 212, 254, 255, 266, 267 Evans, D. D., 209, 264 Evans, G., 208, 267 Evans, R. F., 47, 88 Everett, €1. J . , 158(20),185 Evison, L. If., 112, 117 Evstratov, A. V., 230, 246, 247, 274 F

Farber, C., 15(50), 23(135, 136, 137), 24(137, 138, 139, 140, 141), 26(135, 136),30, 32 Fallona, M. C., 209, 223, 267 Fance, W. J., 41, 84 Fantes, K. H., 201, 267 Farber, S., 258, 270 Fardig, 0. B., 194, 213, 268,270 Farmer, T. H., 193,198,208,266,267 Fatal, B., 148, 152 Fawcett, R., 157(18),L85 Fay, A. C., 91,117 Fay, 1. W., 21(127),31 Feduchy, E., 60,6l, 62,85 Feeney, J . , 289,292 Feigina, M. Yu., 221, 222, 273, 276 Felsenfeld, C., 242, 243, 267 Fencl, Z., 15(57),30 Feryenson, P. E., 38, 87 Fergus, B. J., 194,208,222,265 Feretti, A,, 191,193,264,286,289 Fetzer, IT.,228,411 Fewster, J. A., 19(110, 117).31 Fiechter, A., 35,84, 173, 186 Fields, J. E., 147, 151 Filipello, F., 57, 58, 84 Filosa, J., 35, 83

Finn, R., 172(48),186 Fischer, E., 21(127), 31, 216, 267 Fisher, M. W., 254,255,256,258,261,266 Fisher, W. P., 194, 195,266,267 Flesch, P., 54, 85 Flood, J . E., 125, 136, 151 Floss, H. G., 235, 264 Flyrin, D. J., 175(65),186 Fonirra, I,. A., 213, 228, 274 Forgacs, J., 289, 290 Formanek, S., 154(4), 160(4), 162(4), 171(4),185 Fornachon, J. C. M., 55, 58, 84 Forrester, I. T., 94, 217 Fosdick, L. S., 43,85 Foster, E. M., 64, 84 Fourie, L., 289, 293 Franck, R., 222,265 Frank, H., 103, 119 Frateur, J., 19(115),27(170),31,32 Fratoni, A., 72,87 Freeman, G. G., 288,291 Freeman, R. R., 127, 148, 149, 151, 152 Frenkel, G., 48,83 Frey, A. J., 229,267,268 Friedland, W. C., 160,171(30),185 Friedman, S. M., 94, 108, 117 Frobesher, M. Jr., 149, 152 Frommer, W., 195, 208, 265 Fryer, T. F., 65, 66, 86 Fujii, M., 27(183), 33 Fnjirio, M., 208, 274 Fukui, K., 15(55),30 FuM G . J., 162, 171(33), 178, 178(33), 185,186 Fuller, W., 243, 267 Fulmer, E. I., 12(18), 14(18, 34, 43), 29 Fumiyo, K., 15(55),30 Funk, C., 278,291 Furr, J. R., 104, 117 G

Gaden, E. L., 143, 152, 164(36, 37), 185 Gaurnann, E., 194,212,247,254,255,256, 266,267 Galbraith, M. M., 254, 255, 256,258,261, 266 Galetto, W. G., 61, 62,84,88 Gallo, G. G., 208, 272

30 1

AUTHOR INDEX

Garcia, L. H., 178(76), 186 Gardner, J. N., 203, 273 Gardiner, R. A., 284, 291 Garrod, L. P., 253, 256, 267 Gartside, R. N., 194, 253, 264 Carver, J. C., 172(49), 184(49), 186 Castrock, E . A., 12(20), 14(20), 15(20), 29 Gaudin, A. M., 145, 151 Gaughran, H. R. L., 92, 94, 95,117 Gauze, G . F., 193, 212, 267, 271 Gavrilina, G. V., 193, 212, 267, 271 Geeren, H., 191, 213, 216, 218, 265 Gehring, R. F., 68, 84 Geiger, W. B., 191,275 Gellert, M., 242, 243, 267 Gentilini, L., 58, 84, 87 Gentles, J. C., 281,291 Gerner, R. E., 132, 152, 159(27), 185 Gero, S. D., 221, 266 Ghione, M., 208, 264 Gibbons, N. E., 94, 95, 105,117 Gibor, A., 145, 152 Gil-Av, E., 196, 203, 266 Gilgan, M. W., 288,291 Gill, J. E., 289, 291 Gillette, K. G., 279, 293 Gilliland, R. B., 47, 48, 50, 84, 142, 151 Giolitti, G . , 208, 273 Girotra, N. N., 279, 293 Gjertsen, P., 48,84 Glaz, E. T., 288,291 Glitzer, M. S., 280,289 Glover, G. I., 229,267 Godtfredsen, W. O., 288,291 Godfrey, E. I., 161, 185 Gorlich, B., 12(16),17(99),19(99),21(125), 26(157), 27(99), 29, 31, 32 Gol’berg, L. E., 208, 259, 260, 262, 274 Goldberg, I. H., 244, 267 Coldberg, M. W., 212, 264 Goldblum, N., 148, 152 Goncdves de Lima, O., 208,267 Gonet, A. E., 280, 291 Gordon, H. W., 287, 291, 293 Gore, J. H., 158, 160, 164, 177, 178, 185 Gorrnan, M., 193, 208,267 Gorup, B., 233, 272 Goss, W. A,, 191,255,267 Goto, T., 288,292 Gottlieb, D., 195, 208,266

Could, G. W., 96, 117 Gourevitch, A,, 195, 213, 255, 267, 268, 270 Grabowska, M., 242,267 Gray, B. E., 20,22,26,31 Cream, G . E., 196,209,217,223,266 Greenough, W., 132,151 Gregory, F. J., 201, 258, 267, 270 Grey, J., 105, 119 Grieves, R. B., 144, 145, 151 Griffiths, H., 194, 253, 264 Griot, R. G., 229, 267 Groger, D., 235, 264 Grone, H., 191, 200, 214, 265 Grove, J. F., 281,288,290,291 Grubhofer, N., 197, 198,265 Grussner, A., 11(4), 28 Gualandi, G . , 170(43), 186 Guarino, A. M., 288,291 Guerillot-Vinet, A., 193, 267 Guerillot-Vinet, J., 193, 267 Guimberteau, G . , 51, 53, 54, 86 Guinand, M., 194, 196,208,212,217,218, 264,267 Guseva, V. G., 189, 265 Guymon, J. F., 12(18), 14(18), 29, 47, 51, 52, 58, 61, 83, 84, 85 Guyot, L., 193, 267 H

Hacker, C. H., 167, 186 Hackmann, C., 195, 208, 258, 262, 267, 2 72 Harri, E., 288,291 Hagen, P. O., 94, 95, 105, 112, 117 Haight, R. D., 95,110,117 Haines, W. J., 280,292 Hale, M. B., 140,151 Hale, W. S., 43,83 Hall, G . E., 211,267 Hamada, M., 208,270 Hamamura, K., 279,291 Hamill, R. C., 193,208,267 Hamilton, J. K., 25( 151),32 Hamilton, L. D., 243,267 Hammer, B. W., 64,84 Handler, A. H., 258,270 Hanka, L. J., 280,293 Hansen, N. H., 90, 99, 100, 107, 113, 117 Harada, H., 292

302

AUTIIOR INDEX

Hardlicka, H., 69, 84 Harmon, Y ., 95, 117 Harold, F. M., 249, 268 Harold, F. V., 46, 47, 84 Harper, R. H., 71, 87 Harries, D., 104, 106, 110, 117, 118 Harris, E. J., 248, 266, 268 Harris, N. D., 113, 117 Harrison, D. E. F., 176(70), 177(70), 186 Harrison, G. A. F., 47,48,84 Harrison, J., 280,292 Hartl, A., 47.84 Hartmann, G., 242,269 Hartong, B. D., 45,84 Harvey, R. J.. 67,87 Hasegawa, T., 25(147), 32 Haselkorn, R., 243,268 Hasenpusch, P., 159(27), 185 Hashimoto, N., 49,84 Haskell, T. H., 195, 197,254,264,268 Hassall, C. H., 195, 198, 208, 218, 264, 268 Hata, F., 208,268 Hata, T., 258,267 Hausmann, W. K., 208,270,273 Havens, M . L., 130,152 Hawke, J. C., 67, 68, 84 Hawkins, D., 284, 293 Haworth, W. N., 11, 16(91), 28, 31 Haworth, W. W., 16(91), 31 Haynshi, S., 279, 291 Heater, C. D., 94, 113, 117 Heden C., 110, 117 HedBn, C. G., 158, 163, 181(89), 185, 187 Hegcr, E. N., 156(7), 185 Heim, R., 286, 287, 291 Heinemann, B., 195, 255,268 Heinemann, G., 16(185),33 Heitz, J. E., 51, 84 Helfferich, F., 146,151 Hellbach, R., 157(15), 185 Hellerbach, 211,224,272 Hemming, H. G., 281,288,290 Henderson, J . T., 27(181), 33 Ilenning, J. N., 283, 290, 293 Hensel, €I., 94, 103, 117 Herbert, D., 158, 172(21), 178(79), 179 (21,79), 184(21),185,187 Herbert, R., 11(5), 28

