Advances in Applied Microbiology, Volume 24

ADVANCES IN

Applied Microbiology VOLUME 24

CONTRIBUTORS TO THIS VOLUME

Charles L. Cooney

R. J. Erickson David E. F. Harrison Robert J. Heckly

M. B. Ingle

Nobuo Kato Thomas J. Montville Nduka Okafor Wesley 0. Pipes

M. A, A. Schipper Jun’ichi Shoji Anthony J. Sinskey Yoshiki Tani J. A. Von Arx Hideaki Yamada

ADVANCES IN

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

VOLUME 24

@

1978

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

COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

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LIBRARY O F CONGRESS CATALOG CARD NUMBER:59-13823 ISBN 0-12-002624-4 PRINTED IN THE UNITED STATES OF AMERICA

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

ix

Preservation of Microorganisms

ROBERT J. HECKLY I. 11. 111. IV. V. VI. VII.

Introduction . . . . . ................................ .............. Criteria for Preservation . . . . . . . . . . Preservation Methods . . . . . . . . . . . . . . . . . . . . . . . . Culture Collection Practices . . . . . . . . . . . . . . . . Industrial or Commercial Practices . . . . . . . . . . . . . . . . . . . . . Procedures for Selected Groups . . . . . . . . . . . . . . . . . . . . . . . Summary . _ ., , . . . . , . . . . . . . . . . . . . . . . . . . . . .................................. References.. . . . .

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

.......... ,.........

1 2 5 29 31 34 47 47

Streptococcus mutans Dextransucrase: A Review

THOMAS J.

MONTVILLE, CHARLES

L.

COONEY, AND

ANTHONYJ. SINSKEY I. 11. 111. IV. V. VI. VII. VIII.

................................. . . . . . . . . ......................... Mechanisms of Dext is . . . . . . . . . . . . . . . ..............

The Role of Surface Receptors in Cell Adherence and Aggregation . . . . . . . . . . Distribution of Dextransucrase . , . . . . . . . . . . . . , . . . . . . . . . . . Purification and Properties of Dextransucrase . . . . . . . . . . . . . . . . ........................... Regulation of Dextransucrase . . . . . . . Other Extracellular Enzymes Produced by Streptococcus inutans . . . . . . . . . . . IX. The Heterogeneity of the Species Streptococcus tnutans . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . X. Conclusion . . . . . . . . . . . . . . . . . . References .......................................

55 56 58 61 63 64 75

77 80 82 82

Microbiology of Activated Sludge Bulking

WESLEY0. PIPES I. 11. 111. IV. V.

......................... Introduction . . . . . . . . . . . . . . . . . . . . . . . The Nature of the Bulking Problem ......................... Filamentous Organisms in Activated .................. ................ Case Studies , . . . . . . . . , . , . . . . , . . . . . . . . . . . . . . . Summary and Future Prospects. . . . ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... V

85 86 95 115 123

125

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CONTENTS

Mixed Cultures in Industrial Fermentation Processes

DAVIDE . F. HARRISON I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Traditional Uses of Mixed Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Types of Microbial Interaction IV. Continuous Enrichment Techn V . Mixed Cultures for Single-Cell Protein Production . . . . . . . . . . . . . . . . VI . Other Applications of Mixed Cultures . . . . . . . . . .......................... VII . Future Prospects . . . . . . . . . . . . . . .................................. References ............. ..................................

129 130 132 135 141 157 160 162

Utilization of Methanol by Yeasts

YOSHIKI TANI.NOBUOKATO. I. I1. I11. IV. V.

AND

HIDEAKIYAMADA

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dissimilation and Assimilation of Methanol in Methylotrophs Other Than Yeast Dissimilation and Assimilation of Methanol in Yeasts ...................... Cell Yield and the Metabolic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of Cells and Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 167 170 180 182 183

Recent Chemical Studies on Peptide Antibiotics JUN’ICHI

SHOJI

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. 111. IV. Peptide Lactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

187 188 194 208 212 213

The CBS Fungus Collection

J . A . VON ARX AND M . A . A . SCHIPPER I . Introduction and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. The Collection of Fungi and Actinomycetes in Baarn . . . . . . . . . . . . 111. The Collection of Yeasts in Delft .............................. References .................................................

.. .. .. ..

215 218 233 235

Microbiology and Biochemistry of Oil-Palm Wine

NDUKAOKAFOR I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Microorganisms in Palm Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I11. Biochemical Changes in Palm Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 241 245

vii

CONTENTS

IV. Preservation of Palm Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Survival of Bacterial Pathogens ......................................... VI. Conclusion., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

252 254 254 255

Bacterial a-Amylases

M. B. INGLEAND R. J. ERICKSON I. Introduction . . . . . . . . . . . . . . . . . .

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

11. Thermophilic Amylases . . . . . . . . . . . . . . . . . . ........................ 111. Alkaline Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.................................. .................. .................. VI. Conclusion. ........................... References . . . . .............................. .

257 258

IV. Acidic Amylases . . . . . . . .

SUBJECT INDEX .......................... CONTENTS OF PREVIOUS VOLUMES . . . . . . . . .

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

260 275 276

279 281

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LIST OF CONTRIBUTORS

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

CHARLESL. COONEY, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (55) R.

J . ERICKSON, Research and Development, Industrial Products Group, Miles Laboratories, Inc., Elkhart, Indiana 46514 (257)

DAVIDE . F . HARRISON, Biological Laboratory, University of Kent at Cant e r b u y , Canterbuy, Kent CT2 7 N J , United Kingdom (129) ROBERTJ. HECKLY, Naval Biosciences Laboratory, School of Public Health, University of California, Berkeley, California 94720 (1) M . B. INGLE, Research and Development, Industrial Products Group, Miles Laboratories, lnc., Elkhart, Indiana 46514 (257) NOBUOKATO, *Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan (165)

THOMASJ . MONTVILLE, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (55) NDUKA OKAFOR, Department of Microbiology, University of Nigen'a, Nsukka, Nigeria (237) WESLEY0. PIPES, Department of Biological Sciences and The Environmental Studies Institute, Drexel University, Philadelphia, Pennsylvania 19104 (85) M . A. A. SCHIPPER, Centraalbureau uoor Schimmelcultures, Baarn, The Netherlands (215)

Shionogi Research Laboratory, Shionogi and Company, Ltd., Fukushimu-ku, Osaka, 553 Japan (187)

JUN'ICHI SHOJI,

*Present address: Department of Environmental Chemistry and Technology, Tottori University, Koyama-cho, Tottori-shi, 680 Japan

ix

X

LIST OF CONTRIBUTORS

ANTHONY J. SINSKEY, Department of Nutrition and Food Science, Massachusetts lnstitute of Technology, Cambridge, Massachusetts 02139 (55) YOSHIKITANI,Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan (165)

J. A. VON A m , Centraalbureau v o w Schimmelcultures, Baarn, The Netherlands (215) HIDEAKIYAMADA,Department of Agriculturul Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan (165)

ADVANCES IN

Applied Microbiology VOLUME 24

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Preservation of Microorganisms

ROBERTJ. HECKLY Naval Biosciences Laboratory School of Public Health, University of California, Berkeley, Califwnia I. Introduction . . . . . . . . . . . . . . . .

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

11. Criteria for Preservation. .....................

A. Ability to Reproduce.. . . . . . . .: . . . . . . . . . . . B. Functional Properties ............................... C. Maintenance of Full Genetic Complement . . . . . . . . . . . . . 111. Preservation Methods ................................... A. Direct Transfer on Culture Media .................... C. In Distille

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

E. Dehydrated.. ...................................... IV. Culture Collection Practices . . . V. Industrial or Commercial Practices ....................... VI. Procedures for Selected Groups .......................... A. Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Fungi, Yeasts, and Actinomycetes D . Viruses and Bacteriophages VII. Summary . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . ..............................

1 2 2 3 4

5 5 5 6 6 14 15 29 31 34 34 35 41 42 47 47

1. Introduction Maintenanbe of cultures is a problem common to many areas of microbiology. The microbiologist needs to have a convenient method for maintaining organisms, for without such tools he or she is out of business. Similarly, a number of industries must maintain the cultures used in the manufacture of their product, whether it be beer, wine, antibiotics, bread, or milk products, such as cheese or buttermilk. It would be desirable to be able to define the conditions for optimal survival of each of the various organisms but this is not yet possible. As it is not generally feasible to preserve each organism in a culture collection under more than one condition, there is a scarcity of comparative data on long-term storage of a variety of organisms. The preponderance of publications on optimizing culture preservation methods is written by microbiologists interested in maintaining relatively few species, often only one strain. Those studying the mechanisms of action of freezing and thawing or lyophilization often use only one or two strains in their studies. This author will attempt to summarize information on the various methods used to preserve microorganisms. There are many factors to be considered 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright Q I 1978 by Academic Press, Inc. All rights of r e p d u d i o n m any form reserved. ISBN 0-12402634-4

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ROBERT J. HECKLY

and there is no single “best” method applicable to all situations, but by comparing the results of the various procedures it is hoped that the task of selecting procedures for the preservation of specific cultures can be simplified. Organisms are able to survive under a wide variety of adverse conditions. In fact, they seem to be everywhere and are sometimes difficult to eliminate. However, some organisms are not hardy and preservation of cultures with special characteristics can be a problem. The total property of a culture may depend upon several variants within the culture. Therefore, it is important that all, or at least a representative sample, of the cells be retained in a viable state.

II. Criteria for Preservation A. ABILITYTO REPRODUCE Quantitative measurements, such as colony-forming units (cfu)or plaqueforming units (pfu), provide much information about the quality of the preservation technique. If these assays are made before and after storage, they provide an objective means for selecting the optimum method. Unfortunately, too many investigators do not have the time to make quantitative measurements of viability. Instead, they depend on a growth or no-growth test; i.e., the entire contents of a vial may be transferred to growth media which are then examined for signs of life. Reconstitution of replicate samples can yield a degree of quantitation. For example, Antheunisse (1972)reported survival after various storage periods based on the percentage of vials that yielded viable cultures. Obviously, such survival rates cannot be compared with survival rates based on the number of cells in a sample that have formed colonies. Selection of the proper medium for evaluating ability to reproduce should receive serious consideration. Judging from the number of papers published on injury and repair in bacteria, it appears that a significant proportion of stored cells are injured and are unable to initiate growth under the ordinary conditions. Injury is defined as sublethal or repairable damage, whereas mutation is a permanent change. Some injured cells are no longer able to grow on minimal media but are able to form colonies on complete media, such as trypticase soy agar (Sinskey and Silverman, 1970; Gomezet al., 1973; Gibson et al., 1965). In other instances the injured cells required only supplementation with aspartate (Kuo and MacLeod, 1969) or pyruvate (Baird-Parkerand Davenport, 1965) to grow. A loss of salt tolerance (Morichi and Irie, 1973) or an increase in the lag phase (Beker, 1972) have also been

PRESERVATION OF MICROORGANISMS

3

attributed to cell injury. In other instances, the proportion of injured cells was determined by the use of special selective media (Ray et al., 1971a, b, 1972; Janssen and Busta, 1973a,b). The percentage of injured cells in preserved cultures has been as high as 95% (Janssen and Busta 1973a; Ray et al., 1971a; Gibson et al., 1965).

B. FUNCTIONAL PROPERTIES Viability, even if it is based on number of organisms surviving, is not an entirely satisfactory criterion for evaluating effectiveness of culture preservation. Sirks et al. (1974) demonstrated that the efficacy of two different types of freeze-dried tuberculosis vaccine made from the avirulent bacillus of Calmette and Guerin (BCG) in guinea pigs was not correlated with the percentage of cells surviving the lyophilization and storage. Immunogenic properties of the live vaccine were altered by the drying. Similarly, Heckly et al. (1958) found that although 40% of the lyophilized Yersinia pestis cells survived after 9 months, only 0.3% of the infectivity was retained. Therefore, such preparations would not provide a reliable challenge for testing efficacy of plague vaccines, as apparently there was considerable sublethal damage to the cells. Mitchell and Enright (1957)also demonstrated that the number of viable organisms provided a poor index to preservation. They observed that although the leavening ability of dried yeast was lost, there was no loss of viability. It is particularly important that criteria other than number of viable cells be used in the development of preservation methods if the culture is to be used directly without subculturing. Effectiveness of the preservation method used in the cheese industry has been evaluated by simulating procedures used in making cottage and cheddar cheese (Speckman et al., 1974; Keogh, 1970)or cultured buttermilk (Lamprech and Foster, 1963). Direct measurement of acid development and proteinase activity has also been used (Cowman and Speck, 1965; Speck and Cowman, 1970 Gibson et al., 1966). Gibson et al. (1966) found that although glycerol and dimethyl sulfoxide (DMSO) were effective cryoprotective agents for maintaining high viability of freeze-thawed Streptococcus cremoris, acid production during subsequent incubation was retarded. In contrast, malic acid protected viability and stimulated acid production after thawing. Johannsen (1972) found a good correlation between survival after freezing Lactobacillus leichmannii in malt extract and ability to produce lactic acid but she did not study the effects of other suspending media. Tanguay (1959) used biochemical activity to evaluate the quality of preservation. Even after 12 months storage at -40°C the response of the thawed organisms was identical with that of the nonfrozen control in the assay procedures for lysine, inositol, tetracycline, and various vitamins. Stapert and Sokolski (1968)

4

ROBERT J. HECKLY

found that although only 33%of L. leichmannii survived freezing and thawing, they obtained a normal dose-response curve for vitamin B,, analysis if they used three times the number cells in the test. Davis (1963) also demonstrated that if cells remained viable they were functional. He found that a high percentage of the lyophilized rhizobial cultures were viable after 21 years and capable of effective nodulation of the hosts after having been stored for 21 years. These few examples show that biochemical or biological activity, immediately after recovery from storage, may be an appropriate criterion for evaluating preservation. Viability assays should not be abandoned because they can provide sensitive and quantitative measures of quality control which can be used to predict when the culture should be reprocessed. If the original number of viable cells is in the order of 10s/ml a decrease of even 1 log/year should not be a cause for concern, but if loss in titer approaches 2 or 3 logyear, the culture may easily be lost in 5 years.

c. MAINTENANCE OF FULLGENETICCOMPLEMENT Implicit in the concept of stock culture preservation is the fact that the genetic composition of the progeny is the same as that of the original culture. Ordinarily, cultures are not studied in detail because it is nearly impossible to determine for each culture that there has been no genetic alteration. Under most conditions only gross changes would be noted, such as pigmentation (Servin Massieu and Cruz-Camarillo, 1969). Kubica et al. (1977) concluded that although mycobacteria stored at -70°C for 2-5 years appeared to be sluggish in diagnostic tests, culturing restored their vigor and key differential features were retained. However, Harrison and Pelczar (1963)reported some genetic changes in two lyophilized strains of Bacterwides that had been stored at room temperature for 5 years. They observed changes in morphology, temperature requirements, and fermentation reactions. It was noted that the number of viable cells was probably very low at that time, because after three additional years of storage no viable cells could be recovered. In view of the fact that in many collections only a few organisms might survive storage to provide the inoculum for subculturing, it would seem that there might be a tendency to select mutants. However, properties of most cultures tend to be retained because it has been stated that auxotrophic mutants are not as hardy as the parent prototrophs (Webb, 1969). If it is true that auxotrophic mutants are less stable than the prototrophs, it is extremely important that methods used to preserve mutants, such as the Ames tester strains of Salmonella (Ames et al., 1975), yield maximum survival and do not alter DNA. In studying the preservation of a special mutant of Penicillium chrysogenum, MacDonald (1972) observed that storage of this culture at 4°C

PRESERVATION OF MICROORGANISMS

5

led to the development of a subpopulation with substantiallylower penicillin production. Such spontaneous mutation and selection might be expected in liquid storage. Fortunately, these changes were not observed in cultures stored at -196°C. As a test for mutagenic effects of lyophilization on fungi, Mehrotra et al. (1970)compared the productivity of 100 single-spore cultures before and after the lyophilization of nine different industrial strains. These strains were used to produce organic acids, antibiotics, and enzymes. Since the productivity of lyophilized spores was not significantlydifferent from that of spores that had not been dried, it was concluded that none of the 900 spores suffered a significant genetic change.

111. Preservation Methods A. DIRECTTRANSFERS ON CULTURE MEDIA Agar slants are the most common method for maintaining the working inoculum, but stab or broth cultures are preferred by some workers, particularly for anaerobic organisms. Since there is an increased possibility of mutation with each subculture, frequent transfers are undesirable for long-term preservation of organisms. Mutation frequency in most cultures is low but Watko and Heddleston (1966)reported that in as few as 2 months, agar slants of Pasteurella multocida dissociated to provide a mixture of fluorescent, blue, and sectored colonies.

B. UNDEROIL Many organisms survive reasonably well when agar slant cultures are covered with mineral oil and this procedure is used frequently by teachers to carry cultures for class use. Some cultures that did not survive lyophilization well were maintained under oil in the Agricultural Research Service (ARS) collection (Hesseltine et a l . , 1960; Hesseltine and Haynes, 1974). Nadirova and Zemlyakov (1971) reported %year survival under oil by Pseudomonas, Bacillus, and Escherichia. Mutation during the prolonged storage is possible because it is believed that bacteria can continue to reproduce under these conditions, but Nadirova and Zemlyakov (1971) have concluded that morphological and cultural characteristics, as well as physiological properties, remain unchanged in 3 years’ storage. However, all organisms cannot be stored successfully under oil. Yamasato et al. (1973) reported that the percentage of cells surviving 3.5 years’ storage varied from 77 to 90% for most of the organisms studied but only 25% of the Gluconobacter were recovered. Furthermore, if the slant is not completely covered, it dries up. Since survival for more than 1 year cannot be assured, considerable labor is

6

ROBERT J. HECKLY

involved in making regular transfers. The greatest disadvantage of using oilcovered slants is that it is a messy method. The method described by Antheunisse (1972) may be considered to be comparable to the oil overlay method but it is not as messy. After the cultures had grown out on the agar slant, he simply removed the cotton plug and replaced it with a rubber stopper. He reported 60-100% survival of 36 species for 3 years, with many surviving 10 years. Only Acetobacter, Aerobacter, and Streptomyces failed to survive well. A patent for drying organisms in corn oil was issued to Johnston (1962). In his method, water was removed by bubbling dry air through the oil at 35°C and cells were finallycollected by filtration. Since this author has not seen any application of this method, he presumes that it is not widely used.

C. IN DISTILLEDWATER Many organisms die rapidly when suspended in distilled water. However, cultures of Pseudomonas solanacearum in distilled water have been reported to survive for more than 10 years at room temperature. Surprisingly, these same organisms died rapidly when stored under refi-igeration(Berger, 1970). McGinnis et al. (1974) recommended distilled water for preservation of stock cultures of fungi and presented data showing that 93% of the cultures survived storage for 4 years at room temperature. The fungi that failed to survive were poor sporulators (about 6 4 % of the collection). They also reported that storage in water suppressed pleomorphic changes and that no genetic changes were detected. Tanguay and Bogert (1974) found that both Saccharomyces cerevisiae and Sarcina lutea survived well when suspended in dilute phosphate buffer at 4°C for 4 months, and even after 1 year 2-19% survived. D. FROZEN With the improvements in refrigeration systems and the greater availability of liquid nitrogen, freezing is becoming the method of choice for both short- and long-term storage of viable microorganisms. Disadvantages of liquid nitrogen are that it is relatively expensive and requires constant surveillance if automatic filling and alarm systems are not used. Mechanical refrigeration can also be expensive and it is subject to both electrical and mechanical failure. However, with proper safeguards this is not a serious problem.

1 . Techniques The general practice is to seal ampules by fusing the neck with a flame, but this may leave a small channel (Greiff et al ., 1975). When the ampule is

PRESERVATION OF MICROORGANISMS

7

immersed in liquid nitrogen enough of the liquid may enter during prolonged storage to explode the vial when it is warmed suddenly. Therefore, Simione et al. (1977) used screw-capped plastic ampules to minimize the hazard. These appear to be satisfactory for the preservation of a variety of organisms. In some studies it has been desirable to be able to use frozen material as the inoculum for each experiment and, in these instances, large numbers of vials are required. Instead, bits of culture can be scraped off the frozen surface with a sterile applicator stick without thawing the entire culture (Ames et al., 1975).A more sophisticated tool was described by Bullen (1975) for scraping the surfaces of frozen culture in a closed vial. The purpose of this was to minimize the biological hazard when working with pathogenic microorganisms. Two other techniques should be mentioned because they are convenient and provide relatively uniform inocula. Cox (1968) obtained satisfactory results by freezing drops of culture in liquid nitrogen. Nagel and Kunz (1972) coated small glass beads with cells suspended in the bacterial culture to which an equal volume of horse blood had been added. Sterile forceps were, used to remove a single pellet or bead without disturbing the remaining culture. Both have the advantage of thawing rapidly when dropped into a tube of warm broth. A convenient technique for preserving fungi involves freezing organisms on agar plugs. As described by Dietz (1975), cultures are grown on agar plates and plugs are cut by pressing short sections of sterile paper drinking straws into the culture. By repeating the operation, several plugs of agar, with the overlaying culture, can be collected in each section. These pieces of drinking straw are then placed in a vial and frozen in the gas phase of a liquid nitrogen freezer. For retrieval, a straw is placed in a petri plate and when thawed sufficiently, one plug is removed and the remaining plugs are returned to the freezer. 2. Considerations

An attempt will be made to consider the various factors identified as having affected the survival of frozen organisms, even though these factors are interrelated and cannot be studied as independent variables. a. Age of culture. The physiological condition of microorganisms has been considered by many investigators to be a factor in determining their ability to survive stress. It is generally accepted that cells from the maximum stationary phase cultures are more resistant to damage by freezing and thawing than cells from the early or midlog phase of growth. The percentage of cells surviving is also increased by an increase in cell density, possibly because lyzed cells can yield cryoprotective substance (Bretz and Ambrosini,

8

ROBERT J. HECKLY

1966).This generalization is probably not valid for all organisms, particularly viruses, in view of the observations by Nyiendo et al. (1974), who have found that the percentage survival of lactic Streptococcus bacteriophages is not correlated with original titer before freezing. Heckly (1961) pointed out that nutrition during growth, culture density, and age were equally important factors in the survival of lyophilized bacteria. However, the effects of these functions are not easily separated. By using spent growth media (the filtrate from a stationary phase culture) as a diluent, Packer et al. (1965) demonstrated that for Escherichia coli, the phase of growth, the state of aerobiosis, and the density of the culture had no effect on the degree of susceptibility to death by freezing and thawing. Instead, it appeared that the media were changed by the organisms and that sensitive cells were protected by suspension in the spent growth media. Since the protective effect was demonstrable at relatively high dilutions, some of the observed variations in the killing effect of freezing and thawing may be due to incomplete washing of cells. For practical purposes it makes little difference whether the cells or the media is changed; the fact remains that cells from young cultures do not survive freezing and thawing as well as those in mature cultures at a high cell density.

b. Rate offieexing and thawing. For a number of years it was considered essential to freeze organisms rapidly to obtain high survival. Therefore, methods were developed to achieve ultrarapid freezing 'and it was shown that a variety of organisms survived ultrarapid freezing and thawing (Doebbler and Rinfret, 1963).They calculated that cooling rates of several hundred degrees per second were obtained by spraying the culture into liquid nitrogen from a 26 gage hypodermic needle. Similar warming rates were obtained by slowly sifting the fine pellets into saline at 37°C. However, many investigators who have studied the problem of freezing rate have now found that slow freezing and rapid thawing generally yield the highest number of viable cells (Stalheim, 1971; Johannsen, 1972; Bank, 1973; Mazur, 1966, 1970; Raccach et al., 1975;Torney and Bordt, 1969). Exceptions to this generalized observation have been reported. Levy (1969) found that the highest survival of Mycobacterium leprae was obtained by rapid freezing and slow thawing, but this might not be significant because M . leprae did not seem to be particularly sensitive to the rate of thawing. Furthermore, his data were not entirely consistent. Mazur and Schmidt (1968)found that for yeast, there was an interaction between cooling velocity and warming velocity; i. e., reasonable survival was obtained at high freezing rates if the thawing was rapid enough. Rapidly frozen Pseudomonas fluorescens also survived well when thawed rapidly (Ashwood-Smith and Warby, 1971a).

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9

It has been found that there is an optimum cooling rate for maximum survival that varies with the nature of the cell (Mazur, 1970, 1977; Raccach et al., 1975). The optimum cooling rate for S. cerevisiae was about 7"C/min (Mazur and Schmidt, 1968), but Wellman and Stewart (1973) reported a higher mutation rate of brewing yeasts when the cultures were frozen at 9-17"C/min than when frozen at 1"C/min. Meyer et al. (1975) found that for other yeasts, the optimum rate was between 4.5 and 6.5"C/min. Raccach et al. (1975) have demonstrated that the optimum cooling rate may be dependent upon the final storage temperature. When Acholeplasma laidlawii was cooled to -20°C the optimum rate was between 8 and 1O0C/min,whereas when the culture was cooled to -70°C the optimum rate was about 16"C/min. Despite evidence just cited, cooling at 1"C/min seems to be widely used largely because it is impractical to determine the cooling rate for every organism. Since freezing rate is not a critical factor for survival, a controlled rate freezer, which is relatively expensive, is probably unnecessary. Satisfactory results have been obtained by placing the samples in a slightly insulated container in a -70°C freezer, as described by Swoager (1972).

c. Storage temperature. Liquid nitrogen provides the lowest practical temperature (-196°C) for storing microorganisms and, because viability is preserved so well, it is used extensively for all sorts of organisms (Swoager, 1972; Clark and Klein, 1966; Norman et al., 1970; Hwang, 1970; Butterfield et al., 1974). Most quantitative studies on survival of organisms in liquid nitrogen have involved only short-term storage but prospects for prolonged survival are good. Keogh (1970) stored Streptococcus lactis and S. c r e m d s for 13 months without a significant change in number of viable cells. Norman et al. (1970) preserved some strains of Mycoplasma for as long as 9 years, but they gave no data on viability. Butterfield et al. (1974) reported that practically all of the fungi stored for up to 8 years in liquid nitrogen were viable. They mentioned problems with loss of viability of only two organisms, Paracoccidioides brasilensis and Basidwbolus. These may require special freezing conditions. Clark and Klein (1966) found that although as much as 99% of the original titer of bacteriophages was lost on freezing, there was no further reduction in titer after 3 5 years of storage in liquid nitrogen. It appears that most of the damage leading to the loss of infectivity of phage or viability of other organisms occurs during the freezing and thawing steps and not during the storage period. Therefore, if survival is good after a few months of storage, the samples can be expected to survive for many years. Because liquid nitrogen storage is relatively expensive it would be advantageous for viability to be preserved at higher temperatures. There have been many studies on the effect of temperature on survival of organisms. It is evident that there are marked differences in sensitivity of the various groups

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or kinds of organisms. About 80%of the cells in frozen cultures ofhctobacillus acidophilus were viable after 6 months at either -10, -20, or -60°C (Duggan et al., 1959) and some mycobacteria also survived quite well at -20°C (Gruft et al., 1968;Kim and Kubica, 1972). However, the mammalian tubercle bacilli and a few other species of mycobacteria survived much better at -70°C than at -20°C. Practically 100% of the Mycobacterium tuberculosis culture was viable after 25 months at -70°C (Kim and Kubica, 1972, 1973). About 90%of the L forms of either Neisseria meningitidis or Streptococcus pyogenes survived storage at -196°C for 1 year, but neither survived 6 months at -20 (Stewart and Wright, 1970). Similarly, Cowman and Speck (1965) found that although 7040% of the lactic streptococci survived 2 months at -196"C, fewer than 2% survived at -20°C. Tanguay (1959) reported that a number of different organisms used in microbiological assays appeared to survive satisfactorily for 1 year at -40°C. However, it was subsequently shown that in the absence of cryoprotective agents less than 1%of S. cereuisiae survived 1 year at -40°C (Tanguay and Bogert, 1974). Cox (1968), also without adding cryoprotectants, obtained 80% survival ofE. coli K-12 HfrC after 1 month at -7O"C, whereas at -15°C the preparations were sterile. After an extensive study of 259 strains belonging to 32 genera, Yamasato et al. (1973) concluded that, although many organisms survived well after 4.5 years at -28"C, much higher survival was generally obtained at -53°C. Influenza and syncytial viruses also have been found to survive better at temperatures below -65°C than at -20°C (Rightsel and Greiff, 1967; Law and Hull, 1968).Measles virus appeared to be unusual because there was greater loss of infectivity at -40°C than at -20°C (Greiff et al., 1964);at -65"C, however, the virus was adequately preserved. Using electron microscopy with freeze-fracturing and freeze-etching procedures, Bank (1973) demonstrated that large intracellular ice crystals formed within 30 min when frozen yeast was warmed rapidly from -196 to -40°C. Some recrystalization occurred at temperatures as low as -45°C. As will be discussed in Section 111,D,3, recrystalization of intracellular ice is considered to be lethal for cells. The critical temperature is dependent on a number of factors, but -70°C appears to be sufficiently low to preserve most organisms.

d . Cryoprotective agents. Glycerol has been the most widely used additive or suspending medium for all types of organisms. For example, at the American Type Culture Collection (ATCC), mycoplasma and fungi that do not survive lyophilization are frozen in 10% glycerol (Norman et al., 1970; Hwang, 1968). Brewing yeasts also were well preserved in 10% glycerol

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(Wellman and Stewart, 1973). Glycerol was superior to other substances for preservation of lactic cultures by freezing (Baumann and Reinbold, 1966). Although glycerol was effective without penetrating cells, the presence of intracellular glycerol increased survival on freezing and thawing of E . coli. However, intracellular glycerol alone failed to protect (Nath and Gonda, 1975). The ATCC have used 10% glycerol to preserve bacteriophage (Clark and Klein, 1966) but they have adopted the practice of using rapid freezing without additives because better recovery is obtained for a number of freeze-sensitive phage (Clark and Geary, 1973). Others also have obtained good survival of bacteria, fungi, and yeast without additives (Cox, 1968; MacDonald, 1972; Wellman, 1970; Wellman and Walden, 1971). Keogh (1970) also have considered additives to be unnecessary for S. Zactis and S . cremoris, but this may have been the result of the high cell concentration (1012/ml). The presence of glycerol actually may be detrimental to some organisms. Barnhart and Terry (1971) found that as the glycerol concentration was increased, the percentage of Neurospora crassa surviving freezing decreased from 2 4 3 5 % to less than 1%.However, suspension of the conidia in DMSO increased survival to about 50%. Hwang and Howells (1968), using a less quantitative measure of viability, also found that DMSO was more effective than glycerol for most of the eight fungal cultures tested. Two of the species survived equally well in glycerol and DMSO. A mixture of 10% glycerol with 5% of either lactose, maltose, or raffinose provided the best overall protection for S. cerevisiae, Pseudomonas aureofaciens, Streptomyces tenebrarius, and four species of algae (Daily and Higgens, 1973). Their conclusion was based on the results of testing over 50 compounds and combinations. In addition to the above citation, a few other examples showing that the effectiveness of the suspending media varies with the organisms may be of interest. Yamasato et al. (1973) found that for most species of Acetobacter and Gluconobacter, a 10% solution of honey was superior to 10% glycerol. Syncytial virus was found to be maintained best by freezing in a 44% sucrose solution (Law and Hull, 1968), and calcium lactobionate has been recommended for preserving measles virus (Greiffet al., 1964). Bretz and Abrosini (1966)found that of the various substances from lyzed E. coli cells, only the carbohydrate fraction preserved viability of E . coli. A comparison of its protective effect with sucrose, or other carbohydrates, would have been of interest. Nonionic detergents, as well as glycerol, protected Enterobacter aerogenes from freezing damage (Calcott and Postgate, 1971). The degree of protection offered by the detergents Tween 80, Triton WR 1339, or Macrocyclon decreased as cell concentration was increased. Apparently, the

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protection was dependent upon the ratio of cells to detergent. In contrast, protection by glycerol was dependent upon its absolute concentration and was independent of cell density. It has been shown that using polyvinylpyrrolidone and dextran, cryoprotection increased with molecular weight to a maximum protection at about 90,OOO Daltons (Ashwood-Smith and Warby, 1971b; Vitanov and Petukhov, 1973), but these high molecular weight materials were not compared with other substances, such as glycerol or DMSO. Viability of leptospires was well preserved by freezing in a mixture of 10% rabbit serum with 5% or 10% glycerol (Torney and Bordt, 1969). They did not consider the additives independently. The work of Janssen and Busta (1973a) would indicate that proteins could be protective because most fractions of milk offered some protection for Salmonella anatum. Whey was deleterious even though it contained protein. 3. Nature of Cryoinjury It seems that the problem of identlfying the nature of damage caused by freezing is similar to that of blind men trying to characterize an elephant. The conclusions depend to a large extent on how the subject is approached. Early workers, such as Proom and Hemmons (1949) or Luyet (1951), believed that cells were killed by freezing because ice crystals penetrated the cell wall, but probably this is not true. Mazur (1961) demonstrated clearly that in spite of high mortality (more than 99.99% killed), rapidly cooled yeast cells remained as intact morphological entities when thawed. The cells did lose their vacuole, which was correlated with viability. It now appears that there are at least two types of injury that can result from freezing of cells. Litvan (1972) believes that the injury produced by slow cooling rates is a result of dehydration and that rapid cooling causes membrane rupture. Mazur (1966, 1970, 1977)has identified these as solution effects, caused by slow freezing, and intracellular freezing, which occurs when a cell is frozen rapidly. The intracellular ice per se is not lethal since rapidly frozen cells survive thawing ifwarmed rapidly enough (Mazur, 1966). Furthermore, Farrant et al. (1977), using Chinese hamster fibroblasts, concluded that the damage from intracellular ice occurred during rewarming and was osmotic in nature. On the basis of structural changes in yeast, Bank (1973) also concluded that recrystalization was responsible for death in rapidly cooled cells. For baker’s yeast, he identified the lethal temperatures (recrystalization stage) as being between -40 and -5°C. Slow cooling can prevent formation of intracellular ice but such cells suffer from solute concentration effects. Cryoprotective agents, such as glycerol, act to minimize solution effects. However, as stated by Maxur (1977),“Under-

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13

standing the nature of these solution effects and their role in freezing injury now represents the major challenge in modern cryobiology.” There is considerable evidence that damage to the cell permeability barrier is associated with death in frozen and thawed cells. Calcott and MacLeod (1975a) demonstrated that release of cellular constituents, ultraviolet (UV) absorbing material, potassium, and P-galactosidase were correlated with loss of viability. Also, without being particularly concerned about the mechanics of freeze-thaw damage, Bretz and Kocka (1967) and Ray et al. (1972) found that injury involved the lipopolysaccharide of the cell wall. Since the repair process was not inhibited by either actinomycin, chloramphenicol, or cycloserene, Ray et al. (1972)concluded that the process did not involve RNA, protein, or mucopeptide synthesis; only adenosine triphosphate (ATP)was required. Other evidence that the cell membrane might be involved was provided by Gilliland and Speck (1974) and by Smittle et al. (1972), who found that incorporation of Tween 80 into the growth medium increased the resistance of Lactobacillus bulgaricus to freezing damage. Protection was not conferred when Tween 80 was added to the suspending medium as Calcott and Postgate (1971)had reported earlier for E . aerogenes. However, both groups postulated that the detergent affected the cell membrane to increase its resistance to freezing damage. Along the same lines, Raccach et al. (1975) found that oleic acid enrichment of A. Zaidlawii increased the percentage of cells surviving freeze-thawing which was attributed to a change in the composition of the membrane. The two types of freezing damage demonstrated by Swartz (1970, 1971a,b) were not correlated with the two types of injury mentioned previously. One he identified as being oxygen dependent, which was mediated by free radicals. The radiation-resistant strain, E . coli B/r, could repair this damage in the presence of oxygen but the radiation-sensitive strain, E . coli B,l, could not. The other type of damage was oxygen independent, which he identified as a single-strand deoxyribonucleic acid (DNA) break. However, AshwoodSmith et al. (1972), using slightly different procedures with radiolabeled thymidine, concluded that freezing and thawing did not break DNA of E . coli. Earlier, Ashwood-Smith (1965) concluded that freezing and thawing were not, in themselves, mutagenic to E . coli since he could not demonstrate any reversion from auxotrophy to prototrophy. More recently, Crombach (1973) showed that freezing and storing extracted DNA at -21°C for up to 1year did not affect the thermal denaturation (melting point) or hybridization capabilities, which would indicate that freezing did not break DNA. Small differencesin procedure, such as freezing rate, may be the cause of the conflicting findings regarding the effect of freezing on DNA. Wellman and Stewart (1973) observed that the biochemical properties of Saccharomyces

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uvamm were well preserved when frozen at 1"C/min but when the culture was frozen at 9-17"C/min the percentage of respiratory-deficient mutants increased markedly, despite maintenance of high viability.

E. DEHYDRATED Since water is required for metabolic activity, it is logical that dehydration should prevent changes in microbial cultures. Indeed, this is an effective method for culture preservation, and over the past 60 or 70 years many papers have been published describing techniques to simplify drying methods or to improve survival of organisms. This author will not attempt to review all of these, but the following discussions should provide a basis for selecting applicable methods.

1 . Soil Soil is a natural reservoir for many microorganisms, and they can be recovered after prolonged storage. Sterile soil has been used to induce sporulation of both aerobic and anaerobic bacilli. Azotobacters were recovered from soil stored for 13 years at room temperature in the laboratory (Vela, 1974); Coccidioides immitis was recovered &om soil stored in the laboratory for 8 years (H. B. Levine, personal communication, 1977). Disadvantages are that quantitation is difficult and soil is a variable commodity not easily defined. However, the method continues to be used largely for fungi and anaerobic spore-forming organisms. The fungi and spore-forming bacteria apparently survive in the spore state, but the vegetative cells probably survive because of their low metabolic rate and large amount of stored energy. Despite the applicability to certain requirements, this technique does not appear to be suitable for general use in all cultures.

