Advances in Applied Microbiology, Volume 12

ADVANCES IN Applied Microbiology VOLUME 12 CONTRIBUTORS TO THIS VOLUME M, C. Allwood MiloB Kulhanek P. Margalith T...

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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.

ALL RIGHTS RESERVED N O P A R T O F T H I S BOOK MAY B E REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, W I T H O U T WRITTEN PERMISSION FROM T H E PUBLISHERS.

ACADEMIC PRESS, INC. 1 1 1 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W l X 6BA

LIBRARY OF CONGRESS CATALOG CARD NUMBER:59-13823

PRINTED IN T H E U N I T E D STATES OF AMERICA

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

This Page Intentionally Left Blank

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


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


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


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


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


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


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.


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.


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-


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


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


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.


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,


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-

+


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


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


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.


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).

30

MILOS KULHANEK

49. E. Mikhlin, and I. Rosenberg, Biochimiyu 15,444 (1950). 50. J. Liebster, B. Imkiik, G . Farber, and V. Svoboda, Chem. Listy 50,395 (1956). 51. I(. Stickclwrn, E. Kwenig, C . Kwnetzke, W. Knape, T. Elsaesser, J. Huber, W. Jagemann, H. Stopsack, and G. Junghans, Gernian Patent 33,788. 52. K. Bernhauer, Ergeh. Enzymforsch. 7,246 (1938). 53. F.J. Bates, “Polarimetry, Sacchariinetry and the Sugars,” National Bureau Standards Circnlar 440, 1942. 54. J. Vasilcscu, M. Sternberg, A. Iscovici, and S. Jurubita, Reo. Chim. (Bucharest) 10,87 (1959). 55. Y. Wakisaka, €1. Ishida, K. Fumiyo, H. Kyotani, N. Kawachi, S. Sangen, K. Fukui, and T. Kimura, Ann. Rept. Sltionogi Res. Lab. 15,69 (1965). 56. R. Elsworth, H.C. Telling, and D. N. East,]. Appl. Aucteriol. 22,138 (1959). 57. Z.F e d , J. l‘iibica and V. Miink, Frcnch Patent 1,457,702. 58. hl. J . Kegan, M. S. Belyakova, G . J. Savastyanov, P. M. Kogan, and N. V. Radeckaya, TT.Vses. Nauchno-lssled. Vitamin. lnst. 8,22 (1961). 59. P. S. S. Dawson, Can.]. Microbiol. 9,671 (1963). 60. L. M. Miall, Chem. Process E n g . p . 295 (1965). 61. M. Kulhinek, and Z. SevCikovi, Folia Microhiol. (Prague)7, 288 (1962). 62. M. Kulhlinek,and Z. SevCikwva, Foliu Microbiol. (Prague}10,362 (1965). 63. Y. Wakisaka, F. Kulbota, H. Ishida, H. Kyohni, and T. Kimura, Ann. Rept. Shionoyi Res. Lnh. 15,77 (1965). 64. K. Sato, Y. Yamada, K. Aida, and T. Uemura, A ~ TB. i d . Chem. (Tokyo) 31, 877 (1967). 65. J. T. Cummins, T. E. King, and V. H. Cheldelin,]. B i d . Chern. 224,323 (1957). 66. J. T. Cummins,V. H. Cheldelin, arid T. E. King,J. B i d . Chem. 226,301 (1957). 67. T. E. King, and V. H. Cheldelin,]. B i d . Ckena. 198,135 (1952). 68. T. E. King, and V. H. Cheldelin, Science 115,14 (1952). 69. H. S. Isbell, and J. V. Karabinos,]. Res. Natl. Bur. Std. 48,438 (1952). 70. R. Weidenhagen, and G . Bernsee, Angew. Chem. 72,109 (1960). 71. R. Weidenhagen, and G. Bernsee, Chern. Ber. 93,2924 (1960). 72. 0.Terada, K. Tomizawa, S. Suzuki, and S. Kinoshiti, Bull. Agr. Che7n. Soc. Japan 24,535 (1960). 73. 0. Terada, K. Tomizawa, and S. Kinishita, N i p p o n Nogeikugaku Kuishi 35, 127 (1961). 74. 0. Terada, K. Tornizawa, S. Suznki, and S . Kinishita, Nippori Nogeikuguku Kaislzi 35,131 (1961). 75. 0.Terada, S. Suzuki, and S. Kinoshita, Nippon Nogeikugaku Kuishi 35,178 (1961). 76. 0. Terada, S . Suzuki, and S . Kinoshita, N i p p o l l Nogeikagaku Kuishi 35, 1331 ( 1961). 77. 0. Terada, S. Suzuki, ;ind S. Kinoshita, N i ~ p o nNogeikaguku Kuishi 35, 1336 (1961). 78. 0.Theander, Aduari. Curbohydrule Chem. 17,223 (1962). 79. S. Kinoshita, and 0.Terada, Gerinan Patent 1,171,413. 80. K. Aida, and Y. Yamada, Agr. B i d . Chem. (Tokyo) 28,74 (1964). 81. G . Avigad, and S. Englard,]. B i d . Chem. 240,2290 (1965). 82. S. Englard, and G. Avigad,]. Biol. Chem. 240,2297 (1965). 83. S. Englard, G. Avigad, and L. Prosky, J. B i d . Chem. 240,2302 (1965). 84. G. Avigad, S . Englard, and S. PifkoJ. Biol. Chem. 241,372 (1966). 85. Y. Yamada, K. Aida, and T. Uemura, Agr. B i d . Chern. (Tokyo)30,95 (1966).