Herbert, R. W., 11(5),28 Hermann, S., 17(102,103), 27(103), 31 Herr, R. R., 280, 291 Herrick, H. T., 157(15), 185 Hershey, A. D., 91, 113, 117 Hersiczky, A., 13(26), 29 Hesscltine, C. W., 72, 84 Heuser, L. J., 195,223,266 Heyns, K., 16(94), 31 Hickinbotham, A. R., 57, 58, 84 Hidaka, H., 285,291 I-Iidy, P. H., 279, 293 Higashide, E., 208, 270 Higashikawa, S., 194, 196, 198, 210, 216 Higgen C. E., 193,208,267 Hildebrand, R. P., 46, 47, 84 Hill, H. R., 284, 293 Hilscher, H., 13(29), 29 Hind, H. L., 45,84 Hingst, G., 65, 87 Hinreiner, E., 57, 58, 84 Hirata, Y., 288,292 Hirst, E. L., 11, 28 Hisataka, M., 26(162), 32 Hitchens, A. D., 96, 117 Hitomi, H., 208, 274 Hobbs, I). C., 212,268 Hochster, R. M., 27@75),32 Hodge, E. R., 279,293 Hodges, R., 288,291 Hodgkin, D. C., 208,264 Hoefer, I., 218, 265 Hofer, M., 249, 268 Hoffer, A., 285, 291 Hoffsommer, R. D., 279, 293 Hofmann, A,, 192,210,215,268,274,285, 286, 287,291,293 Holden, J. T., 245, 268 Holdsworth, H., 154, 161, 184 Holme, T., 181(89), 187 Holmlund, C. E., 279, 291 Hooper, I. R., 194, 195,213,255,268,270 Hori, 1.,21(126),26(158),31,32 Horichi, I., 108, 118 Horn, K., 15(89), 31 Hornstein, I., 35, 36, 37, 84 Horvath, I., 195, 268 Hosler, P., 173(52), 178. 186 Hou, C. T., 192, 195,208,271

303

AUTHOR INDEX

Hough, J. S., 46, 84 Howard, E. M., 281, 290 Howell, M. G., 199, 224, 273 Howell, W. H., 37, 84 Hoymann, W. G., 284,291 Hruska, F. E., 214,275 Huang, H. T., 16(95),31,259,268 Huber, J., 13(29), 15(51), 29, 30 Hudson, J. R., 49, 84 Hutter, R., 194,208,265 Huftalen, J. B., 255, 261, 275 Hughes, D. E., 112,117 Hughes, M. S., 208, 275 Hugo, W. B., 104, 117 Humphrey, A. E., 164(36,37,39),175(60), 185,186 Hunt, G. A., 255,267 Hunter, I. G., 43, 84 Hurwitz, J. 244, 268 Hutchinson, H. B. 278, 279,291 I

Iandola, J. J., 106, 108, 117 Iitaka, Y., 209, 230, 269 Il’cheva, N. P., 193, 271 Ingold, W., 173, 186 Ingraham, J. L., 47, 51, 52, 55,85,87,102, 117 Ingram, M., 71, 84 Inoue, M., 208,274 Insalata, N. F., 288, 290 Irreverre, F., 197, 224, 268, 270 Irrgang, K., 19(104), 27(104), 31 Irving, G. M., 157, 185 Isbell, H. S., 15(69), 30 Iscovici, A., 15(54), 30 Iselin, B. M., 233, 272 Ishida, H., 15(55,63), 30 Ishida, N., 194,269 Ismay, D., 281, 291 Isono, M., 16(187), 25(150), 32, 33 Ito, R., 282, 293 Ito, T., 208, 268 Ivanitskaya, L. P., 193, 271 Ivanov, K. K., 193,271 IVdIloV, V. T., 209, 210, 211,218,221,222 225, 227,228, 230,232,233, 246, 247, 254, 264, 271, 272,273, 274, 276

Ives, K. J., 135, 151 Iwasaki, E., 208, 270 I

Jackson, H., 110,117 Jackson, R. W., 159(29), 185 Jackson, T., 162, 185 Jacobs, S., 99, 110, 117 Jagemann, W., 15(51), 30 Jahnke, H. K., 280,291 James, A. M., 141,151 Jansen, H. E., 142, 151 JeIjaszewicz, J., 256, 266 Jenard, H., 47,85 Jensen, L. B., 71,85 Jerchel, D., 54, 85 Jeunehomme-Ramos, C., 4 7 , 8 5 Jirovec, O., 258, 268 Jockusch, H., 102,117 Johannessen, D. W., 195, 254,264 Johnson, A. W., 193, 203, 212, 213, 215, 230,264,266,268 Johnson, D. L., 195, 213, 255, 268, 270 Johnson, J. A., 42, 43, 44, 85, 87 Johnson, J. H., 147, 151 Johnson, M. J., 172(51), 173(52), 174, 175, 176, 179, 180(85), 181(81, 88), 184 (93,94), 186, 187 Johnson, S. M., 246,268 Johnston, J. W., Jr., 39, 83 JollBs, Poiget, G., 199, 224, 228, 268 Jones, A., 132, 151 Jones, N. R., 72, 85 Jordan, R. C., 99, 110, 117 Joslyn, M. A., 53, 57, 85, 87 Judson, G., 132,151 Jung-Sheng Tsai, 189, 268 Junod, A,, 280, 291 Junrat, I. A., 208, 274 Jurubita, S., 15(54), 30 K

Kabler, P. W., 91, 117 Kahan, E., 244, 268 Kahan, F. M., 244,268 Kaiser, S., 212, 264 Kajiri, K., 285, 291 Kalbe, H., 197, 198,265 Kambara, K., 73, 8 7

304

AUTHOR INDEX

Kanwisher, J. W., 175(61), 186 Kanzaki, T., 16(187,188,189), 33,208,270 Kaplan, M. A., 195, 255, 268 Karabinos, J. V., 15(69), 30 Karasawa, K., 189,270 Karle, I. L., 233, 269 Kasemsarn, B., 72, 8 7 Kass, W., 197, 198, 265 Katagiri, K., 191, 194, 195, 202, 236, 239, 256, 259, 260, 268, 269, 272, 276 Kataska, Y., 282, 292 Katz, E., 190, 191, 235, 236,237,238,242, 255,256, 258, 259, 261,262,266,267, 268, 270,272, 275,276 Katznelson, H., 27, 32 K a u h a n n , E., 279,291 Kaul, P. N., 282, 291 Kawachi, N., 15(55), 30 Kawamnta, J., 244,268 Kawamura, Y., 195,270 Kazmierczak, R., 230, 268 Keenan, T. W., 64, 65, 85 Kegan, M. J. 15(58), 30 Keggi, J. J.. 194, 209, 254 Keil, W., 70, 85 Keller-Schierlein, W., 194, 196, 199, 208, 210,212,215,225,227,255,265,266 268,271 Kelley, C., 195, 213, 265 Kellogg, H.,132, 151 Kenipe, L. L., 146, 151 Kepner, R. E., 47, 51, 52, 56, 61, 62, 84, 85, 87, 88 Kern, H., 194,256,267 Kersten, H., 242, 243, 268, 272 Kersten, W., 240, 242, 243, 268,272 Khalilulina, K. Kh., 210, 211, 218, 227, 228,273 Khesghi, S., 19(113), 20(113), 31 Kieser, M. E., 62, 63, 86 Kikuchi, G., 249, 272 Kilborn, D. G., 175(65), 186 Kirnura, A., 195,270 Kirnura, T., 15(5S, 63). 30, 195, 270 King, T. E., 14(3S), 15(65, 66 67, 681, 29, 30 King, W. L., 96, 117 Kingston, I).G . I., 196, 209,217,221,223, 266,269

Kinoshita, S., 15(72, 73, 74, 75, 76, 77, 79, 87), 16(87), 30,31 Kirillova, N. F., 193, 274 Kirk, J. M. 242, 244, 269 Kirsch, E. J., 208, 269 Kiryushkin, A. A., 209,210,211,218,220, 221, 225, 227, 228, 230, 232,233, 254, 264,269, 271,273,276 Kiseleva, 0. A., 230, 264 Kluyver, A. J., 12(13), 19(107), 29, 31 Knape, W., 15(51),30 Knight, S. G., 68, 84 Knobloch, H., 19(105), 27(166), 31,32 Knudsen, M. P., 195, 254, 255, 256, 258, 261,264,266 Kobayashi, Y., 281,292 Kodaira, Y., 194, 196, 198, 210, 216, 254, 261,275 Kobel, H., 286, 287, 291 Koenig, E., 15(51), 30 Koenig, J., 198, 224, 269 Koepsell, H. J., 157(13), 160(13), 172(13), 181(13), 182(13), 185 Kuffler, H., 94, 117 Kogan, P. M., 15(58), 30 Kohn, F. E., 43, 85,88 Komatsu, H. 208,263 Komersova, I., 235, 270 Komoda, H., 63,72,85,88 Kondo, K., 19(116), 27(116, 176, 177), 31,33 Konetzke, G., 15(51), 30 Konikova, R. E., 13(28), 29 Konnert, J., 233, 269 Konstantinova, N. V., 193, 212,265 Koretskaya, T. I., 259, 261, 276 Kosaka, H., 195, 270 Kostetskii, P. V., 211, 228, 271 Kota, M., 102, 117 Kovalenkova, V. K., 193, 212, 267 Kovsharova, I. N., 193, 212, 265 Kowszyk-Gindifer, Z., 195, 272 Koyama, G., 199, 209, 216, 217, 230, 269, 2 70 Kozlow, D., 171,186 KGzu, Y., 208,263 Kradolfer, F., 194, 212, 255, 266 Kraft, K., 11, 28 Gashilina, A. Ya, 259, 261,276