2 . Silica Gel Perkins (1962) first used silica gel for preserving fungi, and the method was since applied successfully to many other organisms. The procedure is simple and requires no special equipment. Silica gel, in small cottonplugged tubes, is dried and sterilized by heating in an oven to 175°C for 1.5-2 hours. Details of the procedure were given by Grivell and Jackson (1969). Most of their cultures survived over 2 years, but three failed: Thiobacillus thipams, Chlamydomonas eugametos, and Euglena gracilis. The procedure used by Parina et al. (1972) differed in that they placed the granules of silica gel into the growth flasks for the last 24 hours of incubation. The silica gel and adherent yeast were then dried in a vacuum desiccator. Nearly 100% survival was obtained after 12 months with all three of the

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15

yeasts tested. More recently, Trollope (1975) reported, on the basis of 33 strains of bacteria and 22 fungi, that the survival period was increased two to three fold by storage of the silica gel at 4°C. However, even at 4"C, fewer than 60% of the bacteria and 36% of the fungi were viable after storage for 4 years. Since he did not count number of viable cells but only growth or no growth, this meant that many strains would have been lost if this were the only collection. Although silica gel and glass are similar, Miller and Simons (1962)reported that after drying on perforated glass beads, only 13of 202 bacterial cultures failed to grow after 21 years at about 10°C.

3. Cellulose A convenient method for drying cultures on filter paper strips was described in detail by Hopwood and Ferguson (1969). They placed thin strips of filter paper, saturated with a suspension of organisms in skim milk, into small tubes (6 x 100 mm). After the neck was constricted to facilitate subsequent sealing under vacuum, the tubes were attached to a manifold and dried at 0.01 torr. After 1-year storage at 37°C there was no apparent loss of viability of Streptomyces. Indications are that the method is applicable to other organisms as well. Annear (1964) followed essentially the same procedure using small tufts of cellulose or quartz wool, except that he suspended the bacteria in 10% sodium glutamate. Up to 59% survival of Salmonella ndolo was reported after 2 years' storage at room temperature. One-inch pieces of cotton string also have been used as a carrier for preserving a variety of organisms (G. D. Searle and Co., 1976). It is difficult to make a quantitative determination of survival on any of these cellulose carriers but it appears that viability is as least as good as in the conventional lyophilization procedure and considerably more convenient.

F. LYOPHILIZED Lyophilization, or freeze drying as some prefer to call the method, is the total process of freezing and sublimation of the water from the frozen preparation. This author will not review the historical developments or the procedure since this has been covered in considerable detail previously (Heckly, 1961). Lyophilization is considered by many microbiologists to be the method for preserving cultures. Indeed, many thousands of cultures are successfully maintained by lyophilization for long periods of time under vacuum. The advantages of lyophilization are that most organisms survive drying and cultures are easily stored. Also, most cultures can easily be shipped at room temperature without significant loss of viability, even though long-term survival may require a lower storage temperature.

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1 . Equipment Except for greater use of direct drive, gas ballasted vacuum pumps there have been few basic improvements in lyophilization equipment in the last 10 years. The equipment and supplies being manufactured by the various companies differ in convenience features but all are roughly comparable. When a system is selected the volume of material to be processed, as well as the nature of the organisms, should be considered. Since many organisms are sensitive to oxygen, it is recommended that a manifold-type unit be selected for drying the cultures. However, a centrifugal system, such as manufactured by the W. Edwards Company, London, is widely used. With secondary drying on a manifold for sealing under vacuum, this system seems to be effective for most organisms. Unfortunately, the commercial equipment is relatively expensive, but elaborate units in attractive cabinets are not essential for successful lyophilization. It is convenient to have condensers cooled by mechanical refrigeration but the condenser temperature may limit the ultimate vacuum attainable. If the pressure is to be reduced below 0.01 torr, the condenser surface must be below -50°C. For this reason, and because the initial costs are lower, dry ice cooled condensers are widely used, especially in “homemade” units. Since only small volumes are needed for stock culture preservation, a small condenser cooled with dry ice is usually adequate. Except for the vacuum pump, a practical lyophilization unit can be assembled in the laboratory (Heckly, 1961). A 3550 liter/min pump is usually adequate, but it should be capable of an ultimate pressure of less than 0.01 torr (10 pm Hg), even though the vapor pressure of water at -40°C is about 0.1 torr. It is desirable, but not essential, to have a vacuum gage in the system. McLeod-type gages are widely used, although a thermocouple vacuum gage is more useful since it can provide a continuous indication of vacuum, which facilitates finding leaks in the system. Heavy wall natural gum rubber tubing (?hinch i.d.) is much more conventient to use than heavy wall pressure tubing. Ampules made by sealing off 10-cm sections of 9-mm standard wall Pyrex tubing are quite satisfactory for culture preservation. A disadvantage is that the 9-mm tubing is a bit difficult to seal under vacuum. This can be overcome by heating and constricting the neck slightly (to about 4 5 mm i.d.) after the ampule is filled. These ampules accommodate 0.1 ml of culture. A novel method for freeze drying without a vacuum system was described by Wagman and Weneck (1963). In their system, cold dry air was forced through a bed of pelletized culture in a closed recirculating system. They suggested that increased viability was obtained because this method permitted more uniform drying by avoiding regional overdrying. The method,

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17

obviously, is not suitable for the usual stock culture preservation, but it may be used in industry for yeast or lactic starter cultures. 2. Factors Affecting Survival

The effects of various factors influencing the survival of bacteria during lyophilization and subsequent storage have been described in considerable detail (Heckly, 1961). The conclusions presented at that time are still basically valid and applicable. Since then, there have been many more reports of the application of lyophilization to the preservation of all types of organisms. Unfortunately, too many of these reports fail to mention details of the methods used. Despite the diversity of methods or approaches, an attempt will be made to summarize the findings. It is hoped that this review will facilitate selection of techniques to meet specific requirements. a . Type of organism. The size and complexity of the organisms are significant factors in determining the ability of the organisms to survive lyophilization, since damage of any vital structure or function is lethal. Although animal tissue culture cells are routinely preserved by freezing in liquid nitrogen, the large cells are extremely sensitive to drying. In only one instance have viable cells been demonstrated to survive lyophilization (Damjanovib et al., 1975). Except for some unicellular forms, algae cannot generally be preserved well by lyophilization, and in the mycelial phase fungi also do not survive lyophilization (Hesseltine et al . , 1960). Because bacteria are larger and have a more complex structure than viruses, it seems that viruses should be the more resistant. However, it appears that viruses are generally more sensitive to lyophilization than are bacteria. This difference may be attributed to the ability of cellular forms to repair damage caused by freezing or drying, whereas viruses and phage cannot. Since viruses depend on the host cells for energy, they must be infective to survive. Among the viruses there are correlations between morphological type and sensitivity to lyophilization. The larger viruses, which were identified as belonging to type A group by Clark and Geary (1973), were demonstrated to be more sensitive to lyophilization than the smaller viruses (group B). Rightsel and GreifT(1967)arranged viruses into eight different groups on the basis of physical and chemical properties. Infectivity of most viruses within a group was similarly affected by freezing and drying. Bacteria can be divided into three broad catagories: spores, gram-positive vegetative cells, and gram-negative bacteria. All types of spores are inherently extremely resistant to dehydration and survive lyophilization well. Steel and Ross (1963),and many others, have observed that gram-positive

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ROBERT J. HECKLY

bacteria survive better than gram-negative organisms when lyophilized and stored under comparable conditions. The ability to withstand drying probably is a genetically stable trait, since there is no indication that resistant strains can be selected for easily. The progeny obtained fiom lyophilized cultures, even after many cycles of lyophilization and regrowth, did not survive drying any better than the original cultures (R. J. Heckly, unpublished). Skaliy and Eagon (1972)found that fieshly isolated cultures of Pseudomonas aeruginosa were no better adapted to withstand the stress of desiccation than were the laboratory strains. Haynes et al. (1955)reported that they were unable to preserve anaerobic organisms by lyophilization, but the problem might have been one of technique. Lupton et al. (1961)reported that with suitable precautions to exclude air, all of the anaerobes processed in a 2-year period grew on reconstitution. White et al. (1974) also reported high survival (up to 10%)of lyophilized anaerobic organisms isolated from bovine rumen. Furthermore, in a more extensive study of 19 strains of strict anaerobes, Phillips et al. (1975)obtained high viability (10-100%). Marine bacteria, in spite of their normal habitat, are probably as resistant to lyophilization as other bacteria. Floodgate and Hayes (1961) reported that all of 45 lyophilized strains tested survived 2 years, and Greig et al. (1970) reported that all but 9% of their dried cultures survived 10 years. Highest survival was among corynebacteria and micrococci, and the lowest was with the vibrios and photobacteria. b . Physiological age. The age of the culture can have a profound effect on survival of bacteria. A well-nourished cell in a culture at the maximum stationary phase is usually the most resistant cell. It is possible that some properties ascribed to mature cultures may have been due to changes in the growth media, as described by Packer et al. (I%), but age of the cell per se must be a significant factor. There are a few instances in which young or log-phase cultures survived lyophilization better than old cultures (Proom and Hemmons, 1949; Amarger et al., 1972; Lingg et al., 1967). Although young cultures of Rhizobium meliloti survived drying better than older cultures, the stationary phase survived storage in the dried state at 30°C better than young cultures (Amarger et al., 1972). Perhaps the results of an experiment summarized in Fig. 1 may help explain some of the contradictory reports regarding the effect of culture age and survival after lyophilization. It is obvious that when frozen rapidly, the mature (24-hour) culture survives markedly better than any of the younger cultures. Young cultures (3-7 hour) were so sensitive that no viable cells were demonstrable when frozen rapidly. In contrast, at least loo0 times as

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4

A g e of c u l t u r e ( h r )

FIG. 1. Effect of culture age and freezing rate on survival of lyophilized Serratiu murcescens. Organisms were grown in chemically defined medium at 30°C with shaking. At intervals, samples were removed and frozen rapidly by immersion in a dry ice-thanol bath or frozen slowly in a -20°C freezer chest. All samples were dried overnight and reconstituted with distilled water for viability assays.

many cells survived when the 12-hour-old culture was frozen slowly prior to lyophilization than when it was frozen rapidly. c . Cell concentration. Although there are some exceptions, as discussed previously (Heckly, 1961),increasing the bacterial cell concentration usually results in an increased percentage of cells surviving lyophilization. A similar concentration dependence was observed with bacteriophage T4 (Shapira and Kohn, 1974). If the preparation contained 1O1O particles per milliliter, as high as 10% survived lyophilization; whereas at an initial lo9 particles per milliliter, less than 0.1% survived. It has been postulated that a high survival rate is obtained with high initial bacterial cell concentrations because the majority of the cells are protected by substances released by the lysis of a few cells. It has been shown that cell lyzates are cryoprotective (Bretz and Amro-

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ROBERT J. HECKLY

sini, 1966) but the observations by Speckman et al. (1974), using lactic bacteria, tend to indicate that the observed effects may not be due to an increase in solutes as a consequence of lysis. They found that with an initial 109/ml,only 10% of the bacteria survived, even in a rich medium consisting of 5% gelatin, 5% sodium citrate, 10% sucrose, and 2% sodium glutamate. In contrast, with an initial concentration of 10"/ml, virtually 100% of the cells survived. Damjanovik and Radulovik (1967) similarly found that the survival of Lactobacillus bifidus increased from 26 to 99% as cell concentration was increased from lo7 to 10g/ml.Their medium, which consisted of 8% sucrose, 5% skim milk, and 1.5% gelatin, was rich and also should have offered protection regardless of cell concentration. Lion (1963) observed that the protective effects of various substances, including glucose and thiourea, were dependent on cell concentration and went on to suggest that protection might be affected by a sort of physical or mechanical oxygen barrier.

d . Suspending medium. The suspending medium is perhaps the most studied factor. The basic considerations have been discussed at length (Heckly, 1961) but there are some additional observations that may merit mentioning. Skim milk continues to be a popular suspending fluid and generally yields satisfactory results. Although a large number of marine bacteria survived in a mixture of 20% dehydrated skim milk with 5%aged sea water, a small group of purple pigmented bacteria failed to survive when lyophilized in milk (Ohye and Gunderson, 1970). These purple bacteria survived when they were lyophilized in a medium containing 25%sea water, 5% peptone, and 5% yeast extract. Fisher (1963)also found that skim milk was inferior for preserving Chromobacterium lividurn. When lyophilized in skim milk, no viable cells were found after 10% years at room temperature, but when suspended in "Mist. desiccans," a term applied by Fry and Greaves (1951) to a mixture of serum and glucose broth, or in nutrient broth or broth containing yeast extract, about lo5 viable cells were recovered. A number of investigatorsfound that the protective effect of skim milk was improved by adding solutes. Addition of either ascorbic acid or thiourea markedly improved survival of S. lactis (Sinha et al., 1974a)and Green et al. (1970) added 5% sucrose and 5% sodium glutamate for Klebsiella. Danilova and Kudryavtsev (1971) improved survival of Serratia marcescens, E. coli, and P. jluwescens by adding 5% sucrose and 5% lactose. Although skim milk protected some strains of blue-green algae, Corbett and Parker (1976)found that only lamb serum gave consistently good recovery. Since there is so little difference between the animal sera, it is difEicult to explain why no viable cultures have been obtained from Synechococcus c e d r m m and other blue-green algae when dried from horse serum, beef

PRESERVATION OF MICROORGANISMS

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serum, or fetal bovine serum. Corbett and Parker (1976) mentioned that resistance of the blue-green algae to freezing appeared to correlate with resistance to lyophilization. As mentioned in Section II1,D on freezing, DMSO is a superior cryoprotective agent and perhaps should be tried for those organisms that are particularly sensitive to lyophilization. However, since concentrated DMSO is toxic to microorganisms, special precautions are required to prevent concentrating DMSO as water is removed. Greiff et al. (1976)found that by maintaining the sample temperature at -50°C using the proper ratio of DMSO to albumin, a dry cake was obtained. Under these conditions the DMSO is apparently immobilized as the water is removed and may not be toxic to organisms. Both glucose and sucrose solutions tend to produce a glazed surface, collapse of the ice structure, and foam but this does not seem to affect the survival of bacteria. After 10 years' storage, Annear (1974) recovered leptospires that were lyophilized in 10% glucose. He mentioned that, although other suspending media were tried, none gave consistently reliable results. Sucrose has been recommended as a general purpose additive replacing skim milk (Heckly, 1961)and, when compared with either lactose or glucose as suspending media, ten times as many mycoplasma have been recovered using sucrose (Yugi et al., 1973). Although sucrose alone conferred signscant protection, even higher survival of P . fluorescens and Salmonella newport was obtained with 0.1 M sucrose plus 0.2 M sodium glutamate and 0.02 M semicarbazide (Marshall and Scott, 1970; Marshall et al., 1974). The rationale for adding semicarbazide was that it could react with carbonyl compounds. Berman et al. (1968) obtained their best results by adding 0.07% glutamate and 2.5% human serum albumin to 8.2% sucrose in 0.01 M phosphate solution. Only a few investigators advocate using sodium glutamate alone for lyophilization of bacteria, but Obayashi (1961)and Annear (1964, 1970b) report greatly enhanced heat stability of bacteria dried in glutamate. Annear (1964) obtained high survival in cultures heated to G"C, but not when heated to 100°C. It was believed that this was due to inadequate drying because stability was increased if drying was completed by immersing the sample in boiling water for 30 min before it was removed from the vacuum system (Annear, 1970b). This additional heating produces a white foam which he thinks is an important indicator. In the foam, both S. ndolo and S . marcescens survived well for 3 days of heating at I0O"C. Sodium glutamate is also an effective suspending media medium for the lyophilization of viruses. Scott and Woodside (1976) found that glutamate alone, or with sucrose, most effectively stabilized pseudorabies virus, and Suzuki (1973b) found glutamate best for vaccinia virus. The media which Calnek et al. (1970) selected contained sucrose, phosphate, and serum albumin in addition to sodium glutamate.

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ROBERT J. HECKLY

In a comprehensive study using 62 strains of organisms, including Streptococcus, Lactobacillus, Escherichia, and Serratia, Morichi (1970)tested the activity of 112 compounds. Good survival after lyophilization was obtained using glutamic, aspartic, and malic acid and several other additives, but poor survival was obtained with glutaric acid, glutamine, proline, and L-threonine. Surprisingly, DL-threonine gave good survival, which was attributed to its greater solubility. Sucrose, lactose, and glucose were among the sugars that protected organisms on lyophilization, but contrary to the findings of Redway and Lapage (1974), inositol was not as effective. On comparing the effects of adding 15 different carbohydrates or mixtures to horse serum, Redway and Lapage (1974) found that addition of arabinose, glucose, and xylose generally offered the least protection. The highest survival was consistently obtained with inositol for all of the organisms tested, which included Bacillus cerus, Haemophilus suis, Neisseria gonorrhoea, and Vibrio met schnikovii. Trehelose and sucrose gave reasonably good survival. This review of the substances that have been studied as additives for increasing the survival of microorganisms is by no means complete, yet it is obvious that this subject presents a complex problem. It appears that most sugars act to protect organisms during the freezing and drying state and that glutamic acid and perhaps other amino acids or proteins serve to protect organisms at elevated temperatures. Another variable decting survival of lyophilized bacteria that has not been confirmed by other investigators concerns temporal relationships. It has been demonstrated that the number of cells that survive lyophilization and storage can vary as a function of the time between mixing of the culture with “additives,” such as ascorbic acid or propyl gallate, and freezing of the sample (Heckly et al., 1967; Heckly and Dimatteo, 1975). This factor may be responsible for some inconsistencies in data since, as is shown in Fig. 2, a delay of as little as 30 sec between freezing of samples can result in more than ten fold difference in the number of cells surviving after lyophilization.

e. Rate of fi-eezing and drying temperature. The rate of freezing can be critical and deserves consideration. As discussed previously in Section 111,D, sensitivity of frozen cells to slow warming is dependent upon the freezing rate. Because the ice temperature in most frozen specimens during drying is usually above -40°C, recrystalization of intracellular ice is probable. Rapid freezing, which may produce intracellular ice, can be recommended only if the vacuum system is adequate to maintain the sample below -50°C. The critical temperature above which recrystallization can take place depends on several factors, as discussed in Section II1,D. If the sample is to look good, i.e., retain the bulk and general shape of the ice cake, the temperature must be maintained below the eutectic temperature of the sample.

PRESERVATION OF MICROORGANISMS

23

0 30 60 120 3M) 600 Seconds between m i x i n g a n d f r e e z i n g

FIG. 2. Effects of propyl gallate on viability of lyophilized Serratia murcescens. Centrifuged cells were resuspended in distilled water and mixed with an equal volume of 0.4% propyl gallate. At the indicated times 0.5-ml samples were rapidly frozen by transferring bottles cooled in dry ice. After all samples were collected they were lyophilized. Half of the samples were rehydrated after 1-day storage and the other half after 7 days at room temperature. (From Heckly et al., 1967, with permission.)

As demonstrated by Fateeva et al. (1970), the effect may not be entirely cosmetic. They determined that the eutectic temperature for yeast was -23°C and that high survival was obtained if it was kept below this temperature during drying. However, their conclusion is not entirely valid, because high survival has been obtained with a number of organisms by drying from the liquid state (Annear, 1970a). Unless contraindicated by experience with a particular system, the recommended procedure would be to freeze slowly and to dry at the lowest practical temperature.

f Extent of drying. Most workers agree that residual moisture content is a factor influencing the survival of organisms, but there still is no agreement as to what constitutes optimum moisture content. This author believes that the highest survival is usually associated with the lowest moisture con-

24

ROBERT J. HECKLY

tent, as has been demonstrated for a number of microorganisms. These included S. murcescens and E . coli (Dewald et al., 1967), phytopathogenic bacteria (Samosudova, 1965),Bacillus popilliae (Lingg and McMahon, 1969), Shigella (Damjanovik, 1974), and vaccinia virus (Suzuki, 1973a; Sparkes and Fenje, 1972). In contrast, influenza virus is apparently most stable at 1-2% residual moisture (GreiE and Rightsel, 1967; Greiff, 1971). Some of the disagreements might be attributable to differences in suspending medium, since Robinson (1972) found that smallpox vaccine was unstable at low residual moisture if the nitrogen content of the preparation was low. Another possible cause for discrepancies in studies on the effect of moisture may be related to the presence of variable amounts of oxygen. The deleterious effect of oxygen on influenza virus was considerably less at 2.5% moisture than when dried to 0.06% (Greiff, 1971). Chen et al. (1966) also found that even though reducing the moisture content of baker’s yeast (S. ceriuiseue) from 8% to 4 4 % greatly improved thermostability in a nitrogen atmosphere, the drier yeast was more sensitive to oxygen. At least in these instances, water tends to increase resistance of cells to oxygen. Most researchers use weight loss on heating, usually to 100°C, to measure moisture but this is not specific since some preparations contain significant amounts of other volatile substances besides water. Problems associated with the various other methods for determining moisture were discussed previously (Heckly, 1961), but a new method which was specific for water was described by Robinson (1972). In his method, water was extracted with benzene and the water content of the benzene was determined by gas-liquid chromatography. Brevelt and van Kerchove (1975) claim superior results using ethanol instead of benzene for the extraction. Since gas chromatographs are now common laboratory tools, this may become a standard method.

g . Storage atmosphere. The effects of oxygen on dried organisms was discussed in considerable detail at an AIBS Symposium in Michigan (Heckly, 1978), but certain aspects will be reviewed. Lion and Bergmann (1961)and Heckly and Dimmick (1968)demonstrated that in the absence of protective additives very small amounts of oxygen were toxic to dry bacteria, and Dewald (1966) found that the rate of inactivation was proportional to the oxygen tension. Although influenza virus was more stable in an inert atmosphere, oxygen was not particularly deleterious (Greiff and Rightsel, 1969). Either the suspending fluid protected the influenza virus or it might have been less sensitive to the effects of oxygen. The addition of almost any substance, such as inositol, sucrose, sodium glutamate, ascorbic acid, skim milk, or serum, reduces the sensitivity of

PRESERVATION OF MICROORGANISMS

25

dried bacteria and viruses to oxygen. As discussed in Section V, antioxidants, such as butylated hydroxyanisole (BHA), can protect dried yeast against the effects of oxygen. Butylated hydroxyanisole has not been used extensively in preserving other organisms. The rate of inactivation of dry bacteria by oxygen immediately after drying is sufficiently slow to permit short exposure to air. However, it has been shown that cells stored under vacuum for as little as 2 weeks, are inactivated extremely rapidly (Heckly and Dimmick, 1968). Fewer than lo6 cells were viable after less than 1 min exposure to air, whereas replicate samples rehydrated under vacuum (no exposure to air) had 108 viable cells. Admittedly, these cells were dried without protective additives, but this pointed up the desirability of rehydrating lyophilized cultures as rapidly as possible after the ampules were opened. Since oxygen is deleterious, it is important that ampules be properly sealed. Grieff et al. (1975)showed that the sealing of gas-filled glass ampules was frequently defective and recommended neoprene dissolved in toluene for sealing the leaks. They do not mention sealing of evacuated ampules, but this author believes that this is not a problem if the thin tip, obtained as the evacuated ampule is separated from the manifold, is heated to fuse the tip into a small ball. However, after a few weeks storage it is well to check the effectiveness of the seal with a high-voltage spark tester. Since this produces ultraviolet light, it is advisable to keep the radiation to a minimum. h. Temperature of storage. As will be discussed in Section 111,F,4, “Accelerated Storage Tests,” the temperature coefficient for the inactivation of organisms is rather high and depends upon a number of fictors. These include moisture content, presence of oxygen, nature of the protective additive, and nature of the organism. Spores generally survive as well at room temperature as at 4”C, but it is advisable to store most organisms at 4°C or lower. Although survival is increased at subzero temperatures, most collections of lyophilized organisms are stored at 4°C because it is adequate and considerably less expensive than -60°C or even -20°C storage.

i. Method of reconstitution. The effects of temperature and composition of the reconstitution have been reviewed previously (Heckly, 1961) and there has been little additional work since then. Choate and Alexander (1967)found that the number of viable cells of Spirillum atlunticum surviving lyophilization was increased almost 10,000-fold by reconstituting the lyophilized cells with 24% sucrose instead of distilled water. They also reported that regardless of the rehydration media, low-temperature rehydration provided the highest survival rate. It has been noted that high osmotic pressure was generally conducive to obtaining maximal recovery, but some

26

ROBERT J. HECKLY

organisms were adversely affected by high osmotic pressures (Heckly, 1961). Unfortunately, it appears that no single method provides for maximal survival of all organisms. Viability assays should be made on all reconstituted cultures for several reasons. (1)Such assays provide a basis for anticipating when to reprocess the culture. (2) Examination of discrete colonies or plaques provides a measure of quality control. Morphological variants or gross contamination can be detected. (3)Initiating a culture from a number of identical colonies insures a pure culture. Since it is possible for the lyophilized sample to become contaminated during any one of the lyophilization steps, cultures should never be initiated by transferring the entire sample to broth. Under some conditions a single contaminant may become the predominant organism in the culture. This consideration is particularly important when only a few of the original organisms persist.

3. Lyophilizatwn Damage As with higher life forms, any one of many events can kill or inactivate a microorganism. In lyophilization, organisms are subjected to possible injury by freezing, slow warming to sublimation temperature, dehydration, and possibly exposure to oxygen. Even in the absence of oxygen, cells stored at room temperature appear to suffer from some sort of solid-state rearrangements (Heckly and Dimmick, 1968). Most of the studies on lyophilization damage have been done with bacteria, probably because more is known about them. Bacteria are certainly easier to work with than are viruses and fungi. There is considerable evidence that lyophilization can damage bacterial membranes (Ray et al. 1971b; Webb, 1960; Calcott and MacLeod, 1975a,b), but proteins and RNA have also been identified as being damaged by the process (Mitie, 1976; Morichi and Irie, 1973). Under some conditions, bacteria damaged by lyophilization can recover, and the variability in rates may reflect differences in the type of injury. The time required for cells to recover from injury varied from about 30 min (Morichi and Irie, 1973; Beker, 1972) to nearly 8 hours (Gomez et al., 1973; Sinskey and Silverman, 1970). Ray and Speck (1972) and Ray et a2. (1971b) demonstrated that repair required ATP synthesis and that temperature was a factor in recovery. At 15"C, it required at least 120 min, whereas repair was virtually complete in about 30 min at 25°C. Israeli and Shapira (1973) concluded that the death of E. coli was not caused by damage to DNA, RNA, or protein synthesis per se; instead, interference with control mechanisms was responsible for cell death. Israeli et al. (1974) demonstrated that lyophilization ofE. coli injured the membrane transport system for 0-nitrophenol P-thiogalactopyranoside.

PRESERVATION OF MICROORGANISMS

27

If oxygen were excluded, the damage could be partially repaired after rehydration. Oxygen also was shown to inactivate nicotinamide adenine dinucleotide oxidase in lyophilized E. coli (Lion and Avi-Dor, 1963). They further showed that the enzyme activity was lost when a cell-free extract was dried and exposed to air. Several workers have demonstrated that cells may suffer genetic damage (Ashwood-Smith and Grant, 1976; Qu6villon et al., 1964; Servin-Massieu and Cruz-Camarillo, 1969; Webb, 1967). At least some of the loss of viability on lyophilization was attributed to DNA strand breakage by Ohnishi et al. (1977),because the strain that could repair radiation-damaged DNA (E. coli B/r) had higher survival than the radiation-sensitive strain (E. coli BPI). However, they went on to point out that cell damage was not restricted to strand breaks of DNA. Unless high viability is retained after lyophilization, it is difficult to differentiate between the mutagenic effect and the selection of spontaneous mutants by lyophilization. Damjanovii. (1972) found no evidence that lyophilization had any influence on the reversion rate of mutant Shigellu vaccines. However, he demonstrated that in a mixed culture, lyophilization effected a great deal of selection (Damjanovii., 1973). Gupta (1975)found that lyophilization of BCG also seemed to be selective. After lyophilization, the percentage of the nonspreading colony types was higher than before drying. Webb (1969) also observed that lyophilization was selective. Since he has found that auxotrophs ofE. coli are more sensitive to damage than the parent prototrophs, extra care should be taken in preserving mutant strains by lyophilization to minimize concentrating spontaneous “back mutations.” Free radicals are probably not a factor in dehydration damage because it has been shown that lyophilization does not produce a significant number of free radicals (Heckly and Dimmick, 1967). However, free radicals are produced by cells exposed to oxygen (Dimmick et d.,1961; Lion et d.,1961; Heckly et al., 1963; Heckly and Dimmick, 1968; Kuznetsov et aZ., 1975). A strong correlation between loss of viability and free radical production has been demonstrated (Heckly and Dimmick, 1968). No free radicals were demonstrable as long as the cells remained viable, even in the presence of oxygen. It is tempting to consider the action of free radicals as the cause of death in dry bacteria exposed to oxygen, but it has been demonstrated that loss of viability precedes free radical formation (Cox and Heckly, 1973). Perhaps bacteria can exclude oxygen as long as they are “alive,” and only after the cells die can oxygen diffuse into the cell and produce free radicals by reacting with cellular components. This is compatible with the concept that at least some of the damage caused by drying is reversible and that oxygen reacts to make the damage permanent (Novick et al., 1972; Israeli

28

ROBERT J. HECIUY

and Shapira, 1973; Israeli et al., 1975). Oxygen can react with many dry biological systems (Heckly, 1972, 1976). Unfortunately, the chemistry involved has not been identified.

4 . Accelerated Storage Tests It usually takes years to observe significant loss of viability of lyophilized preparations, particularly under favorable storage conditions. Therefore, storage at elevated temperatures has been used both for developing or improving methods and for predicting survival. Moisture content is extremely important and can be critical at the higher storage temperatures. At 4"C, Sparks and Fenje (1972) found that at 6.7% moisture smallpox virus was rapidly inactivated, but there was little difference in stability of the vaccine at moisture contents ranging from 0.36 to 4.8%. However at 37"C, there was a graded response, and the highest survival of virus was obtained at the lowest moisture content. Qualitatively the same results were obtained by Suzuki (1973a),who found high survival of vaccinia virus at the lowest moisture content tested (0.97%)at either 37 or 45°C. Suzuki (1973b) also found that at 45"C, glutamate was the most effective additive for vaccinia virus. Recently, Scott and Woodside (1976) similarly observed that glutamate, alone or with other additives most effectively stabilized pseudorabies virus at 20 and 37°C. Unless it is shown that the loss of viability or infectivity fits an Arrhenius equation (a plot of the log of inactivation rate versus ID'), extrapolation or prediction of survival at lower temperatures is not valid. Greiff and Rightsel (1965) described such an accelerated storage test for predicting stability of measles virus. They showed that the activity of measles virus decreased in a reasonably linear manner when stored at 28, 36, and 45"C, and that the data fit a straight line on an Arrhenius plot. Beardmore et al. (1968), using the accelerated storage test of G r e 8 and Rightsel (1965), predicted influenza virus, lyophilized in allantoic fluid, to be stable indefinitely at room temperature. Application of accelerated storage tests to bacterial preparations are more extensive. Although Redway and Lapage (1974)used only 30 and 45°C for B . cerus, H . suis, N . gonorrhoea, and V. metschnikovii, others used higher temperatures. On the basis of short-term incubation of dried L. bififus at 36, 45, and 65"C, Damjanovid and Radulovid (1968) accurately predicted survival after 203 days' storage at 4°C. Survival of L. acidophilus at 4 and 20°C also was predicted accurately on the basis of accelerated storage tests at 50, 60,and 70°C by Mitid et al. (1974). Similarly, Damjanovid (1974) predicted stability of a live Shigella vaccine. Annear (1964) used 37, 45, and 100°C to compare the protective effect of various substances on lyophilized S. ndolo. In a description of the procedures used by the Czechoslovak National

PRESERVATION OF MICROORGANISMS

29

Collection of Cultures, Sourek (1974) mentioned that samples were heated to 75 and 100°C for 30 min and checked for viability. No data on the results of these tests were given, only the long-term survival of a large number of different organisms at 4°C was listed. Presumably, if adequate numbers survived the heating, the lyophilization was considered satisfactory for stock culture preservation. This may not be valid because Maister et al. (1958) found that survival of S. murcescens at 80°C was not correlated with survival at lower temperatures. However, Obayashi et al. (1961) reported a good correlation between results of heating dried samples to 100°C and viability after storage at lower temperature. Even higher temperatures are needed for spores. Molin (1977) heated Bacillus subtilis var. niger, and B. stearothermophilus to 190°C. Although the data fit an Arrhenius plot well only at low moisture content, he predicted a D value (time for 90% reduction) of about 4 years at 0°C. Only one report on the use of elevated temperature for stability testing of dried fungi was found. Rogan and Terry (1973) incubated lyophilized cultures of P. chrysogenum at room temperature (RT), 37, 45, and 60°C. With these data they constructed Arrhenius plots from which they predicted the rates of viability loss at RT. A comparison of predicted values with experimental data at room temperature storage with 12 additives demonstrated that the test permitted reliable estimation of shelflife. The accelerated storage test is a useful tool, but considerably more checking of predicted values for a wider range of organisms needs to be done. Fortunately, as far as stock culture preservation is concerned, inaccuracies have been on the short side; i.e., survival has been better than predicted.

IV. Culture Collection Practices The procedures used to preserve large culture collections are not necessarily optimal, but generally they are adequate. Obviously, it is not possible to determine optimum conditions for each group of organisms. The American Type Culture Collection (ATCC) does have an active research program to improve preservation methods for the more sensitive organisms. Lyophilization is used widely, although some organisms do not survive well. These are frozen. Although the ATCC maintains all of their stock viruses and most phages in liquid nitrogen, specimens are lyophilized for distribution (Clark and Geary, 1969, 1973). The ATCC uses two methods for lyophilization. For hardy organisms a number of tubes is lyophilized in a batch system, only 1 strain per container. The small tubes are then placed in larger test tubes containing a desiccant and label. These are then constricted, evacuated, and sealed. For those organisms that appear to be damaged by even short exposure to air, the ampules are attached individually to a manifold and

30

ROBERT J. HECKLY

sealed under original vacuum by fusing the glass. All are stored at about 4°C. Survival data for 26 strains of Mycoplasma at the ATCC, after up to 10 years’ storage, were reported by Norman et al. (1970) and Norman (1973). In general, better recovery was obtained from the liquid nitrogen than &om the dried preparations, yet 105-106organisms remained viable when lyophilized in 12% sucrose. Clark and Klein (1966) tabulated infectivity data on 26 strains of bacteriophge after 3-5 years’ storage in liquid nitrogen at the ATCC. More recently, Cjark and Geary (1973) reported on preservation of a large number of bacteriophages by lyophilization. Berge et al. (1971) successfully Iyophilized several enteroviruses but they did not present any longterm storage data. Although most of the 4500 strains in the fungal collection of the ATCC are maintained by lyophilization, some do not survive well. Hwang (1968, 1970) reported that 74 of the 104 sensitive strains were successfully frozen in 10% glycerol and survival to 18 months was tabulated These strains were subsequently reported to have survived after 42 months at -196°C (Hwang, 1970). Although DMSO has been shown to be more effective than glycerol for some species (Hwang and Howells, 1968), glycerol may continue to be the standard protective agent. Butterfield et al. (1974) recently summarized results in an extensive table showing survival of lyophilized as well as frozen cultures. Some lyophilized strains survived 32 years of storage but the longest test period reported for storage in liquid nitrogen was 8 years. The Northern Regional Research Laboratory, Peoria, Illinois, has not used liquid nitrogen for preserving their culture collection. Instead, it has relied largely on lyophilization for the preservation of fungi. The procedure, as described by Haynes et al. (1955),was to suspend the organisms in bovine serum and seal the ampules under vacuum. An extensive list showing relative vigor of the lyophilized cultures after 8-17 years’ storage at 4-10°C was published by Hesseltine et al. (1960).Of 363 strains tested, 331 were viable after 17 years. Subsequently, Ellis and Roberson (1968) summarized the results of viability tests on 447 strains. Many of those stored for 23 years were viable. The Indian Type Culture Collection used either calf serum or skim milk as a protective agent. Results after 2 years’ storage of 38 strains were published by Sarbhoy et al. (1974). Only 26 of the 38 strains survived. They did not indicate that there was any difference in survival between those in serum or those in skim milk. The lyophilization method used by the Institute of Tropical Medicine in Antwerp, as described by Bosmans (1974), employs a suspending medium consisting of 10% sucrose, 5% peptone, and 30% ox serum. Details on survival were not given but he did list the fungi and yeasts that had survived 10 years’ storage. He mentioned that other methods, such as covering agar

PRESERVATION OF MICROORGANISMS

31

cultures with oil or freezing cell suspensions were used, but no data were given. The National Collection of Type Cultures, in London, initially used horse serum but it has been superceded by “Mist. desiccans” of Fry and Greaves (1951). Steel and Ross (1963) reported on survival of some 100 strains of bacteria surviving lyophilization and storage for 10 years but only in general terms. Miller and Simons (1962) reported on the standard method used by the Department of Microbiology, Woman’s College of Pennsylvania. Bacteria suspended in defibrinated rabbit or horse blood were dried on perforated glass beads over calcium chloride at room temperature. After 21 years, only 13 of 202 cultures had failed to grow. Considering the presence of oxygen and high temperature, this was a high survival rate. The Department of Microbiology, the Ohio State University, preserves its culture collection in liquid nitrogen using either DMSO or glycerol (Swoager, 1972). No longterm survival data were given. Since 1939, about 3000 cultures have been preserved at the University of Buenos Aires, Argentina, using a simple desiccation system (Soriano, 1970). A small amount of the bacterial culture was introduced into a small tube and closed with a cotton plug, which in turn was placed in a larger tube containing a dehydrating substance, such as potassium hydroxide. The larger tube was fitted with a rubber stopper and capillary tube so that after evacuation to 0.01-0.05 torr, it could be sealed by fusion. Soriano (1970) indicated that despite the fact that room temperature rose to 30°C, some 75%of the tubes retained viability for up to 30 years. The Czechoslovak National Collection of Type Cultures uses a system comparable to that described for the American Type Culture Collection. Sourek (1974)tabulated survival of 122 organisms after storage for as much as 23 years. Skim milk was used in the lyophilization of all microorganisms at the Institute of Microbiology, Academia Sinica, Peking (Research Group of Culture Collection, 1975). Extensive tables showed that reasonably good survival was obtained after 4-8 years, even though the ampules were stored at room temperature. Lyophilization is also used extensively in Russia (Konev and Kuzmina, 1975; Kuznetsov and Rodionova, 1971) and in Turkey (Cetin,

1970). V. Industrial or Commercial Practices Applied aspects of culture preservation can be found in the various industries using microorganisms. Some do not have a culture preservation program. Instead, they use a perpetual culture system. The sourdough French

32

ROBERT J. HECKLY

bread bakeries in San Francisco, California, have been identified as the only bakeries in the United States to carry portions of the sponge as inoculum for the next dough (Reed and Peppler, 1973). However, there are probably many others who use this method because such information is not generally published. The unique flavor of San Francisco sourdough bread has been attributed to a mixed culture of Lactobacillus sanfiancisco and an acidtolerant yeast, Saccharomyces exiguus (Sugihara et al., 1971; Klein and Sugihara, 1971). Efforts are being made to develop a method for preparing these organisms in a dry form that preserves activity and facilitates distribution of the culture. The dried preparation would be used as the primary culture. Although a San Francisco brewery also routinely uses the perpetual culture system, most of the brewing industry uses pure cultures prepared from preserved cultures. For example, Anheuser-Busch prepares the cultures in a central laboratory. Cultures are shipped to plants at approximately weekly intervals. Such practices obviously insure minimizing contaminants in the final fermentation. These breweries, as well as other companies producing yeast, maintain stock cultures in the lyophilized state and on malt agar, either with or without mineral oil overlay. Fresh yeast is supplied to the larger bakeries in metropolitan areas as fresh pressed yeast cake without any requirements for storage. Active dry yeast (ADY), which can be easily stored, is now widely used for home baking and by the more isolated bakeries. However, it is necessary that yeast not only remain viable and able to reproduce without a significant lag period but also remain biochemically active. As with other microorganisms mentioned previously, many factors affect the survival of yeast. These are discussed, with details of the commercial yeast production methods, by Reed and Peppler (1973). Briefly, yeast is grown aerobically to maximize cell yield and nutrients are restricted slightly to increase stability of the cells. After the yeast is washed and concentrated by centrifugation, it is pressed into cakes. Compressed yeast cake loses only about 5% of its initial activity in a week but it cannot be stored for much more than a month. After that time, mold contamination is likely to become excessive. For production of active dry yeast, the filter cake is extruded in the form of thin ribbons and dried in air at 4040°C. Less than 5%of the activity is lost in the drying process but in air, the loss is nearly 7% per month. However, if it is packed in vacuum, or in a nitrogen atmosphere, losses are reduced to about 10% per year. The deleterious effects of oxygen can be minimized by adding an antioxidant, such as butylated hydroxyanisole (BHA), to the yeast before drying (Chen et al., 1966).The optimum concentration, on a dry yeast basis, was 0.1% BHA and 1%sorbitan monostearate. It was also noted that yeast was more active when it was rehydrated with water at 4045°C.