31

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).

32

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131. M. Yamazaki,]. Ferment. Technol. 31,126 (1953). 132. M. Yamazaki,]. Ferment. Techno/.31,230 (1953). 133. M. Yamazaki,J.Agr. Chem. Soc.Jnpnn 28,890 (1954). 134. M. Yamazaki, Japanesc Patent 5331 /'55/. 135. G. Farber, Sb. Cesk. Akad. Zemedel. 23,355 (1951). 136. V. Ettel, F. OEenabek, and G. Firher, Czech. Patent 86,671. 137. G. Firber, 0.Vondrovi, and 13. Luk'sik, Czech. Patent 87,466. 138. .,and Umbreit, W. W. (1959). “Ail Introduction to Bacterial Physiology,” 2nd ed. Freeman, 1,ondon. Pacc, B., and Campbell, L. L. (1967).Proc. Natl. Acad. Sci. U.S. 57,1110-1117. Pethica, B. (1958).J.Cen. Microbid. 18,473-480. . Pheil, C. G., Pflug, I. J., Nicholas, R . C., and Augdstin, J. A. L. (1967). A p r ~ lMicrobid. 15, 120-124. Pnrohit, K., arid Stokes, J. L. (1967).J . Bacteriol. 93, 199-206. Rahn, 0. (1945). Bacteriol. Reu. 9, 2-48. Rahn, 0..and Schroeder, W. R. (1941). Biodynamicu 3, 199. Razin, S.,and Argamon, M. (1963).]. Gem Microbiol. 30,155-172. Hubison, S. H., and Morita, R. Y. (1966). Z.Allgem Mikrobiol. 6, 181-187. Hosc, A. H. (1967). “Tlieriiiobiulogy.” Academic Press, New York. Russcll, A. D. (1965). M f g . Chemist 36, 38-45. Russell, A. D., and Harries, D. (1967). April. Microbid. 15, 407-410. Rnssell, A. D., a i i d Harries, n. (1968).A p ~ dMicrobial. . 16, 1394-1389. Salton, M. H. J . (1953). J . Cen. Micro6iol. 9, 512-523. Salton, M. R. J. (1964). “The Bacterial Cell Wall.” Elsevier, Amsterdam. Salton, hl. H.I., arid Shafa, F. (1858).Nuture 181,132. Saunders, C . F., and Campbcll, L. L. (1965).J . Bacteriol. 91, 332-339. Schmidt, C. F. (1957). In “Antiscptics, Disinfectants, Fungicides and Sterilisatioil” ( G . F. Heddisli, ecl.), 2nd ed., pp. 831-885. Henry Kempton, London. Shaklovskii, K. P. (1965). Bych. E k s p . Hiol. M e d . 59,8640. Sogin, S. J., and Ordd, Z.J. (1967).]. Bacteriol. 94,1082-1087. Stacey, K. A. (1965). 7 5 t h Symp. Soc. Gen. Microhiol., pp. 166-168. Cambridge Univ. Press, London. Stanier, R. Y., Doudoroff, M., and Adelburg, E. A. (1963). “Ceneral Microbiology.” Macni illan, London. Stenesh, J., and Yong, C. (1967).J . Bacteriol. 93, 930-936. Stiles, M. E., and Whitter, L. D. (196,5).J.Dairy Sci.48,677-681. Strange, H. E., and Shon, M. (1964).J . Gem Micsobiol. 34, 99-114. Szybalski, W. (1967). In “Thermobiology,” (A. H. Hose, ed.), pp. 73-122. Academic Press, New York. Thinrann, K. V. (1963). “The Life of Bacteria,” 2nd ed. Macniillan, London. Thomas, W. R., Hdiiibold, G . W., and Nelson, F. E. (1966).J . Milk Food Technol.29, 156- 160. Turri, M., Maccacao, C,. A,, and Dettori, R. (1964). Gert. Microbid. 12,153-161. Upadhyay, J., and Stokes, J. L. (1963).J . Bacteriol. 85, 177-185. Virtanen, A. I., and Pnlkki, L. (1933). Arch. Microbiol. 4, 99-122.

THERMAL INJURY IN NONSPORULATING BACTERIA

Warren, C. H., and Grey, J. (1963).Proc. SOC. Exptl. BioL Med. 114,439-444. Weidel, W., Frank, H., and Martin, H. H. (1960).J . Gen. Microbiol. 22, 158-166. Wills, B. A. (1957).J . Pharm. Phurmacol. 9,864-876. Wise, E. M., and Park, J. T. (1965). Proc. Natl. Acad. Sci. U.S. 54, 75-81. Wood, T . H. (1956). Aduaa. Biol. Med. Phys. 4, 119-164.

119


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


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


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


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


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


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


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.


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 “


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


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


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.


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.


151

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.

152


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%


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


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


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


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


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

185

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.

FERMENTOR DESIGN

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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


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)


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


TABLE I (Continued)


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


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


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

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