AUTHOR INDEX

Krenova, R. A,, 14(40), 29 Kroll, C. L., 154(4), 160, 162, 171, 185 Kruglyak, E. B., 193, 212, 265 Krylova, Yu. 1.,210,211,218,229,230,274 Kubeczka, K. H., 68,85 Kubo, H., 282,292 Kudaka, M., 14(44), 22( 129), 29, 31 Kudrina, E. S., 189, 265 Kugel, R. E., 289,293 Kulbota, F., 15(63), 30 Kulhanek, M., 12(7), 15(61, 62), 21(125), 23(130), 24(130), 25(155), 28, 30, 31, 32 Kulka, D., 19(118),31 Kuninaka, A., 36,85 Kunkee, R. E., 54, 55, 56, 85, 86 Kunrat, I. A., 259, 260, 262, 274 Kunstmann, M. P., 194,197,198,208,276 Kuo, C. H., 279,293 Kuo, J. F., 246, 279, 291, 296 Kupryszewski, G., 190, 230, 268, 269 Kuroiwa, Y., 49, 84 Kuroya, M., 198, 204,269 Kurtzman, C. H., 38, 85 Kushner, D. J., 94, 95, 105, 117 Kusurnoto, M., 208, 263 Kuyama, S., 194, 196, 198, 210, 216, 228, 269,275 Kybal, J., 235, 270 Kyotani, H., 15(55, 63), 30, 195, 270 1

Lacey, M. S., 191,269 Lackner, H., 214, 222, 225, 243, 255,265 Lafon-Lafourcade, S., 53, 54, 56, 85, 8 6 Lagoda, H. A., 159(29), 185 Laine, B., 35, 83 Laland, S., 245, 267 Lambert, A. E., 280,291 Lambion, R., 55, 85 Langridge, P., 95, 111, 117 Largier, J. F., 148, 151, 152 Larkin, R. L., 289, 293 La Sala, E. R., 212, 264 Laskin. A. I. 257, 269 Lautenbach, A. F., 48,49,83,88 Lavrova, M. F., 193, 212, 265 Lawrence, R. C., 65, 66, 67, 85,86

305

Lawrence, W. C., 48,85 Lawson, W. B., 199, 212, 215, 273 Lechowich, R. V., 107, 116 Lederer, E., 190, 194, 208, 212, 218, 221, 264,266,267,269 Lee, F. A., 69, 8 7 Leefers, J. L., 12(17), 29 Leemam, H. G., 229,274 Lees, K. A., 195, 276 Lees, T. M., 195, 274 Leesment, H., 65,86 Lehninger, A. L., 249,265 Lein, J., 194, 195, 213, 255, 267, 268, 270 Lerncke, R. H., 100,117 Lemich, R., 143, 152 Lengyrel, Z. L., 176(69), 186 Lennox, E. S., 104, 117 Leon, S. A., 280,281,290, 292 Lepeshkina, G. N., 259,272 LeQuesne, P. W., 194,210, 217, 265 Leung, A. Y., 287, 292 Lev, A. A., 246,269 Levchenko, T. N., 62,87 Levin, G. V., 145, 152 Levy, C. P., 288, 290 Levy, H. B., 245,269 Lewin, L. M., 49, 85 Libbey, L. M., 66,86 Liebster, J., 15(50),24(138, 139,140,142), 30,32 Liersch, M., 242, 269 Light, M., 106, 117 Lillehoj, E. B., 288, 290 Lilly, M. D., 175(65), 186 Lincoln, R. E., 144, 151 Lindahl, T., 109, 11 7 Lindsay, R. C., 64, 65, 66, 85, 86 Link, K. P., 284, 291 Linko, Y. Y., 43, 44, 85 Lins Lacerda, A., 208, 267 Lipis, B. V., 61, 85 Lippe, C., 247, 269 Lipscomb, H. S., 284, 293 Liston, J., 72, 8 7 Lizete Lins de Oliveira, 208, 267 Lloyd, B., 55, 58,84 Lockwood, L. B., 12(19,20), 13(23,27),14, 15(20,23,27),19(108), 29,31 Loeffler, W., 288,291

306

AUTHOR INDEX

Logiiiova, N. F., 213, 228, 274 LnGrippo, C. A,, 147, 152 Lomakina, N. N., 189, 193, 208, 212, 265, 274 Longsworth, L. G., 149,152 Lord, T. H., 44,87 Losse, G., 190, 210, 225, 228, 238, 269 Lovrekovitch, I., 195, 268 Lowe, D. 288,290 Ludescher, U., 240,272 Lubke, K., 190,272 Lugli, A. M., 208, 273 Lukas, C., 60, 61, 62, 85 Lukin, M., 208,265 Lrikbik, B., 15(50), 23(137), 24(137, 138, 140, 141),30,32 Lumb, M., 157(18), 185 Lyaslenko, V. A,, 259, 274 Lynn, W. S., 249, 250

209, 213,215, 216, 217,224, 230, 254, 269,270,273,275 Maesawa, T., 282,292 Maggiora, L., 62, 88 Magyar, K., 195,268 Mahony, N. C., 137, 152 Maillard, M. A., 245, 262, 270 Majer, J., 235, 270 Malachta, S., 24(143), 32 Malenkov, G. G.. 230, 246, 247.274 Malmgren, B., 181(89), 187 Mamakova, Z. A., 61,85 Manaker, H. A,, 201,270 Manegold, J. H,, 195, 208, 213, 214, 225, 239,240, 242, 255,265 Mania, D., 199, 203, 213, 215, 273 Mano, F., 63,85 Mansfeld, V., 12(7), 28 Marasas, W. F., 288,290 Marcilla, J. A., 60, 61, 62, 85 M Maresca, L., 48, 86 Mabbitt, L. A,, 65, 66,85 Maretzki, A,, 195, 197, 254, 264, 268 McBride, T. J., 254, 255, 256, 257, 267 Margolis, A. A., 244, 264 Maccacao, G . A,, 101, 118 Marlier, S., 12(8), 23(8), 28 McCann, E. P., 156(1l), 161, 162, 163(ll), Marquis, R. E., 107, 117 171(11), 172, 177(11), 185 Marshall, R., 238, 270 McCapra, F., 285, 292 Martakov, A. A., 62, 87 McCarthy, R. E., 252, 269 Martin, C. H., 90, 99, 117 McClintock, M., 280, 292 Martin, H. H., 103, 119 Macdonald, C. G., 210,218, 272 Martin, J. H., 208,270,273 MacDonald, J. C., 195,235,238,239,249, Martin, S. M., 157(16), 158,170,185 269 Maskell, M. A., 141, 152 Macek, K., 20(119), 31 Masschelein, C. A., 47, 85 McErlean, B. A,, 279,292 Mathieson, A. McL., 213, 218, 270 McCugan, W. A,, 66,84 Matsakura, G., 14(44), 29 Mach, E., 212,264 Matsubara, S., 38, 83 Machado, M., 208,267 Matsui, M., 246, 270 Machlowitz, R. A., 195,266 Matsumoto, A., 25(148), 32 MacInnes, D. A., 149,152 Matsumura, S., 194, 269 McKeon, J. E., 194,209,254,275 Matsuura, S., 259, 260, 270 Mackereth, F. J. H., 175(64), 176(64),186 Matsuzaki, M., 285, 291 McI,ean, R. A,, 71, 85 Mattick, L. R., 69, 70, 8 6 , 8 7 Maclennan, D. G., 176(71), 186 Matz, S . A., 42, 85 Macleod, R. A., 106,117 Mauger, A. B., 191, 203, 212, 213, 215, McMillan, J., 281, 291 230,237,243,264,268,270,271,276 MeMorris, T. C., 209, 223, 267 Maurer, K., 12(15), 29 McMurray, W. C., 247,269 Maxon, W. D., 156(8, lo), 158(10, 19), McNutt, S. H., 279,292 160, 163(8), 171(8), 173(8), 174, 177 Maddock, C. L., 258,270 (8),178(8, 80), 179(85), 180(85), 185, Maeda, K., 191, 195, 198, 199, 203, 208, 186, 187

307 Maxwell-Savage, R. H., 96, 117 May, 0. E., 157(15), 185 Mayama, M., 194, 195,269,270 Means, C. W., 157(13), 160, 172(13), 181, 182(13), 185 Mecke, R., 214, 255, 265 Meienhofer, J., 222, 270 Meitus, R. A., 288,292 Mekler, L. B., 242, 270 Meloni, M. L., 236, 268 Meltsner, B. R., 212, 264 Mendillo, A. B., 288,291 Mendlik, I?., 142, 151 Mervyn, L., 201, 270 Meskhi, R., 55, 85 Meyers, E., 256, 257,270 Meynell, E., 102, 118 Meynell, G. G., 102, 118 Miall, L. M., 15(60), 30 Michael, S. E., 281, 290 Michaels, S., 94, 109, 110, 117 Michailovic, M. Lj., 199, 212, 215, 268 Micheel, F., 11,15(89),28,31 Michel, G., 194, 196, 208, 212, 217, 218, 264,267 Michiyo, M., 73, 8 7 Mikhaleva, I. I., 228, 230, 246, 247, 271, 2 74 Mikhlin, E., 14, 30 Miki, T., 20(122), 24(144, 145), 25(146, 147), 31,32 Mikolajak, E. M., 102,117 Mill, P. J., 142, 152 Miller, B. S., 42, 43, 44, 85, 87 Miller, 0. C., 159, 161, 171, 173(28), 185 Miller, T. L., 177(73), 183(73), 186 Minghetti, A,, 191,193,264,286,289 Mirocha, C. J., 279,280,288,290,292 Mirrington, R. N., 285, 292 Misiek, M., 213, 270 Mitchell, P., 97, 105, 118 Mityushova, N. M., 14(36, 46), 29 Miura, T., 194, 195, 196, 208, 275 Miyake, A., 208, 274 Miyamoto, K., 22(129), 31 Mizuno, D., 108, 118 Mizuno, K., 208, 270,274 Mogey, G. A., 286, 290 Mold, J. D., 195, 254, 264 Moncrieff, R. W., 36,37, 38,39,40,85,86