PRESERVATION OF MICROORGANISMS

33

The dairy industry is dependent on preserving microorganisms because the likely contamination with bacteriophage makes perpetual culture uneconomical. Not only is a variety of organisms needed, but bacteria with different phage types must be available to maintain production. Since few production plants can afford a microbiology staff, several companies have been established to provide starter cultures. Chr. Hansen’s Laboratory, Inc., has been providing starter cultures to the industry since 1893 (Sellars, 1975). Initially, liquid cultures were delivered to plants in their immediate area, but expansion of business required some form of preservation to insure that the starters were active. They now use liquid nitrogen. Cultures frozen in liquid nitrogen survive well, and in some instances stored cultures were slightly more active than before or immediately after freezing (Sellars, 1975). The Marshall Division of Miles Laboratories, Inc., Madison, Wisconsin, ships dairy cultures frozen in dry ice with a recommendation that they be stored between -48°C and -80°C. Microlife Tecnics, Sarasota, Florida, preserves their stock cultures in liquid nitrogen, but activity of the commercial starter cultures is retained satisfactorily at -29°C. Christensen (1977) commented that the Marshall Dairy Laboratory’s old freeze-dried culture program was inadequate, but the DPL Culture Service, San Francisco, California, has a thriving business lyophilizing lactic cultures for the manufacture of buttermilk, cheese, and yogurt. Use of special proprietary additives is claimed to yield dried cultures that can be stored with little loss of activity. Advantages of lyophilization are that cultures can be stored and shipped economically and activity is not lost if a shipment is delayed. Industrial strains of bacteria, molds, and yeasts are maintained by the Marshall Division of Miles Laboratories, Inc., at Elkhart, Indiana, by lyophilization in double-strength skim milk (C. E. Brownewell, personal communication, 1977). These are lyophilized on a manifold system after rapid freezing and are stored at 44°C. Pfizer, Inc., Groton, Connecticut, also preserves the majority of its stock cultures by lyophilization using skim milk and the double-tube system similar to that used by the ATCC. After 20 years of storage a lyophilized culture of Streptomyces rimosus still produced oxytetracycline in the same quantity as before (L. H. Huang, personal communication, 1977). Prior to 1963, stock cultures at Squibb Institute for Medical Research, New Brunswick, New Jersey, were lyophilized (Fortney and Thoma, 1977). Because survival of frozen S. griseus stored at -40°C was higher than in lyophilized cultures, storage over liquid nitrogen was instituted. Under these conditions there was not loss of viability. Ross Laboratories, in Columbus, Ohio, also maintain their cultures in liquid nitrogen using the method described by Swoager (1972).

34

ROBERT J. HECKLY

The Upjohn Company, Kalamazoo, Michigan, maintains stock cultures of actinomycetes and fungi on soil and others over liquid nitrogen. The freezing procedures, as described by Dietz (1975), are of interest. For bacteria, broth cultures or distilled water suspensions of agar growth are dispensed into small ampules and frozen without additives. Fungi and related organisms are grown on agar plates and plugs are cut and frozen, as described in Section III,D, with no added fluids. There have been no viability problems to date and cultures hnction normally in fermentations and bioassays. Unfortunately this section is not complete because a number of companies have declined to provide any information on culture preservation methods.

VI. Procedures for Selected Groups A. ALGAE The preservation of algae has received little attention. Tsuru (1973) reported a high percentage (6045%) survival of a variety of algae at -196"C, with glycerol and DMSO being equally effective as protective agents. He concluded that the addition of suspending agents to algal cultures resulted in greater viability for most of the green algae but showed little effect on the blue-green algae. Algae varied markedly in their resistance to freezing; in fact, nearly 1,OOO times as many ChZoreZZa protothecoides cells survived freezing and thawing as C. fusca (Morris, 1976a). However, in the exponential growth phase, the resistant species was damaged by cooling from 25 to 0°C as well as by freezing and thawing. A study of the effect of cooling rates, from 25 to O"C, showed that maximum survival was obtained at a cooling velocity of about 4"C/min. Morris (1976b) went on to show that the growth temperature affected freezing tolerance. Cells grown at 20°C were the most sensitive to freezing and the highest survival was obtained at 4°C with 24-day incubation. Morris (1976~) found that resistance of Prototheca spp. to freezing also was affected by the growth temperature. Incubation at 4°C yielded the most resistant cells but even the most resistant cells failed to survive rapid freezing, whereas 90% survived slow freezing (O.S"C/min). Some lyophilized Nostoc m u s c m m algae can be stored for 5 years at 25°C with no loss of viability (Holm-Hansen, 1967)but other algae were not that resistant. Corbett and Parker (1976) reported consistently good recovery of various b;ue-green algae when suspended in lamb serum. Since they reported only growth or no growth, it is difficult to evaluate their data. They did test a number of other additives, including skim milk, which yielded no viable cells when rehydrated. Tsuru (1973), using 10% skim milk with 1% monosodium glutamate, obtained 0.03-0.08% survival of six different algae

35

PRESERVATION OF MICROORGANISMS

TABLE I STORAGE DATAON SELECTED ALGAE

Storage Organism and preservation method

Anabaena sp. Lyophilized in lamb serum Dried on silica gel in milkb Chlorella sp. Lyophilized in milk Lyophilized in milk + 1% glutamate Frozen in 10% DMSO or 10% glycerol Nostoc m u s c m m Frozen in 10% glycerol or DMSO Lyophilized in milk + 1% glutamate Lyophilized in milk Stichococcus bacillaris Lyophilized in milk Synechoccus cedrorum Lyophilized in lamb serum

Temp. (“(2)

Years

Viable“

References

26 2 4

0.25 2

25 5

0.25

- 196

0.25

85%

- 196

0.25 0.2 5

60-70%

Tsuru (1973)

+5 25

0.05%

Tsuru (1973)

25

5

26

0.25

100%

+

5

0.007% 0.08%

+

10-5% to 10-3s 100%

Corbett and Parker (1976) Grivell and Jackson (1969) Holm-Hansen (1967) Tsuru (1973) Tsuru (1973)

Holm-Hansen (1967) Holm-Hansen (1967)

Corbett and Parker (1976)

=Percentage of original viable cells or qualitative measure of growth (+). *In all instances “milk’ refers to skim milk.

when rehydrated after 3 months’ storage. Takano et al. (1973)failed to obtain any survival of a rather sensitive blue-green alga, Spirulina platensis, when suspended in any of 18 different materials, including bovine albumin, skim milk, and sucrose. They did obtain viable cells with gum arabic using a modification of Annear’s peptone-plug method (Annear, 1956). Dried algae, as well as other organisms, are adversely afLected by oxygen (Holm-Hansen, 1967). No significant effect of storage temperature (-26, 4, and 26OC) on survival of two of the algae tested was found. Some data on the preservation methods used with algae and results obtained are summarized in Table I. This is not intended to be a complete list but it should serve as a useful guide. B. BACTERIA

Since so much of the discussion on preservation methods has involved bacteria as test organisms, it seems redundant to consider these again. Furthermore, the lyophilization of bacteria has been reviewed in considerable

36

ROBERT J. HECKLY

detail (Heckly, 1961). Table I1 summarizes some of the data on preservation of bacteria that has been published since 1961. Survival data using different preservation methods are given for a few organisms largely for comparative purposes. Several investigators have published extensive tables showing survival characteristics of many bacteria under various conditions. Trollope (1975) reported on the use of anhydrous silica gel for preserving 33 bacterial species. Cultures were stored at 4°C and at room temperature for up to 3.7 years. Antheunisse (1972)tabulated survival data of 36 species stored on agar slants in sealed tubes. These were stored at room temperature for up to 10 years. Although the storage period was only 2 months, the report by Sinha et al. (1974b) may be of interest. They compared the survival of 23 strains of lyophilized lactic acid bacteria held in both air and vacuum at 30°C. Iijima and Sakane (1973)reported on survival of 16 genera dried from the liquid state and stored at 5 and 37"C, However, the longest storage period was only 6 months. The Research Group of Culture Collection (1975) reported on the survival of organisms lyophilized in skim milk. Only a few of the cultures stored for 16 years at 5°C failed to grow. Most of the data, however, pertained to bacteria stored 4-7 years at 536°C. Survival data on 122 TABLE I1 STORAGE DATAON SELECTED BACTERIA Storage Organism and preservation method"

Temp. ("C)

Archromobacter spp Under parafin oil Frozen 15% glycerol Lyophilized in mist. desiccansc Frozen 15% glycerol Lyophilized in mist. desiccans" Amtobacter Dried on silicagel in milk On agar in sealed tubes Cysts on agar slow dried Cysts in soil Bacillus popilhe Lyophilized in 5% glutamate 0.5% tragacanth

+

Years

Viableb

1

2

+

-29

2 2

10%

RT -29

RT

10 10

RT RT

2 3-10 10 10

RT

0.5

2 4 RT

+ %

0.02%

+ + +

References

Floodgate and Hayes (1961) Floodgate and Hayes (1961) Greig et al. (1970) Greig et al. (1970)

78%

Grivell and Jackson (1969) Antheunisse (1972) Vela (1974)

2 -25

Lingg et al. (1967)

37

PRESERVATION OF MICROORGANISMS TABLE I1 (continued) STORAGEDATAON SELECTEDBACTERIA Storage Organism and preservation methoda Lyophilized and mixed with soil RH < 22% Bacteriodes (8 spp.) Lyophilized in horse serum 7% glucose Burdetella pertussis Frozen in 15% glycerol Curynebacterium spp. Under oil

+

Frozen in 15% glycerol

Temp.

(“C)

Viableb

References

1

10%

Lingg and McMahon (1969)

4-6

3

10-100%

-70

4

1

2

-29

RT

Years

Phillips et al. (1975)

2

+ + +

Floodgate and Hayes

RT

2

40%

Floodgate and Hayes

RT

10

Eckert and Flaherty (1972) Floodgate and Hayes

(1961)

(1961) Lyophilized mist. desiccansc Lyophilized mist. desiccans‘ Dried from liquid in 5% peptone + 5% glutamate Escherichia coli Frozen-no additive Lyophilized in milk 5% sucrose 5% lactose Agar slant under oil

+

+

Dried from liquid in 0.1 M PO, + 3% glutamate Klebsiella Lyophilized in sucrose + glutamate polyvinyl pyrrolidone (5% each) Lactobacillus acidophilus Lyophilized in 3% glutamate Lyophilized in 3% glutamate Lyophilized in 8% lactose + 1.2% peptone Frozen concentrate in whey

+

(1961)

37

4

-70

RT

2 2

RT

3

2-100%

+++ 30% 50%

+

Greig et al. (1970) Annear (1970a)

cox (1968) Danilova and Kudryavtsev (1970)

Nadirova and Zemlyakov (1971) Iijima and Sakane (1973)

0.5

42%

0.9

6-22%

Green et al. (1970)

37

0.5

0.01%

Obayashi et al. (1961)

5

1.0

100%

20

1.6

5%

0.6

86%

5

RT

-20

Miti6 et al. (1974) Duggan et al. (1959)

..

I

38

ROBERT J. HECKLY

TABLE I1 (continued) STORAGEDATAON SELECTEDBACTERIA Storage Organism and preservation method"

Temp.

(T)

Lactobacillus bijidus Lyophilized in 8% sucrose 5% milk 1.5% gelatin Leptospira canicola Frozen 10% rabbit serum Leptospira pomona Frozen 10% bovine Leptospira interrogans Frozen in 10% glycerol Dried on quartz fibers in 10% glucose Mycobacterium (BCG) Lyophilized in 1.5% glutamate Mycobncteriuin leprae Frozen-no additive Mycobacterium tuberculosis Frozen in milk Frozen in milk Mycobacterium tuberculosis Frozen in milk Frozen in milk Frozen in various media Mycobacterium tuberculosis Lyophilized in 1% glutamate Lyophilized in serum and 10% lactose Myocbacterium (9 strains) Lyophilized in milk On agar slants sealed in tubes Mycoplasmu (L form) Frozen-no additive Lyophilized-no additive Frozen-no additives Lyophilized in 2% bovine albumin

+

4

+

Years

Viable*

References

Damjanovib and Radulovib (1968)

0.6 60%

- 196

2

22%

Torney and Bordt (1969)

- 196

2

0.7%

Torney and Bordt (1969)

- 196

4

0.5 10

1% 919

Stalheim (1971) Annear (1974)

37

0.1

50%

Sirks et al. (1974)

-60

0.23

25%

-20 -70

3 3

Levy (1971)

5% 100%

Kim and Kubica (1972)

Gruft et al. (1968) Kim and Kubica (1973) Kubica et al. (1977)

-20

4

+

-70

3 5

100% 100%

RT

16

44

Slosarek et al. (1976)

RT

18

515

Slosarek et al. (1976)

RT RT

5

3-10

2433 80-100%

1 1

10% 0.02%

Stewart and Wright (1970) Stewart and Wright (1970)

3.5 3.5

>W%

Addey et al. (1970) Addey et al. (1970)

-70

-70 -20 -70

4

100%

Gruft et al. (1968) Antheunisse (1972)

39

PRESERVATION OF MICROORGANISMS

TABLE I1 (continued) BACTERIA STORAGEDATAON SELECTED Storage Organism and preservation method" Lyophilized in 2% Lyophilized in milk M y c o p l a m sp. Lyophilized in 12% sucrose Frozen culture medium Frozen culture medium Neisseria Dried from liquid in 5% glutamate 5% peptone Pseudomonas Agar slant under oil Agar slant in sealed tubes Dried from liquid in 0.1 M PO4 + 3% glutamate Dried in silica gel in milk Dried from liquid in 5% peptone + 5% glutamate Lyophilized in milk 5% sucrose + 5% lactose Salmonella Dried from liquid in 5% peptone and 5% glucose Lyophilized in 0.1 M sucrose + 0.2 M glutamate + 0.02 M semicarbazide Dried on cellulose tufts in 10% peptone + 10% glutamate Sarcina lutea Suspended in 0.02 M PO, buffer Frozen in 15% glycerol SerratM murcescens Dried from liquid in 3%

Temp. ("C) 37 -26

4 -20 -70

Viableb

3.5 4

1%

8-10

(est.) 10%

0.2 0.2

10-4% 1%

+

References Addey et al. (1970) Kelton (1964) Norman (1973) Raccach el al. (1975)

4

++

3 3-10 0.5

95% 52%

Antheunisse (1972) Iijima and Sakane (1973)

4

+ +++

Grivell and Jackson (1969) Annear (1970a)

2

40%

Danilova and Kudryavtsev (1970)

25

2

90%

Annear (1970a)

25

5

80%

Marshall and Scott (1970)

2

96

35

1

19%

-40

1

2%

0.5

74%

25

+

+

Years

RT RT

5 2 4 25

RT

RT

5

2

Annear (1970a)

+

Annear (1964)

Tanguay and Bogert (1974)

Iijima and Sakane (1973)

continued

40

ROBERT J. HECKLY

TABLE I1 (continued) BACTERIA STORAGE DATAON SELECTED __________________

______-

~

~

Storage Organism and preservation method'

+

glutamate 0.1M PO, Streptococcus cremoris Lyophilized in fortified milkd Streptococcus lactis Frozen in skim milk Frozen in skim milk Lyophilized in fortified milkd Frozen at 10"/ml Streptococcus spp. Frozen in milk Frozen in milk Frozen in milk at pH 7 Thiobacillus ferrooxidans Frozen-no additive Vibrio Under oil

Frozen in 15% glycerol Lyophilized Frozen in 15% glycerol Lyophilized in mist. desiccansc Dried from liquid in 0.1 M PO4 + 3%glutamate Yersinia pestis Lyophilized in 0.01 M PO4 2.5% albumin + 8% sucrose

+

Temp. ("C)

Years

Viableb

References

37

0.5

0.5%

30

0.2

0.3-7%

0.2 0.2 0.2

1.6% 90% 50%

Speck and Cowman (1970) Speck and Cowman (1970) Sinha et al. (1974a)

- 196

1

100%

Keogh (1970)

-23 - 196 -20

0.5 0.5 0.7

-20 - 196

30

330% 76-91% 1546%

Sinha et al. (197413)

Gibson et al. (1966) Gibson et al. (1966) Lamprech and Foster (1963)

150

3

1%

Manchee (1975)

1

2

f

Floodgate and Hayes (1961) Floodgate and Hayes (1961) Floodgate and Hayes (1961) Greig et al. (1970) Greig et al. (1970)

+, -

-29 RT -29 RT 5

-20

0.1% 10 10

-

<10-88

0.5

0.6%

Iijima and Sakane (1973)

1

100%

Berman et al. (1970)

"In all instances "milk refers to skim milk. bPercentage of original viable cells; or qualitative results are indicated by (-) for no growth to (+ +) for heavy growth; or fractions, such as 3/4, indicate that growth was obtained in three of four samples tested. e"Mist. desiccans" is a mixture of 3 parts serum and 1part broth with 7.5% glucose added (Fry and Greaves, 1951). dMilk fortified with ascorbic acid, thiourea, and ammonium chloride (0.5% each).

+

PRESERVATION OF MICROORGANISMS

41

strains of bacteria in the Czechoslovak National Collection of Type Cultures were tabulated by Sourek (1974). Some of these bacteria were still viable after 23 years at 4°C. Survival data after 2 years’ storage of 45 lyophilized and frozen cultures of marine bacteria were first tabulated by Floodgate and Hayes (1961). Lyophilized cultures were stored at room temperature and frozen cultures were held at -29°C. Subsequently, Greig et al. (1970) reported on the survival of these cultures after 10 years of storage. C. FUNGI, YEASTS,

AND

ACTINOMYCETES

Some members of this diverse group of microorganisms are difficult to preserve, but most are extremely hardy. H. B. Levine (personal communication, 1977) found viable C . immitis in soil samples from Woodland, California. These had been stored in screw-capped bottles at room temperature for more than 8 years. The record for “naturally preserved’ organisms is probably that described by Seaward et al. (1976).They obtained viable actinomycetes from samples that were 1,890 years old. Therefore, it is not surprising that lyophilization is widely used for preserving fungi and has been the only method used in some laboratories. Because some fungi in their collection failed to survive the freeze drying, the ATCC began storing cultures in liquid nitrogen 8 years ago (Butterfield et al., 1974). Wellman and Stewart (1973) also mention that since brewing yeasts tend not to survive lyophilization well, they have used liquid nitrogen storage and reported high viability after 3 years. Similarly, Squibb Institute for Medical Research discontinued the use of lyophilization in favor of freezing and storage over liquid nitrogen (vapor phase) for preserving fungi (Fortney and Thoma, 1977). Hesseltine et al. (1960) stated that fungal cultures without spores never survived lyophilization, but they reported that 15 cultures failed despite the presence of spores. Sarbhoy et al. (1974) found that 12 of their lyophilized cultures failed to grow after 1 year of storage. These results indicate that it is extremely important that viability of lyophilized fungal cultures be checked at rather frequent intervals to preclude loss of the culture, at least until it is found that numbers of viable cells remain high. Several procedures have been used to freeze fungi. The method used by Wellman (1970) and by Wellman and Walden (1971) was to grow the organisms on agar slants in the small cryogenic ampules which were heat sealed and rapidly frozen without additives by immersion into liquid nitrogen. They considered this to be necessary for the osmotic mutants of Neurospora that were glycerol sensitive (80-97% survival versus 1839%) when frozen in 10%glycerol solution. The procedure used by the ATCC for preserving cultures not amenable to lyophilization, as described by Hwang (1968, 1970), was to transfer small plugs of fungal growth with agar to am-

42

ROBERT J. HECKLY

pules containing 10% glycerol. These were frozen slowly (about l"C/min), and cultures were thawed rapidly by swirling the ampules in a 3840°C water bath. The use of glycerol and rapid thawing may not be necessary for fungi because Dietz (1975) has obtained satisfactory results using an agarplug method without glycerol and with a slow thawing procedure. Addition of suspending liquids to any of a number of rust spores before freezing was harmful, and one of these, Pucciniu stri$wmis, was so sensitive to moisture that to retain viability it had to be vacuum dried before freezing (Cunningham, 1973). Barnhart and Terry (1971) found that with N. crassa conidia, the rate of freezing was not critical provided samples were thawed rapidly. They also reported that survival of conidia was improved 50-fold by using DMSO instead of glycerol as the protective agent. Hwang and Howells (1968)also found that DMSO was generally superior to glycerol but observed that the relative efficacy of protective agents depended on the species involved. Storage of fungi and yeasts in water has been advocated because it is convenient and inexpensive and minimizes or prevents pleomorphism (McGinnis et aZ., 1974; Bosmans, 1974). In this procedure, pieces of culture were placed in distilled water and stored at room temperature in screwcapped vials. McGinnis et aZ. (1974) claimed that 92-94% of cultures were viable after a year. Those that failed to survive belonged to the following genera: Madurella, Paracoccidioides, and Trichophyton. Bosmans (1974) reported viability of some dermatophytes after 4 years in distilled water. Other methods used for preserving fungi, and the results obtained, are summarized in Table 111. This does not include data from extensive tables on lyophilized cultures showing preservation for up to 32 years that have been published by Hesseltine et al. (1960),Ellis and Roberson (1968), Butterfeld et al. (1974), Antheunisse (1973),and the Research Group of Culture Collection (1975). Survival data of about 23 strains of fungi stored on silica gel were tabulated by Trollope (1975). N o genetic changes were detected in P . chrysogenum cultures stored in liquid nitrogen, but 18% of the conidia of P . chrysogenum stored at 4°C formed a subpopulation with substantially lowered ability to produce penicillin (MacDonald, 1972). Except for one report of decreased pigmentation in three lyophilized strains (Kuznetsov and Rodionova, 1971), morphological characters were retained after lyophilization and storage for up to 17 years (Semenov, 1975; Sarbhoy et al., 1974; Hesseltine et al., 1960; Kapetonovii: and Pavletik, 1972; Ellis and Roberson, 1968). D. VIRUSESAND BACTERIOPHAGES In a comprehensive report, Rightsel and Greiff (1967) classified a large number of viruses into eight different groups on the basis of their nucleic

43

PRESERVATION OF MICROORGANISMS TABLE 111 REPRESENTATIVE DATAON SELECTED FUNGI

Storage Organism and preservation method Aspergillus Lyophilized in goat serum Lyophilized in bovine serum Agar slant in sealed tube Sealed in H,O Candida Dried on silica gel in spent media Agar slant in sealed tubes In distilled water Neurospora erassa Agar slants-no addition Agar slants frozen-no additive Penicillium Lyophilized in goat serum Lyophilized in bovine serum On agar slant in sealed tube Agar slants frozen-no additive Streptomyces griseus Lyophilized in goat serum Lyophilized in bovine serum Lyophilized in growth medium Frozen in growth medium Dried from liquid in 0.1 M PO, + 3%glutamate Sheptomyces Agar slants in sealed tubes Agar slants in sealed tubes

Temp. ("C)

Years

Viable"

RT

2

>90%

7

23

+

References

Mehrotra et al. (1970) Ellis and Roberson (1968)

RT RT

3-10 4

RT

1

RT RT

3-10 2

97% 212

Antheunisse (1972) McGinnis et al. (1974)

25

0.5 5

5% 98%

Wellman and Walden (1971) Wellman and Walden (1971)

RT

2

90%

Mehrotra et al. (1970)

7

23

+

- 196

80% 111

+

Antheunisse (1972) McGinnis et al. (1974) Parina et al. (1972)

Ellis and Roberson (1968)

3-10

33%

Antheunisse (1972)

- 190

4

68%

MacDonald (1972)

RT

2

80%

Mehrotra et al. (1970)

RT

7

10-13

5

7

- 100

5

5

RT RT

++

Hesseltine et al. (1960)

2.5%

Fortney and Thoma (1977)

100%

Fortney and Thoma (1977)

0.5

65%

Iijima and Sakane (1973)

13 3-10

67% 0%

Antheunisse (1972) Antheunisse (1972)

"Percentage of original viable cells; or qualitative results are indicated by (-) for no growth to (++ +) for heavy growth; or fractions, such as 314, indicate that growth was obtained in three offour samples tested.

44

ROBERT J. HECKLY

acid content, solvent sensitivity, and membrane and pH lability. Most of the viruses in each group had similar resistance to freezing or lyophilization and the effect of additives varied from one group to another. On the basis of trends indicated by the sparse data, they anticipated it to be ultimately possible to correlate physicochemical characteristics of viruses and the effects of freezing on lyophilization. Clark and Geary (1973)also demonstrated a correlation between morphology of bacteriophage and sensitivity to both freezing and drying. No additives were used by Rightsel and Greiff (1967), who noted that the titer of syncytial virus, probably the most sensitive of those tested, decreased markedly while frozen for 1month at -65°C. However, essentially no loss in titer after 28 months at -70°C was observed when the syncytial virus was stabilized with 44.5% sucrose (Law and Hull, 1968). It was unlikely that the difference in temperature was a significant factor, even though Greiff et al. (1964) demonstrated that measles virus was less stable at -40°C than at either -76 or -20°C. Freezing has been used extensively for the preservation of bacteriophage. Although freezing in 10% glycerol may reduce the titer of some preparations by as much as 99.9%, Clark and Klein (1966)reported that the titer of almost half of the 26 different bacteriophages tested were unaffected by freezing and storage for 4 years at -196°C. None of the preparations suffered more than a one log loss in titer during storage. Nyiendo et al. (1974) also found that in 15%glycerol, all of a number of lactic streptococcus phages were resistant to freezing. As mentioned before, glycerol or other substances are necessary for preserving viability of bacteria and higher forms but this may not be needed for bacteriophages. A few years ago, the ATCC adopted a rapid freezing method with no protective substances for the preservation of bacteriophages (Clark and Geary, 1973). Better recovery of the freeze-sensitive phages was obtained than with glycerol and, furthermore, samples could be lyophilized directly for mailing without further manipulations. It might seem that lyophilization are not a suitable method for preserving phage because less than 1% was recovered (Clark and Geary, 1973). However, the work of Imshenetskii et al. (1970) shows clearly that even though the sensitive T2 coliphage lost 96.5% of the original titer on lyophilization, there was little additional loss during storage at 4°C for 4 years. Grivell et al. (1971) described a silica gel method for preservation and reported active preparations of lettuce necrotic yellow virus after 2% years' storage at 4°C. At 30°C, none of the preparations was viable after 12 weeks. This method probably results in a relatively high moisture content, which may account for the high temperature coefficient for inactivation of the virus. Residual moisture appears to be critical, particularly at the higher storage temperatures (Sparkes and Fenje, 1972; Suzuki, 1973a). See Section II1,F for a discussion of the effects of moisture.

45

PRESERVATION OF MICROORGANISMS

As with other microorganisms, a wide variety of substances has been tried as protective additives for lyophilization of viruses. Rightsel and Greiff (1967) demonstrated a synergism between calcium lactobionate and serum albumin for poliovirus. Even with optimum concentrations, 1%albumin with 3.8% lactobionate, about 97% of the titer was lost on dehydration. Berge et al. (1971) also found that after lyophilization in bovine serum albumin, dextran, polyvinyl pyrollidone, or polyethylene glycol, less than 10%of the poliovirus infectivity was retained. However, when electrolytes were removed by dialysis or ultrafiltration, there was virtually no loss of poliovirus titer on lyophilization. After 1month of storage, less than 0.5 log loss was observed. Comparable results were observed with other enteroviruses. In contrast, Fellowes (1968)found that purification significantly reduced the stability of foot-and-mouth disease virus. Scott and Woodside (1976) tried 15 media for stabilizing pseudorabies virus during lyophilization and concluded that peptone was the least effec-

STORAGE

TABLE IV DATAON SELECTED BACTERIOPHAGES Storage

Organism and preservation methodn

Temp. (“C)

Corynebacteriophage (14 strains) -25 Lyophilized in 3% peptone, 1.7% sucrose, 0.3% glutamate Escherichia coli phage T-2 4 Lyophilized lyzate Lyophilized in 10% milk Dried in lyzate from liquid E . coli phage MS-2 4 Lyophilized in lyzate E . coli phage 5d Lyophilized in lyzate 4 E . coli phage (14 types) Frozen in 10% glycerol - 196 Mycobacteriophage (11 strains) RT Lyophilized in 10% glutamate 1% gelatine Serratia marcescens phage Frozen in 10% glycerol - 196 Streptococcus phage (15 types) Frozen in 15% glycerol -22

Years

Viable (%)

References

+2.5

10-100

Came and Greaves (1974)

0 0

0.5 0.1 7

lmshenetskii et a2. (1970) Clark and Geary (1973) Iijima and Sakane (1973)

4

0.1

Imshenetskii et al. (1970)

+

all instances “milk’ refers to skim milk.

4

4

100

Imshenetskii et al. (1970)

34

10-100

Clark and Klein (1966)

2

>10

Engel et al. (1974)

3

40

Clark and Klein (1966)

2.5

30-100

Nyiendo et al. (1974)

46

ROBERT J. HECKLY

TABLE V STORAGEDATAON SELECTEDVIRUSES Storage Organism and preservation method Foot and mouth disease Lyophilized in various media Lettuce necrotic yellow virus Dried on silica gel in milk Measles virus Frozen in 2% DMSOb Polio virus Lyophilized in 1 M tris buffer Pseudorabies virus Lyophilized in 5% sucrose 1%glutamate 4% dextran Respiratory syncytial virus Frozen in 30% sucrose Frozen in 44.5% sucrose Frozen in 44.5% sucrose Vaccinia virus Lyophilized in 5% peptone buffer Lyophilized in 5%peptone or 5% glutamate

+

+

Temp. (T)

Years

Viable'

References

37

0.1

4

2.5

+

Grivell et al. (1971)

-76

0.5

100%

Greiff et al. (1964)

4-10

1

16%

Berge et al. (1971)

4

1

1%

-70 -70

1 3 0 % Fellowes (1968)

Scott and Woodside (1976)

-20

2 2 0.3

5% 100% 0.1%

Law and Hull (1968) Law and Hull (1968) Law and Hull (1968)

437

0.5

100%

Sparks and Fenje (1972)

45

1

14%

Suzuki (1973a)

+

indicates some infectivity. bDimethyl sulfoxide.

a

tive medium. They found that the best suspending medium for preserving infectivity was a mixture of sucrose, phosphate, and glutamate, to which albumin had been added. Calneket al. (1970), as well as Scott and Woodside (1973),also found the above mixture to be the best for herpesvirus. Peptone is not necessarily detrimental because infectivity of vaccinia virus was retained at a high level when lyophilized in media containing 5% peptone (Sparks and Fenje, 1972; Suzuki, 1973a). Corynebacteriophage also was stable when lyophilized in a mixture of peptone, sucrose, and sodium glutamate (Carne and Greaves, 1974). Fairly extensive tables showing the effects of freezing and lyophilization on bacteriophage were presented by Clark and Geary (1973) and the effects of storage at -196°C of a number of ATCC bacteriophages were given by Clark and Klein (1966). Table IV summarizes results that others have ob-

PRESERVATION OF MICROORGANISMS

47

tained with bacteriophages and similar data on preservation of viruses are given in Table V.

VII. Summary If cost is not considered a factor, storage in liquid nitrogen is probably the best method for preserving all microorganisms. For some viruses it may be better to freeze the sample rapidly, but slow freezing with a cryoprotective agent is desirable for all other organisms to retain maximum viability or infectivity. Storage at -70°C is nearly as effective as -196°C (liquid nitrogen). Only a few organisms survive well at higher temperatures, such as -20 or -40°C. For short-term preservation (3-12 months) agar slants or stab cultures in tightly capped tubes are usually adequate. For long-term storage of those organisms that can withstand dehydration, lyophilization is the most economical and reliable method. Oxygen is harmful to some organisms; therefore cultures should be sealed in glass ampules under vacuum. Although many lyophilized organisms survive well at room temperature, all cultures should be refrigerated. Since excellent survival has been demonstrated at 4"C, the additional expense of using lower storage temperatures is probably not warranted. Certain problems associated with lyophilized cultures must be recognized. Dehydration may be mutagenic and, in addition, the relative number of spontaneous mutants can increase in the rehydrated culture. Some revertants have been shown to survive lyophilization better than the mutant strain. Reconstituted cultures should be diluted and plated to ascertain colonial morphology, and by initiating the new culture from a few of the colonies the chances of propagating a mutant or a contaminated culture are minimized. ACKNOWLEDGMENT This work was supported by the Office of Naval Research. I wish to thank J. H. Quay for her dedicated assistance in the preparation of this manuscript.

REFERENCES Addey, J. P., Taylor-Robinson, D . , and Dimic, M . (1970).J. Med. Microbiol. 3, 137-145. Amarger, N . , Jacquemetton, M., and Blond, G. (1972). Arch. Mikrobiol. 81, 361-366. Ames, B. N . , McCann, J . , and Yamasaki, E. (1975). Mutat. Res. 31, 347-364. Annear, D. I. (1956).j. Hyg. 54, 487508. Annear, D. I. (1964). Aust. J . E r p . Biol. Med. Sci. 42, 717-722. Annear, D. I. (1970a). In "Culture Collections of Microorganisms" (H. Iizuka and T. Hasegawa, eds.), pp. 273-279. Univ. Park Press, Baltimore, Maryland.

48

ROBERT J. HECKLY

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Streptococcus mutans Dextransucrase: A Review

THOMASJ. MONTVILLE, CHARLES

L.

COONEY,

AND

ANTHONYJ. SINSKEY Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 I. Introduction

.........

11. The Properties of Dext 111. Mechanisms of Dextran Synthesis

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

IV. The Role of Surface Receptors in Cell Adherence and Aggregation ................... ........ V. Distribution of Dextransucrase . . . ........ VI . Pur6catiou and Properties of Dextransucrase VII. Regulation of Dextransucrase ..... VIII. Other Extracellular Enzymes Produced by Streptococcus mutans ................................ cies Streptococcus mutans . . . . . . . . . IX. Heterog X. Conclusion ..................... References ............................................

55 56 58 61 63 64

75 77 80 82 82

1. Introduction Streptococcus mutans occupies a central role in the etiology of dental caries. Streptococcus mutuns is readily isolated from carious lesions (Keyes, 1960) and induces caries when inoculated onto the tooth surfaces of germfree rats. The two factors that contribute to the cariogenic potential of S. mutuns are its ability to produce high yields of lactic acid from a variety of carbon sources and its ability to adhere to hard surfaces by the action of its extracellular polysaccharides. The lactic acid produced by S. mutans and other members of the oral flora, by demineralizing the tooth surface, is directly responsible for the formation of carious lesions. The formation of the extracellular polysaccharide, dextran, is of particular importance in the initiation of smooth surface caries. Streptococcus mutans produces both a water-soluble and a "sticky" waterinsoluble dextran. Insoluble dextran appears to mediate the attachment of s. mutuns cells to the tooth surface. Strains of S. mutuns that are low producers of insoluble dextran fail to adhere to hard surfaces (Johnson et al., 1974; 55 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0 - i z - 0 0 ~ 2 4 4

56

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

Tanzer et al., 1974)and are unable to produce caries (Tanzer et al., 1972). As the extracellular dextrans accumulate, they provide a matrix in which other acid-producing oral bacteria may be entrapped. It has been postulated that in addition to mediating cellular adherence the production of dextran may be a means by which S. mutans cells protect themselves from acid. This would be achieved by the diversion of the glucosyl moiety of sucrose away from the normal fermentative pathway and into polymer formation. Newman et al. (1976), for example, showed that cells cultured in sucrose broth could survive low pH better than cells cultured in a broth having the same concentration of glucose. They postulated that this was due to the protective effect of dextran production. This role is not likely. Only 18%of the sucrose in a broth is converted to dextran, while the rest is fermented to lactic acid (Tanzer et al., 1972). It is more likely that the cell-associated dextran produces a diffusion barrier so that pH within the plaque drops lower than the pH of the surrounding area. Hojo et al. (1976) found that the pH within the plaque of a wildtype, insoluble dextranproducing S. mutans strain dropped from pH 7 to pH 4 during continuous culture. The pH of the culture medium and plaque from a mutant deficient in insoluble dextran production dropped only one pH unit from 7 to 6. That acid accumulates within the plaque is further supported by radiographs of the pattern of S. mutuns growth within a plaque deposit (Tanzer and Johnson, 1976). These show that only the cells on the surface and edges of the plaque mass are viable. The cells in the interior of the mass were not viable, presumably because the plaque acted as a diffusion barrier to the entrance of nutrients and exit of waste. Gibbons (1968)has suggested that dextrans may also serve as an extracellular polysaccharide reserve. The extracellularplysaccharides of S. mutuns are synthesized by dextransucrase (EC 2.4.1.5).This enzyme polymerizes the glucose moiety of sucrose into water-soluble and water-insoluble polyglucans with the liberation of fructose. (It should be noted that dextransucrase is also known as glucosyltransferase.)Attempts at isolating and characterizing the enzyme(s)responsible for these different products have been inconclusive, with one to seven enzymes being identified (see Section VI on purification and properties). Research on S. mutans has been hindered by the fimdamental heterogeneity of the species. This makes it difficult to compare work done on various strains. The remainder of this review will examine in depth the nature and properties of dextrans and dextransucrase, briefly elaborate on other enzymes of S. mutans that can complicate studies of dextransucrase, and present information emphasizing the heterogeneity of the S. mutans species.

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

57

II. The Properties of Dextran Guggenheim and Schroder (1967), using periodate oxidation studies, determined that dextrans were a(l46)linked polyglucans with a(1+3) linked branches and a small number of a(l+4) linkages. Dextrans are heterogeneous. Different researchers, using precipitability in water and various concentrations of alcohol (Guggenheim and Schroder, 1967) or solubilities in different concentrations of NaOH (Gibbons and Nygaard, 1968), have been able to isolate at least four different dextrans. Precipitation of water-soluble dextrans with alcohol yields a product that subsequently may be water-insoluble. The dextrans of S. mutuns are also heterogeneous with respect to molecular weight. Gibbons and Nygaard (1968), for example, showed that of the water-soluble dextrans produced by S. mutuns GS-5, 35% had a molecular weight > lo6, 60% < lo6, and 4% < lo5. One of the insoluble dextrans had a distribution 93% > lo6, 7% < lo6, and 0% < lo5. These values were strain dependent and dextran fractions from the same strain had markedly different values. Guggenheim (1970)suggested that the solubility of dextrans was governed by their proportion of a(1+3) linkages. This was confirmed by Usui et u2. (1975). Using 13Cpulse Fourier-transform NMR spectra of water-soluble and water-insoluble dextrans produced by S. mutans JC-2, they showed that water-soluble dextrans contained 60% a(l+6) linkages and 40% a(1+-3) linkages. In contrast, water-insoluble dextrans had 48% a(l-6) linkages and 52% a(1+3) linkages. These results were supported by Nisizawa et ul. (1977), who used methylation analysis to determine that water-insoluble dextrans had a higher proportion of a(1-3) linkages than water-soluble dextrans. Ebisu and Misaki (1974) analyzed the water-insoluble dextrans of S. mutuns OM7 176 by methylation analysis and found 70% a(1+3) linkages and 30% a(1+6) linkages. Unfortunately, they did not analyze the watersoluble dextran for comparison. Figure 1 illustrates the probable structure of dextran. Using only the water-soluble dextrans from three strains of oral streptococci, Pearce (1976) correlated the structural properties of the dextrans with their adsorption characteristicson hydroxyapatite. Hydroxyapaptite (HA) is similar in composition to the tooth surface and adsorption to HA is a widely used indicator of the potential of materials for adsorption to the tooth. Pearce determined that while the total amount of dextran adsorbed to HA was related to the size of the molecule, the strength of the adsorption was related to the proportion of a(1-3) linkages in the molecule. This observation would have been more definitive if performed with water-soluble and -insoluble dextrans from one S. mutuns strain, rather than the soluble dextrans of several strains, since it

58

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

FIG. 1. The structure of dextran.

is generally acknowledged that soluble dextrans are not important contributors to the adherence of S. mutuns. Further evidence for the importance of insoluble dextrans had been obtained by electron micrographs of adherent and nonadherent S. mutuns cells. Guggenheim and Schroder (1967)observed that insoluble dextran appeared in electron micrographs as fibrils about 20 A thick. Soluble dextrans were more amorphous and globular. In comparing cariogenic S. mutuns GS-5 with two of its nonadherent mutants, Johnson et al. (1974) observed development of the fibril, insoluble dextran early in the fermentation of the adherent strain. In contrast, the nonadherent strain GS-511 showed only synthesis of the globular water-soluble dextran. De Stoppelaar et uZ. (1971) described a mutant similar to GS-511 which did not induce caries in animals. Barnett’s (1973) electron micrographs of cells attached to oral epithelium showed that this attachment was mediated by the fibril dextrans.

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

59

111. Mechanisms of Dextran Synthesis Dextransucrase is highly specific for sucrose and does not catalyze the polymerization of free glucose or glucose from other disaccharides (Jordon and Keyes, 1966;Tanzer et al., 1972; Germaine et al., 1974). Germaine et al. (1974) have shown that, while dextransucrase from glucose-grown cultures is only mildly stimulated by the addition of soluble dextran, upon purification by HA chromatography dextransucrase becomes highly dextran dependent. This is because exogenous dextran is thought to serve as a glucosyl receptor or primer of dextransucrase. In the absence of added dextran, autopriming occurs, in which sucrose serves as both glucosyl donor and acceptor. However, the affinity of dextransucrase for dextran is 1OOO-foldhigher than its affinity for sucrose. In the case of autopriming, there is a lag of 2 4 hours before rapid dextran synthesis occurs. The rate of autoprimed dextran synthesis never achieves that of the primed reaction. Germaine et al. (1974) demonstrated that the dextran molecule must be at least eight glucose moieties long to have primer activity. The catalytic effect increases with the length of the molecule up to 32 glucose moieties, at which point the stimulation remained constant. Neither glycogen, amylose, inulin, nor isomaltose functioned as a primer. Chludzinski et al. (1976) found that 0.1 M maltose, isomaltose, and isomaltotriose inhibited dextransucrase activity by about 30%. Sharma et al. (1975) have found that the maltose inhibition is competitive. Two possible mechanisms of dextran synthesis by Leuconostoc mesenteroides have been proposed by Ebert and Schenk (1968)and are illustrated in Fig. 2. Both mechanisms require a glucosyl acceptor site and a glucosyl donor site on the enzyme. Chludzinski et a2. (1976) suggest that in the absence of primer dextran, dextran synthesis proceeds by what Ebert and Schenk (1968)refer to as an insertion mechanism. In this model, the enzyme and the polymer chain remain in contact throughout the growing cycle and the new glucosyl unit is inserted between the enzyme and the growing chain. In the unprimed reaction dextran synthesis is slow, since sucrose is an inefficient glucosyl acceptor. The reaction proceeds by Eq. (1)(Fig. 2), and the isomaltodextran eventually attains sufficient size to have a catalytic effect. Chludzinski et al. (1976) suggest that if primer dextran is present, the reaction proceeds by a stepwise mechanism. This is unlikely since the stepwise mechanism suggests that all of the enzyme is present as enzyme or enzyme-substrate complex. In fact, the enzyme is agreed to exist almost entirely in the enzyme-polymer complex form (Mukasa and Slade, 1974b; Kuramitsu, 1975; Chludzinski et al., 1974; Ciardi, 1976).

60

T. J. MONTVILLE, C. L . COONEY, A N D A. J. SINSKEY

S =sucrose

E = enzyme P = polymer (dextran) A = acceptor

F = fructose

A. Insertion

1. E + S * E S - E P , + F

2. EP,

+S

ESP,

- EP.,, + F - + ES

t + A AESP. AP,,

3.

B. Stepwise

4. E + S = E S 5. ES

+ P.

- Pn+]+ E + F

FIG.2. Mechanisms of dextran synthesis. (Ebert and Schenk, 1968).

The applications of Leuconostoc models to S . mutans must be qualified. Equation (1) (Fig. 2) does explain autopriming, and Eq. (3) (Fig. 2) can linkages, if explain how one enzyme can synthesize both ( ~ ( 1 4 6and ) (~(143) the C-3 hydroxyl serves as an acceptor. One might think that, with the addition of soluble dextran at concentrations above those required to prime the reaction, there would be an increased amount of branching and more synthesis of insoluble dextran. In hct, more soluble and less insoluble dextran is synthesized. This could still be consistent with an insertion model if the C-6 hydroxyl of the added dextran were a better glucosyl acceptor than the C-3 hydroxyl of the endogenous dextran. Equation (2) would then be favored over Eq. (3), when exogenous dextran is added to the system. Using two criteria, Germaine and Schachtele (1976) suggest that S. mutans synthesizes dextran from the nonreducing end of the polymer. Since they could reduce the reducing end of dextran with sodium borohydrate without affecting its primer ability, they concluded that synthesis of dextran must have been from the nonreducing end. Experiments using dextran T-10 and dextran T-2000 as primer were also performed. Both of these dextrans have similar proportions of (~(143) branch points, so that the molar concentrations of reducing ends should differ by 200 while the molar concentrations of nonreducing ends are equal. Since both types of dextran had the same primer efficiency, it was concluded that synthesis must have been from the nonreducing end.

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

61

Robyt and Corrigan (1977) modified the nonreducing ends of dextran with trypsyl chloride, making them unavailable for reaction. This modified dextran still activated S. mutans dextransucrase. They suggested that instead of acting as a classical primer, exogenous dextran displaced the growing polymer from the enzyme and formed an a(1-3) link with the polymer. This would allow the active site of the enzyme to react rapidly with sucrose to give rise to new dextran chains. That large insoluble dextran attached to the enzyme slows the rate of reaction and that displacing it allows for more rapid dextran synthesis is plausible, but it is inconsistent with the slow initial rate of dextran synthesis by purified dextransucrase in the absence of “primer” dextran. Robyt and Corrigan (1977) provide the alternate explanation that the dextran may bind the enzyme, causing a conformational change that activates it. Recently, Harlander and Schachtele (1978) have found that phosphoglycerides stimulate dextransucrase activity by binding at a distinct site on the enzyme. This supports the idea that the conformational state of the enzyme may be an important determinant of its activity. It is also possible that dextran is synthesized from the reducing end, as it is with L. mesenteroides dextransucrase (Robyt et al., 1974). This is less likely since the dextransucrase from L. mesenteroides is less specific, is inducible (Kobayashi and Matsuda, 1974), and requires divalent ions (Tsumuraya et al., 1976), while dextransucrase from S. mutans does not.

IV. The Role of Surface Receptors in Cell Adherence and Aggregation Mukasa and Slade (1973, 1974a,b)did a series of studies to determine the mechanism by which S. mutans attaches to smooth surfaces. The model they developed again emphasized the role of insoluble dextran and suggested that there was a specific dextran binding site on the cell surface. Heat-killed cells mixed with crude enzyme preparation (CEP), sucrose, or dextran failed to adhere to glass surfaces. However, when mixed with CEP plus sucrose, the cells did adhere. Viable cells mixed with sucrose would also adhere, which was undoubtedly due to the presence of cell-associated dextransucrase activity. Cells incubated with CEP, washed, and then incubated with sucrose also adhered to glass surfaces. This indicates a CEP binding site on the cell which is not inactivated by heat. Kuramitsu (1974a) confirmed that heat-killed cells failed to adhere to dextran-coated glass surfaces. Freedman and Tanzer (1974) demonstrated that the ability of cells to aggregate was distinct from their ability to form water-insoluble dextran. When cells were preincubated with an antibody that reacted against both a and d serotypes before incubation with CEP plus sucrose, no adherence occurred (Mukasa and Slade, 1973). The a-d antibody did not affect dextran-

62

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

sucrase activity. Addition of anti-CEP did not prevent the enzyme from being adsorbed on the cell. From these studies, Mukasa and Slade (1973) concluded that adherence required a dextran receptor site that was near or part of the a-d antigen. In a subsequent work, Mukasa and Slade (1974a) showed that binding of dextransucrase was inhibited by both antidextran globulin and dextranase. This suggests that cell-bound dextran may be the dextransucrase binding site. Heat-killed glucose-grown S. mutuns cells were found to contain 2.6% of their cell dry weight as dextrans. It was also determined that soluble dextrans inhibited the synthesis of insoluble dextran and the binding of dextransucrase to the cell. Mukasa and Slade (1974a) have postulated that the &nity of dextransucrase for dextran is so high that it cannot bind with the dextran-like receptor site on the cell. Soluble dextran inhibition of dextransucrase binding would also occur if insoluble dextran directly mediated the binding, since soluble dextran would inhibit the synthesis of insoluble dextran. These two effects could be separated by using an inhibitor of dextransucrase activity, such as inaltose. Germaine and Schachtele (1976) built upon this work and, using CsCl gradients, found that the enzyme-dextran complexes contained 25% dextran. This corresponds to 150 enzyme molecules (MW = 40,000) per dextran molecule (MW = 2 x lo6). I

L n DEXTRAN

0

DEXTRANSUCRASE

0 7 I

.

Y?

PROTEIN with dextran-like D.S. binding site POLYSACCHAR,DE or dextran binding site TElCHOlC ACID

FIG. 3. Model of dextran and dextransucrase binding by S. mutans. (Modified from the drawings of Germaine and Schachtele (1976), and Slade (1976).)

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

63

Figure 3 is a composite model of the models of Germaine and Schachtele (1976) and Slade (1976). Note that cell-cell aggregation can occur in the absence of dextransucrase. McCabe and Smith (1975) demonstrated that toluene extraction of cell-bound dextransucrase did not adversely affect dextran-induced aggregation. They also found, as did Mukasa and Slade (1974a), that trypsin treatment resulted in a partial block of cell aggregation. This indicates that the nature of the dextran receptor is at least partly proteinaceous. With strain K1-R McCabe and Smith (1975) found that divalent calcium ions were required for both sucrose- and dextran-induced aggregation. The Ca2+was postulated to activate the cell receptor site to form specific intercellular Ca2+bridges. In a later work, McCabe and Smith (1976) purified a protein thought to be the proteinaceous dextran receptor. They found that dextran-induced aggregation of cells was inhibited when the partially purified protein was added to the cell suspension prior to adding the inducing dextran. This inhibition could be reversed by increasing the concentration of added dextran. The addition of dextransucrase did not adversely affect the ability of the cells to aggregate under the same conditions. Janda and Kuramitsu (1977) have provided additional support for a nondextransucrase dextran receptor site on the cell surface. They found that a variant of S. mutuns GS-5 that possessed cell-associated dextransucrase activity similar to the parent had greatly reduced dextran binding capacity and could not adhere to smooth surfaces. Their data suggest that the receptor is a heat stable, pronase-sensitive protein or that two receptors, one nonprotein and the other protein, are involved. It is interesting to note that dextran will mediate the aggregation of S. mutans and other oral bacteria, such as Actinomyces viscoses (Bourgeau and McBride, 1976). The mechanism of attachment of dextran to A. viscoses is probably different from that of dextran attachment to S. mutans, since the ability of A . viscoses to bind dextran is heat sensitive. Other interspecial studies (Liljemarkand Schauer, 1977)have observed no competition for binding sites by S. mutans, Streptococcus miteor, and Streptococcus salivarius on saliva, dextran, or saliva plus dextran-coated HA, except between S. miteor and S . salivarius on saliva-coated HA. In this case, the competition was for related but daerent sites.

V. Distribution of Dextransucrase Kuramitsu (197413) studied the cell-associated dextransucrase of S. mutuns strain GS-5. He found that intracellular, cell-associated extractable and extracellular dextransucrase had similar gel filtration properties, pH and temperature optima, and kinetic constants. From this he concluded that there

64

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

was only one enzyme under investigation. The “cell-associated extractable” enzyme was obtained by washing cells in 1N NaCl, eluting the dextransucrase from the cell surface binding sites. Mukasa and Slade (1974a) reported that cell-associated dextransucrase from strain HS-6 could not be eluted even in 6 N NaC1. Kuramitsu also failed to find a salt-extractable dextransucrase with strain HS-6. This indicates the strain dependency of this phenomenon and suggests that the exact nature of the dextrddextransucrase binding sites may be strain or serotype variable. Gibbons (1968) hypothesized that cell-associated dextransucrase was due to the presence of trace amounts of sucrose in the growth medium. This theory has gained wide credence in the literature and the presence of cellassociated dextransucrase is now almost universally attributed to medium sucrose (Robrish et al., 1972; Kuramitsu, 1974a; McCabe and Smith, 1973). McCabe and Smith (1973) reported that S. mutans K1-R had only low levels of cell-associated dextransucrase activity in a trypticase broth but that dextransucrase became highly cell associated when sucrose was added to the medium. Spinel1 and Gibbons (1974) reported similar results for S. mutuns 6715. This observation is not universally valid, as demonstrated by Montville et al. (1977b), who have shown that S. mutans strain GS-5 has substantial cell-associated activity in a medium devoid of sucrose. The addition of sucrose to the medium caused no change in the distribution of the dextransucrase. This ‘again demonstrates strain variability, and suggests that both sucrose-dependent and sucrose-independent mechanisms of cell association may exist. The dynamic equilibrium of dextransucrase between insoluble and soluble activities and cell associated and supernatant forms has been schematically summarized by Montville et ul. (197713) and is presented in Fig. 4. Dextran T-10 has been shown to mediate shifts from insoluble to soluble activity but the reverse shift has not been observed in the presence of any compound. The distribution of dextransucrase between the cell surface and the supernatant can be affected by sucrose, which causes a shift to the cell associated forms of the enzyme, and sodium chloride, which causes a shift to the supernatant forms. This model is generalized; the fidelity with which specific S. mutans strains adhere to it remains to be seen. Further study of the regulation of dextransucrase distribution may be valuable, since the cell may wash out of the oral cavity more easily if dextransucrase activity can be dissociated from it. VI. Purification and Properties of Dextransucrase

The most complex and confusing aspect of S. mutans literature is undoubtedly that which pertains to the purification and properties of dextran-

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW CELL

ASSOCIATED

65

DEXTRANSUCRASE

t DEXTRAN T - I 0 INSOLUBLE ACTIVITY//

SOLUBLE I \ACTIVITY / -

-- .. Ca

- PANTOTHENATE + I N NaCl

I5

-lq

I

I

%

T-I0

t DEXTRAN T-I0

- /

SOWBLE ACTlVlN

’

SUPERNATANT

I

J

\\

-\ INSOLUBLE ACTIVITY

DEXTRANSUCRASE

FIG.4. Schematic illustrating the dynamic equilibrium of dextransucrase between soluble and insoluble activity and between the cell associated and supernatant forms. Conversion experimentallydemonstrated; A conversion experimentallydisproved; conversion may take place.

-

sucrase. Investigators in this area tend to use different strains, different growth media, and different purification procedures which, not surprisingly, yield different and conflicting results. The presence of levansucrase, invertase, and dextranase compounds the confusion. It should be noted that, except for McCabe and Smith (1977), contaminating dextranase activity is probably contained in all of the “purified’ large molecular weight dextransucrase preparations. (See Section VIII for the details of dextranase action.) As it is impossible to reconcile all findings, the literature on purification and properties will be reviewed in roughly chronological order with methods and annotations suggesting the problems of each method. Guggenheim and Newbrun (1969) admitted that the properties of their dextransucrase varied from batch to batch and that the assignment of different dextransucrase activities to different pools was somewhat arbitrary. Figure 5 shows their basic procedure. Their results bring to mind several

66

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

Strain: OM2176

5 liters culture supernatant

I

+ 2 liters H,O

+ 250 In1 HA

Sediment Slurry packed in column

Supernatant Discard

I

Elute stepwise with 0.2 and 0.5 M PO I

0.2 M fraction 2 peaks 13%of the original activity

0.5 M fr.iction 1 peak 18%o1;ictivity

I

I I PAGE IEF

IE7 PAGE

FRACTION Supernatant HA Adsorbed 0.2 M Elute 0.5 M Elute

SA ( I U h g )

PURIFICATION

YIELD (%)

0.008 -

1

100

-

0.35 0.92

41 108

86 21

pH4.2 21% 7.23 6.0 none

pH5.0 34% 0.98 7.0 215

pH5.6 25% 3.37 6.0 1-2

7

2 4

I 4

21

Results of isoelectric focusing IEF Fraction Frequency K m (d) pH opt. No. of enzymes on PAGE No. of proteins on PAGE

FIG. 5. The purification method of Guggenheim and Newbrun (1969).

problems applicable to many of these purifications. First, since only half of the enzyme applied to the HA column is recovered, what has happened to the rest of the activity? Are specific dextransucrases retained? Second, a reducing sugar assay for dextransucrase has been employed, which does not discriminate between dextransucrase, fructosyltransferase, and invertase activities. Third, 15-20% of the supernatant activity was not bound to the HA and might represent unique dextransucrases, but this fraction was not further characterized. Purifications of dextransucrase are plagued by poor yields. If polyacrylamide gel electrophoresis (PAGE) of each preparation

67

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

were performed at each step of purification, one could determine if specific proteins, or a fixed amount of each, were being lost. This information is essential to a definitive study of dextransucrase. Chludzinski et al. (1974) used strain 6715 in their purification, as diagrammed in Fig. 6. Their procedure entails the use of ammonium sulfate, HA chromatography, gel filtration, PAGE, and isoelectric focusing (IEF). Some sequence of these steps is typical of most dextransucrase purifications. Upon dialysis 14% of the dextransucrase was precipitated and it was not further characterized. Again, yield after HA chromatography was poor. After elution, 57% of the enzyme was still bound to the HA. The activity which did elute from the HA did so as one distinct peak. Isoelectric focusing of this protein yielded one band at pH 4. Polyacrylamide gel electrophoresis of this

Strain: 6715 (NH,),SO,, 55% of saturation

I

Sediment Dissolve in 0.01 M PO, buffer, pH 6

Supernatant Diacard

I

Dialyze

I

14% of the activity precipitated

HA chromatography

I

Elute with buffer 0.014.15 M

I

Fractions concentrated with (NH,),SO, and dialyzed I PAGE

I

IEF

I

I

GEL FILTRATION

PAGE

FRACTION

SA (IU/mg)

PURIFICATION

Supernatant (NHJzSO, (NH4hS04,dialyzed HA IEF

0.oO0206 0.00747 0.00871 0.200 0.306

36 42 970 1,515

1

FIG. 6. The purification method of Chludzinski et al. (1974).

YIELD (%) 100 95 69

30 13

68

T. J . MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

band yielded four distinct proteins, while PAGE of the HA fraction yielded ten. In each case, only two bands had dextransucrase activity, with one band containing 90% of the activity. Gel filtration of the HA peak yielded one peak with a molecular weight corresponding to 94,OOO. This enzyme was characterized as having a broad temperature optimum of 3442OC, a pH optimum of 5.5, and a K , of 3 mM. It was noted that m unprimed assays, enzyme activity was lost rapidly upon storage at 4°C. If the assay was primed with soluble dextran, the activity remained constant for 97 days. This suggested the presence of a contaminating dextranase which destroyed the enzyme-bound dextran that could act as a primer. This study concluded that there were at most two dextransucrases. Streptococcus mutuns HS-6 grown in a complex dialyzed medium with a controlled pH of 7 was used as the source of dextransucrase by Fukui et ul. (1974) (see Fig. 7). They used a reducing sugar assay for dextransucrase and assumed that the invertase activity was removed by anion-exchange chromatography. Their purified dextransucrase had only one component on sodium dodecyl sulfate (SDS) gel electrophoresis with a molecular weight corresponding to 170,000. Polyacrylamide gel electrophoresis also demonstrated the purity of this preparation, which had a K , of 2 mM and optimal activity at pH 5.7. The product formed by this soluble dextransucrase was 94% a(1+6) linked and 6% a(l43)linked. An “invertase-like” activity was noted in this preparation but was not further characterized. The insoluble dextransucrase found in the high molecular weight shoulder of the gel filtration study was not further characterized. It can be concluded from this study that there is at least one dextransucrase. Mukasa and Slade (197413)(Fig. 8) studied the dextransucrase of S. mutans HS-6 cultured in Todd Hewitt Broth, a medium known to promote the cell association of dextransucrase. hvansucrase activity was noted in all of their fractions. Immunodfision tests of anti-CEP against each fraction showed only one band for AH2 but two or possibly more bands in AH,, CEP, and HF. These bands may be due to the levansucrases or dextranases. Polyacrylamide gel electrophoresis showed that in each case dextransucrase and levansucrase were the major proteins, with other proteins present in “negligible” amounts. The ability of the enzyme fraction to cause the adherence of cells in the presence of sucrose ran parallel to its ability to form insoluble dextran. Kuramitsu (1975) has also shown that when dextransucrase was fiactionated on the basis of molecular weight, the larger aggregated enzyme contained the insoluble dextransucrase activity. Kuramitsu’s purification uses strain GS-5 and is outlined in Fig. 9. Almost all of the activity migrated as fraction A, but when soluble dextran was added to the growth medium most

69

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW Strain: HS-6

I

Invertase

Culture supernatant

I

I

Soluble dextransucrase Concentrate, dialyze

I

'i"

Elute with NaCl gradient Single peak

I I HA I Single symmetrical peak

PAGE

Purified dextransucrase

I

SDSPAGE

I

PAGE

FIG. 7. The purification method of Fukui et al. (1974).

of the activity migrated as fraction B, which synthesized only soluble dextran. Addition of invertase to the growth medium also caused a shift to the B peak. Incubating fraction A with dextranase and a(1+3) glucanase caused some shift to the lower molecular weight fraction. 14C-Solubledextran synthesized by fraction B could be converted in limited amounts to insoluble dextran, when incubated in the presence of fraction A and sucrose.

70

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

Strain: HS-6

(NH 4 ) 2 S 04, dialyzed, dissolved in buffer

, Crude en!zyme preparation (CEP)

HA

Elute 0.01-0.8 M buffer

I

Single active fraction concentrated and dialyzed

I

HA

I

H F = single active fraction 1,100-fold purification

+

B Lyophiliw

1,Bio Gel A

0.5m

Single peak insoluble activity

I

1 I

Single peak soluble activity

HA Eluted with 0 . 0 1 4 3 5 M buffer

A AH2

AH1

FIG. 8. The purification method of Mukasa and Slade (1974b).

Fractions A and B had similar pH optima and K,s. Whereas the addition of primer dextran resulted in a twofold increase in the activity of fraction A, however, it resulted in a 12-fold increase in the activity of fraction B. This suggests that an endogenous dextran may be bound to the large molecular weight activity of fraction A. Fraction B was shown by PAGE and SDS gel electrophoresis to consist of a single protein with a molecular weight corresponding to 45,000, although IEF resulted in two peaks at pH 4.3 and 6.5. Sodium dodecyl sulfate electrophoresis of fraction A suggested molecular weights of 190,OOO and 235,000. Three enzyme activities were detected by PAGE. It was likely that dextranase was present in fraction A. Kuramitsu concluded that fractions A

71

STREPTOCOCCUS MUTANS DEXTRANSUCRASE: A REVZEW

(NH,).$O,,

501,then dialyzed

CEP

I

Bio Gel A-15

I

Concentrated

i’ FRACTION Supernatant (NH4)2S04 A B HA-A HA-B

HA Eluted with 0.01-1 M PO, buffer

SA (IU/mg)

O.OOO9 0.0174 0.213 0.0076 0.985

0.756

PURIFICATION

YIELD (%)

1 19 236 8 1,094 840

100 48 4 2.8 0.3 32

FIG. 9. The purification method of Kuramitsu (1975).

72

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

(a) Primary strain: 6715 Culture supernatant

I

Concentrated sequentially with Amicon PM 10,XM lOOA and XM 300membranes I

I

Filtrate

1 Retentate Adjust to 75% Ethanol

I

SedimLnt

Supernatant

I

Dissolve in buffer

Unfrhtionated enzyme preparation (UFE)

I

PAGE

I SDS gel electrophoresis

I Sediment at 3350% EtOH Soluble product enzyme (SPE)

I

I

Alcohol added

I

Sediment at 5040% EtOH Insoluble product enzyme (IPE)

I

PAGE

PAGE

111 J FIG. 10. (a) The pudcation method of Ciardi et al. (1976).(b)PAGE of UFE from serotypes a-e. Black bands and shaded areas represent insoluble activity; bulges represent soluble activity.

ing into the gel. No insoluble dextransucrase was detected in the SPE. The UFE contained an “invertase-like” activity. This could be due to an exohydrolytic dextranase. When the UFE was placed on gels of different acrylamide concentrations, a plot of the log of the relative mobilities of the enzyme bands versus increasing acrylamide concentration for the nine bands revealed the following. There were five bands of insoluble activity with a

STREPTOCOCCUS MUTANS DEXTRANSUCRASE: A REVIEW

73

molecular weight of 160,000 but different net charge; two bands of 200,000 and 225,000 molecular weight insoluble activity with different charge; and two bands of soluble activity with similar charge, but molecular weights of 185,000 and 272,000. When the UFE was incubated with dextranase, a loss of insoluble dextransucrase activity occurred and a new PAGE band appeared. Comparative studies by PAGE of the UFE from dextransucrases of serotypes a-e showed similarities between serotypes a and d and between c and e, but each strain had a distinctive pattern with multiple bands (see Fig. lob). It is interesting to note that this is the only study in which insoluble dextransucrase activity has migrated deep into the gels. This study concluded that there were two major fractions of dextransucrase (SPE and IPE) and suggested that its multimolecular forms might be due to its nature as glycoprotein, since glycoproteins occurred in many multimolecular forms differing only in their carbohydrate moieties (Pazur and Aronson, 1972). This may also explain the multiple PAGE bands obtained from preparations of other groups judged to be pure by criteria other than PAGE. Speculation that dextransucrase is a glycoprotein is based on the observation that the enzyme has a high carbohydrate content that is not separated by ammonium sulfate precipitation, gel filtration, or electrophoresis. Whether the enzyme is a glycoprotein, as suggested by Germaine et al. (1974), or a pseudo-glycoprotein in which the enzyme has a strong a n i t y for dextran, remains to be determined. Janda and Kuramitsu (1976) have found that several sugar analogs known to inhibit glycoprotein synthesis have no specific effect on dextransucrase production, suggesting that the enzyme is not a true glycoprotein. Osborne et al. (1976) also performed PAGE on CEP obtained by ammonium sulfate precipitation of dextransucrase from several S. mutans strains. They found between 12 and 20 protein staining bands and two to seven polysaccharide synthesizing bands for each strain. Again, each strain produced distinctive patterns with two types of polysaccharide synthesizing enzymes and one “invertase-like” enzyme for each strain. Bulkacz and Hill (1977) compared the dextransucrases of strain 6715 and its nonadherent mutant SM23. They performed isoelectric focusing on the CEP obtained by salting out with 50% ammonium sulfate. The parent strain produced nine protein bands and 14 dextransucrase bands. The mutant SM23 had 13 protein bands but only eight dextransucrase bands. The parent’s enzymes focused over pHs from 4.5 to 7.2. The mutant showed no enzyme focusing below pH 5.7 or above 6.4. It is also interesting to note that while the gross activity (IU/ml) of both strains was similar, the enzyme from the parent had a specific activity two to seven times higher than the mutant.

74

T. J. MONTVILLE, C. L. C O N E Y , AND A. J. SINSKEY

This study is significant because it shows that, unlike S19, the hyperproducing mutant of 6715 that simply produces more of the same enzymes (Schachtele et al., 1975b), the nonadherent mutant SM23 produces enzymes that differ phenotypically from those of the parent. The most innovative purification for dextransucrase is that of McCabe and Smith (1977). This procedure (Fig. 11)was designed to yield dextransucrase free of dextranase, invertase, and levansucrase. Polyacrylamide gel electrophoresis was performed on the a5nity chromatography fractions and on the two hydrophobic chromatography fractions. The A n i t y chromatography fraction had three protein bands, two of which represented dextransucrase activity. The minor inactive protein band was recovered as the major protein band of fraction 2. Fraction 1had only one dextransucrase band near the top of the gel. Fraction 2 had two poorly separated dextransucrase activities, both near the top of the gel. Strain: 6715-49 Culture supernatant (NH4)30,, 50%

1

supe’matant Discard

Sediment Mixed with slurry of insoluble dextran and Bio Gel P-2

I

Elute with 0.5% clinical dextran

I

PAGE

Hydrophobic chromatography

I

Dextransucrase eluted with NaCl gradient

I

I

I

Major peak 2

Minor peak 1

I

I

PAGE

PAGE FRACTION Supernatant (NHdW4 Af6nity chromatography Hydrophobic Chromatography

SA (IU/ml)

PURIFICATION

YIELD (%)

0.014 0.027 4.05

1 2 289

100 95

14.8

1.057

65

FIG. 11. The purification method of McCabe and Smith (1977).

75

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

75

Both fractions 1 and 2 synthesized soluble and insoluble dextran. When fraction 1was incubated in the presence of soluble dextran, soluble dextransucrase activity increased at the expense of insoluble dextransucrase activity. When fraction 2 was incubated with soluble dextran, insoluble dextransucrase activity remained constant while soluble activity was greatly increased. The type of response exhibited by fraction 1has been repeatedly observed in the culture supernatants of several S. mutans strains (Montville et al., 1977a). To summarize all of these findings would be impossible. One can say with certainty that large molecular weight aggregated dextransucrase is responsible for the synthesis of insoluble dextrans. This may be due to the copurified dextranase, but insoluble dextran is also formed by the dextranase-freepreparation of McCabe and Smith (1977).Soluble dextransucrase characteristically has lower molecular weights. Some evidence exists that these activities may be interconverted, since insoluble dextransucrase treated with dextranase or soluble dextran loses insoluble activity and synthesizes more soluble dextran. None of these investigations has definitively answered the question of how many unique enzymes are responsible for the different dextransucrase activities.

VII. Regulation of Dextransucrase Embarrassingly little research has been done on the regulation of dextransucrase, considering the wide interest in dextransucrase and the large number of articles being published about it. Unlike the dextransucrase of L. mesenteroides, which is inducible (Kobayashi and Matsuda, 1974), the dextransucrase of Streptococcus sanguis has been classified as constitutive (Carlsson and Elander, 1973). Streptococcus mutans dextransucrase is also constitutive, since large amounts of dextransucrase are produced in media devoid of sucrose (Gibbons, 1968; Guggenheim and Newbrun, 1969). Schein (1976) noted that dextransucrase could be produced in a medium which contained glucose as the only sugar and ammonium bicarbonate as the sole nitrogen source. She noted that dextransucrase was produced in every medium in which S. mutans grew. Her studies ofS. mutans PR89 showed no evidence for carbon catabolite repression or ammonia repression. The only real study of regulation of dextransucrase produced by an oral streptococcus was by Carlsson and Elander (1973), using S. sunguis. They found that, in batch culture in complex media, enzyme activity increased with a linear differential rate of synthesis during the logarithmic phase of growth. Schein (1976), Montville et al. (1977a), and Janda and Kuramitsu (1976) have confirmed that the production of dextransucrase by S. mutuns is growth associated.