Money, T., 209,223,267 Montegut, J., 193,267 Moore, C., 248,251,270 Moore, G. E., 159,161,185 Morgan, M. E., 64,65,66,85,86 Mori, M., 164(35), 178(35), 185 Morice, I. M., 202, 211, 235, 252, 264 Morieson, A. S., 46, 47, 84 Moriguchi, S., 73, 87 Morita, K., 197, 224, 268, 270 Morita, R. Y., 95, 105, 110, 111, 113, 117, 118

Morrison, R. B., 148, 152 Morrison, R. I., 289, 291 Mortimer, A. M., 195, 276 Mortimer, P. H., 211, 252, 266 Mortimer, R. K., 150, 152 Morton, R. B., 195, 208, 268 Moses, A. J., 255, 267 Moss, F. J., 172(50), 176(68), 177(68), 179(68),186 Moss, M. O., 289,292,293 Motizuki, K., 16(187,188),33 Mueller, P., 246,270 Muller, W., 232, 240, 242, 243, 266, 270 Munchhausen, F., 285, 292 Muggleton, P. W., 281, 290 Mular, A. L., 145, 151 Muller, R., 147, 152 Mullholland, T. P. C., 281, 291 Mulvany, J., 139, 140,152 Mundkin, B. B., 284, 292 Munk, V., 15(57),30 Munson, R. J., 110, 116 Muramatsu, I., 208, 265, 270 Muraveiskaya, V. S., 259, 260, 262, 274 Murav’eva, L. I., 193, 212, 265 Murray, C., 279, 292 Murray, P. J., 46, 47, 84 Murrell. W. G . , 96, 97, 118 N

Nadkarni, V., 280, 292 Naef-Roth, St., 194, 256, 267 Nagdnawa, H., 199, 209, 216,217,270 Nagasaka, N., 26(162), 32 Nagatsu, T., 285, 291 Nager, U., 193, 198, 210, 211, 216, 218, 225,254,267,271 Nagi, S., 73, 8 7

308

AUTHOR INDEX

O’Connell, P. W., 280, 292 Nakamura, H., 141, 142,152 Nakamura, S., 189, 199,203,213,215,270, O’Conrior, R. F., 145,151 Oda, T., 208, 275 2 73 Ogati, E., 248, 270 Nakanishi, I., 16(187),33 Oyawa, H., 208,268 Nakanishi, T., 25(150), 32 (&Ward, H., 199,209,216,217,270 Nakata, H., 292 Nakatani, T., 26(158),32 Ogihara, Y., 195, 208, 268 Oginsky, E. L., 100, 118 Nakazawa, K., 208,270,274 Ohashi, S., 281,287,292 Nara, K., 16(188),33 Ohkubo, Y., 208,270 Nardnjo, P., 281,288,292 Ohnishi, M., 213,270 Neipp, L., 194,208,212,255,265,266 Nelson, F. E., 64,84,91,92,100,118 Oka, Y., 246,270 Nelson, G. H., 279,280,288,290,292 Okabe, K., 223, 270 Nelson, H. A., 156(8), 160, 163(8), 171(8), Okami, Y., 195, 208, 270, 275 Okamoto, K., 19(111), 31 173(8), 177(8), 178, 185 Okamoto, S., 195,270 Nelson, J. W., 280, 292 Okazaki, H., 16(187, 188, 189), 33 Nemek, P., 254, 258, 264 Oladkina, V. A., 221,273 Neuenschwauder, J., 284, 293 Oldshue, J. Y., 156, 185 Neville, D., 242, 243, 267 Olson, J. C., 64, 84 Newlin, T. E., 130, 152 Omelianski, V. L., 40, 86 Newson, 1. H., 144, 152 Omori, R., 64, 88 Ng, H., 43,84 Omori, T., 64, 86 Nicholas, R. C., 96, 118 Ondetti, M. A,, 196, 201, 211, 225, 265, Nichols, A. A., 280, 292 271 Nichols, R. E., 283, 290, 293 Ondrejickova, 237,266 Niemczyk, H., 195,272 Oparysheva, E. F., 208,274 Niinome, Y., 194, 269 Oppexiauer, R., 11(4),28 Niketic, G., 69, 86 Orci, L., 280,291 Niniivaara, F. P., 71, 86 Nishibori, A., 208, 270 Ordal, E. J., 70,86,149,151 Nishimura, H., 195, 270 Ordal,Z. J,, 106, 108,112,117,118 Nishimura, J. S., 237, 270 Orlando, M. D., 148, 152 Oterholm, A., 70, 86 Nitta, K., 287, 292, 294 Niven, C. F., 71, 84 Otsuka, H., 199, 212, 214, 224, 260, 271, Nolting, H., 233, 272 2 74 Nordstrom, K., 50, 86 Ott, H., 268 Norris. C. L. F., 288,290 Ough, C. S., 57, 58, 60,86 Northrop, J., 97, 118, 184(96), 187 Ovchinnikov, Yu. A,, 190, 209, 210, 211, Novdk, L., 198, 224, 269 2 18, 221, 222, 225, 227, 228,230,232, Nozawa, R., 108,118 233,239,247,254,255,264,269,271, Nueschul, P., 19(103), 27(103), 31 272, 273, 274,276 Nunley, C. E., 130, 1 5 1 , 152 Owades, J. Id., 48, 86 Nyiri, L., 176(69), 186 Owaki, K., 63, 86 Owen, S. P., 181(88), 187 0 Oxford, A. E., 281, 292 Omki, M., 282, 292 Obrist, W., 247, 267

o.,

OEenaBek, F., 23(136), 26(136), 32 Ochoa, S., 54, 86

P

Pace, B., 94, 108, 118

AUTHOR INDEX

Pache, W., 246, 271 Pack, M. Y., 65,86 Pagano, J. F., 195,223, 266 Paisley, H. M., 288,292 Paladino, S., 154(3), 161(3), 162(3), 185 Palmer, H. T., 222, 240, 271 Palmer, R. A,, 222, 240, 271 Pampus, G., 214,255,265 Pangborn, R. M., 36, 37, 38, 40, 83, 8 6 Park, J. T., 104, 119 Parker, A., 156(11), 161, 162, 163(11), 171(11), 172(11), 177(11), 185 Parmentier, G., 211, 239 Pascal, CI., 262, 266 Pasternack, R., 17(100), 20, 21(100), 22, 31 Paszkiewicz, A., 195,272 Patki, G., 105,116 Patrick, W. C., 111, 127, 148,152 Patterson, E. L., 194, 197, 198, 208, 276 Patton, S., 66, 68, 86 Paul, A. G., 287,292 Paulsen, H., 16(94),31 Pavelka, J., 69, 84 Pavlenko, I. A., 193, 212, 213, 228, 267, 2 74 Pavzner, N. S., 208,274 Peck, G. Yu., 210,228,232,271 Peck, M. M., 289,293 Pedersen, K. O., 240,271 Pederson, C. S., 69, 70, 83, 86, 8 7 Pellerat, J., 245, 262, 270 Pence, J. W., 43, 83, 84 Pendleton, I. R.. 148, 152 Pennelh, P., 191, 193,264, 286,289 Percival, E. G. V., 11(5), 28 Pereira, R. L., 66, 86 Perlman, D., 26(156), 32, 191, 192, 195, 208,230,239, 256, 257,270,271 Peron, Y., 95, 117 Perone, V. B., 288,292,293 Perrett, C. J., 181(90), 187 Perrin, D. D., 191, 271 Perry, R. P., 244, 271 Perry, S., 132, 151 Peterson, M. H., 160, 171(30), 173(53), 185,186 Peterson, W. H., 14(31),29

309

Pethica, B., 105, 118 Petrzilka, T., 271, 274 Pevzner, N. S., 212,271 PB-W6n Liu, 194, 271 Peynaud, E., 51, 53,54,55,56, 57, 58,85, 86 Pfaffmann, C., 36,86 Pfeifer, V. F., 156(7),185 Pfennig, N., 191, 194, 195, 265 Pfenninger, H. B., 46, 86 Pflug, I. J., 96, 118 Pheil, C. G., 96, 118 Philippe, J., 208, 271 Philips, F. S., 272 Phillips, D. A. S., 198, 264 Phillips, D. H., 170, 174,175,176(67),186 Phillips, K. L., 170, 186 Phipps, P. J., 158(21), 172(21), 179(21), 184(21), 185 Pickering, A., 66, 86 Pickles, D., 156(11), 161(11), 162(11), 163(11), 171(11),172(11), 177(11),185 Pictet, R., 280, 291 Pienta, P., 191, 261, 268 Piersma, H. D., 288, 290 Pifio, S., 15(84), 30 Pigman, W. W., 17(101),31 Pilone, G . J.. 54, 55, 56, 86 Pinkerton, M., 213, 271 Pioda, L. A. R., 246,276 Piquet, G., 59, 83 Pirt, S. J., 173(55), 176(70, 71), 177(70), 186 Pisarnitskii, A. F., 62,87 Plastourgos, S., 71,86 Plattner, P1. A., 193, 198, 210, 211, 216, 218, 225,227, 254,267,271 Plengvidhya, P., 249, 271 Plotnikova, V. T., 13(22), 29 Podojil, M., 193, 272 Pohl, K., 158(24), 171, 185 Pokras, L. S., 193, 265 Polatanick, J., 148, 151 Pollard, A., 62, 63, 86 Polson, A., 148, 152 Porges, N., 12(20),14(20), 15(20),29 Porter, H. F., 125, 136, 151 Porter, J. N., 194, 197, 198, 208, 276