76

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

Janda and Kuramitsu noted that throughout the period of logarithmic growth a constant fraction of the dextransucrase synthesized by S. mutans GS-5 had the potential to form insoluble dextran when incubated with sucrose. Further enzyme synthesis ceased when the culture reached stationary phase. Addition of chloramphenicol or rifamycin to the culture immediately inhibited further accumulation of dextransucrase. A close coupling between dextransucrase synthesis and excretion was also indicated. Carlsson and Elander (1973) observed that in the transition of S. sanguis from growth in defined media to complex media, there was a transient overproduction of dextransucrase. Schein (1976) observed transient overproduction of dextransucrase when a steady-state chemostat was pulsed with glucose. This observation indicates that dextransucrase production is subject to some control mechanism. Maltose and soluble dextran have been shown to be competitive and noncompetitive inhibitors, respectively, of insoluble dextran synthesis (Sharma et al., 1975; Newbrun et al., 1977). This inhibition appears to be due to their ability to act as glucosyl acceptors. Fructose is also known to inhibit dextransucrase activity (Gibbons, 1968). McCabe and Smith (1975) reported that sodium fluoride at a concentration of 0.4 M inhibited dextransucrase activity, while Gibbons (1968)reported that it did not. The unpublished results of Montville also show no effect of sodium fluoride on dextransucrase activity. Again these discrepancies may be due to strain variability. Ciardi (1976) noted that significantly higher yields of dextransucrase were obtained in glucose-limited cultures grown at 30°C, rather than 37"C, if the pH was maintained above 5.7. There has been a number of papers in recent years suggesting that plasmids mediate the ability to produce insoluble dextran. Greer et al. (1971) demonstrated that eight strains of cariogenic S. mutans lyzed after induction by ultraviolet light and released viruses with similar morphology. Neither lysis nor the virus could be induced with several noncariogenic strains. The group of Dunny et al. (1973) has reported the isolation of a plasmid from S. mutans LM7. The plasmid had a molecular weight of 3 x 10 and was present at 16 copies per chromosomal genome equivalent. The group of Higuchi et al. (1976) continued this work and detected satellite bands of plasmid DNA in cell lyzates from cariogenic S. mutans PK-1 and JC-2. Mutants of these strains that had reduced ability to form insoluble dextran had no detectable satellite band of DNA. Using strain PK-1 and its mutant in further studies (1977a), Higuchi et al. observed phage particles in PK-1 but not in its mutant. Free phage was isolated from S. mutuns PK-1 and was used to transform the mutant cells to the adhesive cariogenic strain. In addition, the transformants could produce ammonia from arginine, whereas neither the parent nor the untransformed mutant

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW

77

exhibited this characteristic. Finally, free phage from S. mutans PK-1 was used to transform S. sanguis ATCC 10556, thereby increasing its ability to synthesize insoluble dextrans and adhere to glass surfaces (Higuchi et al., 197%). The transformants in this case could not produce ammonia from arginine, although the parent S. sanguis cells could. It is doubtful, however, that plasmids code for the production of insoluble dextransucrase per se. In each case it should be noted that the phageless or untransformed strain already had low levels of insoluble dextransucrase activity. To date, no S. mutans strain has been reported that is totally lacking the ability to synthesize insoluble dextran. Since transformed strains have increased insoluble dextransucrase activity, it is more likely that the phage codes for some other molecule. This phage-coded molecule may be an activator, a cell surface receptor, or possibly even the enzyme dextranase, which has been shown to affect the solubility of the products formed by dextransucrase (see Section VIII, “Other Enzymes, ” for details). That plasmids do not mediate dextransucrase production is also supported by a recent survey of the extrachromosomal gene pool of S. mutans by Marcianaet al. (1977). In their study, only two out of 50 human cariogenic S. mutans isolates were found to contain plasmid DNA. Although there have been many reports of mutants of S. mutans with decreased insoluble dextransucrase activity, there has been only one report of isolation of a hyperproducing strain. After irradiating with ultraviolet light, Schachtele et al. (197513) obtained a mutant of strain 6715 which produced six times more dextransucrase than its parent. Kinetic studies indicated that the mutant simply produced more of the same enzyme than the parent.

VIII. Other Extracellular Enzymes Produced by Streptococcus Mutans Studies of dextransucrase may be complicated by the presence of invertase (EC 3.2.1.26), levansucrase (EC 2.4. lo), and the hydrolytic enzyme dextranase (EC 3.2.1) in the culture broths of some strains. Invertase interferes with the reducing sugar assay for dextransucrase, in which activity is measured by the amount of reducing sugar liberated. Obviously, the presence of invertase would lead to erroneously high activities. For this reason, the reducing sugar assay is no longer commonly used or is used only in conjunction with simultaneous invertase assays. The invertase of S. mutans GS-5 has been characterized by Kuramitsu (1973) as having a pH optimal activity at 37”C, a molecular weight of 47,000, and an affinity for sucrose similar to that of dextransucrase. Fukui et al. (1974) determined the K, for invertase to be 20 mM.

78

T . J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

The ability of some S. mutuns strains to produce levans (polyfructoses) was one of the first criteria used to subdivide the heterogeneous species. Streptococcus mutans belonging to the HS group produced both dextrans and levans (Wood and Critchley, 1966). The role of levans in the etiology of dental caries has not been extensively investigated. Levansucrase can interfere with dextransucrase assays that determine the incorporation of glucose from UL[ 14C]-sucrose.The presence of levansucrase will result in production of methanol-insoluble levans, resulting in erroneously high reports of dextransucrase activity. In this case, the more expensive ([ 14C]-glucose)sucrose must be used. The use of UL [14C]-sucroseis justified with strains that are levansucrase negative. Fortunately, both invertase and levansucrase activities are lost on ammonium sulfate precipitation of dextransucrase. Comparing the amounts of these sucrose metabolizing enzymes in S. mutans 10449, Chassy et al. (1976) found 83 IU dextransucrase, 13 IU levansucrase, and 1001 IU of invertase in the broth of a 6-liter culture. Similar proportions were found for the other levan-producing strains. Studies on the role of dextranase in oral ecology are beginning to appear in the literature. It has long been known that dextranases from such fungi as Penicilliumfunicilium can degrade soluble dextran by attacking the a(1+6) linkages. Insoluble dextrans are resistant to dextranase degradation because of their higher proportion of a(1+3) linkages (Guggenheim, 1970). In 1972, Walker isolated dextranase from an oral isolate of Streptococcus mitis 439, a strain that did not synthesize dextrans. She postulated that dextranases regulated synthesis of dextrans by other oral streptococci. If dextranase was added early in the incubation period, synthesis of insoluble dextran was inhibited. Walker’s data supported an insertion mechanism of dextran synthesis, with the first step: Sucrose

+ sucrose + 6-a-glucosylsucrose + fructose

The 6-a-glucosylsucrose is a better glycosyl acceptor for dextransucrase than sucrose. This trisaccharide is very susceptible to dextranase. Walker demonstrated that the first five to six glucosyl moieties attached to the receptor are exclusively a(1+6) linked. Therefore, the presence of a a(1-6) degrading dextranase can inhibit the synthesis of all dextrans. Staat et al. (1973) developed a medium for the detection of dextranase activity from oral isolates. All subjects tested were found to harbor dextranase-positive strains at a level of 0.5% of the total viable oral isolates. These dextranase-producing strains were identified as Actinomyces and Bac-

STREPTOCOCCUS MUTANS DEXTRANSUCRASE: A REVIEW

79

teroides. One of these isolates was capable of producing acid from dextran. This supports the hypothesis that dextrans serve as a carbohydrate reserve. Using defined strains of S. mutans, Staat and Schachtele (1974) detected dextranase activity in ten out of 13 strains. Dextranase-positive strains included S. mutans 10449, Ingbritt, LM-7, GS-5, FA,, and OMZ161, which are widely used in dextransucrase research. The dextranase was found to be endohydrolytic and to produce free dextrans but not glucose. Schachtele et al. (1975a) confirmed the effect of dextranase on insoluble dextran synthesis. They found that as little as 0.002 IU/ml of fungal dextranase would inhibit insoluble dextran synthesis and that 0.005 IU/ml reduced the adherence of S. mutans by 80%. These results may explain why it is difficult to implant S. mutans in the human mouth, even though S. mutans rapidly induces caries in germ-free rats (Jordan et al., 1972; Krasse et al., 1967). Germaine et al. (1977)purified extracellular enzymes of S . mutans 6715 by ammonium sulfate precipitation. The resulting high molecular weight aggregates were separated into two fractions by anion-exchange chromatography. The (Y fraction produced water-insoluble dextrans and contained dextranase activity. The p fraction produced water-soluble dextrans but contained no dextranase activity. When the (Y and p fractions were mixed in the absence of primer dextran, the dextranase in the a fraction could modify the product of the p fraction. Water-soluble dextran synthesized by could be converted into insoluble dextrans when incubated with the (Y fraction. The dextran fragments produced by the dextranase are apparently reincorporated into the polymer as branches, by Eq. (3) of the insertion mechanism. The fact that the dextranase copurifies with the large molecular weight dextransucrase aggregates upon gel filtration poses serious problems in the interpretation of past papers on the purification of dextransucrase. The long-standing observation that high molecular weight dextransucrase synthesizes insoluble dextran could be due in part to the presence of dextranase in this fraction. Recently, McCabe and Smith (1977) have isolated a dextranase-free dextransucrase which synthesizes insoluble dextran. Hence, the formation of insoluble dextran is not due exclusively to the action of dextranase. Ellis and Miller (1977) have found that the presence of sucrose or the addition of sucrose in glucose broth produced elevated levels of dextranase in S . mutans 6715, whereas the addition of dextran or glucose alone produced only basal levels of dextranase. This suggests that the metabolic role of dextranases may be more closely related to synthesis than to degradation of dextrans. The presence of endohydrolytic dextranase should not af€ect primed dex-

80

T. J . MONTVILLE, C. L . COONEY, AND A. J. SINSKEY

transucrase assays, which measure the retention of methanol-insoluble ([14C]-glucose)-dextranon paper disks, since polymers as small as five glucosyl moieties are retained on the disks (Germaine et al., 1974).

IX. The Heterogeneity of the Species Streptococcus mutans Throughout this review discrepancies between the results of different investigators have been reconciled by reference to strain variability. In fact, the heterogeneity of S. mutans strains is well documented in the literature. Brathall (1970) recognized that S. mutans could be divided into five serotypes (a, b, c, d and e) on the basis of cell wall antigens. Perch et al. (1974)found two additional serotypes f and g. The use of Brathall's serotypes is widely accepted by S . mutans researchers, but its use is hampered by untypable strains and strains that crossreact. Brathall's serotype designations will be used in this section to distinguish different S. mutans biotypes. Routine serotyping of S . mutans is hindered by the lack of widely available, pure, fresh, type-specific antibodies. Recognizing this, Shklair and Keene (1974)proposed a biochemical scheme for the separation of S. mutans into five biotypes which correspond to Brathall's serotypes. This scheme was based on the ability of S. mutans to ferment mannitol (with and without bacitracin), sorbitol, raf€inose, and melibiose and to produce ammonia from arginine. If one considers biotypes differing in only one of these characteristics to be closely related, and those differing in two or more to be unrelated, one can subdivide S . mutans serotypes into three overlapping groups. These are the a, c, e; c, b; and d, e groups. This can be easily visualized by the diagram :

In this scheme, biotypes a, d, and b are very distinct from each other. Russel (1976) classified a number of S. mutans strains by SDS gel electrophoresis of the washed cells. He found that the electrophoretic patterns were virtually identical for members of the same serotype but could differ widely between serotypes. From his results, he was able to group S. mutans into four biotypes: a; b; c, e, f; and a, d. Tanzer et al. (1977) did a comparative study of the invertases of 13 S.

81

STREPTOCOCCUS MUTANS DEXTRANSUCRASE:A REVIEW TABLE I COYKENDALL'S PROPOSED DIVISIONOF s. MUTAA'S

INTO

FIVE SPECIES

Proposed species ~

S. mutans Representative strains

GS-5, 10449

DNA G + C (mole %)

3638

Distinctive biochemical traits

-

S. rattus FA1, BHT 4143 Ammonia from arginine, growth at 45°C

~

~

~

S. cricetus

S. sorbrinus

AHT, OMZ61

6715, K1R

4244 Basitracin sensitive

S. ferw HA2, 8s 1

4446

4345

Fail to

Fail to ferment raffinose, produce H*O*

ferment rdnose, basitracin sensitive

mutans strains, representing the five Brathall serotypes. On the basis of the electrophoretic mobility of the invertases, their molecular weights, and the effect of inorganic phosphate on their kinetics, he found that S. mutans could be divided into four biotypes containing the serotypes a; b; c, e; and d. Smith and Taubman (1977) studied the antigenic relatedness of dextransucrases. They found, for example, that antisera prepared from the dextransucrase of serotypes a or d inhibited the dextransucrase from serotypes a, d, and g, but not from serotypes b, c, and e. Antibody inhibition also showed that c and e were related biotypes. It was suggested that serotypes a, d, g; b; and c, e may comprise distinct biotypes of S. mutans. Based on his earlier work, which showed S. mutans to be heterogeneous using the criteria of DNA homology (Coykendall, 1971), Coykendall (1977) TABLE I1 PROPOSED GROUPINGS OF S. inutuns BIOTYPES Criteria DNA homology G + C ratios (Coykendall, 1971) SDS gel electrophoresis (Russel, 1976) Invertases (Tanzer et al., 1977) Dextransucrases (Smith and Taubman, 1977) Biochemical traits (Shklair and Keene, 1974)

Serotypes with common traits c,e,f

b

a

d, g

c,e,f

b

a

d, g

c, e

b

a

d

c, e

b

a,dg

b,c

a,c,e

d,e

C

82

T. J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

proposed that S. mutans be divided into five species. His criteria are presented in Table I and compared in Table 11 with the subgroupings discussed in this review. These studies present strong evidence against considering S. mutans to be one homologous species.

X. Conclusion Although considerable research has been conducted on S. mutans dextransucrase, many problems remain to be solved. Specific questions concerning mechanisms of dextran synthesis, role of surface receptors in cell adherence and aggregation, and distribution and regulation of dextransucrase, as well as the fundamental properties of the enzyme, remain to be answered. ACKNOWLEDGMENT

We thank Marie Ludwig for her assistance throughout the preparation of this manuscript. REFERENCES Barnett, W. (1973).J. Dent. Res. 52, 1160. Bourgeau, G., and McBride, B. C. (1976). Infect. Immun. 13, 1228-1234. Brathall, D. (1970). Odontol. Reoy. 21, 143-152. Bulkacz, J., and Hill, J. H. (1977).Arch. Oral Biol. 22, 119-123. Carlsson, J., and Elander, B. (1973). Caries Res. 7, 89-101. Chassy, B., Ereall, J. R., Bielawski, R., Porter, E. V., and Donkersloot, J. (1976). Infect. Zmmun. 14, 408-414. Chludzinski, A., Germaine, G., and Schachtele, C. F. (1974).J. Bacteriol. 118, 1-7. Chludzinski, A., Germaine, G. R., and Schachtele, C. F. (1976). J. Dent. Res. 55, 675-687. Ciardi, J. (1976).In “Immunologic Aspects of Dental Caries” (W. Bowen, R. Genco, and T. O’Brien, eds.), p. 101. Inforination Retrieval, Inc., Washington, D. C. Ciardi, J., Hagrage, G. J., Jr., and Wittenberger, C. L. (1976).J. Dent. Res. 55, (37x98. Coykendall, A. (1971). J. Bacteriol. 106, 192-196. Coykendall, A. (1977). Int. J . Syst. Bacteriol. 27, 2630. De Stoppelaar, J., Konig, K., Plasschaert, A,, and van der Hoeven, J. S. (1971). Arch. Oral B i d . 16, 971-975. Dunny, G. M., Birch, N . , Hascall, G., and Clewell, 0. B. (1973).]. Bacteriol. 114, 1362-1364. Ebert, K. H., and Schenk, G. (1968). Ado. Enzymol. 30, 179-219. Ebisu, S., and Misaki, A. (1974). Carbohydr. Res. 38, 374381. Ellis, D., and Miller, C. (1977). J . Dent. Res. 56, 57-69. Freedman, M. L., andTanzer, J. M. (1974). Infect. Immun. 10, 189-196. Fukui, K., Fukui, Y., and Moriyama, T. (1974). J. Bacteriol. 118, 796-804. Germaine, 6. R., and Schachtele, C. F. (1976). Infect. Immun. 13, 365472.

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Germaine, G . R., Chludzinski, A. M., and Schachtele, C. F. (1974).]. Bacteriol. 120,287-294. Germaine, G . R . , Harlander, S. K., Leung, W. S., and Schachtele, C. F. (1977). Infect. Immun. 16, 637-648. Gibbons, R. J. (1968). Caries Res. 2, 161-171. Gibbons, R. J., and Nygaard, M. (1968).Arch. Oral Biol. 13, 1249-1262. Greer, S. B., Hasiang, W., M u d , G., andZimmer, D. D. (1971).]. Dent. Res. 50, 1594-1640. Guggenheim, B. (1970). Helu. Odontol. Acta 14, 89-108. Guggenheim, B., and Newbrun, E. (1969).Helv. Odontol. Acta 13, 84-97. Guggenheim, B., and Schroder, H. E. (1967). Helv. Odontol. Acta 11, 113-152. Harlander, S. K., and Schachtele, C. F. (1978).Infect. Immun. 19, 450456. Higuchi, Mi., Araya, S., and Higuchi, Ma. (1976).]. Dent. Res. 55, 266-271. Higuchi, Mi., Araya, S., and Higuchi, Ma. (1977a). Infect. Immun. 15, 938-944. Higuchi, Mi., Rhee, G., Araya, S., and Higuchi, Ma. (1977b). Infect. Immun. 15, 945-949. Hojo, M., Higuchi, Ma., and Araya, S. (1976).J. Dent. Res. 55, 169. Janda, W. , and Kuramitsu, H. (1976). Infect. Immun. 14, 191-202. Janda, W., and Kuramitsu, H. (1977). Infect. Immun. 16, 575586. Johnson, M. C., Bozzola, J. J., and Shechmeister, I. L. (1974).J. Bacteriol. 118, 304311. Jordan, H. V., and Keyes, P. H. (1966).Arch. Oral B i d . 11, 793-801. Jordan, H . V . , Englander, H. R., Engler, W. O., and Kulczyk, S. (1972)./. Dent. Res. 51, 515-518. Keyes, P. H. (1960).Arch. Oral Biol. 1, 304320. Kobayashi, M., and Matsuda, K. (1974). Biochim. Biophys. Acta 370, 441449. Krasse, B., Edwardsson, S., Svensson, I., and Trell, L. (1967).Arch. Oral Biol. 12, 231-236. Kuramitsu, H. (1973).J. Bacteriol. 115, 1003-1010. Kuramitsu, H. (1974a).Infect. Immun. 9, 764-765. Kuramitsu, H. (1974b). Infect. Immun. 10, 227-235. Kuramitsu, H. (1975).Infect. Immun. 12, 738-749. Liljemark, W. F., and Schauer, S. U . (1977).]. Dent. Res. 56, 157-165. McCabe, M. M . , and Smith, E. E. (1973). Infect. Immun. 7, 829-838. McCabe, M. M., and Smith, E. E. (1975). Infect. Immun. 12, 512520. McCabe, M. M., and Smith, E. E. (1976). In “Immunologic Aspects of Dental Caries” (W. Bowen, R. Genco, and T. O’Brien, eds.), p. 111. Information Retrieval, Inc., Washington, D. C. McCabe, M. M., and Smith, E. E. (1977). Infect. Immun. 16, 760-765. Marciana, F. L., Reider, J. L., Virgili, S. L., and Kopecko, D. J. (1977). Infect. Immun. 17, 215-266. Montville, T. J., Cooney, C. L., and Sinskey, A. J. (1977a).J . Dent. Res. 56, 983-989. Montville, T. J . , Cooney, C. L., and Sinskey, A. J. (197713). Infect. Zmmun. 18, 629435. Mukasa, H., and Slade, H. (1973).Infect. Immun. 8, 555-562. Mukasa, H . , and Slade, H. (1974a).Infect. Immun. 9, 419429. Mukasa, H., and Slade, H. (1974b). Infect. Immun. 10, 1135-1145. Newbrun, E., Finzen, F., and Sharma, M. (1977). Caries Res. 11, 153-159. Newman, N. H . , Donohue, H. D., and Britton, A. B. (1976). Microbios 15, 113-125. Nisizawa, T., Imai, S . , and Araya, S. (1977).Arch. Oral Biol. 22, 281-285. Osborne, R. M., Lambert, B. L., Meyer, T. S., and Roush, A. H. (1976).I . Dent. Res. 55, 77-34, Pazur, J. H., and Aronson, N. N. (1972).Ado. Carbohydr. Chem. Biochem. 27, 301304. Pearce, E. (1976).Arch. Oral Biol. 21, 545549. Perch, B., Kjems, E., and Rawn, T. (1974).Acta Pathol. Microbiol. Scand. 82, 357370. Robrish, S., Reid, W., and Krichevsky, M. (1972). Appl. Microbiol. 24, 181-190.

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T . J. MONTVILLE, C. L. COONEY, AND A. J. SINSKEY

Robyt, J. F., and Corrigan, A. L. (1977). Arch. Biochem. Biophys. 183, 726-731. Robyt, J. F., Kimble, B. K., and Walseth, T. F. (1974).Arch. Biochem. Biophys. 165,634440. Russel, R. (1976). Microbios 2, 5 5 5 9 . Schachtele, C. F., Staat, R. H., and Harlander, S. (1975a). Infect. Zmmun. 12, 309-317. Schachtele, C. F., Germaine, G . , and Harlander, S. (1975b). Infect. Immun. 12, 934-937. Schein, C. H. (1976). M. S. Thesis, Massachusetts Institute of Technology, Cambridge. Sharma, M., Holbrook, S., and Newbrun, E. (1975).J. Dent. Res. 54,1.10. Shklair, I. L., and Keene, H. J. (1974). Arch. Oral Biol. 19, 1079-1081. Slade, H. D. (1976).In “Immunologic Aspects of Dental Caries” (W. Bowen, R. Genco, and T. O’Brien, eds.), p. 21. Information Retrieval, Inc., Washington, D. C. Smith, D., and Taubman, M. (1977). Infect. Immun. 5, 91-103. Spinell, I). M., and Gibbons, R. J. (1974). Infect. Immun. 10, 1448-1451. S t a t R., and Schachtele, C. F. (1974). Infect. Immun. 9, 467469. Staat, R., Gawronski, T., and Schachtele, C. F. (1973). Infect. Immun. 8, 1009-1016. Tanzer, J. M., and Johnson, M. C. (1976). Arch. Oral B i d . 21, 555-559. Tanzer, J. M., Chassy, B. M., and Krichevsky, M. I. (1972). Biochem. Biophys. Acta 261, 379387. Tanzer, J. M., Freedman, M. L., Fitzgerald, R. J., and Larson, R. H. (1974). Infect. Zmmun. 10, 197-203. Tanzer, J. M., Brown, A. T., McInerney, M. F., andwoodiel, F. M . (1977).Infect. Zmmun. 16, 318327. Tsumuraya, Y . , Nakamura, N . , and Kobayaski, T. (1976). Agric. Biol. Chem. 40, 1471-1477. Usui, T., Sugiyama, H., Seto, S., Araya, S., Nisizawa, T., Imai, S., and Kosaka, K. (1975).J. Biochem. {Tokyo) 78, 225-227. Walker, G. (1972).J . Dent. Res. 51, 409414. Wood, J. M., and Critchley, P. (1966). Arch. Oral Biol. 11, 1039-1042.

Microbiology of Activated Sludge Bulking

WESLEY0. PIPES Department of Biological Sciences and The Environmental Studies Institute, Drexel University, Philadelphia, Pennsylvania

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

................. A. Activated Sludge Process Operation . . . . . . . . . . . . . .

11. The Nature of the Bulking Problem.

B. The Relationship Between Bulking and Floc Formation . . 111. Filamentous Organisms in Activated Sludge. A. Filamentous Bacteria. ............................... B. Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Blue-Green Algae . . . . . . .......... D. Fungi ........................ IV. Case Studies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nonfilamentous Bulking ............................. B. Filamentous Bulking ................................ V. Summary and Future Prospects . . . . . . . . . . . References . . . . . . . . . . . . . . . . .......

85 86 86 93 95 95 113 113 114 115 115 117 123 125

1. Introduction In Volume 9 of this series this author reviewed the information available about bulking and pointed out that there were several other types of activated sludge separation problems which were frequently confused with bulking (Pipes, 1967a). This makes the literature on the subject difficult to interpret. Most cases of bulking are due to prolific growths of filamentous microorganisms which interfere with the compaction of the sludge, but nonfilamentous bulking is also sometimes observed. There had been no attempts to identify any of the filamentous organisms in activated sludge reported in the literature between 1940 and 1967, and it had become customary to call any filamentous microorganism seen in activated sludge “sphaerotilus” without any attempt at a specific identification. Over the last 10 years, more attention has been paid to defining the exact nature of the bulking phenomena and identlfying the microorganisms involved. The purpose of this article is to review these recent investigations (1967-1977) and summarize what is now known about the physical properties of bulking sludge, the microbiology of bulking, why bulking occurs, and how it sometimes can be controlled. It is only fair to the reader to state at this point that communication between microbiologists and sanitary engineers is poor and a general solution to the bulking problem is still a very long way off. 85 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12aO2624-4

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It. The Nature of the Bulking Problem There is a widespread misconception that bulking activated sludge settles so slowly that it does not separate in the final settling tanks and thus is carried out with the effluent. It is true that bulking sludge settles more slowly than normal sludge at the same suspended solids concentration but it still settles rapidly enough to be separated. The problem with bulking sludge is that it compacts poorly and the thickened sludge in underflow from the settling tank has a low solids concentration. It is expedient to start out with an explanation of how bulking interferes with the proper operation of an activated sludge process so that certain concepts and terms can be used without additional explanation in the rest of this article. Also there is complex relationship between bulking and the formation of activated sludge floc particles which needs to be explored. A. ACTIVATED SLUDGEPROCESSOPERATION

Figure 1 is a diagram of a conventional activated sludge process. The influent waste water contains soluble and particulate organic matter. Settleable solids have been removed (in most cases) by primary sedimentation so that the particulate matter is colloidal or at least the particles are so small that they do not settle out of the waste water in a few hours. The organisms in the aeration tank convert most of the organic matter into a settleable form. This conversion of the soluble organic matter is called absorption and adsorption and that of the particulate organic matter is called floc formation. The activated sludge organisms themselves can grow either in a dispersed or in a flocculated form. In the final settling tank the organic matter and organisms

" " '

RETURN

SLUDGE

WASTE

SLUDGE

w, su FIG. 1. Schematic diagram of a conventional activated sludge process.

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

87

which are aggregated into particles large enough to settle are separated and the effluent carries off the organic matter and organisms which are not flocculated. Most of the sludge is returned from the final settling tank to the aeration tank in order to maintain the population of microorganisms but some sludge must be removed continuously. The mixture of sludge and waste water in the aeration tank is called mixed liquor.

1. Process Variables The variables which are used to describe the system quantitatively are the rates of flow, the sizes of the aeration and settling tanks, the amounts of sludge, and the concentrations of organic matter. The amount of waste water to be treated is measured as the influent flow rate (Q in m3/day) and the strength of the waste water as the 5-day, 20°C biochemical oxygen demand (BOD, or Lo in mg/liter). The organic matter treated each day is expressed as QL, (gmlday) which is a misleading parameter because BOD, is a rate of oxygen consumption not an amount of organic matter. The use of BOD, as a measure of the total amount of organic matter treated makes sense only if one assumes that the rate of oxidation of organic matter is proportional to its concentration in the waste water (which all sanitary engineers do assume). The concentration of organic matter in the effluent is measured by the effluent BOD, (L, in mg/liter). The volume of the aeration tank is V (m3)and the aeration time is t = Q/V (days). The concentration of sludge in the aeration tank is called the mixed liquor suspended solids (MLSS or Su in mg/liter). The concentration of microorganisms in the mixed liquor is something less than the MLSS and is most often measured as the mixed liquor volatile suspended solids (MLVSS in mglliter) which is not particularly valid but is as good as anything else which has been suggested. If the ratio of MLVSS to MLSS isf, then a measure of the total amount of microorganisms in the aeration tank isfs,V (grams). The amount of organic matter removed from the waste water each day per unit weight of microorganisms in the aeration tank is called the sludge loading ratio or the food to microorganism ratio and is formulated as

The FIM ratio has units of per day and is a convenient parameter used for design and to compare different systems. Theoretically, the higher the FIM ratio the faster the activated sludge organisms must grow in order to assimilate the organic matter which is being separated from the waste water in the aeration tank. Another, and probably better, way to get a relative measure of the rate of growth of the activated sludge microorganisms is to calculate the sludge

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residence time (SRT), which is sometimes called the mean cell residence time. The SRT is defined as the total weight (kilograms) of sludge in the process divided by the rate of sludge wasting (kilograms per day) and has the units of per day. In most cases the weight of sludge in the settling tanks can be neglected and the SRT is the weight of sludge in the aeration tank divided by the rate of sludge wasting. The amount of new sludge formed each day is proportional to the BOD, reduction (Lo - L J . In order to keep the MLSS constant the rate of wasting has to be equal to the rate of formation of new sludge. Thus, for an activated sludge system treating a constant flow of a constant strength waste water at a constant MLSS, the SRT is proportional to the reciprocal of the FIM ratio. However, the only systems with constant waste water flow, constant waste water strength, and constant MLSS are laboratory systems. In treatment plants not only are there almost continuous variations in the variables that are used to calculate the FIM ratio and the SRT but also the weight of sludge in the settling tanks may not be negligible in comparison with the weight of sludge in the aeration tank. In a case of bulking there may be more sludge in the settling tank than in the aeration tank. Full-scale activated sludge systems are never operated at an equilibrium condition and the FIM ratio and SRT are related only by averages over many days of operation. The SRT theoretically is a better measure of sludge growth but the FIM ratio is a better measure of what is accomplished by the process (BOD, reduction). Both parameters can be used simultaneously to give useful information about the process, but the FIM ratio is more widely known and applied than the SRT. The operator of an activated sludge system has to use the aeration tank volume which the design engineer has provided and to treat whatever amount and strength of waste water comes in. The only control one has over the FIM of the process is one’s control of the MLSS. If the suspended solids in the influent and the growth of new solids in the aeration tank are ignored (which approximation is usually accurate to within 10%)the MLSS may be calculated from the return sludge rate (R in m 3/day)and the underflow solids concentration (S, in mdliter) from

s,

R R+Q’,

=-

If the operator knows the value of S, he or she can set R at some fraction of Q to maintain the MLSS at some desired value. Usually S, is three to five times greater than S, and R is 50-25% of Q; however, the range of variability of these ratios is much greater than that. The final settling tank is designed to provide an area adequate for separation of the sludge solids from the effluent. The parameters used for design

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

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are the overflow rate (Q/As in m/day) and the solids loading (SL in gmlm2/ day),

where A, is the effective area of the settling tank. All particles with a settling velocity greater than the overflow rate should settle out in the settling tank. The thickening of the sludge (how high a value of S, is obtained) is in some way a function of the solids loading. However, exactly how S, is related to SL has not been formulated in a manner useful to the plant operator (Schafher and Pipes, 1978)so the operator has to depend upon a laboratory settling test for the prediction of S,.

2 . The Settling Curve In the standard control test a sample of the mixed liquor is poured into a 1-liter graduated cylinder and allowed to settle. Usually the MLSS are high enough that a clear interface is formed between the settling sludge mass and the supernatent within a few minutes after the sample is placed in the cylinder and thereafter the volume occupied by the sludge can be easily read at any time. Typical curves that are obtained from samples of normal activated sludge (curve A) and samples of bulking sludge (curve B) are depicted in Fig. 2. There is a short period while the turbulence produced by pouring the sludge into the cylinder dissapates, followed by relatively rapid zone settling, followed in turn by a transition to a compaction phase. During zone settling the particles of sludge are moving downward together through the water as a mass. During compaction the particles rest on each other and water is squeezed out by the weight of the particles as they fit closer together. The volume occupied by the sludge after 30 min settling is 100 ml for curve A and 400 ml for curve B. The volume occupied by 1gm of sludge after 30 min settling is called the sludge volume index (SVI). If both sludges A and B started out at MLSS = 2000 mglliter, then the SVI of sludge A would be 50 ml/gm and that of sludge B would be 200 ml/gm. The SVI is used to define bulking. An SVI of less than 100 mllgm is considered to be normal, between 100 and 200 mllgm is considered to indicate moderate bulking, and of greater than 200 ml/gm is considered to be severe bulking. It is obvious that the name, bulking sludge, comes from the fact that the sludge is bulkier than a normal sludge; i.e., it occupies more volume per unit weight. Notice that the defination of bulking sludge depends neither upon the zone settling velocity nor upon the concentration of suspended solids in the effluent from the settling tank.

WESLEY 0.PIPES

I

I

5

10

15 20 TIME (rnin- 1

25

30

35

FIG. 2. Typical batch settling curves for activated sludge (curve A is for normal sludge; curve B is for bulking sludge).

A graduated cylinder may be calibrated in centimeters just as well as in milliliters. If the ordinate on Fig. 2 had been specified in centimeters the slopes of the second parts of the two curves would be the zone settling velocities in centimeters per minute. The zone settling velocity of any activated sludge is a function of the suspended solids concentration. As depicted in Fig. 2 the zone settling velocity of a bulking sludge at 2000 mg/liter will be considerably less than that of a normal sludge at 2000 mg/liter and this would be true for all solids concentrations. However, it is very, very unusual for a bulking sludge to have a zone settling velocity less than the overflow rate of the settling tank. Activated sludge settling tanks are designed with overflow rates of between 15 and 35 m/day which is roughly 1 to 2.5 cm/min. For a bulking sludge to have a settling velocity of less than 3 cm/min at the MLSS concentration, the return sludge rate would have to be over 300%of Q. This is just beyond reasonable design. Low values of S, keep the MLSS low enough so that the zone settling velocity of the mixed liquor is always less than the overflow rate of the settling tank. It would, of course, be possible to design a settling tank with a small area and a very large underflow rate so that this would not be true, but it would be a foolish thing for an engineer to do.

40

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

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3 . Bulking and Solids Lossfi-om the Settling Tank

Figure 3 is a schematic diagram of the settling tank. In general, there will be some suspended solids in the effluent and the mass balance of suspended solids into and out of the settling tank will be

dM dt

=

(Q

+ R)S, - (R + W)S,

- (Q - W)S,

where W is the waste sludge flow rate and S, is the effluent suspended solids concentration. Usually S, is less than 30 mglliter, S, is in the range of 10004000 mglliter, and S, is in the range of 2oOO-l6,0Oo mglliter, so the last term of the mass balance equation may be neglected. One important goal of operation of the settling tank is to keep dMldt from becoming positive, i.e., to avoid a solids buildup. If dMldt is positive for a day or two, the entire settling tank fills up with solids which then overflow into the effluent making S, very large. In order to avoid a solids buildup in the settling tank it is necessary to keep (R W)S, 3 (Q R)Sa. It is clear that the best way to do this is to obtain a high value of S, and then adjust R until it is just high enough to keep dMldt = 0. The critical question is how high can S, be? Notice that the SVI is a reciprocal solids concentration. An SVI of 100 ml/gm implies that a liter can hold 10 gm of sludge; an SVI of 200 mYgm implies that a liter can hold 5 gm of sludge; and so forth. A 1-liter cylinder is not a very good settling tank and 30 min is an arbitrary selected time, so it is usually possible to get a underflow solids concentration somewhat greater than that implied by the SVI. Using the SVI value to calculate the underflow solids concentration and then setting R to keep dMldt = 0 is a conservative procedure which assures that no sludge buildup occurs. Of course, there is an upper limit on R which is designed into the system with the selection of the capacities of return sludge pumps and piping. If the mixed liquor suspended solids are 2000 mg/liter and the underflow solids concentration is limited to 4000 mglliter (SVI = 250 ml/gm) then R must be almost equal to Q to prevent solids buildup in the settling tanks. There are some activated sludge systems designed so that R can be as large as Q but it is more usual to find R limited to about half of Q . In this case it is impossible to maintain a MLSS of 2000 mglliter with an SVI of 250 mllgm. A bulking sludge is one that compacts poorly; i.e., it does not give a high underflow solids concentration. The only time that bulking results in high effluent suspended solids is after the settling tank has filled up with solids to the extent that they overflow into the effluent. If there is excessive loss of

+

+

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FIG.3. Diagram ofan activated sludge settling tank with variables for making a mass balance on the suspended solids.

suspended solids in the effluent during a bulking episode, this is a sure sign of poor operation and/or inadequate design of the system. There are types of activated sludge which produce excessively high effluent suspended solids because some of the sludge does not settle at all (Pipes, 1969).These types include floc formation problems (pinpoint floc and deflocculation) and sludge density problems (floating sludge, rising sludge, and septic sludge). The confusion of these types of sludge with bulking sludge has greatly impeded the understanding of what bulking is and how certain types of microorganisms are involved in bulking. Searching for a solution to a sludge separation problem should always start with a determination of whether the cause of the excessively high effluent suspended solids is poor compaction or one of the other problems. This is not always an easy determination; it usually requires several days of good operating data in addition to the results from laboratory settling tests. Sometimes two or more types of sludge separation problems occur simultaneously, making the determination of the exact nature of the problem even more difficult. The role of filamentous organism in bulking is that they prevent the sludge from compacting very well. Activated sludge floc particles without filaments

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

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fit together and squeeze out the water from between them so that the compacted sludge has a high solids concentration. The floc particles with filaments do not fit together well because the filaments hold them apart; thus, there is a lot of room for water between the particles in the compacted sludge and the solids concentration is kept low. This is a mechanical effect. Any filaments which hold the particles apart keep the solids concentration of the compacted sludge low, so any filamentous organism which can grow in large numbers in the sludge can be expected to cause bulking.

BETWEENBULKINGAND FLOC FORMATION

B . THE RELATIONSHIP

There are usually some suspended solids in the effluent from an activated sludge process. These are particles which are so small that they have a settling velocity less than the overflow rate for the settling tank and larger particles with a density less than that of water. Floc formation is the process of small particles coming together to form large particles. A well-flocculated sludge is composed mostly of large particles and a poorly flocculated sludge mostly of very small particles. The mechanism of floc formation of activated sludge is very poorly understood. There are several different phenomena which may be involved but most of the recent work has centered on the nature and properties of the extracellular polymers produced by the bacteria which appear to have a major role in floc formation. Harris and Mitchell (1973) have reviewed the work on the role of polymers in the aggregation of microorganisms, including the work which has been done with waste water treatment systems. They point out, among other things, that there is no generally accepted method for measuring how well flocculated a microbial suspension is and the numerous methods which have been used give qualitative rather than quantitative evidence of the state of aggregation. They also point out that floc formation in an activated sludge system is a complex physiochemical process involving interactions among organic and inorganic materials, microbial cells, and soluble surface-active substances. It is not likely that this complex process will be easily unraveled, particularly since good measurement techniques have not yet been developed. Floc formation is definitely related to the settling properties of activated sludge. For the same density, larger particles will settle faster and for the same size, denser particles will settle faster. Individual bacterial cells or even aggregates of few bacterial cells do not settle rapidly enough to be separated from the effluent in the settling tank of an activated sludge process. When there is an appearance of enough small particles to make the effluent turbid this is usually called “deflocculation” because it looks as

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though the sludge particles are breaking up. There are also occasions when particles large enough to be visible but not heavy enough to settle are seen in the effluent from an activated sludge system. These are called “pinpoint floc.” Deflvcculation and pinpoint floc are phenomena quite distinct from bulking. Pinpoint floc never occurs simultaneously with bulking but bulking and deflocculation may occur together (Pipes, 197813). There is also a relationship between floc formation and compaction of the sludge. It stands to reason that, of the particles which settle, the dense ones should give a higher sludge solids concentration and the lighter ones should give a lower sludge solids concentration. Heukelekian and Weisberg (1956) described a case of nonfilamentous or “zoogloeal” bulking and ascribed the lower solids concentration in the settled sludge to bound water. Bisogni and Lawrence (1971) reported that in the absence of filamentous organisms the SVI of a laboratory activated sludge was an inverse function of the solids retention time (SRT). Forster (1971) reported that increases in the carbohydrate content of activated sludge was associated with increased in the surface charge on the floc particles and with increases in the SVI in the absence of filamentous organisms. Steiner et al. (1976) confirmed this work using samples from four other activated sludge systems. Magara et al. (1976) also reported that at higher F/M ratios an increase in the extracellular polysaccharide content of activated sludge was accompanied by an increase in particle size, a decrease in particle strength, and an increase in the SVI. Enough investigators have studied enough cases of nonfilamentous bulking to establish that such a phenomenon does occur and is distinct from filamentous bulking. There is, or at least has been, a school of thought which holds that bulking is caused by physiochemical changes in the nature of the floc particles and that the filamentous organisms are more or less irrelevant. Forster (1971) quotes Finstein and Heukelekian (1967)on the existance of this school of thought, which is somewhat ironic because the major conclusion of their paper is that the SVI in the four activated sludge systems which they have examined is directly proportional to the total length of filaments which can be measured microscopically. Sezgin et al. (1978) also presented data vn measurements of filaments in activated sludge and showed that the proportionality between SVI and total filament length held only for SVI values greater than 100 mVgm. The investigators of nonfilamentous bulking have not done any microbiological studies so there is no information on what microorganisms are involved. Those who have studied filamentous bulking have not made the physical measurements necessary to define the properties of the floc particles. Thus, it is not clear exactly what the relationship between filamentous organisms and floc formation is, although it is clear that bulking can occur in the absence of filamentous organisms due to changes in the size and density of the flvc particles.

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111. Filamentous Organisms in Activated Sludge There are four main groups of filamentous microorganisms which may cause bulking of activated sludge; namely, filamentous bacteria, actinomycetes, filamentous blue-green algae, and fungi. The actinomycetes are, of course, classified as bacteria but they are so easily differentiated from other filamentous bacteria by their branching hyphae that they are treated separately here. Some of the colorless blue-green algae are also classified as bacteria but observations of filaments in activated sludge include pigmented as well as colorless blue-green algae. The fungi are a distinctly different group but they only rarely cause bulking and little effort has been expended toward identifying genera of fungi in activated sludge.

A. FILAMENTOUS BACTERIA As previously noted, between 1940 and 1967 these were all identified as Sphaerotilus natans by cursory microscopic observation. There are now between 20 and 30 types of filamentous bacteria which have been observed in activated sludge. Some of them have been isolated in pure culture and partially characterized; however, many have not. The dependence upon microscopic observations to differentiate between the various filamentous organisms makes the situation quite canfusing and considerably more work is needed to straighten it out. 1 . Methods for Observations on Sludge Samples

All investigators have used phase-contrast microscopy of activated sludge samples as the starting point for identification of the filamentous bacteria. Observations to determine the presence or absence of a sheath, cell inclusions, and motility of filaments in a hanging drop preparation are widely used as are measurements of the cell dimensions and staining reactions of the filaments in dried smears. Both van Veen (1973) and Eikelboom (1975) recommend examination by electron microscopy as being essential for proper determination of cell dimensions and the presence or absence of a sheath. Farquhar and Boyle (1971a) published methods for examination of activated sludge samples for identification of the filamentous organisms in situ. They used a lysozymedetergent test (Romano and Peloquin, 1963)to demonstrate the presence of a sheath, an iron oxidation test, a manganese oxidation test (Mulder and van Veen, 1963),a lipid staining test (Roulf and Stokes, 1961),a sulfur deposition test (Skerman et a l . , 1957), and a series of stains (gram stain, acid fast stain, spore stain, and a methylene blue simple stain) to characterize the organisms. Most of their tests were originally developed to demonstrate characteristics of Sphaerotilus and Farquhar and Boyle used

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them mainly to demonstrate that some of the filamentous organisms were not Sphaerotilus. This approach seems to have definite promise for practical applications.

2 . Isolation Techniques The isolation of any of the filamentous bacteria from activated sludge is difficult. All of them grow slowly and several of them have not been grown at all on solid media. Many of them have slimy surfaces to which other bacteria adhere. Attempted isolations on rich media usually fail because the filamentous bacteria do not grow or they do not grow as filaments or they are rapidly overgrown by single-celled bacteria. The media used for isolation of filamentous bacteria from activated sludge are low in organic content. This author (Pipes, 1967b)had the most success with an agar medium containing 0.5 gm glucose, 0.1 gm peptone, and 0.1 gm yeast extract per liter (GPY agar). Farquhar and Boyle (1971b) used the casitone-glycerol-yeast extract agar (CGY agar) which Dondero et al. (1961) had developed for isolation of SphaerotiZus, trypticase soy agar (TCS agar), glucose-trypticase soy agar (GT agar), and glucose-trypticase soy -azide agar (GTA agar). Van Veen (1973) used a trypticase soy-casitone-yeast extractnutrient broth agar (D medium), a glucose-ammonium-salts agar with added vitamin BIZand thiamine (I medium), and a sucrose-casitone-yeast extracttrypticase soy agar with added vitamin BIZ and thiamine (SCY medium). Eikelboom (1975) used van Veen’s I medium and SCY medium plus a glucose-lactose-casamino acids-peptone-yeast extract agar with added vitamins (C medium) and media prepared by hydrolyzing activated sludge in sodium hydroxide. There is really not much difference among these media (except perhaps the hydrolyzed activated sludge) and it is possible that some of the other filamentous bacteria can be isolated from activated sludge if some imagination is used in devising other media to try. Farquhar and Boyle (1971a), van Veen (1973), and Eikelboom (1975) all use some method of gently washing and diluting activated sludge in sterile water and then spreading it over the surface of agar plates. This author (Pipes, 1967b) had better luck with heavy streaks of the undiluted sludge from which the filaments would grow outward. The agar plates are incubated at about 20°C at high humidity to keep the surface of the agar moist. The plates are examined microscopically at frequent intervals to detect growth of the filamentous bacteria. It often requires several weeks for the filamentous bacteria to form colonies large enough for transfers to be attempted. Protozoa reproducing on the surface of the plates and feeding on the bacteria are often a problem, as are fungi which overgrow the bacterial colonies and prevent isolation. It is also possible that additional success in isolation of some of the other filamentous organisms might be gained by trying other conditions of incubation (e.g., lowered O2 tension).

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3. Organisms Described To facilitate comparisons among the results reported by various investigators this author has prepared tables for each of the papers. The column headings of the tables are the characteristics used by Farquhar and Boyle (1971b)because they had the best organization and the greatest amount of information, both negative and positive. The tables presenting the results of other investigators may appear to be skimpy in some cases simply because they did not report negative information. Hunerberg et al. (1970) were attempting to identlfy filamentous bacteria associated with specific cases of bulking in the Berlin waste water treatment plant. They found six different types of filamentous bacteria (Table I) solely on the basis of microscopic observation of sludge samples and did not attempt isolations. Farquhar and Boyle (1971b) made a survey of 11 activated sludge systems (nine waste water treatment plants, one laboratory system, and one experimental system). They reported 15 different types of filamentous bacteria plus actinomycetes and fungi (Table 11). They showed a tendency to lump different types into few genera, identfiing two types of Sphaerotilus, three types of Thiothrix, and four types of Vitreoscilla. They isolated only the two types of Sphaerotilus, Beggiatoa, and the lactic acid bacteria. Van Veen (1973) examined samples of activated sludge in an attempt to isolate and identlfy the filaments from bulking sludge but gave no information about the number and source of the sludge samples. The descriptions he gave were limited to the organisms which he was able to isolate except for the species of Sphaerotilus which oxidized manganese slowly (Table 111). Eikelboom (1975) examined approximately lo00 different samples from about 200 waste water treatment plants. Most of the samples were bulking sludge. H e published descriptions of 24 types of filamentous bacteria (Table IV) and mentioned that he also found blue-green algae, actinomycetes, and fungi; he was able to isolate only 12 of he types which he described, however.

In addition to these four papers on attempts to differentiate the several types of filamentous bacteria in activated sludge, there are a few reports of a more casual nature. Pasveer (1969) described a case of bulking which he ascribed to an overgrowth of the sludge by a filamentous growth form of Escherichia coli. Cyrus and Sladka (1970) mentioned that in addition to Sphaerotilus and Beggiutoa they had seen a number of other filamentous bacteria in activated sludge including Lineola longa, Leucothrix, Pelonema subtilissimum, Peloplaca, members of the family Vitreoscillaceae, Spirulina albida, and Achronema. Benedict and Carlson (1971) undertook a comparison of the major genera of aerobic heterotrophic bacteria in an activated sludge system. Although they do not comment on which organisms are

TABLE I FILAMENTOUS BACTERIA OBSERVED BY HUNERBERC et (11. (1970) Designation

Trichome

Sheath

False dichotomons branching

Present

Type A

Unbranched

Rarely visible

TypeB

Unbranched, tapered

Absent

Sphaerotilus

Motility

0.5-1.0 p m wide, 1 pm long

Refractile granules, four per cell

Gram negative

Bacterial cells adhere to filaments

Ca. 2 pm wide, at least twice as long as wide; basal cells wider than apical cells

Often numerous motile granules

Gram negative

1.8-2 pm wide; shorter than wide

Unbranched

>1 pm i f S granules present; <1 p m othenvise

Free cells very motile

Other Growth increased by glucose and lactate

clear constrictions on cell walls

Not visible

Physiology Gram negative, slime layer stains with methylviolet

Unbranched or occasional false branches, slightly bent

Long coiled filaments

Intracellular deposits Refractile granules

3 p m wide, 5-6 pm long;

(D W

Thiothrix

Cells

1 p m wide, up to 20 pm long, free cells 2 p m long

Gram negative

S granules formed in 15 min in presence of H,S and air

S granules

sometimes recognizable

Gram negative, slime layer stainswith

methylviolet

Growth of free cells stimulated by glucose

TABLE I1 FILAMENTOUSBACTERIA DESCRIBEDBY FARQUHAR AND BOYLE(1971b)

Designation

Trichome

Sheath

sphaerotilus

500 pm, Ezse branching rare

Present, stained deeply by crystal violet

Trichomes sessile, cells motile

S phaerotilus SP. Form I1

loo0 pm, false branching common

Present, stained deeply by crystal violet

Toxothrix sp.

200 pm,

SP. Form I

Motility

Cells

Intracellular deposits

Physiology

Other

Rods, 1 pm Lipids; does diam., 3-5 fim not deposit S long

Gram negative, Fe deposited, Mn not deposited

Debris attached to sheath

Trichomes sessile, motile cells not observed

Rods, 1 . 5 p m diam., up to 5 pm long

Lipids, does not deposit S

Gram negative, Fe deposited, Mn not deposited

Debris attached to sheath

Present but not easily visible

Trichomes sessile

Cell walls not visible by phase contrast; length up to 5 pm

None observed

Gram negative, slight Fe deposition, No Mn deposition

Germlings are spherical bodies

W

(D

unbranched

Unidentified Form I1

%pm, rigid, unbranched

Septate residue after lysozyme treatment

Trichomes not motile

1 pm diam., 5-10 pm long; cylindrical to barrel shaped

None observed

Gram negative, Fe deposition questionable

Unidentified Form I

500 pm, unbranched

None

Trichomes not motile

Discoid 0.5-1 pm diam., 2 pm long

No lipids, no S deposited

Gram negative

Metachromatic granule in center of cell

continued

TABLE I1 (continued) FILAMENTOUS BACTERIA DESCRIBED BY FARQUHAR AND BOYLE(1971b)

Designation

Trichome

Thiothrix sp. Form I

100 p m , unbranched, rigid, segmented, tapered

None

lo00 pm, unbranched, rigid, segmented, tapered

Sheath, present, stained poorly

Trichomes sway

Thiothrix sp. Form 111

500 pm, unbranched

None

Trichomes sway

Vitreoscilla Form I

100 pm, occasional false branch, curved or coiled

None

Trichomes moved jerkily

Thiothrix sp. Form I1

Sheath

Motility Trichomes sway jerkily

Cells

In tracellular deposits

Physiology

Other

Lipids, S rapidly deposited

Gram negative, no Fe deposited

Debris on on trichome

S rapidly deposited, lipids uncertain

Gram negative, no Fe deposited

Rosettes formed

Rods 0.5-1.0 pm diam., 2 5 p m long

S rapidly deposited, no lipid deposits seen

Gram negative, no Fe deposited

Coccoid to rod shaped,

Lipids, no S deposits

Gram negative, no Fe deposited

Base cells

2 4 pm diam., 0.5-2 pm long; tip cells 1-2 pm diam., 5-10 pm long Base cells

2-3 p m diam., 10 p m long; tip cells 1-2 pm diam., 2 pm long

1-2 p m diam., 1-2 p m long

TABLE I1 (continued) FILAMENTOUS BACTERIA DESCRIBED BY FARQUHAR AND BOYLE(1971b)

Sheath

Trichome

Vitreoscillu Form I1

200 pm, unbranched, straight, flexible

None

Trichomes moved slowly by gliding

Barrel shaped 1-2 p m diam., 2 4 p m long

Lipids, no S deposited

Gram negative, no Fe deposited

Vitreoscillu Form I11

100 p m , unbranched, straight, flexible

None

Trichomes moved slowly by gliding

Cylindrical, 1 p m diam., 5-10 p m long

Lipids, no S deposition

Gram negative, no Fe deposited

Vitreoscillu Form IV

Several 100 pm. unbranched

None

Trichomes slightly motile

Coccoid rods, 1-2 p m diam., 2 5 p m long

Lipids, no S deposited

Gram negative, no Fe deposited

Beggiatoa

300 pm, unbranched flexible

None

Trichomes glide

2 p m diam., 10 pm long

Lipids and S

Gram negative, no Fe deposited

Mimoscillu

50 p m ,

None

Very active whiplike motility

Septation not visible; 0.5pm diam.

None

Gram negative, no Fe deposited

None, lysozyme treatment had no effect

Trichomes not motile

Rods, 0.5 p m diam., 1.0 p m long

Lipids, no S deposited

Gram positive or variable, no Fe deposited

unbranched, extremely flexible Lactic acid bacteria

500 p m , unbranched, rigid but curled and entwined

Motility

Cells

Intracellular deposits

Designation

Physiology

Other

Metachromatic granules at 5-pm spacing continued

TABLE I11 BY FILAMENTOUS BACTERIA ISOLATED

Designation Group I

c N 0

Trichome

Sheath

Motility

VAN

VEEN(1973)

Cells

Intracellular deposits

Present

Physiology Gram negative

Sphaerotilus natans

Cells flagellated

1.2 pm diam., subpolar1y flagellate '

Do not deposit Mn

Sphaerotilus SP.

Individual cells motile

0.6-1.2 pm diam.

Deposit Mn slowly

0.6-1.4 pm diam .

Deposit Mn rapidly

0.35-0.45 Small elecpm diam., tron-dense 3.5 pm long; globules no flagella

Gram negative, no Fe or Mn deposition

S p haerotilus discophorus

Hvaline, hardly visible, 0.5-0.8 pm wide

Haliscomenobacter hydrossis (van Veen et al.

1973) Group I1 resembles Flavobacterium

Not motile

Nonmotile

lo00 pm

Other

Carotenoid pigments

Gram negative

0.71-1.1 pm diam., 3-10 p m long

Carotenoid pigments

TABLE 111 (continued) FILAMENTOUS BACTERIAISOLATEDBY VAN VEEN(1973)

Designation

Trichome

Motility

Cells

Intracellular deposits

0.6-1.0 pm diam., 2-5 pm long

shorter than group I1 organisms

Group IV

Chains of coccoid cells

Nostocoida limicola

600 to 900 p m , occasional branch

Physiology

Other

Gram negative

Gliding

Group I11 resembles Flexibacter and Mirroscilla

Sheath

May have carotenoid pigments Gram positive

Nonmotile Coccoid to rod shaped, 0.7-1.5 p m diam., 0.30.6 p m long

None observed

0.34.7pm diam., 0.71.5 pm long

Electrondense granules

No pigments

Group V Microthrix paroicella

Irregular winding

None

Gram positive

Autolyzed cells can be mistaken for sheath

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filamentous, they report finding Bacillus, Breuibacterium, C ytophaga, Flauobacterium, Hyphomicrobacterium, and Sphaerotilus, all of which have filamentous species.

4 . Comparisons among Reports Despite the large variety of generic and specific names and unidentified types used by the several investigators, it would seem likely that they were all finding more or less the same organisms. Table V is an attempt to find correspondence among the descriptions given in the various reports. All of the filamentous organisms reported by van Veen fit nicely into descriptions given by Eikelboom. They both mention that the organism which they identlfy as Microthrix parvicella is the one which Pasveer (1969) called a filamentous form of E. coli. Since they both have had the opportunity to go back to the source used by Pasveer it is quite likely that they are correct. The placing of the six types described by Hunerberg et al. (1970) in Table V is what Eikelboom (1975) has suggested. This placing should be accepted because Eikelboom had a personal communication with one of the investigators who did the earlier work. There is, however, one exception to this placing which this author suggests. The photomicrograph of type B in the paper by Hunerberg et al. shows the formation of enlarged spherical bodies on the filaments. Eikelboom’s description of type 0961 does not mention this nor do any of his other descriptions. This author has observed this phenomenon in several different sludge samples and it is so striking that it is hard to believe that it can have been overlooked. The spherical enlargements occur on the filaments after they have been under anaerobic conditions for an hour or two. Upon resumption of oxygen supply the filaments break at the points of the spherical enlargements. If the sludge is fed peptone and aerated, this filamentous bacterium can be propagated indefinitely in mixed culture and retains the characteristic of forming the spherical enlargements under anaerobic conditions. However, this author has not obtained a pure culture and the taxonomic value of this characteristic remains questionable. Eikelboom (1975) quarreled with most of the identifications made by Cyrus and Sladka (1970). He equates their Leucothrix cohaerans with his type 021N but rejects their name because Leucothrix is halophylic and this organism clearly is not. He places their P . subtilissimum and Peloploca in his group I (ensheathed, gram-negative filaments) but notes that they do not provide enough descriptive information to permit a clear comparison with his types. He also placed the organism that they called Achronemu in his group VI. This author believes that this placing is a result of his misreading their paper. They write about seeing members of the Vitreoscillaceae in activated sludge in the same paragraph in which they mention also finding Achronema and Spirulina alba. It seems to this author that their descriptions

TABLE IV FILAMENTOUS B A C T E%PORTED ~

Designation

Trichome

Group I

Sheath

Motility

BY

EIKELBOOM (1975)

Cells

Intracellular deposits

Physiology

Other

Gram negative

Present

1 Sphaerotilus natans

lo00 pm, false branching frequent

Cylindrical, 1 . 2 3 . 0 pm diam., 1.55.0 pm long

PHB granules

Bacterial cells on filaments

2 Type 1701

200 pm, hlse branching incidental

Cylindrical, 0.5-0.9 pm wide, 2.53.5 p m long

PHB granules

Some deposit Bacterial Mn slowly cells on filaments

3 Type 1702

150 pm, unbranched

0.4-0.5 pm diam., 2.44.0 pm long

No PHB granules seen

4 Haliscomenobacter hydrossis

Small, straight, unbranched

0.2-0.35 p m wide, 2.54.0 pm long

Electrondense granules, no PHB

5 Type 0321

Small, straight

Clearly visible by phasecontrast microscopy

Fibrillar sheath

0.3 pm diam.

H. hydrossis could not be isolated when this was present continued

TABLE IV (continued) REPORTED BY EIXELBOOM (1975) FILAMENTOUS BACTERIA Designation

Trichome

Sheath

Motility

Cells

Intracellular deposits

Physiology

Other

Gram positive

Group I1

Present

6 Type 0041

Several 100 Pm, occasional false branching

End cells not enclosed

7 Type 0675

Resembles type 0041

0.6-0.8 pm wide, 1.54.0 p m long

8 Type 1851

300 pm, slightly bent

Cylindrical, 0.5-0.7 p m wide, 1.73.5 p m long

Group 111

Curled

9 Type 021N

Several mm, tapered, slime layer present

Hormogonia formed

Barrel shaped, 1.01.4p m wide, 0.7-2.3 p m long

Bacterial cells adhere to filament Resemble bluegreen algae

None Basal cells discoid, 1.82.9 pm wide, 0.4-0.7 pm long, apical cells cylindrical, 0.60.8 p m wide, 2.03.0 pm long

Volutin granules, S not normally present

Gram variable

Necroid cells present

TABLE IV (continued) FILAMENTOUS BACTERIA REPORTED BY EIKELBOOM (1975)

Designation

Trichome

Sheath

Motility

Cells

lntracellular deposits

Ovoid, 0.71.5 p m diam., 0.30.6 p m long

Physiology

Other

Gram positive

10 Nostocoida limicola

200 pm, occasional branch

Group IV

Slender, coiled, unbranched

12 Microthrix parvicelh

500 pm, thin, strongly coiled

Cylindrical, 0.3-0.6 pm diam., 0.71.8 pm long

Gram positive

13 Type 0581

300 pm, slender, coiled

0.3 p m wide, 0.7-1.6 pm long

Gram negative

14 Type 0192

100 pm, thin, slightly bent

0.154.25 p m wide, 1.5-3 p m long

Volutin probably present

None

Metachromatic granules, electrondense granules

Vohtin

continued

TABLE IV (continued) BACTERIAREPORTED BY EIKELBOOM (1975) FILAMENTOUS Designation Group V

Trichome Straight, unbranched

Sheath

Motility

Cells

Cylindrical, 0 . 6 4 . 8 pm wide, 0.93.0 p m long

16 Type 1091

0.6 p m diam., 0.71.6 p m long

17 Type 0092

Slime layer

0.4-0.6 p m diam., 1.42.0 pm long

18 Type 0961

Several 100 pm, straight or smooth, coiled

Cylindrical, 1.1-1.5 p m diam., 0.93.0 pm long

19 Type 0914

None Slightly bent

Physiology

Other

Gram negative

None

15 Type 0803

Group VI

Intracellular deposits

Electrondense areas at septa

Volutin

Intercalary regions suggest sheath Trichomes surrounded by slime layer

Polyphosphate distributed throughout cells

Gliding Slight gliding 0.8 p m wide, S deposits present 0.6-1.0 p m movement long

Gram positive

TABLE IV (continued) REPORTED BY EIKELBOOM (1975) FILAMENTOUS BACTERIA

Designation

Trichome

Sheath

Motility

20 Beggiatoa spp.

0.8 pm diam. or 1.9 prn diam.

Distinct gliding movement

21 Type 1111

100 p m , septated, bent

Slight but distinct gliding

Flexibacter 22 Type 1501 Flexibacter or Microscilla

SO pm, small, extremely flexible

Cells

0.54.8 p m

diam .

Intracellular deposits

Physiology

S deposits present

Gram negative

No S deposits present

Gram negative

0.5 p m diam.

Gram negative

Group VII 23 Type 1863

Short chains of cells

None

Coccoid 0.8 prn wide, 0.5-1.0 p m long

Gram negative

24 Type 0411

Short, strongly coiled

None

0.5-0.7 pm wide, 1.54.7 p m long

Gram negative

Flexibacter

Other

Electrondense granules

TABLE V POSSIBLE CORRESPONDENCE BETWEENTHE VARIOUSREPORTS Eikelboom (1975) Group I 1. Sphaerotilus natans 2. Type 1701 3. Type 1702 4. Haliscomenobacter hydrossis 5. Type 0321

van Veen (1973)

Group I Sphaerotilus natans Sphaerotilus sp. Sphaerotilus discophorus Haliscoinenobacter hy drossis

Farquhar and Boyle (1971b)

Sphaerotilus form I1 Sphaerotilus form I

Hunerberg et al. (1970)

Sphaerotilus Type A

Toxothrix sp.

-

Unidentified form I1

-

-

Thiothrix form I

Group I1 6. Type 0041 7. Type 0675 8. Type 1851 Group I11 9. Type 021N 10. Nostocoida limicola Group IV 12. Microthrix parwicellu 13. Type 0581 14. Type 0192 Group V 15. Type 0803 16. Type 1091 17. Type 0092

Group IV Nostocoida limicola

Group V Miwothrix paroicellu

-

-

Thiothrix

-

TABLE V (continued) POSSIBLECORRESPONDENCE BETWEENTHE VARIOUS REPORTS Eikelboom (1975)

van Veen (1973)

Farquhar and Boyle (1971b)

18. Type 0961

-

Group VI 19. Type 0914 20. Begiutoa sp. 21. Type 1111 Flexibacter 22. Type 1501 Microscilla or Flexibacter

Group I11 Flexibacter Group 111 Microscilla or Flexibacter

Group VII 23. Type 1863 24. Type 0411 Flavobacterium

Group I1 Flavobacterium

Beggiatoa Vitreoscilla Types I, 11, 111, IV Microscilla

-

-

Thiothrix form 11 Thiothirx form 111 Lactic acid bacteria Unidentified form I

Hunerberg et al. (1970)

Type B (?)

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of organisms which fit his group VI refer to Vitreoscillaceae and that they imply that Achronema and S. alba are something different. Cyrus and Sladka did not give enough information about the organisms which they called Pelonema, Peloploca, Achronema, and Spirulina to support any conclusions about what they might have seen or how they might correspond to what Eikelboom described. The biggest problem is reconciling the descriptions of Farquhar and Boyle (1971b) with those of Eikelboom (1975). Eikelboom suggested that their Sphuerotilus form I and form I1 and Toxothrix corresponded to organisms in his group I as shown in Table V. This seems reasonable and their unidentified form I also appears to be a gram-negative, ensheathed organism which fits into this group somewhere, although it does not correspond with any of Eikelboom’s descriptions. Eikelboom states that Farquhar and Boyle’s Thiothrix form I and form I1 are obviously identical with his type 021N but rejects the name Thiothrix. It seems to this author that their descriptions are adequate to accept them as separate forms and that they have a reasonable basis for calling at least form I Thiothrix. Both Eikelboom and van Veen seem to have rejected the idea of using the name Thiothrix for any activated sludge organism but neither reports having tried Farquhar’s and Boyle’s H2Soxidation test. Farquhar and Boyle clearly have demonstrated that the organisms they call Thiothrix are different from Sphuerotilus because the organisms they call Sphaerotilus do not deposit S in their cells. Their Thiothrix form I1 could well be the or ganism which led Skerman et al. (1957) to report that Sphaerotilus did oxidize H2S. Their Thiothrix form I11 also appears to be an organism different from anything described by van Veen or Eikelboom. Eikelboom (1975)states that Farquhar’s and Boyle’s lactic acid bacteria are obviously identical with M . parvicella. However, Farquhar and Boyle obtained some pure cultures of these organisms and called them lactic acid bacteria because they produced large amounts of acid and lowered the pH of liquid media rapidly. Eikelboom was unable to obtain a similar reaction with his M. parvicella. This author also has isolated some of these organisms from the BrooHeld, Wisconsin, activated sludge plant. They do not form long filaments in pure culture and they do lower the pH of liquid media rapidly. These organisms develop much more rapidly and develop much larger colonies than what van Veen (1973) has described for M. parvicella. Dondero (1975) expressed his opinion that the filamentous organism which Smit (1934a,b) isolated from activated sludge and called Sphaerotilus paludosus was a heterofermentative lactic acid bacterium, so there might be a precident. Farquhar’s and Boyle’s Vitreoscilla forms I, 11, 111, and IV fit somewhere in Eikelboom’s group VI but it is not clear exactly where. The organisms in

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

113

this group, other than Beggiatoa, need a lot of work and much more information is needed before any definite identifications can be accepted. Farquhar’s and Boyle’s unidentified form I is somewhat reminiscent of Eikelboom’stype 021N and Cyrus and Sladka’sL. cohaerens but there are some differences in the morphological descriptions. It is clear that there are many different types of filamentousbacteria which can be seen in activated sludge. It is also clear that much work remains before all of these organisms can be properly characterized and identified. There is no assurance that all of the filamentous bacteria which have been observed in activated sludge are bulking organisms nor that all of the bulking organisms have been reported in the literature even in vague descriptive terms.

B. ACTINOMYCETES The actinomycetes are separated from the other filamentous bacteria because they are a well-defined, distinct group and because this author is now of the opinion that they are not bulking organisms. They have been suggested as bulking organisms by this author (Pipes, 1967a, 1969) and by Carter and McKinney (1973). They probably are always present in activated sludge at least in small numbers. When present in large numbers they do cause some increase in the sludge volume index which is the usual measure of bulking. However, they grow as small, compact colonies of highly branched filaments which are not really long enough to hold floc particles apart and they do not cause the SVI to rise above 200 ml/gm. The problem which they cause is a persistant, viscous, brown scum which floats instead of settling (Pipes, 1978a).The increase in the SVI due to the excessive numbers of actinomycetes in the sludge is not related to the loss of the sludge solids in the effluent. The increased effluent solids is the result of the floating scum. Lechevalier (1975) did a survey of actinomycetes in activated sludge systems. He found the scum problem to be very common in municipal waste water treatment plants. All of his isolates were identified as members of the genus Nocardia and the most common isolate was described as a new species, N. amarae (Lechevalierand Lechevalier, 1974). Farquhar and Boyle (1971b)and Eikelboom (1975) also called their isolates Nocurdia but did not study them in any detail.

c. BLUE-GREENALGAE Agersborg and Hatfield (1929), Viehl et al. (1%3), and Singh and Saxena (1969) all have reported cases of activated sludge bulking caused by an overgrowth of pigmented (and thus presumably photosynthetic) blue-green

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algae. Eikelboom (1975) reported that some blue-green algae filaments were present in most of the sludge samples which he examined and that in one sample of bulking activated sludge the dominant filamentous organisms was a blue-green resembling Lyngbya . Apparently bulking caused by filamentous blue-green algae is relatively rare. It can be a very serious problem when it develops. The Southerly Wastewater Treatment Plant in Columbus, Ohio, has had an extremely difficult bulking problem during 1976 and 1977 (Sykes, 1977). The dominant filamentous organism in the activated sludge was originally identified as Phormidium and later as Schizothrix; however, no practical solution for the bulking problem has been developed.

D. FUNGI Both Farquhar and Boyle (1971b) and Eikelboom (1975) mentioned observing fungi in the activated sludge samples which they examined but neither found a case of fungus bulking. Cooke and Pipes (1970) reported a survey of the fungi which were isolated from 64 sets of samples from 19 different waste water treatment plants. The most commonly isolated fungi were the yeastlike organisms Trichosporon, Geotrichum, Candida, and Rhodotorula but a large variety of filamentous, imperfect fungi were also found. Although many of the samples were from plants with bulking sludge only one sample showed fungus bulking. This was a species of Cephalosporium growing in a municipal waste water made acid (PH < 4.0) by an industrial discharge and the sludge was deflocculated as well as bulking. Nash et al. (1977)reported that fungus growths proliferated in both oxygen and air activated sludge systems at the Newtown Creek Plant in New York City during the winters of 1972-1973, 1973-1974, and 1974-1975 and interfered with the proper operation of the system. They mention that the dominant filamentous form during the winter of 1974-1975 was Geotrichum. Although not mentioned in the article, this author was responsible for the fungus identifications. Also present in large numbers were Trichosporon, Oididendron, and Candida, as well as a variety of strictly filamentous fungi. Geotrichum, Trichosporon, Oididendron, and Candida can grow as single cells and as filaments. The fungi in the activated sludge were also found in large numbers in the influent waste water so it was clear that there was a continuing heavy innoculation of the activated sludge with spores of these organisms. The prolific fungus growths caused moderately high SVIs and prevented the attainment of the MLSS concentrations which were desired for the oxygen activated sludge system. However, the effluent BOD and suspended solids concentrations were held to reasonable levels in spite of the problems of maintaining the desired MLSS concentrations.

MICROBIOLOGY OF ACTIVATED SLUDGE BULKING

115

IV. Case Studies Surveys of many different activated sludge systems for identification of the filamentous organisms present are helphl in providing information about what organisms need to be studied. However, just because a filamentous microorganism is isolated from activated sludge does not prove that it can cause bulking. It needs to be shown to be the predominant filamentous organism in a bulking sludge and the reason for its predominance in that sludge needs to be elucidated. Isolations from activated sludge are quite likely to result in finding organisms which have not yet been described. Even when an organism which has been described previously is isolated, it is quite difficult to determine exactly which one it is because the literature descriptions are so sketchy and imprecise. Before microbiologists can make real contributions to practical solutions to bulking problems it is necessary to get beyond the stage of arguing about what to call the filamentous organisms and into the stage of providing physiological and ecological information about them. The engineer’s approach to solving a bulking problem is to skip the microscopic part and try to correlate the SVI with some operational parameter, such as FIM ratio or oxygen supply, or some waste water characteristic, such as the presence of carbohydrates, H2S, pH, or low nitrogen content. This approach occasionally does result in the solving of a particular bulking problem but there is never any clear evidence as to exactly why the SVI has decreased and the solutions found are, in general, not applicable to other bulking problems. What is needed at the present time is a number of detailed case studies of individual bulking problems. These case studies should include isolation and identification of the filamentous organisms and studies of their physiology as well as a thorough examination of the waste water characteristics and the parameters of the activated sludge system. Only in this way can it be possible to determine exactly why those particular organisms grow so prolifictly in the sludge and how to eliminate them. There have been few such studies but there is some information in the literature about the physiology of some of the bulking organisms which may give a clue as to why they may predominate in activated sludge. This material is reviewed below. It is quite incomplete but this attempt at organizing the available information may make apparent exactly what information is missing. A. NONFILAMENTOUS BULKING

Nonfilamentous bulking has been reported to be the result of an increase in the bound water content of the floc particles (Heukelekian and Weisberg, 1956) and a decrease in the particle density (Magara et al., 1976) which are

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apparently two different ways of measuring the same thing. Bisogni and Lawrence (1971), using a laboratory activated sludge system treating a synthetic waste water which had glucose as its main organic substrate, showed that SVI was inversely proportional to the solids retention time in the absence of filamentous organisms. In their system moderate bulking occurred at an FIM ratio of approximately 0.5 per day (SRT = 8 days) and severe bulking occurred at an FIM ratio of approximately 0.6 per day (SRT = 4 days). At FIM ratios above 1.6 per day (SRT = 1 day) the sludge deflocculated. Using the same type of laboratory system, Magara et al. (1976) found SVI to be an approximately linear function of FIM ratio at values &om 0.25 per day to 1.0 per day and that bulking occurred at an FIM ratio of approximately 0.75 per day. Nonfilamentous bulking does occur in municipal treatment plants (Forster, 1971; Steiner et al., 1976) in which the carbohydrate content of the waste water may be presumed to be low. This needs to be confirmed by additional studies, but it appears that increasing the FIM ratio above approximately 0.3 per day for an activated sludge system causes an increase in the SVI which is independent of the growth of any filamentous organisms in the sludge and when the FIM ratio gets too high the sludge deflocculates. It was known long ago that a higher organic loading (FIM ratio) resulted in a poorer quality effluent (Fair and Thomas, 1950). It has never been established unambiguously whether the poorer quality effluent is due to lack of removal of the organic matter of the influent or to the loss of activated sludge solids due to deflocculation or bulking or both. The study by Bisogni and Lawrence (1971)strongly suggests that the deterioration in effluent quality at higher FIM ratios is due to nonfilamentous bulking and deflocculation but their study has been carried out using a laboratory system which does not simulate all of the factors of a full-scale activated sludge system. In recent years, most of the work on using activated sludge systems at FIM ratios of greater than 0.5 has centered on the question of oxygen supply. Wuhrmann (1964)was responsible for the suggestion that when the interior of an activated sludge floc particle becomes anaerobic it has a tendency to break up. Mueller et al. (1966)worked out a method of calculating the upper limit on an activated sludge floc particle as a function of the dissolved oxygen concentration in the mixed liquor. They found that a dissolved oxygen concentration of 0.2 mgAiter would limit the floc particles to a diameter of 20 p m , while a dissolved oxygen concentration of 4 mg/liter would alIow ffoc particles of up to 115 pm diameter (assuming that Wuhrmann’s theory is correct). An increase in the FIM ratio results in an increase in the rate of oxygen uptake per unit weight of activated sludge and thus to a lower mixed liquor dissolved oxygen concentration unless the rate of oxygen supply to the

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aeration tank is increased. This provides a possible explanation of the observed deflocculation at higher FIM ratios. It is clear than an activated sludge system can be operated at an FIM ratio between 0.5 and 1.0 per day, if the dissolved oxygen concentration in the mixed liquor is maintained above 2.0 mglliter. Kalinske (1976)contends that it makes no difference whether the oxygen in the mixed liquor is dissolved from air or from a pure oxygen supply as long as the minimum 2.0 mglliter concentration is maintained. Chapman et al. (1976) disagree and maintain that a pure oxygen activated sludge system will produce a sludge of superior settling characteristics (and thus produce better effluent quality) primarily because a lower level of turbulence is needed to maintain the desired dissolved oxygen concentration in the pure oxygen system. They depend to some extent on the paper by Parker et al. (1972) which shows that higher turbulence levels can physically tear activated sludge particles apart. However, Parker and Merrill(l976) tend to agree more with Kalinske than with Chapman et al. Kalinske, Chapman et al., and Parker and Merrill all present their information about the settleability of activated sludge in terms of the initial zone settling velocity as a function of the MLSS. This gives no information about deflocculation (which requires measurement of the amount of suspended solids ;eft behind after the sludge settles), but does give some qualitative indication of bulking. The hypothesis here is that an FIM ratio above 0.5 tends to produce a lighter, more fragile, larger particle sized floc because the activated sludge organisms accumulate more extracellular hydrated organic material. These particles compact poorly and thus give a higher SVI. They have a higher oxygen consumption per gram of dry weight. They may break up into smaller particles if the dissolved oxygen concentration in the mixed liquor becomes so low that all the cells in the particles cannot get an adequate oxygen supply. This tendency toward nonfilamentousbulking and deflocculation can be overcome by maintaining an adequate oxygen supply but, if maintaining the higher dissolved oxygen concentration requires excessive turbulence, the turbulence itself can cause breakup of floc particles. All of the papers cited in this section support this hypothesis but none of them proves that it is correct. B.