310

AUTHOR INDEX

Powell, E. O., 178(78), 187 Preiss, B., 281, 290 Prelog, V., 194, 196, 199, 212, 215, 255, 266, 268 Prentice, N., 284, 292 Preobrazhenskaya, T. P., 193, 212, 267, 271 Pressman, B. C., 247, 248, 251, 266, 268, 270, 271 Pretlow, T. G., 131,152 Preud'homme, J., 212, 271 Pringle, R. B., 208, 276 Probine, M. C., 192, 201, 202, 211, 264 Prockop, U.I., 237, 268 Proshylakova, V. V., 270 Prosky, L., 15(83), 30 Protiva, M., 12(9), 13(9), 28 Pruss, M. P., 280,289 Ptociennik, Z., 195, 272 Puchkov, V. A., 221, 222, 264,272, 276 Pugh, L. H., 242,255, 256, 258, 259, 262, 267, 272 Puglisi, T., 255, 267 Pulkki, L., 96, 118 Purohit, K., 94, 118 Prirwin, P., 279, 292 Pyimesi, J., 288,291 Q

Quitt, P., 210, 211, 224,225,227,228,271, 272,274 R

Rabinowitz, M., 244, 267 Radeckaya, N. V., 15(58), 30 Radler, F., 54,86 Rahn,0.,92,99,111,118 Rainbow, C., 27(182), 33 Rainey, D. P., 284, 292,293 Raistrick, H., 281, 290, 292 Rakieten, M. L., 280, 292 Rakieten, N., 280, 292 Rankine, B. C., 51, 52, 59, 60, 86 Rapoport, H., 229, 267 Rasmussen, H., 248, 270 Ratner, D. I., 232, 240, 241, 266 Haue, H., 210, 225, 228, 238, 269

Rauen, H. M., 242, 272 Ravdel, G . A., 228, 239,255,274 Raymond, S. A,, 39, 83 Razin, S., 104, 106, 118 Razumovskaya, Z. G., 11, 13(28), 14(38, 39,41,46), 27(171), 28,29,32 Redfield, B., 238, 270 Redfield, E. G . , 237, 275 Redin, C., 194, 197, 198, 208,276 Reich, E., 190, 243, 244, 267, 272 Reichstein, T., 11, 20(124), 28, 31 Reilly, H. C., 258, 272 Reimer, C. B., 130, 152 Reinbold, G. W., 100, 118 Reisman, H. B., 158, 160, 164, 177, 178,

185 Reiter, B., 65, 66, 86 Heitler, R., 280, 281, 290, 292 Hennie, F. W., 125, 136, 151 Renold, A. E., 280,291 Rentschler, H., 59, 86 Resnick, M. A., 150, 152 Reusser, F., 157(13), 160(13), 172(13), 181(13), 182(13), 185 Reusser, P., 194, 212, 255, 266 Reynolds, D. M., 191, 275 Rhone-Poulenc, S. A,, 208, 272 Ribeiro, A. M., 71, 86 Ribkrcau-Gayon, J., 53, 54, 55, 56,57, 58, 86

Rice, A. C., 55, 86 Riches, P., 194, 253, 272 RiEica, J., 15(57),30, 172(46),186 Riedl-Tfimovi, E., 19(114),31 Rieman, H., 90,99, 100,107,113,117 Riggs, N. V., 284,288,290,293 Riley, J. M., 148, 152 Rinehart, K. L., 284,290,291 Ritchie, E., 285, 292 Hittel, W., 233, 272 Robert, J., 199, 224, 228, 268 Roberts, €I.R., 19(113), 20(113),31 Robertson, H. V., 197, 268 Robinson, A,, 105, 116 Robinson, F. V., 288,292 Robinson, H. J., 258,262,272 Robinson, R. J., 43,44,85,87 Robison, S. H., 95, 105, 113, 118 Roch, L. A., 46,48, 83 Roche, B. H., 284,291,292

AUTHOR INDEX

Rodopulo, A. K., 62,87 Roe, E. T., W19, 20), 14(20), 15(20), 19(108), 29, 31 Roephe, M. H., 149,152 Roessler, E. B., 36, 37, 38, 40, 83 Rogers, M. A., 281, 291 Ronaldson, J. W., 288,291 Rose,A. H.,93, 112,117,118 Rose, H., 147, 152 Rosenberg, I., 14(49), 30 Rosenberger, R. F., 172(47), 186 Rosensteel, J. F., 178(76), 186 Rosinov, B. V., 222, 272 Rossolimo, 0. K., 259, 260, 262, 272, 274 Roth, S., 254, 267 Rothlin, E., 285, 292 Rothwell, A., 194, 253, 272 Rotman, B., 179(84), 187 Roussos, G. G., 213, 214, 272 Routien, J. B., 185, 274 Roux, L., 193,267 Rowatt, E., 70,87 Roxburgh, J. M., 158,170(44),185,186 Roy, H. E., 148,151 Rozinov, B. V., 276 Rubin, E., 143,152 Rubin, G., 48,86 Rubin, M., 39,83 Ruczaj, Z . , 195,272 Rudin, D. O., 246,270 Rudinger, J., 198,224,269 Rudlich, E., 281,292 Rumpf, P., 12(8), 23(8), 28 Rushton, J. H., 158, 185 Russell, A. D., 96, 101, 104, 105, 106, 107, 109, 110, 113,116,117,118 Russell, D. W., 190, 191, 192, 194, 197, 198,202,208,209,210,211,215,216, 218,235,236,238,252,253,264,265, 266,272 Rutschmann, J., 268 Ruttenberg, M. A., 240,272 Ryabova, I. D., 211,227, 254, 273 Ryan, V. J., 57, 58, 84 Ryder, A,, 195,254,264 S

Sabashi, Y., 24(144), 32 Sabol, S. L., 232, 240, 266

311

Sacks, L. E., 148,152 Sadoff, H. L., 164, 185 Saeed, M., 172(50), 186 Sahashi, Y,, 25(146, 147), 32 Saisithi, P., 72, 8 7 Sakabe, N., 289,292 Sakai, F., 288,293 Sakai, H., 282,288,293 Sakasai, T., 73, 83,88 Sallans, H. R., 158, 170(44), 185, 186 Salton, M. R. J., 103, 104, 105, 118 Salzman, L., 238, 272 Salzrnan, L. A., 235, 272 Sandegren, E., 48, 49, 50, 8 7 Sandine, W. E., 65, 67, 85,86, 87 Sangen, S., 15(55), 30 Sano, T., 208,268 Sarber, R. W., 254,255,256,258,261,266 Sarin, P. S., 196, 209, 217, 223, 266, 269 Saruno, R., 63,88 Sasajima, K., 16(187, 189), 25(150), 32, 33 Sasaki, K., 63,88 Sato, H., 249,272 Sato, K., 15(64, 88), 24(145), 30, 31, 32, 194, 269 Saunders, C . F., 94, 108,118 Savage, G. M., 157(13), 160(13), 172(13), 181(13), 182(13), 185 Savastyanov, G. J., 15(58), 30 Sawyer, F. M., 38,87 Sazerac, R., 12(11),29 Schade, J. E., 39,87 Schaffner, C. P., 195, 208, 266, 287, 291, 293 Schanderl, H., 54, 60,61,62,87 Schaufus, C. P., 139,140,152 Scheel, L. D., 289,292,293 Scheibler, H., 216,267 Schein, P., 280,293 Schiedt, B., 12(15),29 Schlingman, A. S., 254, 255, 256, 258, 261,266 Schmid, J., 195, 208, 272 Schmidt, C. F., 99,118 Schmidt-Kastner, G., 191, 195, 202, 208, 213,235,255,265,272 Schoen, H. M., 143,152 Schon, K., 17(98), 19(98),31 Schoenewaldt, E. T., 16(185), 33 Schoental, R., 288,293

312

AUTHOR INDEX

Schoppee, C . W., 285,292 Schramm, W., 243, 265 Schroder, E., 190,272 Schroeder, W. R., 111,118 Schulz, H., 208,272 Schulz, M. E., 65, 87 Schwartz, E., 46, 84 Schwartz, H. S., 259, 272 Schwyzer, R., 233,240,272 Scott, A., 262, 272 Scott, A. I., 285,292 Scott, de B., 288,293 Scott, W. E., 212, 264 Scott, W. J., 96, 118 Scotti, T., 190, 208, 264, 274 Seebeck, E., 20(124), 31 Sen, A. K., 198, 203, 224, 273 Sensi, P., 208, 272 Sevcik, V., 193,272 SevEikovB, Z., 15(61, 62), 30 Sgarzi, B., 208, 273 Shah, F., 104, 118 Shaklovskii, K. P., 104, 118 Shands, R. G., 284,293 Shank, J. L., 71, 87 Shannon, J. S., 192, 194, 201, 202, 208, 209,210,211,218,221,222,264,265, 269,272 Shapovalonva, S. P., 212, 271 Sharpe, M. E., 65,66,86 Shavit, N., 252, 264 S h y , A. J., 208, 273 Shchelkunova, S. A., 14(42), 29 Shchelokov, V. I., 209,210,211, 218,229, 230,264,273,274 Shchukina, L. A , , 221, 228, 239, 255,273, 2 74 Shedlovsky, L., 143,152 Sheehan, J. C., 197, 198, 199, 203, 212, 213, 215,224, 273 Sheehan, J. T., 201, 211, 265 Shemyakin, M. M., 189, 190, 209, 210, 211, 213, 218,221,222,225, 227,228, 229, 230, 232, 233, 239, 246, 247, 254, 255, 264, 269, 271,272, 273,274,276 Sheppard, R. C., 229, 274 Shenvood, I. R., 13(24),29 Shibata, M., 208, 270,274 Shigemi, M., 73, 87

Shimmin, P. D., 66, 83 Shiratori, O., 194, 239, 259, 269, 272 Shito, E., 46, 83 Shizume, Y., 279, 293 Shkrob, A. M., 209, 210, 211, 218, 222, 229,230, 246, 247,273,274,276 Shoemaker, R. N., 26(160, 161), 32 Shcji, J., 194, 199, 212, 214, 224, 260, 269,271,274 Shon, M., 97, 106, 107,118 Shorin, V. A., 208, 259, 260, 262,274 Shu, P., 194, 197, 198,208,273,276 Shu-Fong Liang, 189,268 Shuval, H. I., 148, 152 Shvetsov, Yu. B., 213, 228, 274 Siebenberg, J., 48, 83 Sieber, P., 233,272 Sigg, H. P., 288,291 Sikyta, B., 179(82), 187 Silliker, J. H., 71, 87 Simola, P. E., 279, 293 Simon, W., 246, 276 Simonant, P., 281, 292 Simonart, P., 19(115), 27(170),31,32 Singer, H., 167, 186 Singer, R., 287, 293 Sinka, Y. K., 282, 290 Sinskev, T. J., 132, 152 Sirsi, M., 193, 194, 257, Inc. Sjostrom. L. B., 38,85 Skovholt, O., 42, 87 Slapovalova, S. P., 259, 274 Slater, G. P., 238, 239, 269, 274 Slates, H. L., 279, 293 Slepecky, R. A., 97, 118 Smalley. E. B., 283, 284, 288, 290, 291, 292,283 Smith, A. H., 287, 292 Smith, C. E., 242, 243, 267 Smith, D. A,, 256, 257, 270 Smith, D. E., 43, 44, 87 Smith, E. J., 49, 85 Smith, E. L., 201, 266,274 Smith, F., 11(5), 25( 151),28,32 Smith, R. B., 229, 267 Smith, R. M., 195,254,255,256,258,261, 264,266 Smith, S., 203, 210, 274 Smith, W., 278, 279, 291, 293

313

AUTHOR INDEX

Snell, B. K., 196, 197, 198, 199, 201, 203, 212, 215, 266 Sobin, B. A., 195, 212, 266, 274 Sodergren, J. E., 259, 272 Sogin, S. J., 104, 112, 118 Sokiyama, F., 197, 224, 270 Sokoloski, W. T., 280,293 Sokolova, A. I., 193, 274 Soirns, J.. 36, 8 7 Solomons, G. L., 154,185 Sorkin, E., 20(124), 31 Spalla, C., 190, 274 Spatz, H-C., 240, 242, 243, 270 Specht, H., 46 47,84 Speck, M. L., 64, 84 Spector, W. S., 287, 293 Spencer, H., 239, 274 Spencer, J. F. T., 158, 170(44), 185, 186 Spoon, M., 244,266 Springorum, M., 218,265 Squires, R. W., 178, 186 Stacey, K. A,, 108, 118 Stacey, M., 11(5), 28 Stadhouders, J., 67, 83 Stadtman, F. H., 70,84 Stihelin, H., 289,291 Stamkoff, G., 224,276 Standiford, J., 195, 254, 264 Stanier, R. Y., 93, 118 Stanislavskaya, M. S., 259, 260, 262, 272, 274 Staudenrnayer, T., 59,87 Steams, T. W., 149, 152 Steel, R., 156(10), 158(10),176(72), 185, 186 Stefanac, Z., 246,276 Steinhardt, R. G., 38,87 Steinrauf, L. K., 213,271 Steir, E. F., 38,87 Stenesh, J., 94,108,118 Sternbach, L. H., 212,264 Sternberg, M., 15(54),30 Sternberg, S. A,259,272 Sternhall, S., 285,292 Stevens, R., 46,49,84 Stewart, F. H. C., 225,274 Steyn, P. S., 288,293 Stezak, J., 1?9(82), 187 Stich, K., 229,274

Stickdorn, K., 15,30 Stiles, M. E., 108,118 Stob, M., 279,293 Stock, C. C., 258,272 Stock, J. A., 198, 199, 203, 213, 215, 224, 273 Stoessl, A., 209,223,267 Stokes, J . L., 94,112,118 Stoll, A., 190, 192,201,210, 211,215,274, 285,286,293 Stoll, C., 288,291 Stolpnik, V. G., 193,208,264 Stone, C. J.. 141, 152 Stoutharner, A. H., 27(178, 180), 33 Strange, R. E., 97, 106, 107, 118 Strating, J., 46, 47, 85, 8 7 Strong, F. M., 284,288,290,291,292,293, Strukov, I. T., 13(22),29 Stubbs, J. J., 12(19, 201, 14(20), 15(20), 19, 29, 31 Studer, R. O., 210,211,225,227,228,271 272,274 Stumm, W., 142, 143,151 Subramonyan, S. S., 14(48), 29 Suda, H., 285,291 Sugita, N., 73,87 Sugiura, K., 191, 256, 258, 259, 260, 268, 275 Sulzbacher, W. L., 71,85 Suppel, W. L., 288,290 Suzuki, A., 210,275 Suzuki, S., 15(72, 74, 75, 76, 77, 87), 16(87), 30, 31 Svoboda, V., 15(50), 30 Swartling, P., 64, 87 Swift, M. E., 194, 197, 198, 208, 276 Sylvester, J. C . , 160, 171(30), 173(53), 185,186 Synder, J. J.. 284, 291 Synge, R. L. M., 191, 240,271,272 Szent-Gyorgyi, A., 11(1),28 Szybalski, W., 109, 118, 243, 268 T

Tabachnick, J . , 57, 87 Tabak, H. H., 91,117 Tabenkin, B., 19(108), 31 Taber, W. A., 190,194,195,197,201,275, 285, 286,293

3 14

AUTHOR INDEX

Tachima, A., 282, 292 Tadra, M., 12(7),20(119), 25(149), 28, 31, 32 Taguchi, H., 175(60),186 Takagi, Y., 25(1.52, 153, 154), 32 Takahashi, K., 249,272 Takahashi, S., 14(44),29 Takahashi, T., 26, 27(165),32 Takashiina, M., 288,293 Takeda, R., 25(150),32 Takeuchi, S., 189, 276 Takeuchi, T., 208,275, 285,291 Takeya, K., 285, 291 Takura, C., 287, 292 Tamm, C., 288,291 Tamura, S., 194, 196, 198, 210, 216, 228, 269,273 Tanaka, N., 189,245,256,262,270,275 Tanaka, Yu., 195,270 Tanenbaum, S. W., 27(167, 168),32 Tanner, F. W., 41,87 Tanret, C., 190, 27,5 Tarr, H. C. A., 71, 87 Tarridec, P., 212, 271 Tatum, E. L., 27( 167), 32 Taub, D., 279,. 293 Tawara, K., 195, 270 Taylor, A., 191, 192, 201, 202, 211, 218, 221,235,252,264,266,269,272,288, 291 Taylor, F. H., 39,83 Taylor, W. C., 285, 292 Tchelistcheff, A., 55.87 Te-Ch‘un Ch’iu, 194, 271 Teitel, S., 212, 264 Telling, R. C., 15(56), 30, 141, 152, 178 (79), 179(79),187 Tempest, D. W., 158(21),172(21),179(21), 184(21),185 Tengerdy, R. P., 16(96),31 Te Piao King, 240, 272 Terada, O., 15(72, 73, 74, 75, 76, 77, 79, 87), 16(87),30, 31 Terada, Y., 16(189),33 Teramoto, S., 21(126),31 Teranishi, R., 35, 36, 37, 84 Terent’eva, E. I., 259, 261, 276 Terlain, B., 199, 224, 228, 268 Theander, O., 15(78),30 Theron, J. J., 288,293