FILAMENTOUS BULKING

In this author’s experience (Pipes, 1978b) filamentous bulking is a phenomenon occurring at low organic loading (FIM < 0.2 per day), but some activated sludge systems with very low FIM ratios produce sludges with very good compaction characteristics. Thus, the low FIM ratio cannot

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be the cause of the excessive growth of filamentous microorganisms. A low

FIM ratio implies a low growth rate of the entire microbial community and a low concentration of soluble organic substrate in the mixed liquor and a high

F/M ratio implies a higher growth rate and a higher concentration of soluble organic substrate. By and large, the filamentous microorganisms involved in bulking grow more slowly than the single-celled, heterotrophic bacteria and thus it is not surprising that they are more likely to be found in large numbers in an activated sludge system with a low F/M ratio. To expect a more explicit relationship than that is probably asking too much. This author has seen filamentousbulking at F/Mratios greater than 0.3 per day (Pipes, 1978b);however,these have been cases of the filamentous bulking causing the high FIM ratio rather than vice versa. The scenario goes as follows: Filamentous bulking occurs initially at a low F/M ratio, the underflow solids concentration decreases to the point at which the maximum return rate designed into the system cannot maintain the desired MLSS, the decreasing MLSS increases the FIM ratio, and when the FIM ratio becomes high enough the floc characteristics change and eventually the sludge deflocculates. This scenario assumes that filamentous and nonfilamentous bulking are distinct phenomena but that they can occur concurrently in the right conditions. Undoubtedly, the some 20-odd different filamentous microorganisms which have been found in activated sludge have a variety of different physiological adaptations. It is likely that when they are studied thoroughly some will be found to be adapted to low dissolved oxygen concentrations and others to high dissolved oxygen concentrations, some to low temperatures and others to moderate temperatures; some will require organic nitrogen sources while others will be able to utilize inorganic compounds as their sole nitrogen source, some will be adapted to carbohydrate substrates while others will do better on protenaceous substrates, some will require growth factors and others will not, and so forth. It is unlikely that there will be any one set of conditions which will “cause” filamentous bulking. What is likely is that there will be a variety of Werent conditions under which one or more of the filamentous organisms will flourish. It will take a lot of very careful work to sort all this out.

1 . Sphaerotilus It is quite clear that filamentous bulking is sometimes due to gramnegative, ensheathed, rod-shaped bacteria which do not oxidize H2S and deposit S in their cells, but do deposit small amounts of ferric oxides in their sheaths. This organism fits the description of Sphuerotilus natans in Bergey’s manual (Mulder and van Veen, 1974). A similar organism but without the ability to deposit iron in its sheath is listed in Bergey’s manual as Streptothrix

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hyalina. van Veen et al. (1973)isolated this second organism from activated sludge and renamed it Haliscomenobacter hydrossis on the grounds that the name Streptothrix was used earlier to describe a fungus. van Veen also recognizes Sphaerotilus discophonrs which can deposit Mn slowly in its sheath as an activated sludge organism. Eikelboom recognizes yet another type in this group and Farquhar’s and Boyle’s unidentified form I1 also probably belongs here. Very little is known about the physiology and ecology of any of these organisms except for Sphuerotilus nutans. Whether there are two or three or more valid species in this group must await further microbiological investigations. In the meantime, it must be accepted that clear demonstration of a sheath, iron deposition, and the absence of ability to deposit S in the cells is not adequate for a firm identification of Sphaerotilus natans. Dondero’s (1975)comment about the Sphuerotilus-Leptothrix group was “. . . .There are obvious gaps in the information needed for classification.” There are two main theories to explain why Sphaerotilus natans can predominate in activated sludge; namely, (1)its filamentous growth form gives it an aredvolume ratio higher than that of flocculated bacteria and thus it is able to compete successfully for low concentrations of soluble organic substrates or dissolved oxygen if either of these factors are limiting and (2) when the waste water has a high C/N or C/P ratio it is able to compete more successfully for the organic substrate which it accumulates as a food reserve (intracellular granules of poly-P-hydroxybuterate) and as extracellular polysaccharide. Nothing has been published in the past 10 years which would modify these theories. Mulder (1964) had compared the growth in pure culture of Arthrobacter glob$mmes and S. natans and reached the conclusion that Sphaerotilus grew better in poorly aerated cultures while Arthrobacter grew better in well aerated cultures. Adamse (1968a) found that species of Arthrobacter dominated a laboratory activated sludge developed on an artificial dairy waste (powdered milk and tap water). He then studied the competition between Arthrobacter-like (flocculated)bacteria and S . natans in mixed culture on the artificial dairy waste and reached conclusions similar to Mulder’s (Adamse, 1968b). There is no doubt about the identities of Mulder’s cultures or about Adamse’s identification of the Arthrobacter species which he has obtained in pure culture. However, Adamse gave no information which would establish that the filamentous organism in his laboratory activated sludge culture actually was S. natans. Since this work was done during the period when all filamentous bacteria in activated sludge were routinely called “Sphaerotilus,” the assertion that he was working with S. natans must be viewed with suspicion; since he was just confirming Mulder’s pure culture work, however, this suspicion does not change the basic conclusion.

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Dias et al. (1968a) found that dilute sewage (BOD, = 20 mglliter) with added glucose (20 mg/liter) selected for S. natans out of a mixed culture of attached, filamentous microorganisms. In a later paper, Dias et al. (196813) found that a low dissolved oxygen concentration had less of an adverse effect on S. natans than on competing organisms. There is no doubt that they know how to differentiate S. natans from other filamentous bacteria but the physical setup of their enrichment cultures leaves questions about the applicability of their observations to activated sludge systems. Rao and Washington (1968)observed that S. natans was more prevalent in underloaded (low FIM ratio) laboratory activated sludge units than in those with normal or high loadings. They used the CGY medium of Dondero et al. (1961)for making Sphaerotilus colony counts. They use the term “organisms morphologically similar to S. nutans,” so it appears that they are aware of the possibility of confusing other filamentous organisms with Sphuerotilus but the lack of detail about how they have identified Sphaerotilus leaves doubt about the organisms they are observing. 2. Eikelboom’s Type 021N

Chudoba et al. (1973) studied the effect of mixing on the bulking of laboratory activated sludge units treating a synthetic waste water (peptone and starch in tap water plus inorganic nutrients) at F/M ratios between 0.28 and 0.48 per day. They reported that the completely mixed unit bulked with a mixture of Leucothrix and Sphaerotilus, while the unit with an intermediate degree of mixing bulked with Leucothrix alone and two units set up to simulate plug flow did not bulk in spite of the fact that Sphaerotilus and Leucothrix were both present in small amounts. Since all of their units were the same size and received the same waste water at the same rate, the FIM ratio for each unit was inversely proportional to the MLSS and they produced a very fine example of how a high SVI causes an increase in the FIM ratio. In later experiments (Chudobaet a l . , 1974)the F/M ratio was purposefully varied from 0.5 per day to about 2.0 per day and they observed that the SVI increases with increased FIM ratio in the plug flow units but decreased with FIM ratio in the completely mixed units. No information was given about identities of the filamentous organisms in the later paper. Eikelboom (1975) rejects the identification of Leucothrix in activated sludge by Cyrus and Sladka (1970) on the grounds that Leucothrix requires NaCl for growth. Apparently, this is the identification which Chudoba et al. (1973) were using for the dominant filamentous organisms in their culture. They do not give enough information about it to reach any firm conclusions but neither do Cyrus and Sladka. It may be presumed to be one of the colorless blue-green algae but not much more can be concluded about the organism until it is studied in pure culture and in an actual case of bulking in a waste water treatment plant.

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3. Thiothrix Farquhar and Boyle (1972) found Thiothrix (their forms I and 111)bulking in a pilot plant activated sludge system treating municipal waste water. Their pilot plant was a tanks-in-series unit quite similar to the one Chudoba et al. (1973) used to simulate plug flow conditions. They found that sulfide in the waste water was the limiting hctor for the growth of Thiothrix and were able to eliminate the sulfide fi-om the waste water and the Thiothrix from the activated sludge by preaeration. When preaeration was stopped the sulfides were again carried into the aeration tank, the Thiothrix returned, and the SVI increased again. Resumption of preaeration again eliminated the sulfides, Thiothrix, and bulking. They were able to get the Thiothrix bulking to return by adding NazS continuously to the mixed liquor in the pilot plant receiving preaerated waste water. All in all, it was a very convincing demonstration of the importance of sulfide in this particular type of bulking. They maintained constant influent and return sludge flow rates and their data are another good example of filamentous bulking causing a high FIM ratio. Merkel (1975) observed Thiothrix bulking in a full-scale activated sludge system treating waste water from an anaerobic lagoon which had high sulfide concentrations. He provided enough information to make it reasonably certain that he was observing Farquhar’s and Boyle’s Thiothrix form I1 (the one with the sheath). He stated that he had confirmed Farquhar’s and Boyle’s results on eliminating the Thiothrix bulking by eliminating the sulfides from the waste water but gave no details of the experimentation. This author (W. 0. Pipes, unpublished data) has observed two cases of Thiothrix bulking. The organisms in both cases were gram negative; showed only slight, swaying motility; and had S deposits (ethanol-soluble granules) in their cells, but this author did not collect enough information to associate them with one of Farquhar’s and Boyle’s three forms. In both cases sulfide was present in the influent waste water but so were large numbers of Thiothrix which evidently were growing in the sewers. In both instances, chlorination of the influent waste water oxidized the sulfide and apparently killed the Thiothrix and eliminated the bulking problem. As previously noted, both van Veen and Eikelboom refuse to accept Thiothrix as a name for any of the filamentous organisms which they have found in activated sludge. However, there is no clear evidence that either one of them has examined activated sludge from a treatment plant with a situation which may lead to Thiothrix bulking and the descriptions by Farquhar and Boyle (1971b) fit easily within the description of Thiothrix in Bergey’s manual (Brock, 1974). The identification problem cannot be resolved until pure cultures of these organisms are obtained and comparative studies are made. In the meantime, it is useful to use the name Thiothrix for those filamentous organisms in activated sludge which oxidize sulfide and

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deposit sulhr in their cells but do not show the characteristic motility of Beggiatoa because there really is not any other generic name which fits.

4 . Microthrix parvicella Pasveer (1969)published a description of an attempt to make a complete case study of a bulking problem in a type of modified activated sludge system which he called an oxidation ditch. He obtained adequate operational data to define what the problem actually was; an attempt was made to isolate and identify the filamentous microorganism; he advanced a hypothesis to explain why the filamentous organism was predominant in that particular activated sludge system; and he found a practical solution to the bulking problem. Unfortunately, the attempt to isolate the filamentous microorganisms failed and he got sidetracked by the fact that E. coli grew as filaments at pH values of less than 6.0 with high COz concentrations. His hypothesis about how a filamentous E . coli could predominate activated sludge depended upon some very improbable differences in conditions between the liquid and the interior of the floc particles and the misapprehension that E. coli had a nucleus. He did find an association between the Occurrence of nitrification and bulking in the oxidation ditch and was able to eliminate the bulking by converting from continuous to fill and draw operation. The fill and draw operation produced periods when the rate of oxygen consumption was very high and the mixed liquor dissolved oxygen concentration fell so low that denitrification occurred. Changing from continuous to fill and draw operation is analogous, in some respects, to changing from complete mixing to plug flow. There is no information in Pasveer’s paper which allows one to determine if it is the anaerobic periods, the occurrence of denitrification, or the change in mixing conditions which has been critical in eliminating the filamentous organisms. Both van Veen (1973) and Eikelboom (1975) have attempted to isolate filamentous organisms from oxidation ditches (which are popular in the Netherlands but are not widely used elsewhere). They both isolated a slender, coiled, filamentous microorganism to which van Veen gave the provisional name Microthrix parvicellu. Van Veen pointed out that oxidation ditches are very underloaded (low F/M ratio) activated sludge systems and notes that Microthrix has been eliminated from these systems by removal of half of the MLSS as well as by discontinuous operation. However, neither van Veen nor Eikelboom provide any hrther information which can be used to explain why Microthrix grows in the sludge under the conditions occurring in oxidation ditches.

5 . Unidentifwd Organisms There is a number of papers which describe cases of filamentous bulking in which no attempt is made to identify the responsible organisms. Most of

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these are of little value in the context of this article. They just report that chlorination of the return sludge reduced or did not reduce the SVI, or that decreasing the MLSS helped or that increasing the MLSS helped, or that increasing the oxygen supply eliminated the filamentous organisms, or that holding the return sludge under anaerobic conditions eliminated the filamentous organisms, and so forth. Clearly, there must have been difFerent filamentous organisms responsible for these different cases of bulking or there would not be so many different ways to eliminate them. Case studies of this type can have continuing value only when the filamentous organisms are identified and the procedures necessary to eliminate a particular organism are demonstrated. There is one study, however, which sheds some light on the occurrence of filamentous bulking at high FIM ratios even though the filamentous organisms are not identified. Ford and Eckenfelder (1967) studied activated sludge treatment of a brewery waste, a petrochemical waste, and municipal sewage in pilot plant units. In all three cases the SVI values of the sludge produced were at a minimum at FIM ratios of about 0.2-0.3 per day. Bulking occurred at FIM ratios greater than 0.8 per day for the municipal sewage, above 0.9 per day for the brewery waste, and above 0.7 per day for the petrochemical waste. They made bound water determinations and published photomicrographs of the sludges. The high FIM ratio bulking in the cases of the domestic sewage and of the brewery waste clearly was filamentous bulking and there was no increase in the bound water in the sludge. In the case of the petrochemical waste there was an increase in the bound water in the sludge with an increase in the FIM ratio and some filamentous organisms were also present. This appears to be a case of simultaneous filamentous and nonfilamentous bulking. Unfortunately, they gave no information on how well flocculated the sludges were and it was not possible to use their data to test the hypothesis that bulking at high FIM ratios led to deflocculation. V. Summary and Future Prospects A great deal more information about the various types of filamentous microorganisms which may be found in activated sludge is available in 1977 than was known in 1967. It is now widely recognized that many different types of filamentous microorganisms can cause bulking. There is even a reasonable start on an organized presentation of morphological information which may enable treatment plant personnel to categorize, if not class& and identify, the filamentous organisms which cause bulking in their plants. However, the terminology used to designate these organisms is very conhsing due to the lack of completeness of the microbiological studies. There is enough published information to establish nonfilamentous bulking as a phenomenon which is probably distinct from filamentous bulking. All

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that remains to be done to prove absolutely that the two phenomena are distinct is a determination of what microorganisms are present when nonfilamentous bulking occurs and an investigation of how the filamentous bulking organisms change the chemical and physical nature of the floc particles. Nonfilamentous bulking is a high FIM ratio phenomenon and if the FIM ratio becomes high enough the sludge deflocculates. The FIM ratio at which the sludge deflocculates depends upon the dissolved oxygen concentration in the mixed liquor and the level of turbulence in the aeration tank. The cure for nonfilamentous bulking is chlorination of the return sludge which changes the surface properties of the floc particles and allows them to compact better, followed by building up the MLSS so that the FIM ratio is reduced. Filamentous bulking is most frequently observed to occur in activated sludge systems with very low FIM ratios. Filamentous bulking is sometimes observed at high FIM ratios, but the reasons for this are not clear. Some of the cases of high FIM ratio bulking are due to the fact that the filamentous bulking causes a high FIM ratio because it forces a reduction in the MLSS. However, there are also cases of filamentous bulking occurring at high FIM ratios when the MLSS have been under control. There is at least one documented case of filamentous bulking occurring at a high FIM ratio at the same time that there have been changes in the bound water content of the sludge which are indicative of nonfilamentous bulking. The possible simultaneous occurrence of filamentous and nonfilamentous bulking in activated sludge systems with high F/M ratios and its relationship to deflocculation of the sludge is deserving of considerably more attention than it has received so far. An obvious hypothesis for investigation is that the filamentous bulking organisms occurring in systems with high FIM ratios are different &om those occurring in systems with low FIM ratios. Of course, in investigating this hypothesis it would be necessary to eliminate those cases in which the filamentous bulking caused a high FIM ratio or at least to find out what the FIM ratio was when the bulking first occurred. None of the investigators who have made surveys of filamentous organisms in activated sludge has published information on the FIM ratios of the systems from which they have obtained their samples, so it is not possible to test this hypothesis using the available published information. At the present time it is not, in general, possible for a microbiologist to come in on the investigation of a particular case of filamentous bulking, identlfy the organism responsible, explain why it is growing in that system, and formulate a solution for the bulking problem. The one clear exception to this is Thiothrix bulking due to sulfides in the influent waste water. Other cases of filamentous bulking have been solved empirically but without an unequivocal demonstration of which filamentous organism has been responsible and why it has been responsible. The future prospects for finding

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specific solutions are bright. The requirements for finding such specific solutions are as follows,

1. Sanitary engineers and treatment plant personnel must pay much more attentinon to describing exactly the nature of sludge separation problems. Bulking is poor sludge compaction and the associated operational problem is inability to obtain a high enough solids concentration in the underflow from the settling tank. This must be clearly distinguished from deflocculation pinpoint floc, floating sludge, rising sludge, and the other sludge settling problems. Simultaneous occurrence of bulking and another sludge separation problem can be confusing but the various problems can be sorted out by careful collection and analysis of operational data. 2. Microbiologists need to develop better methods for isolating filamentous organisms. Only about half of the filamentous organisms which have been described as occurring in activated sludge have been isolated in pure culture and only a few of those have been studied to the extent that there is significant information about their physiology in the literature. Different types of media and different cultural conditions for the isolation plates need to be tried. Aerated enrichment cultures are often useful for propagating the filamentous organisms in the laboratory while different isolation techniques are tried. If several aerated enrichment cultures are maintained under different conditions to the laboratory, the reactions of the mixed microbial populations can be followed by microscopic observations and this can provide clues to which factors stimulate the growth of the filamentous organisms. 3. Comparitive pure culture studies are badly needed to provide much better methods of differentiating among the large variety of filamentous microorganisms which are associated with bulking. The morphological descriptions, staining reactions, and physiological tests applied to sludge samples are obviously not adequate to sort out all of the problems of identification. However, in the long run some simplified identification procedures which can be used by plant personnel who are not well-trained microbiologists are clearly desirable. 4. Studies of the competition between activated sludge organisms, both filamentous and floc forming, in the laboratory are needed. Such studies should be used to test hypotheses derived from observations of plant operation about why a particular filamentous organism predominates in a particular activated sludge system. Such studies should, of course, be preceded by pure culture studies on the individual organisms. REFERENCES Adamse, A. D. (1968a). Water Res. 2, 665-671. Adamse, A. D.(1968b). Water Res. 2, 715-722.

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Agersborg, H. P. K., and Hatfield, W. D. (1929). Sewage Works J. 1, 411424. Benedict, R. G., and Carlson, D. A. (1971). Water Res. 5, 1023-1030. Bisogni, J. J., Jr., and Lawrence, A. W. (1971). Water Res. 5, 753-763. Brock, T. D. (1974).In “Bergey’s Manual of Determinative Bacteriology” (R. E. Buchanan and N. E. Gibbons, eds.), 8th ed. pp. 118-120. Williams & Wilkins, Baltimore, Maryland. Carter, J. L., and McKinney, R. E. (1973). J . Enoiron. E n g . Dio. Am. Soc. Cio. Eng. 99, 135-152. Chapman, T. D., Matsch, L. C., and Zander, E. H. (1976).J. Water Pollut. Control Fed. 48, 2486-2510. Chudoba, J., Ottova, V., and Madera, V. (1973). Water Res. 7, 1163-1182. Chudoba, J., Blaha, J., and Madera, V. (1974). Water Res. 8, 231-237. Cook, W. B., and Pipes, W. 0. (1970). Mycopathol. Mycol. Appl. 40, 249-270. Cyrus, Z., and Sladka, A. (1970). Hydrobiologiu 35, 383396. Dias, F. F., Okrend, H., and Dondero, N. C. (1968a).Appl. Mhobiol. 16, 276-278. Dias, F. F., Dondero, N. C., and Finstein, M . S. (1968b). Appl. Mimobiol. 16, 1191-1199. Dondero, N. C. (1975).Annu. Reo. Microbiol. 29, 407428. Dondero, N. C., Phillips, R. A., and Heukelekian, H. (1961).Appl. Mimobiol. 9, 219-227. Eikelboom, D. H. (1975). Water Res. 9, 365-388. Fair, G. M., and Thomas, H. A., Jr. (1950). Znst. Sewage Purif., J. Proc. Part 3, pp. 235-264. Farquhar, G. J., and Boyle, W. C. (1971a).J. Water Pollut. Control Fed. 43, 604422. Farquhar, G. J., and Boyle, W. C. (1971b).J. Water Pollut. Control Fed. 43,779-798. Farquhar, G . J., and Boyle, W. C. (1972).J. Water Pollut. Control Fed. 44, 14-24. Finstein, M. S., and Heukelekian, H. (1967).I. Water Pollut. Control Fed. 39, 3340. Ford, D. L., and Eckenfelder, W. W., Jr. (1967).J. Water Pollut. Control Fed. 39,1850-1859. Forster, C. F. (1971). Water Res. 5, 861670. Harris, R. H., and Mitchell, R. (1973). Annu. Reo. Mimobiol. 27, 2750. Heukelekian, H., and Weisberg, E. (1956).J. Water Pollut. Control Fed. 28, 558567. Hunerberg, K . , Sarfert, F., and Frenzel, H. J. (1970). Gas.Wasser, Abwasser 111, 7-10. Kalinske, A. A. (1976).J. Water Pollut. Control Fed. 48, 2472-2485. Lechevalier, H. A. (1975). “Actinomycetes of Sewage-Treatment Plants,” Rep., Program 1B8043, Grant No. R802003. U.S. Environ. Prot. Agency, Washington, D.C. Lechevalier, M. P., and Lechevalier, H. A. (1974). Int. J . Syst. Bacteriol. 24, 278-286. Magara, Y., Nambu, S., and Utosawa, K. (1976). Water Res. 10, 71-77. Merkel, 6. J. (1975). Water Res. 9, 881-885. Mueller, J . A,, Voelkel, K. G., and Boyle, W. C. (1966).J . Sanit. E n g . Diu. Am. SOC. Ciu. E n g . 92, No. SA2, 9-20. Mulder, E. G. (1964). J . Appl. Bacteriol. 27, 151-173. Mulder, E. G., and van Veen, W. L. (1963).Antonie van Leeuwenhoek 29, 121-153. Mulder, E. G., and van Veen, W. L. (1974). In “Bergey’s Manual of Determinative Bacteriology” (R. E. Buchanan and N. E. Gibbons, eds), 8th ed., pp. 128-133. Williams & Wilkins, Baltimore, Maryland. Nash, N., Krasnoff, P. J., Pressman, W. B., and Brenner, R. C. (1977).]. Water Pollut. Control Fed. 49, 388400. Parker, D. S . , and Merrill, M. S. (1976).J . Water Pollut. Control Fed. 48, 2511-2528. Parker, D. A., Kaufman, W. J., and Jenkins, D. (1972).]. Sanit. Eng. Dio. Am. SOC. Cio. Eng. 98, No. SAI, 79-99. Pasveer, A. (1969).J . Water Pollut. Control Fed. 41, 1340-1352. Pipes, W. 0. (1967a). Ado. Appl. Microbiol. 9, 185-234. Pipes, W. 0. (1967b). “Ecology of Sphaerotilus in Activated Sludge,” 3rd Annu. Rep. Dep. Civ. Eng., Northwestern University, Evanston, Illinois.

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Pipes, W. 0. (1969). J . Water Pollut. Control Fed. 41, 714-724.' Pipes, W. 0. (1978a). I . Water Pollut. Control Fed. 50, 628-634. Pipes, W. 0. (197813).J. Water Pollut. Control Fed. 50 (in press). Rao, S. S., and Washington, D. R. (1968). Appl. Mimobiol. 16, 942-943. Romano, A. H., and Peloquin, J. P. (1963).J. Bacteriol. 86, 252-265. Rod, M. A., and Stokes, J. L. (1961). J. Bacteriol. 83, 343358. Schather, M. W., and Pipes, W. 0. (1978).J. Water Pollut. Control Fed. 50, 2030. Sezgin, M., Jenkins, D., and Parker, D. S. (1978).J. Water Pollut. Control Fed. 50, 362-381. Singh, V. P., and Saxena, P. N. (1969). Hydrobiologiu 34, 503512. Skerman, V. B. D., Dementijeva, G., and Carey, R. J. (1957). J . Bacteriol. 73, 504512. Sladka, A,, and Ottova, V. (1974). Hydrobiologia 43, 285-294. Smit, J. (1934a). Sewage Works 6, 1041-1053. Smit, J. (1934b). Arch. Mikrobiol. 5, 550-360. Steiner, A. E., McLaren, D. A,, and Forster, C. F. (1976). Water Res. 10, 2530. Sykes, R. B. (1977). Department of Civil Engineering, Ohio State University, Columbus (personal communication). van Veen, W. L. (1973). Antonie van Leeuwenhoek 39, 189-205. van Veen, W. L., van der Kooij, D., Geuze, E. C. W. A,, and van der Vlies, A. W. (1973), Antonie van Leeuwenhoek 39, 207-216. Viehl, K., Mudrack, K., and Neumann, H. (1963). GesunoL-lng. 84, 260-268. Wuhrmann, K. (1964).Ado. Appl. Microbiol. 6, 119-150.

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Mixed Cultures in Industrial Fermentation Processes

DAVIDE. F. HARRISON Biological Laboratory, University of Kent at Canterbury, Canterbury, Kent CT2 7N], United Kingdom I. 11. 111. IV.

Introduction ........................................... Traditional Uses of Mixed Cultures . . . . . . Types of Microbial Interaction ........................... Continuous Enrichment Techniques ...................... A. Methods . . . . . . . . . . . . . . . . B. Turbidostat . . . . .

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141 A, Rationale ..................................... B. Examples.. ........................................ C. Advantages of Mixed Cultures for SCP Production . . . . . . D. Problems Related to the Use of Mixed Cultures . . . . . . . . VI. Other Applications of Mixed Cultures . A. Vitamin B,, Production.. ............................ B. Production of Organic Acids C. Metal Leaching ................... . . . . . . . . . . . . . . . . . D. Steroid Transformations ............................. VII. Future Prospects . . . . ...... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141

142 152 156 157 157 158 158 159 160 162

1. Introduction The use of mixed microbial cultures in industrial processes is a subject which is, at the same time, too large to be covered sensibly in one chapter and somewhat slight in scientific content. The number of Occurrences of mixed cultures in industrial processes is legion if both accidental and deliberate mixtures are considered. However, the number of mixed cultures in commercial use which are well studied and understood, and deliberately constituted, is extremely small. Furthermore, the complexity of physiological and biochemical interactions in such cultures is likely to be such that, if our understanding of them were complete, each culture would provide material for a whole volume. Therefore, in this article, this author will attempt only to instance the scope and possibilities of mixed culture. To this end this author will briefly review the role of mixed cultures in traditional processes and define types of microbial interaction. Then, to illustrate the interactions in mixed microbial systems, specific mixed cultures, recently developed for single-cell protein (SCP) production from natural gas, will be described in some detail together with methods for obtaining them. The advantages and disadvantages of such cultures will be discussed. Finally, this author will 129 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1878 by Academic Press, Inc. All rights of r e p d u c t o n in m y form reserved. ISBN 0-1243262.4-4

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consider some other recent uses of mixed cultures and review future possibilities.

II. Traditional Uses of Mixed Cultures Traditional microbial processes were almost invariably based on mixed cultures, although not through deliberate choice. To catalog all mixed microbial systems used in such processes would fill a very large volume. Fermentations were carried out in open vats and were unprotected from contamination so that many of the organisms that grew in the cultures contributed nothing to the overall process or were actually harmful to it. However, many of these traditional processes depend on precise and complex cooperative action of many different species in the cultures, the development of which has owed much more to serendipity than invention. Certainly, there was no question of deliberately mixing species in these early processes and, in fact, not until the last decade was this technique attempted. One interesting example of a traditional process depending in a mixture of organisms is the ancient Japanese process for the manufacture of soy sauce. The first step in the complicated processes of making both soy sauce and saki is the production of “Koji” (Yong and Wood, 1974; Murakami, 1972). This is accomplished by the action of Aspergillus wyzae. The mold is grown on polished rice for 5 days and then mixed with soaked, boiled soybeans and with roasted, crushed wheat. The mixture is spread onto shallow trays and incubated for 72 hours at 30°C to form the koji. For soy sauce the koji is mixed with a 20% salt brine and allowed to undergo a very slow fermentation, which in the best quality soy sauce takes from 1 to 3 years. The fermentation is accomplished by a succession of a salt-tolerant lactic acid bacteria, Pedwcoccus soyae, followed by yeast. The bacteria cany out a homofermentative process and thereby lower the pH, allowing the yeast to grow. A common yeast found in saki fermentation is termed Saccharomyces rouxii, but as many as 300 yeast strains are reported to have been isolated from soy sauce fermentations (Yong and Wood, 1974). Thus the biological transformations involved in these ancient but sophisticated processes require classes of microorganisms grown under carefully controlled conditions; it is difficult to imagine such a process being developed from a monoculture basis. Brewing is probably as ancient as civilization itself. Wines and beers were undoubtedly originally all products of mixed cultures. Today, bottom fermented beers are commonly brewed using monocultures, but top fermented beers are still largely produced from mixed yeast cultures. Traditional wine making always involves a succession of yeast species. These

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have been elucidated into three successive populations (Goswell, 1967): initially, alcohol-sensitive species predominate, including Kloekera apiculata, Hanseniuspora quillemndii, and Candida pulchewima, followed by such species as Saccharomyces rosei, S. veronae, and S . cerevisiae var ellipsoideus, and, when the alcohol content rises above lo%, the predominant species are S. cerevisiae var ellipsoideus, S . ov$n-mis, S . chevaleiri, and S . italicus. Also in certain full-bodied wines, such as Burgundy, lactic acid bacteria play an important role in reducing the malic acid content (Pitone and Rankine, 1974). Acetic acid production from ethanol by the acet&er process is undoubtedly a mixed culture system but is largely undefined. Dairy fermentations in their various forms are all based on mixtures of microorganisms; yoghurt is produced by the combined action of Lactobacillus bulgaricus and Streptococcus thermphilus, kefir by a yeast Saccharomyces kefr together with the bacteria Lactobacillus bulgaricus and Streptococcus lactis, and the many, many varieties of cheeses arise from the action of various combinations of streptococci, lactobacilli and filamentous fungi. The biological treatment of wastes will, of necessity, be by mixed culture (Pike and Curds, 1971). Only by the combined action of many species can complex mixtures of organic compounds be effectively degraded. These mixed cultures, however, arise fortuitously from inoculation from the waste stream or from “sludge” obtained from other plants. An “adaptation” period may be required while the appropriate microbial population develops in the treatment plant but there is no deliberate tailoring of populations to meet the requirements of the waste stream. Indeed, the populations developed are so complex, consisting of bacteria and protozoa, that our understanding of the interactions can only be said to be rudimentary. However, one example of the cooperative effect of two bacterial species in waste treatment is worthy of further mention: namely, the denitrification of waste water by Nitrosomonas and Nitrobacter species. Both species must be present in activates sludge for an aerobic treatment plant to effectively oxidize ammonia to nitrate. Nitrosomonas is responsible for initiating the attack on ammonia but can oxidize it only as far as nitrate (McCarty and Haug, 1971). This latter compound is highly toxic and Nitrosomonas is itself inhibited by this product. Nitrobacter sp. serve to oxidize the nitrite through to nitrate but cannot, themselves, oxidize ammonia. Thus a balanced population of both organisms must be present in a ‘‘nitrfiing” activated sludge plant. The increasingly important process for the anaerobic digestion of sludge and organic materials to produce methane is achieved by mixed bacterial populations. This process and the related topic of the rumen have been much studied in recent years (Zeikus, 1977). One section of the bacterial population is responsible for the degradation of complex organic molecules

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DAVID E. F. HARRISON

to organic acids and hydrogen and another group uses the products of fermentation to produce methane. The interaction between H,-producing organisms and methane-producing species has recently been large elucidated (Wolin, 1975; Taylor et d.,1975). The methanogenic species are dependent on nonmethanogenic species for a supply of Hz which they use to reduce CO, to methane. The removal of H,, in turn, permits nonmethanogenic fermenters to oxidize NADH to NAD+ with the production of Hz, a process which proceeds only very slowly if Hz accumulates but which is energetically more ivorable than the alternative pathways (Wolin, 1975). Bacterial cultures capable of producing methane from lactate and ethanol have been reconstituted (Wolin, 1975), and the commercial exploitation of specially reconstituted mixtures would seem to be a definite future possibility. In spite of the dependence of so many traditional processes on the cooperative action of many microbial species, it is undeniable that the modern fermentation industry owes its existence to the development of pure culture techniques. The microbial production of antibiotics, organic acids, vitamins, amino acids, etc., could not ‘have been contemplated without the development of specific strains of microorganisms and the techniques to keep cultures pure. Indeed, since the time of Koch, microbiology has been concerned almost exclusively with pure cultures and a monoculture philosophy was firmly established early in the growth of the modern fermentation industry: The first step toward developing new processes is invariably to attempt to obtain pure cultures of appropriate organisms. It is generally assumed, with much justification, that in order to maintain consistency of produce quality and production rate it is necessary to grow monocultures. Recently, however, the deliberate use of mixed cultures for the production of single-cell protein from methanol and methane has been developed by workers of the Shell Laboratories in England (Harrison et aZ. 1976).

111. Types of Microbial Interaction In order to discuss the potential use of mixed cultures it is convenient to classlfy the types of microbial interaction that may occur. These are commonly divided into the following seven categories (Meers, 1973).

1. Competition. This occurs when the growth of two or more species is limited by a common factor, which may be a substrate. Competition does not normally lead to a stable population. The organism which can grow fastest eventually “take over.” 2. Predation. Predation in microbial systems usually involves protozoa, myxobacteria, or myxomycetes preying upon bacterial species, although

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there have been reports of bacteria preying upon filamentous fungi (Chet et al., 1971). Predator-prey relationships can, depending on the conditions and kinetic interrelationships, give rise to instability, leading to collapse of the population; or to stable oscillations in populations; or to stable situations. Tsuchiya et al. (1972) defined the conditions required for stability of populations in continuous cultures of predatory protozoa and bacterial prey. It is difficult to envisage an industrial exploitation of predator-prey mixed cultures except perhaps if it can be shown that protozoa heave clear advantages over bacteria as a basis for single-cell protein. This seems unlikely as the advantage of protozoan biomass over bacterial biomass must be very large to offset the diminished conversion efficiency caused by moving up the food chain. Ciliate protozoa are most important, however, in removing bacteria and other particles from suspension in waste water treatment systems (Pike and Curds, 1971). 3. Parasitism. Apart from Bdellovibrio, bacteria also play hosts to bacteriophages. Cultures of phage-infected bacteria may be termed mixed cultures and undoubtedly have applications in terms of genetic manipulation of industrial strains. Discussion of this topic is outside the scope of this article. Yeasts are also subject to attacks by viruslike particles, which may be important in a negative sense because of possible harm to or infection of industrial strains, but are not yet exploitable. 4. Amensalism. Amendism is an interaction in which the growth of one organism interferes with, and inhibits, the growth of another. This effect may be mediated by the removal of essential nutrients, altering the physical or chemical environment, e.g., changing pH or producing a toxic metabolite. Antibiotic production is an example of the latter. Amensalism will not normally lead to a stabilized population; obviously the “aggressor” organism must predominate and therefore it is not a relationship that is likely to find application in continuous culture processes. However, traditional batch fermentations, such as sauerkraut and saki production, depend on amensalism for creating a succession of species leading to a multistep fermentation. The amensal effect in batch fermentations is often mediated through pH and dissolved oxygen changes. There appear to be no new processes developed to exploit amensal relationships in this way, probably because similar effects can be produced by electronic feedback control of the chemical and physical environment. 5 . Neutralism. True neutralism probably rarely occurs; it is unlikely that two or more species ever coexist in the same environment without interacting in some way. However, it may be that such interactions are sufficiently weak for species to coexist with little effect on each other. Lewis (1967) studied a synthetic cheese starter culture of lactobacilli and streptococci grown both as discrete monocultures and as mixed cultures. She reported

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DAVID E. F. HARRISON

that growth of both species was quantitatively the same whether they were grown separately or in association. This would certainly indicate a high degree of neutralism but this work was carried out in an artificial medium. In milk there is evidence that the relationship is symbiotic and synergistic (Pette and Lolkema, 1950). When the component organisms of a mixture utilize separate limiting substrates, all common substrates being in excess, then the relationship may approach true neutralism. Such neutral mixtures may find application for the production of SCP from mixed substrates. Neutral mixtures with species utilizing separate nutrients provide a possibility of exploiting mixed substrates more fully. Netrdism provides the basis for stable populations and so such mixtures are amenable to continuous culture. 6. Commensalism and mutualism. These types of interaction both involve one organism’s conferring benefit on another. In a commensal relationship the benefit is one sided but in mutualism both (or all) component species derive benefit from the association. The term “symbiosis” is often used of such relationships although, pedantically, it would only be applied to cases of obligate mutualism. Such relationships are by nature usually stable. The benefit derived is often in the form of the production of a growth factor by one organism which stimulates the growth of the second. In a mutualistic relationship an exchange of growth factors may occur. For example, in a mixture of certain strains of Lactobacillus plantarum and Streptococcus faecalis, the former provided folic acid for the latter which, in turn, supplied phenylalanine to the Lactobacillus (Nurmikko, 1956) so that although neither could grow in basal salts media in monoculture, they could grow together, symbiotically. In commensal relationships it is often the case that one organism has no direct access to a source of carbon and energy except through the action of a second organism. This is common where photosynthetic organisms supply energy and carbon to heterotrophic bacteria, e.g., lichens are associations of algae and fungi. An example of this type of association among bacteria is the mixture of Rhodospseudomonas capsulatus and Azotobacter vinelandii studied by Okuda and Kobayashi (1963). Symbiotic associations commonly occur between yeasts and bacteria in which the yeast depends on the bacteria for providing essential vitamins, such as biotin and thiamine. Symbiosis may also be mediated by the removal of one organism of a compound which is toxic to the other. The ulitization of methanol by Hyphomicrobium sp. in the methane-utilizing mixture studied by Wilkinson (1972) falls into this category. Synergism is a particular form of mutualism or commensalism which leads to a greater growth or production in the mixture than can be achieved by the component species individually. Presumably, mixed cultures used by preference for a commercial process would demonstrate synergism. An example of synergism in a commercial mixed culture is that of the

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135

yoghurt starter consisting of Lactobacillus bulgariczrs and Streptococcus thermophilus. In this culture S. thermphihs depends upon the L. bulgaricus to hydrolyze milk proteins and thus supply amino acids (I. Miller et al., 1964)while S. thermophilus in turn provides L. bulgaricus with essential formate by fermentation of lactose (Veldkamp, 1976; Galesloot et al., 1968; Veringa et al., 1968) leading to improved growth and acid production. Of various types of relationship possible in mixed cultures, the types likely to be of most use in commercial processes are those based on symbiosis. Stability of the population is most important, particularly in continuous culture systems, and such stability results from a codependence of component organisms. As we have seen, amensal relationships can be exploited to achieve a succession of species in batch cultures but generally amensalism, parasitism, predation, and competition are basically unstable systems which are not normally beneficial in commercial processes. The question is whether mixed cultures can solve process problems which cannot be solved by monocultures. A philosophy of using mixed cultures rather than monocultures was adopted by a team working at Shell Research Laboratories. This approach was adopted after failure to obtain monocultures which could grow as well on methane and methanol as did mixtures obtained by continuous enrichment (Harrison et al., 1976).