Thielen, R., 258, 267 Thimann, K. V., 96, 97, 118 Thomas, J. O.,218, 268 Thomas, J.-P., 199,224,228,268 Thomas, P. L., 211, 225, 271 Thomas, W. A., 195, 208, 268 Thomas, W. R., 100, 118 Thompson, R. L., 149, 152 Thompson, R. Q., 208, 275 Thorne, R. S. W., 142,152 Thoukis, G., 62, 87 Tiboni, O., 238, 263 Tieffenberg, M., 246, 264,275 Tietze, H., 27(166), 32 Tiller, F. M., 135, 152 Timmis, G . M., 203, 210, 274 Tippetto, R. D., 150, 152 Tirunarayanan, M. O., 193, 194, 257, 275 Tisch, D. E., 246,248,255,261,266 Tishler, M., 218, 275 Titus, D. S.,39,87 Todd, A. R., 193, 196, 197, 198, 199, 203, 209, 212,215, 217,221, 223,266,269 Tognoli, L., 190, 274 Tomizawa, K., 15(72, 73, 74), 30 Tonolo, A,, 191,193,264,286,288,293 Tookey, H. L., 288,294 Tori, K., 199, 224, 274 Toropova, E. G., 193,271 Tosteson, D. C., 246, 264, 275 Tovarova, I. I., 230,264 Townsend, R. J., 288,288,293 Townsend, S. B., 281, 290 Traexler, G . , 218, 265 Troll, W., 244, 264 Trolle, B., 48, 84 Tsukada, M., 279, 291 Tsn-Yuan Su, 189, 268 Tuite, J., 279, 293 Tullet, A., 281,290 Turri, M., 101, 118 Tyler, V. E., 287, 290 Tyrell, A. A., 195, 266

u Uchimoto, D., 58, 87 Udenfriend, S., 237, 268 Ueda, M., 62.87 Ueda, R., 73,87

315

AUTHOR INDEX

V

Verwey, W. F., 149,152 Victor, T. A, 214,275 Vining, L. C., 190, 193, 194, 195, 197,201, 208,211,213,214,217,220, 266,270, 272,275,276,285,286, 293 Vinogradova, E. I., 213,221,222,228,239, 255, 264,273, 274, 276 Virtanen, A. I., 96, 118 Visco, S., 72, 8 7 Vissert Hooft, F., 12(14), 29 Viswamitra, M. A,, 208,264 Vogler, K., 210, 211, 224, 225, 227, 228, 271,272,274 Vojnovich, C., 156(7), 185 Vondrovti, O., 23(137), 24(137, 138, 139, 140, 141), 32 von Skramilk, E., 36, 87 von Stedingk, L-V., 249,264 Vorbeck, M. L., 6 9 , 8 7 VotoCek, E., 24(143), 32 Vrtiskova, A., 193, 272

Vacheron, M. J., 208, 267 Vanas, D., 195, 255, 268 Vanderhaeghe, H., 211,275 Van der Hoeven, M. G., 189, 275 Van der Merwe, K. J., 288,293 Vandeputte, J., 195,208,275 Vanderzant, W. C., 94, 113, 117 Van Dijck, P., 195,211,254,256,266,275 Van Dum, H., 67,83 Van Ekenstein, W. A., 20, 31 Van Engel, E. L., 48, 83 Vangedal, S., 289, 291 Van Halsema, G., 254,255,256,257,267 Van Wyk, C. J., 52, 56, 87 Vasilescu, J., 15(54), 30 Vasileva, 0. A., 27(171), 32 Vaughn, R. H., 55, 70, 71, 84, 85, 86, 87, 180(86), 187 Vavra, J. J.. 280, 293 Vazquez, D., 190,256, 257,275 Vedamuthu, E. R., 65, 67, 86, 87 Venema, A., 46,87 Venezia, M., 58, 87 Venstrom D., 40, 83 Vernet, C., 35, 83 Vernon, L., 280, 293 Vero, L., 191,193,264,286,289 Vertogradova, T. P., 259,260,262,274

Wade, R., 243, 270 Wagner, J. R., 39, 87 Wahab, A., 5 7 , 8 7 Wainwright, L. K., 245, 275 Wainwright, S. D., 245, 275 Waisvisz, J. M., 189, 275 Wakisaka, Y., 15, 27(173), 30,32 Waksman, S. A., 191, 194, 201, 218, 242, 255, 256, 258, 259, 262, 270,272,275 Waldron, C. R., 236, 268 Walker, J. A. H., 154, 161, 184 Walker, J . R. L., 67, 87 Walker, T. K., 19(118), 31 Wang, D. I. C., 132, 137,152 Wang, H. L., 72,84 Wang, S., 145, 151 Ward, A., 19(108), 31 Ward, V., 194, 253, 264 Waring, W. S., 288,291 Warner, D. T., 245, 275 Warren, G . H., 105, 119 Waser, P. G., 286,293 Wasserman, H. H., 275 Watanabe, K., 212,262,275 Watennan, H. J., 12(12), 29 Waters, W. R., 157(16), 158, 170, 185 Waterworth, P. M., 253,256,262,267,272

Ueki, H., 279, 291 Uemura, T., 15(64, 85, 86, 88), 30, 31 Ugi, I., 228, 275 Ugolini, F., 154(3), 161(3), 162(3), 185 Ukholina, R. S., 193, 212, 267 Ulrich, K., 159, 161, 185 Umbreit, W. W., 100, 118 Umeda, M., 282, 293 Umezawa, H., 189, 195, 199,208,209,216, 217,230,245,256,262,269,270,275, 285,287,291,293 Underkofler, L. A., 12(18), 14(18, 31, 34, 43), 29 Undstrup, S., 48, 84 Upadhyay, J., 112,118 Urry, D. W., 213, 270 Urry, W. H., 279, 293 Uspenskaya, T. A., 193,212,265 Utech, N. M., 245,268

W

3 16

AUTHOR INDEX

Waxham, F. J., 209,266 Wilson, C. H., 288,293 Webb, A. D., 51, 52,53, 55, 56,57,58,61, Wilson, G. D., 71, 84 62, 84, 85,86,87,88 Wilson, R. E., 172(48), 186 Wilt, F. H., 245, 276 Webb, F.C., 175(65), 186 Weber, S., 279, 293 Winkle, K. C., 179(83), 187 Winter, L. B., 278, 279, 291, 293 Webster, B. R., 221, 264 Weidel, W., 103, 119 Wipf, H. K., 246,276 Weidenhagen, R., 15(70,71), 30 Wise, E. M., 104,119 Weimberg, R., 19(112), 31 Wise, M., 238, 268 Weinstein, I. B., 94, 108, 117 Wiseblatt, L., 42, 43, 44, 85, 88 Weisinyer, D., 289, 291 Witkop, B., 197, 224, 229, 237, 266, 268, 270, 276 Weissbach, H., 191, 235, 237, 238, 268, 270,271,272,275,276 Witter, L. D., 70,86 Witzke, W., 57, 87 Wells, P. A,, 12, 14(20), 15(20), 29 Wells, R. D., 242, 276 Witzman, J. S., 288, 290 Welvaert, R., 12(21), 29 Wogan, G. N., 288,294 Wendland, W., 140, 152 Wolfrom, M. L., 17(101), 31 Wendler, N. L., 279, 293 Woll, E., 59, 88 West, D. B., 47, 48, 49, 83,88 Wolstenholme, W. A., 211, 212, 218, 220, West, I. C., 173(53), 186 264,276 West, J. M., 154(4), 160(4), 162(4), 171(4), Wood, A. B., 288,292 185 Wood, T. H., 90, 97, 99, 100, 109,119 Weston, J. K., 254, 255, 256,258, 261,266 Woodbine, M., 110, 117 Weunnan, C., 47,85 Woodruff, H. B., 194,275 Weyrauch, L., 70,85 Woolley, D. W., 208, 276 Whaley, H. A., 194, 197, 198, 208,276 Woznicka, W., 195,272 Whermeister, H. L., 279, 293 Wright, D., 62, 8 7 White, E. P., 191, 272, 288, 288, 290, 291 Wright, D. G., 156(11), 161(11), 162(11), White, H. R., 100, 117 163(11), 171(11), 172(11), 177(11),185 White, J., 42, 88 Wtodzimierz Kurytowicz, 189, 268 White, L. A., 106, 117 Wul’fson, N. S., 221, 222, 264, 272, 276 White, N. H., 208, 267 Wyckoff, R. W. G., 110,117 Whitfield, J. F., 195, 276 Wyse, B. M., 280,291 Whiting, G. C., 15(172), 27(172), 32,62,88 Y Whiting, R. A., 15(90), 31 Whitlock B. J., 284, 293 Yarnada, M., 63, 72, 85, 88 Whitney, J. G . , 197, 224,273 Yamada, S., 208,263 Whitter, L. D., 108, 118 Yamada, Y., 15(64, 80, 85, 86, 88), 30, 31 Wickens, A. J., 94, 117 Yamaguchi, H., 245,256,262,275 Widholm, J. M., 244, 276 Yamaguchi, M., 281, 287,292 Widmer, C., 14(35), 29 Yamaguchi, T., 27(176), 33 Wielarid, T., 289, 293 Yamaki, H., 245, 256,262,275 Wilker, E. L., 173(56), 186 Yamamoto, A., 63, 64, 86, 88 Wilkin, G. D., 195, 276 Yamamoto, I., 281, 287, 292, 294 Wilkinson, S., 286, 293 Yamamoto, K., 208, 270 Williams, D. H., 196, 209, 217, 221, 223, Yamamoto, Y., 287, 292, 294 266,269 Yamano, T., 208,263 Williams, R. P., 284, 293 Yamashita, S., 164(35), 178(35), 185 Wills, B. A. 113, 119 Yamatodani, S., 208, 263, 285, 286, 287, Wilson, B. J., 288,293 289,292

AUTHOR INDEX

Yamazaki, M., 19(109), 20(122), 23, 25 (146), 26(159), 28(133), 31,32 Yanagihara, T., 72,88 Yasui, H., 64,86,88 Yates, S. G., 288,294 Yokotsuka, T., 73, 83, 88 Yokoyama, S., 208,270 Yonehara, H., 189, 270,276 Yoneyama, H., 72,88 Yong, C., 94, 108,118 Yoshida, T., 194, 195, 202, 236, 237, 239, 269,272,276 Yoshino, H., 16(187), 33 Yoshizawa, K., 48, 88 Yu, A., 209, 227, 228, 230, 269, 273, 274 Yudintsev, S. D., 208, 259, 260, 262, 274 Yurkowski, M., 72, 88 2

Zachau, H. G., 199, 212, 215,273

317

Zahner, H., 194, 208, 212, 255, 265, 266 Zaikin, V. G., 222, 276 Zalta, J. P., 245, 266 Zamenhof, S.,,96, 117 Zangari, V., 255, 267 Zaretskii, I. I., 259, 261, 276 Zaretskii, V. I., 222, 276 Zelinsky, N., 224, 276 Zhdanov, G. L., 211, 227,254,273 Zhdanov, V. M., 242, 270 Zhdan-Pushkina, S. M., 14(37,39,40,45), 29 Zimmermannova, H., 198,224,269 Zohner, H., 246,271 Zoumut, N. F., 44,88 Zsadanyi, J. 195, 268 Zyakoon, A. M., 222, 276

This Page Intentionally Left Blank

SUBJECT INDEX Citreoviridins, 288 Citrinin, 288 Colisan, 288 Colistin, 281 288 Cyclodepsipeptides optical rotation and toxicology, 250,251

A

Actinomycinic acid, 192 Actinordin, 204 Actinomycin degradation, 191, 192 Actinomycins, 193-198, 200, 201, 202, 204, 213, 214, 222, 225, 235, 237, 238-245,255,258,259 Activity from microorganisms ACTH-like from BacUZus subtilis, 280 from Nocardia asteroides, 280 from Serratia marcescens, 280 antiinflammatory, 281 antiprotozoal, 280 antispasmodic, 280 cardiotonic, 282 diabetogenic, 280 emetic, 283 epinepherine-like, 284 from Bacillus anthracis, 284 insulin-like from bacteria, 279 from Streptomyces griseus, 279 from yeast, 279 salivation-inducing, 283 Aflatoxins, 289 Angolide, 194, 197, 209, 215, 216 /3-Alanine, 196 D-Alanine, 196 Althiomycin, 206 Amanitins and phalloidins, 289 Amidomycin, 195 Amphotericin, 283 D-2-Aminobutyric acid, 196 a-Aminobutyrylpeptidolipin-NA, 205 Antibiotic 894, 199,211 Arsimycin, 205 Aspartocin, 205 Avenacein, 205

Crotociny

D

Datemycin, 205 4-Dimethylaminophenylalanine, 199 3,4-Dimethyl-2-methylaminovaleric acid, 198 Destruxins, 194, 196, 198, 207, 210, 254 Dihydrodestruxin B, 216 Doricin, 198,199 E

E-129 B, 196,212 Echinomycins, 193, 194, 195, 196, 198, 206,212,215,255,259,260,262 Endomycin, 283 Enniatins, 193, 194, 198, 210, 214, 216, 225, 246,247,254 Ergocornine, 210 Ergocristine, 211 Ergokryptine, 210 Ergoscaline, 205 Ergosine, 210 Ergot, 192,193,215,228,285,286 Ergotamine, 190, 191,210 Espirine, 205 Estrogenic activity from Fusaria, 279 from Gibberella zeae, 279 Ebmycin, 192, 193, 195, 197, 199, 200, 211,212,215,245,254,261 Eurocidin, 282 F

Fermentor design aeration-agitation system, 157-159 agitation shaft seals, 159-161 air filtration, 164-169 antifoam addition equipment, 171-172 aseptic operations, 161-164. carbon dioxide measurement, 174-175 continuous fermentation equipment, 178-187

B

Baccatin A, 211 Beauvericin, 193, 205, 216 Butenolide, 289 C

Candicidin, 287 Card(c)inophyllin, 205

319

320

SUBJECT INDEX

equipment suppliers, 187- 188 geometry of fermenters, 155-157 materials of construction, 157 mechanical defoamer, 169-171 nutrient addition equipment, 171-172 oxygen measurement equipment, 175177 pH measurement equipment, 172-174 temperature measurement equipment, 177 pressure measurement equipment, 177 Flavor from microbial metabolites baked products, 40-45 chemistry, 75-82 dairy products, 64, 68 fermented beverages, 45-64 oriental foods, 72-74 pickles, 68-71 Fmctigenin, 205 Funyichromin, 283 Fusasiu, emetics from, 284 Fusaric acid, 285 G

Clumamycin, 205, 246 Griseococcin, 206 Criseofulvin, 281, 287 Griseoviridin, 190,209,217,223,254 H

Hamycin, 282 Hydroostreogrycin A, 217 ~-3-Hydroxyleucine,198 cis-3-Hydroxyproline, 197 t~uns-3-Hydroxyproline,197 do-~-3-Hydroxyproline,197

Leucinamycin, 206 D-Leucine, 197 M

Mass spectroscopy of peptide-lactones, 218 Matamycin, 206 Melanosporin, 207 L-N-Methylalanine, 196 L-2-Methylaminophenylacetic acid, 199 L-N-Methylcystine, 199 BMethyl-3-hydroxy-4-amino-5(pyridy1-3’) valeric acid, 199 L-N-Methylisoleucine, 198 L-N-Methylleucine, 198 P-M ethyl tryptop han, 199 L-N-Methylvaline, 198 Microbial cells collection by biphase liquid extraction, 147-148 by centrifugation differential centrifugation, 122- 124 equipment for, 125-126 viruses, 126-132 zonal centrifugation, 124-125 by electrophoresis, 148-150 by filtration, 132-141 by flocculation, 141-143 by foam fractionation, 143-146 by ion exchangers, 146-147 Mikamycin B, 212 Monamycins, 195, 198,206 Monorden, 285 Muconomycins A and B, 288 Muscarine, 285,286

N I

Isariin, 194, 197,211,217,221 Isarolides, 206 D-Isoleucine, 198 D-UbIsoleucine, 197 K

L-Ketopipecolic acid, 197 1

Lagosin, 283 Lateritiin, 193,206

Neotelomycins, 193, 198,207 Nystatin, 282 0

Ochratoxin A, 288 Oneostqtin C, 19s Oosponal, 287 Oospolactone, 287 Oosponglycol, 287 Ostreogrycins, 190, 195-198, 199, 201, 209,212,223,245,254,255,262

32 1

SUBJECT INDEX

P

PA-114 A and B, 195,209,212 Patulin, 281 Pentamycin, 283 Peptide-lactones antibacterial properties, 253 antitumor properties, 258,259 association phenomena with DNA, 239 biosynthesis, 234-258 conformation studies, 228-234 effect on membranes, 245 mechanism of action, 257 nonenzymic synthesis, 224 occurrence on surface of fungal spores, 252,253 structure determination, 203 synergism in microbial tests, 256, 257 toxicity, 254,258,259 toxicology, 239 Peptidolipin NA, 194, 196, 197, 212, 217 L-Phenylglycine, 198 Pimaricin, 282 Pithomycolide, 194, 196,210,216,217 Pristinomycins I and 11,209,212 D-Proline, 196 Psilocin, 286 Psilocybin, 286 Psoralens, 288 Pyridomycin, 195, 199, 209, 216, 217, 230,254 Q

Quinomycins, 195,199,212,236,260 Quinoxaline antibiotics, 191

Siomycin, 207 Slaframin, 283 Sorbose fermentation, 12-16 Sporidesmins, 289 Sporidesmolides, 192, 194, 197, 198, 202, 210,211,216,218,238,254 Staphylomycins, 195,209,211,254 Stendomycins, 207 Streptogramin, 195 Streptozotocin, 280 T

Teleocidin, 288 Telomycin, 197, 198, 199, 213, 255, 261 Thermal injury to bacteria, repair mechanism, 112,114 Thermal inactivation of bacteria by moist heat, 97-98 survival curves of heated bacteria, 98103 possible types of damage to bacterial cells, 103-112 Thermosensitivity of various bacteria thermophilic bacteria, 93,94 psychrophilic bacteria, 94,95 bacterial spores, 95 Thiostrepton, 195,207 Toxic fungal metabolites, 288 Trichomycin, 282 Trichothecin, 288 Triostin A, 212 Triostin C , 198, 199,212,214 Triostins, 194, 196,259 U

R

Ussamycin, 208

Radicicolin, 207 Roridins A to E, 288 Rubratoxins A and B. 288

S Sambucinin, 207 Saramycetin, 207 Scirpene, 289 Sedatives from microorganisms, 285 D-Serine, 196 Serotonin from fungi, 286 Serratamolides, 194,209,217,254

V

D-Valine, 197 Vale-peptidolipin-NA, 208 Valinamycin, 191, 195,212,216,246,247, 248,249,255 Vernamycin A, 209 Vernamycin B, 196,212,225,226,227 Vernamycins, 200,201 Verrucarins A to J, 288 Viridogrisein, 195 Virginiamycin M, 209

322

SUBJECT INDEX

Virginiamycin S, 211 Vitamin C calcium-2-keto-~-idonate,18,20,21,22, 23,25 calciun1-5-keto-D-ghcon~te,19, 20, 22,

Reichstein’s synthesis, 11-16 W

Wildfire toxin, 208,216

x, y, z

24 calcium-bidonate, 20,24,25 e-keto-~-idonicacid, 26,27 5-keto-~-idonate,17

x-53,212 X-948,212 Zearalenone, 279,280