IV. Continuous Enrichment Techniques

A. METHODS For commercial processes which are to be based on continuous-flow culture, there are obvious advantages to using continuous enrichment techniques to obtain the process organisms. The more usual batch-culture enrichment followed by isolation of pure cultures on solid media was found to produce cultures which did not perform well under continuous-flowcultivation (Harrison et al., 1976). Continuous-flow enrichment provides a method for directly selecting cultures adapted to continuous cultivation, and it is possible to arrange the enrichment to correspond closely with predicted process conditions. A main strategical problem in using continuous culture as a selection technique is to apply sufficient selection pressure on the initial inoculum to allow rapid ascendancy of organisms with desired characteristics, without washing out the inoculum before an adapted culture has become established. Two methods may be employed to achieve this, the first based on a turbidostat and the second on two-stage chemostat culture.

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DAVID E. F. HARRISON

B.

TURBIDOSTAT

A turbidostat is a continuous culture system in which the cell concentration is controlled by feedback regulation on culture absorbance or any other population-dependent parameter (Watson, 1972). It contrasts with the chemostat in that all substrates are in excess and the growth rate is close to pmax(Pirt, 1975). The culture vessel, in which pH and temperature are controlled at appropriate values, is charged with medium and inoculated with a soil infusion or some other appropriate native source of microbes. It is often found desirable to enrich the initial infusion in shake-flask culture for 48 hours before use as inoculum. The medium used should be optimized as far as possible. The medium supply pump is started at a modest dilution rate of about 0.1 per hour and the culture run for about 48 hours before switching to turbidostatic control. The turbidostat is set to control the optical density of the culture at about 75%of the maximum expected value; e. g., if methanol is the carbon source and is fed at 4 g/liter then, assuming a yield coefficient of 0.5 g celYg methanol, the turbidostat should be set to control at an optical density equivalent to about 1.5 gm cellsfliter. In a simple system where the medium feed pump is operated simply by an on or off basis, when the optical density exceeds the set value, the pump is started. The pumping rate should be equivalent to about twice the expected maximum dilution rate so that the culture washes out until it reaches the lower set point, whereupon the pump is switched off. The length of time the pump is on increases as the growth rate of the culture increases and the overall dilution rate can be obtained by measuring the volume of medium pumped over a time period. The dilution rate is numerically equal to the specific growth rate of the culture (for theory, see Pirt, 1975). A more accurate control system would be achieved by proportional feedback control of the pumping speed, but quite adequate performance can be obtained by the simple on or off system. The culture dilution rate quickly reaches the maximum growth rate attainable by the organisms present; those organisms unable to compete and grow at this rate are lost, and the selection pressure for growth rate is maintained as long as the culture is run, which should normally be in excess of lo00 hours.

C.

TWO-STAGE

CHEMOSTAT

When growth of a culture is limited by a component of the gas phase then a turbidostatic method cannot conveniently be employed for selection. When a gas-phase component, such as oxygen or methane, limits growth, the relationship between cell concentration and dilution rate is as shown in Fig. 1. For a constant gas supply rate, the cell concentration falls rapidly with

MIXED CULTURES IN INDUSTRIAL FERMENTATION

137

20 1816-

.-

-:

14-

\ Dl

12-

a D

per hour

FIG. 1. Predicted relationship between organism concentration and dissolved oxygen concentration with dilution rate in an oxygen-limited continuous culture system in which oxygen is fed in the gas phase (model of H. H. Topiwala, 1971, private communication).

dilution rate in contrast to the case of a culture limited by a component in the liquid medium (Harrison, 1972). Thus a turbidostat control set at a constant cell concentration would not automatically control at maximum growth rate. The problem is overcome by running the culture as achemostat at a fixed high dilution rate but preventing early washout of the inoculum by employing a continuous inoculation. The first stage of a two-stage chemostat system is used as a continuous inoculum to the second stage. A soil infusion is inoculated into the first stage which may simply be a large bottle of basic salts medium bubbled with the appropriate gas. This is fed into a second stage, which is a fully controlled, agitated culture vessel, thus supplying a continual dilute inoculum. The dilution rate in the second stage is initially set close to the maximum expected and the optical density is recorded. Establishment of a “fit” culture is denoted by an increasing optical density in stage 2, and when this occurs the dilution rate can be set higher. At this point the firststage culture may be omitted.

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DAVID E. F. HARRISON

D.

THEORY

In a culture consisting of many different species, of microorganisms, the change of population with time will depend on the growth rate of all the component species:

where x is the total cell concentration; ql), x ( ~ ) ,x , represent the concentrations of individual component species; and p, p(*), are the respective specific growth rates. Therefore, the population changes in a single-stage continuous culture with a dilution rate D would be:

so that any organism for which pmax < D will have a negative value for dxldt and will be lost from the culture. For organisms where p,,, > D the relative change in populations may be expressed as:

Integrating gives:

If the two organisms are competing for a common limiting substrate, then the total amount of organism biomass that may be formed is limited by the supply of that substrate. If the concentration of the limiting substrate in the medium feed (SJ is very large compared to that remaining in the culture (So) then:

MIXED CULTURES IN INDUSTRIAL FERMENTATION

139

Where y(l)and y(2) are the yield coefficients for the limiting substrate of the respective organisms. Therefore [ql) q2Japproaches a limiting value:

+

But, if p ( l )> /.q2,, then from Eq. (4), the ratio X ( ~ J X ( ~will ) increase, and as r,,Jy approaches Si,x ( ~approaches ) zero, i.e., washes out. Thus, for organisms competing for the same substrate, the organism with the highest growth rate under the prevailing conditions will completely replace all competitors. According to the Monod growth hypothesis, the growth rate of a microorganism is given by the relationship: P = Pmax

S

(7)

This may be modified by including a term for “maintenance” as proposed by Pirt (1975): P =

Pmax

S V s -

where m = the “maintenance” substrate requirement, independent of growth rate and YG = growth yield corrected for maintenance. Note: The negative term may also include cells lost by death and autolysis, in which case part of the term mYGcould be replaced by an experimental death rate constant K , i.e.,

From this it follows that for two organisms competing for a limiting suband maintenance ratio of the two organisms is approxistrate, if the pmax mately equal, then the organism with the lowest K , (or highest S n i t y ) for the substrate has the highest growth rate and therefore replaces the other. Equation (8) may be further expanded. Assume that the specific rate of utilization of the limiting substrate (q) approximates to Michaelis-Menton kinetics:

140

DAVID E. F. HARRISON

If the growth yield, corrected for maintenance, is given by YG, then

From Eq. (10) it can be seen that a chemostat selects for:

1. 2. 3. 4.

Low values of K , High values of qmax High values of YG [m < Low values of m

< p m d / ( K s+ s)]

For single-cell protein (SCP) production the single most important process parameter is yield coefficient (Harrison et al., 1972). Thus selections for high YG and low m are both desirable for SCP production. It is also necessary that the K , be low as this leads to more complete utilization of substrate, but a reduced K, at the expense of yield (arising, for instance, &om an energy-dependent transport of substrate) is detrimental. In practice it seems that cultures selected in this way do have high yields but this does not eliminate the possibility of selecting against high-yielding organisms that have high qmaxand low K, values. For a turbidostat culture, s > > K,; therefore, Eq. (8) simplifies to:

Therefore, a turbidostat culture selects for:

1. Highq,, 2. High YG 3. Lowm Thus, although selection pressure for maximum growth rate is high, if organisms have similar qmaxvalues, the one with higher yield is favored. From the above considerations, it can be predicted that if a chemostat or a turbidostat culture is run for sufficient length of time, with only one source of carbon and energy, then eventually one organism must replace all others, as the likelihood of two or more organisms having identical values of pmm,K,; YG, and m must be small. This would apply for all cases where the organisms are in competition for the same substrate.

E. PRACTICE In the course of selecting cultures suitable for production of SCP by means of continuous enrichment, in no case has it been reported that a pure culture

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141

was selected even after many thousand hours of cultivation (Malashenko et

al., 1971; Harrison, 1976; Sheehan and Johnson, 1971). Instead, cultures develop which usually consist of one organism which can utilize the primary, supplied carbon source and a number of other, nonmethylotrophic organisms, obviously living in symbiosis with the primary methylotroph (Malashenko et al., 1973; Harrison et al., 1976). When attempts were made to isolate the methylotroph and grow it in pure culture, the performance in terms of growth rate, yield, and culture stability was invariably poor compared to that of the enriched mixtures (Sheehan and Johnson, 1971; Harrison et al., 1976). In the case of methane-grown cultures, it was often very difficult or impossible to grow the methane utilizer in pure culture at all (Malashenko and Romanovskaya, 1973; Wilkinson et al., 1974; Harrison et

al., 1976). V. Mixed Cultures for Single-Cell Protein Production A. RATIONALE

The use of mixed microbial cultures to produce food is by no means novel; such traditional fermented foodstuffs as cheese, yoghurt, sauerkraut, and soy sauce are products of the concerted action of several microbial species. However, it was commonly held that to obtain the consistency of control required for the acceptance of any new food or animal feed product, it would be necessary to use pure (mono-) culture systems. This prejudice was, in fact, implicit in early attempts to frame international guidelines for single-cell protein (SCP) production. If the only alternative to pure cultures were a completely undefined and uncontrolled mixture, then arguments for monocultures would be hard to refute on the basis of safety and consistency of the product. However, a new aspect of the approach employed in the SCP process developed at the Shell Laboratories in England was that, although a mixture of microorganisms would be used, the culture would be completely defined in terms of species present and the quality of the product could be controlled. The term “structured mixed cultures” was used to denote the fact that the cultures were obtained by careful recombination of species rather than by crude enrichments. There was a real incentive to develop mixed culture technology for SCP production from natural gas because the maintenance of pure continuous cultures on these substrates had proved troublesome, while mixed cultures were easily maintained (Malashenko et al., 1970; Sheehan and Johnson, 1971). Moreover, natural gas is a mixture of substrates for, while the main component is methane, ethane, propane, and butane may also be present in significant quantities. A multicomponent culture may be expected to better exploit such a mixture of substrates. Work on mixed cultures as a basis for

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DAVID E. F. HARRISON

SCP production from natural gas has been carried out in the United States (Vary and Johnson, 1967; Sheehan and Johnson, 1971), in the USSR (Malashenko et al., 1970, 1973; Malashenko and Romanovskaya, 1973), and in the United Kingdom (Wilkinson et al., 1972; Harrison et al., 1976; Harrison and Wren, 1977; Barnes et al., 1976). As the technology required for SCP production is one of the most advanced among modern industrial fermentations, it is perhaps not surprising that the use of mixed cultures for the production of biomass from natural gas and its derivatives has been studied in some depth. These studies provide a good illustration of the principles of defined mixed cultures, and so the following account looks in some detail at some of this work.

B. EXAMPLES 1 . Methane-Utilizing Mixture ( i ) (Obligate Symbiosis)

This culture was originally isolated by Hamer et al. (1967) in batch culture and extensively studied in continuous culture by Wilkinson (1972). The mixture, which was extremely stable, consisted of just four organisms: a methane-utilizing gram-negative, rod-shaped organism; a Hyphomicrobium sp. capable of growth on methanol in minimal media but incapable of growth on methane; and two gram-negative rod-shaped organisms incapable of growth on methane or methanol but which grew on a wide range of complex organic media. These latter two were identified as an Acinetobacter sp. and a Flavobacteria sp. The methane-utilizing bacterium was isolated on minimal medium agar in a methane-air atmosphere but did not grow at all in liquid medium bubbled with methane-air mixtures unless the Hyphomicrobium sp. was also present (Wilkinson, 1972). To obtain growth rates and cell densities equal to that of the crude enrichment, all four organisms were required to be present. Attempts to grow the methane-utilizing bacterium in shaken-flask cultures on medium containingthe spent liquor from a culture ofHyphomicrobium grown on methanol failed. This suggested that the Hyphomicrobium was not supplying an essential growth nutrient but rather removing an inhibitory substance. In fact, hyphomicrobia have frequently been found in enrichment cultures for methane-utilizing organisms although they canot utilize methane directly themselves (Harder and Attwood, 1975). As the Hyphomicrobium sp. obviously played an important role in the mixed culture, this organism was studied in more detail (Wilkinson and Hamer, 1971). This organism demonstrated a maximum specific growth rate of 0.17 per hour under fully aerobic conditions. The yield coefficient expressed on an oxygen basis @,) was found to increase when the dissolved oxygen tension fell below 30 mmHg. (Wilkinson and Hamer, 1971). This is

143

MIXED CULTURES IN INDUSTRIAL FERMENTATION

attributable to a switch from oxygen to nitrate as the electron acceptor for methanol oxidation. Nitrate reductase activity is a characteristic of hyphomicrobia species (Attwood and Harder, 1972). This ability of Hyphomicrobium to use nitrate rather than oxygen as electron acceptor even at quite high oxygen tensions is of particular significance to its role in mixed cultures. Methane utilization is a highly aerobic process and the utilization of nitrate rather than oxygen at low dissolved oxygen tensions by Hyphomicrobium would prevent this organism's competing for oxygen with the primary methane utilizer. Wilkinson et al. (1974) reported results of steady states for the complete mixed culture grown in continuous cultures supplied methane as the sole carbon and energy source. Table I summarizes the results. Under all conditions the methane-utilizing bacterium constituted over 90% of the total cell numbers present, although the actual numbers varied between oxygen and methane-limited conditions. Furthermore, as the cells of the methaneutilizing organism were considerably larger than those of the other symbionts, it was estimated that possibly 99% of the biomass was derived from this one organism. The oxidation route for methane in bacteria is generally accepted as being: CH,

+ CH,OH

+ HCOH + HCOOH

+C02

Cell mass

Thus methanol is an intermediate and, in fact, has been shown to accumulate in pure cultures of methane-utilizing organisms (Higgins and Quayle, 1970). Therefore, Wilkinson et al. (1974)considered methanol to be the most likely inhibitory substance produced by the methane-utilizing bacterium and removed by the Hyphomicrobium. When small amounts of methanol were TABLE I RELATIVECELL COUNTSOF SPECIESCOMPRISING A MIXED BACTERIAL CULTUREGROWNIN CHEMOSTAT CULTUREON METHANEAS SOLECARBONSOURCE(AFTER WILKINSONet a l . , 1974) Percent of population"

Limiting substrate

Dilution rate (per hour)

Methane-utilizing bacterium

Hyphomiwobium SP.

Acinetobacter sp. plus Flawbacterium sp.

Oxygen Methane

0.344). 15 0.0344.12

94.2 90

4.7 8.8

1.1 1.2

"Numbers of methane-utilizing bacteria were estimated by microscopic counts, those of Hyphomicrobiurn and heterotrophic bacteria by agar plate technique.

144

DAVID E. F. HARRISON

added to continuous cultures of the mixed population growing on methane, methane utilization was immediately inhibited and the culture began to wash out. During this washout period, however, the cell count of the Hyphomicrobium increased although that of the methane utilizer fell rapidly. The Hyphomicrobium grew at the expense of the methanol and, after about 20 hours, when the methanol concentration had fallen to below 0.16 mM (0.005 gm/liter), methane oxidation recommenced and the culture regained its original steady state. Experiments of this type demonstrated that methane oxidation was inhibited completely by methanol concentrations as low as 1 mM and that the Hyphomia-obium sp. could remove methanol added to a mixed culture. However, if the Hyphomicrobium sp. is to grow by scavenging methanol produced by the primary methane-oxidizing organism, then it must be able to use methanol at the concentrations likely to accumulate from methane oxidation. To demonstrate this, Wilkinson and Harrison (1973)estimated the affinity for methanol of both a pure culture of Hyphomicrobium sp. and the methane-utilizing mixed culture, in which the methane-utilizing organism was predominent. The results are shown in Table 11. The Hyphomicrobium sp. was found to demonstrate a remarkably low value of K , for methanol, 8 /.LM compared with 20 /.LM for other methanol-utilizing bacteria (Harrison, 1973). The methane-utilizing mixed culture demonstrated a two-phase Lineweaver-Burke plot in response to methanol (Table 11)with one K , of2.9 mM and V max of 24 /.LM hour and another with a lower V max of 6.6 /.LM/hour and a much lower K,<300 mM. It is reasonable to assume that the higher K, value appertains to the predominant organism of the mixture, the methaneTABLE I1 VALUES O F

K,

AND

pmax FOR METHANEAND METHANOLOXIDATIOP BY

MIXED CULTURE GROWNON METHANE AND A

A

PURE CULTURE OF THE

METHANOL-UTILIZINGMOIETY, Hyphorniwobium WILKINSON AND HARRISON, 1973)

SP. (AFTER

Substrate

Mixed culture

Kn(mM) vmax

(Mkmhour)

Hyphomicrobium sp.

Methane

Methanol

Methanol

0.019

2.9 (c0.3") 24.0 (6.6")

0.008

4.0

21.5

=A two-phase Lineweaver-Burke plot was obtained. These values represent the second linear portion.

MIXED CULTURES IN INDUSTRIAL FERMENTATION

145

utilizing pseudomonad. From these results it would be expected that the methane-utilizing organism, growing on methane, would accumulate methanol to a level of about 1 mM sufficient to inhibit methane oxidation. The high affinity of the Hyphomicrobium sp. for methanol would enable it to scavenge the methanol down to a level of a few micromolar, thus relieving inhibition of methane oxidation. The roles of the two heterotrophic organisms in the culture were not defined but it was suggested that they served to remove complex products of growth and lysis (Wilkinson et al., 1974). This mixed culture is a good example of obligate symbiosis. It seems that the methane-oxidizing bacterium is quite incapable of growing in liquid medium in the absence of a methanol utilizer: Methane oxidation appears to be its sole source of energy but methane oxidation of necessity leads to the organism’s poisoning itselfwith methanol unless this is removed by a second organism. The Hyphomicrobium sp. is not obligately symbiotic but is ideally suited for its role in this relationship, being able to scavenge methanol at very low concentrations and, furthermore, under oxygen-limited conditions, to utilize nitrate as an electron acceptor, thus not competing for oxygen with the methane-utilizing organism. A culture based on similar trophic relationships of a methane oxidizer, methanol utilizer, and heterotrophic bacteria was described by Namsaraev and Zavarzin (1972).

2. Methanol-Utilizing Mixed Culture: Synergism By means of continuous enrichment of a mud sample on methanol (Harrison et al., 1974), an extremely stable mixed culture was obtained which did not alter in constituent organisms over many thousands of hours of cultivation (Harrison et al., 1976). The stable mixture was found to consist of only one methanol-utilizing bacterium, an obligate methylotroph designed E N because it was a new species, and four other bacteria which would not grow on, or oxidize, methanol but which used a variety of other substrates (Table

111). TABLE I11 ORGANISMS COMPRISING A

MIXED CULTURE GROWNON METHANOL(HARRISON et d., 1976)

Organism

NCIB No.

Colony type

Growth substrates

1. Pseudvmonas sp. 2. Curtobacterium sp. 3. Pseudvmonas sp. 4. Aciyetvbacter sp. 5. EN

11019 11021 11022 11020 11040

Mucoid Flat, grey Yellow Grey Entire edges white

Heterotrophic Heterotrophic Heterotrophic Heterotrophic Obligate methanol utilizer

146

DAVID E. F. HARRISON

The primary organism, EN, unlike the previous example, was not an obligate symbiont but grew readily in pure culture, but with lower maximum growth rates and yield coefficients than were obtained for the complete mixture (Table IV). Adding just one of the heterotrophic organisms improved the growth of the culture but the complete mixture was required to maximize growth rate and yield (Table IV). One method used to investigate the trophic relationships between the organisms was to grow the different species in chambers separated by a dialysis membrane (Harrison et al., 1976). Each chamber was fitted with 25 ml of mineral salts medium and 0.2%methanol and then inoculated with a pure culture of the methanol-utilizing organism EN or a mixture of the nonmethylotrophic organisms, or it was uninoculated. The results obtained after incubation on an orbital shaker at 42°C (Table V) show that growth of organism EN only occurs when it is grown in contact with the mixture of nonmethylotrophic organisms, indicating either that some small, dialyzable molecule(s) is (are) produced by the nonmethylotrophs which promotes growth of the organism EN or that a dialyzable toxic substance has been removed. Further, it is obvious that at least one of the nonmethylotrophic organisms can grow on a dialyzable product of the methylotroph. The products of the methylotroph have recently been identified (Wren, 1978) and are shown in Table VI. Besides these products, the supernatent of a pure culture TABLE IV YIELD COEFFICIENT O F A PURE CULTURE OF ORGANISM EN AND WITH MIXTURESCONTAINING (A) O N E AND (B) ALL T H E HETEROTROPHIC SYMBIONTSFROM AN ENRICHED MIXEDCULTURE (AFTER HARRISONAND W R E N , 1976)

EFFECTO F

GROWTH RATE ON

Yield coefficient (gm dry biomasdgm methanol)" Dilution

rate (per hour)

0.06 0.10 0.16 0.19 0.24 0.55 0.64 0.66

(1) Pure culture

of EN N.D. 0.29 0.30 Washed out

Complete mixture

0.35 N.D. 0.35 0.35

0.31 N.D. 0.46

Washed out

N.D. 0.43 0.45 0.54 Washed out

0.16 ~~

Organism EN plus Curtobacterium sp.

~~

~~

"N.D. = not determined.

0.19

0.64

MIXED CULTURES IN INDUSTRIAL FERMENTATION

147

TABLE V EXPERIMENT TO INVESTIGATE THE INTERDEPENDENCE OF ORGANISMS COMPRISING A METHANOL-UTILIZING MIXEDCULTURE (AFTER HARRISON AND WREN, 1976)" Experiment 1. Inoculum

final o.d. 2. Inoculum final o.d. 3. Inoculum final o.d.

Chamber 1

Chamber 2

Organism EN

Four heterotrophic species (see Table IV)

2.05

0.55

Organism EN 1.70 None 0.0

None 0.0 Four heterotrophic species 0.06

"Twogrowth chambers separated by a dialysis membrane were both charged with a mineral salts medium to which was added 2.0 gmlliter methanol, and inoculated as indicated. The culture optical density was measured after incubation on a shaker at 42°C for 48 hours.

E N has been found to contain nondialyzable substances, such as proteins and nucleic acids, which are also removed during growth of the complete mixture (Table VI). Studies on the population of the complete mixture revealed a remarkable stability. Table VII shows the effect of changing growth rate (dilution rate) on the relative numbers of the various species in the mixture grown methanol limited in chemostat culture. Steady states were maintained for at least 2 days at each dilution rate and the culture was maintained, in total, over 200 hours. At dilution rates below 0.6 per hour the primary methanol-utilizing bacterium comprised at least 90% of organisms present. As with the mixed methane culture this organism was the largest organism of the mixture SO that of the resulting biomass, more than 90% and probably more than 95% was derived from the one species. At a dilution rate of 0.6 per hour the methanol-utilizing organism comprised only 64% of the culture, but this dilution rate was close to the washout value and wall growth may have contributed significantly to the population (Topiwala and Hamer, 1971). 3. Methane-Utilizing Mixture (ii)

The third example of a mixed methylotrophic culture is of interest because it provides an example of yet another type of trophic relationship. In contrast to the first sample, discussed above, in this mixture the primary methylotrophic organism can be grown in pure culture, and unlike the second example, the yield coefficient and maximum growth rate is not greatly improved by growth of the complete mixture. The primary advantage in his case lies with the stability of the culture and lack of foaming. This mixture was isolated from a tropical duck pond using continuous flow enrichment in

148

DAVID E. F. HARRISON

ANALYSIS OF

TABLE VI CULTURE SUPERNATANTOF METHANOL-

LIMITEDCHEMOSTAT CULTURES OF THE OBLIGATE METHANOL-UTILIZING BACTERIUM NCIB 11040 IN PURE AND MIXED CULTURES (DATA OF WREN, 1978) Pure culture

Mixed culture

Dilution rate (per hour)

0.08

0.34

0.20

0.44

Total culture carbon (gm/liter)

0.85

0.85

0.86

0.86

Supernatant (gm/liter) Total carbon Dialyzable carbon Protein Nucleic acid Lactic acid Pyruvic acid Formaldehyde Formate

0.15

0.06 0.10

0.05 0.21

0.007 o.oo00 N.D.“

0.14 0.08 0.06 0.03 0.011 0.006 o.oO03 N.D.O

“N.D. = not detectable.

chemostat culture with a basal salts medium and methane as the sole source of carbon and energy. The culture, as originally isolated, included two methane-utilizing bacteria (Methylococcus sp. NClB 11083) and eight nonmethylotrophic bacteria. This was reconstituted to form a mixture comprised of one methylotrophic organism, a Methylococcus type, and four heterotrophic organisms identified as: Pseudomonas sp. NClB 11310, Mycobacterium sp. NClB 11307, Moroxella sp. NClB 11038, and Pseudomonas sp. NBlB 11309 (Harrison et a l . , 1976). Details of this and similar mixtures have been published (Harrison et al., 1974, 1976; Harrison, 1976). The main methylotrophic organism, Methylococcus sp. in common with many isolated from mixed cultures, grew very poorly on solid medium and special techniques had to be used to enumerate the cells. These consist of spreading the organisms over a lawn of the nonmethylotrophic organisms on basal salt agar and incubating in an atmosphere of methane-air (D. E. F. Harrison and H. Doddema, unpublished; Malashenko and Romanovskaya, 1973). Continuous cultures of the mixture, under optimal growth conditions, gave yield coefficients of 0.85 gm celldgm methane and grew at specific growth rates of up to 0.25 per hour at a temperature of 45°C. Pure cultures of the Methylococcus sp. were grown at growth rates up to 0.2 per hour (Linton and Buckee, 1977) but foamed copiously and were remarkably less stable

149

MIXED CULTURES IN INDUSTRIAL FERMENTATION

TABLE VII RELATIVE POPULATIONS OF THE CONSTITUTIVE ORGANISMS OF A METHANOL-UTILIZING MIXEDCULTUREGROWNIN CHEMOSTAT CULTURE(FROM HARRISON AND WREN, 1976) Cell numbers ( x Dilution rate (per hour)

0.12 0.20 0.32 0.45 0.55 0.60

Organism

EN 7.5 4.0 6.0

5.0 5.5 0.25

Curtobacterium SP. 11021 0.15 0.05 0.05 0.04 N.D. 0.04

rnlliter)=

Pseudomonas sp. 11019

Pseudomoms sp. 11022

0.20 0.15 0.10 0.05 0.03 0.05

0.30 0.18 0.15 0.15 0.05 0.05

aCell counts were obtained from agar spread plates. The methanol-utilizing organism was counted using minimal agar plus 1% methanol and the other organisms on lab lemco agar.

than the mixture, washout occurring if the culture steady state were perturbed for even short periods of time. Productivities of up to 9 gmtliterhour were obtained (Harrison, 1976) with the complete mixture, while in pure cultures productivities greater than 1 gm/liter/hour were not maintained over long periods. Studies of the trophic relationships in this culture were reported by Linton and Buckee (1977). It was found that the concentration of carbon in the supernatent of the pure culture was greater than that of the mixture, especially at low growth rates (Table VIII). Analysis of the organic material in the culture supernatent failed to detect any methanol, but protein and nucleic acid were detected (Table VIII). In fact, the protein content of the organic matter in the supernatent was similar to that of harvested cells, which led to the speculation that the supernatent organic matter was derived largely from cell autolysis. This was further confirmed by comparing the protein bands obtained by SDS -polyacrilamide gel electrophoresis of culture supernatent and cell-free extracts of whole bacteria. These indicated a similar range of proteins. As neither the proteins nor the nucleic acids were detected in culture supernatent of the mixture, it seemed likely that the heterotrophic species present were able to utilize the cell lysis products of the primary methane utilizer. To investigate this, Linton and Buckee (1977) examined the range of extracellular enzyme activity produced by the constitutive species of the mixture. These are summarized in Table IX. It can be seen that between them the four organisms possessed the ability to utilize all the major organic compounds present in the culture supernatent. These results, depicted in Table IX, are incomplete as they do not include organic acids or

TABLE VIII MATTER IN CULTURE SUPERNATANTS OF A MONOCULTURE AND A MIXEDCULTURE GROWN CHEMOSTAT WITH METHANEAS SOLE CARBON SOURCE (AFTER LINTONAND BUCKEE, 1977)

THE CONCENTRATION IN A

OF ORGANIC

Supernatant carbon (gm/liter)

Ratio of supernatant carbon to bacterial carbon

0.36

0.28 0.16

Culture

Dilution rate (per hour)

Monoculture of Methylococcus sp.

0.08 0.20

Mixed culture

0.08

0.12

0.20

0.12

Supernatant protein (gm/lOo gm organic matter)

Supernatant nucleic acid (gml100 gm organic matter)

40

4 8

~~

"Not reported.

0.18

0.10 0.11

55 u

-

-

151

MIXED CULTURES IN INDUSTRIAL FERMENTATION

TABLE IX EXTRACELLULAR ENZYMEACTIVITYOF ORGANISMS COMPRISINGA RECONSTITUTED METHANE-UTILIZING MIXED CULTURE (AFTER LINTONAND BUCKEE,1977) Extracellular enzyme activity Protease (neutrd)

Organism

-

Methylococcus sp. Pseudomonas sp. NCIB 11308 Pseudomonas sp. NCIB 11309 Pseudomonas sp. NCIB 11310 Mycobacterium sp.

+

+ -

NCIB 11:307 Complete mixture

+

DNAse

RNAse

Lipase

Peptidase

-

-

-

-

+ +

-

-

+

+ + +

-

-

-

+ +

+ + + +

-

+

carbohydrates released by cell autolysis. However, it is clear that the presence of the four heterotrophic bacteria allows the lytic products of the methylotrophic organism to be degraded to monomers which can then support growth of the component species. Foaming of the pure culture might thus be attributed to lytic products of the cell. Amino acids inhibit the growth of methane-utilizing bacteria (Eccleston and Kelly, 1972; Eroshin et al., 1968), so that extensive cell lysis may be expected to cause instability in the pure culture which can be prevented in TABLE X THE EFFECTOF ADDEDAMINO ACIDS ON GROWTHIN PUREAND MIXEDCULTURESUTILIZING METHANE AS SOLE CARBON AND ENERGYSOURCE (AFTER MALASHENKO et d . , 1974) Organisms" Methylomonus methanica (1") (pure culture)

M . ucruinicus

M . nethunica

Rh. glutinis

P . fluoresccns

p-Alanine

++ +

DL-Aspartic DL-Clutamic DL-Methionine L-Cysteine DL-Tryptop han None

+ ++ ++ ++

+ + ++ + ++

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

Amino acid (added at 0.01%)

Methylococcus ucrainicus (159) (pure culture)

Glycine

aKey: -, no growth;

+

+, satisfactory growth; ++, good growth.

+

152

DAVID E. F. HARRISON

the complete mixture. Instability in pure cultures of autotrophic and methylotrophic organisms appears to be common and may reflect the inability of such organisms to produce the necessary extracellular enzymes to deal with their own lysis products. Malashenko et al. (1974) also tested the growth of a number of methylotrophic bacteria in the presence of a number of organic molecules in pure and “artificial” mixed cultures. Some of the results are shown in Table X. It can be seen that the mixed culture is less sensitive than the pure culture to most of the compounds tested. Thus, improved stability appears to be a characteristic of mixed cultures grown on natural gas.

4 . Other Exampks Other mixed cultures for SCP production have been described. It was suggested by T. L. Miller et al. (1964) that mixed cultures might be advantageous for producing SCP from n-alkanes and a Japanese patent (Kanegafuchi et al., 1972) describes the use of mixed yeast cultures for this purpose. In this system the vitamin requirement of yeasts was overcome by growing a combination of species. By culturing biotin-requiring yeasts, such as Candida novellus, C . tropicalis, or Pichia sake, with B,requiring species, such as Trichosporon pululans or C . lypolytica, the inventors claimed that good growth could be obtained in simple media without any added vitamins, the yeast species presumably supplying each other’s vitamin requirements. Mixtures gave improved yields and productivities over the pure cultures and lower concentrations of oxidizable substances in the culture supernatent. Furthermore, the numbers of contaminating bacteria were reduced by up to Another application of defined yeast cultures for SCP production was described by Akaki (1965),who tested various combinations of seven strains of yeast for growth on sulfite waste liquor. The highest sugar consumptions and yields obtained were with a mixture of Candida utilis and a Mycotorula sp. Akaki (1965)found that culture filtrate ofC. utilis enhanced the growth of Mycotomla sp. when added to the growth medium but no enhancement of C. utilis growth was obtained fiom Mycotorula sp. supernatent. He also carried out dialysis*ulture experiments which gave similar results, a dialyzable product of C. utilis stimulating growth of Mycotorula sp. C. ADVANTAGESOF MIXED CULTURES FOR SCP PRODUCTION

1 . Yield In the second example above, the mixed culture grown on methanol gave a substantial improvement in yield over the pure culture (Table IV), which

MIXED CULTURES IN INDUSTRIAL. FERMENTATION

153

was too large to be accounted for by the conversion of extracellular products to cell mass by the nonmethylotroph. However, in more recent work with this culture Wren (1978)obtained much higher yields with the pure culture (0.46).The improvement was possibly due to adaptation of the organism to pure-culture growth and better optimization of growth conditions. Other pure cultures of methanol-utilizing organisms have been reported to give yield coefficients approaching those obtained in the mixed cultures discussed here (Chalfan and Mateles, 1972; Mateles and Battat, 1974), while other workers have obtained diminished yields in mixed cultures compared with monocultures (Haggstrom, 1969; Namsaraev and Zavarzin, 1972). However, these low-yielding mixtures were obtained via batch enrichment, not continuous selection. Nonetheless, as pointed out by Goldberg (1977), high yields are not necessarily a unique attribute of mixed cultures. More modest yield improvements can be predicted from the conversion into biomass of organic matter leaking from the cells of the primary substrate utilizer into the culture supernatent. In the case of methanol- and methaneutilizing monocultures, up to 10% of the methanol carbon supplied may appear in the supernatent as organic matter (Goldberg, 1977; Linton and Buckee, 1977). Assuming the heterotrophic organisms convert this to biomass with a carbon conversion efficiency of 60%, then if the yield from methanol for the monoculture is 0.45,the mixture would improve the yield by 11% to 0.50. Certainly a yield improvement of this magnitude would make a significant saving in the cost of the SCP (Harrison et al., 1972). Improved yield coefficients for mixed cultures over pure cultures have also been claimed for yeasts growing on n-alkanes (Kanegafuchi et al., 1971) and a sulfite waste liquor (Akaki, 1965).

2 . Growth Rate As discussed above, there are many cases of methane-utilizing organisms which grow very poorly or not at all in pure cultures (Wilkinson et al., 1974; Harrison et al., 1974; Malashenko and Romanovskaya, 1973). These are not available for exploitation other than by growth in mixed cultures. Other organisms, such as organism EN grown on methanol (Harrison et al., 1976), only attain their maximum growth rate when grown in mixed culture. This is not to say that mixed cultures are always necessary to achieve high growth rates in continuous culture on C , compounds. Goldberg (1977) quotes growth rates of pure cultures as high as 0.49 per hour. Growth rate is, in any case, not of primary importance in SCP production since productivity is almost certainly to be limited by gas transfer rate. Productivity (W) (gm/ literhour) in continuous culture is given by:

w = Df

154

DAVID E. F. HARRISON

where D = dilution rate and f = steady-state organism concentration. Thus maximum productivity can be achieved by increasing the cell concentration rather than by increasing growth rate (= D). 3 . Stability

A characteristic of pure cultures of methane-utilizing bacteria is a tendency for instability in continuous culture, particularly at high cell densities (> 5 gm/liter) and high growth rates (> 0.2 per hour) (Sheehan and Johnson, 1971). In fact, reports of productivities greater than 1 gm/liter/hour in methane- or natural gas-grown cultures all seem to refer to mixed cultures (Vary and Johnson, 1967; Sheehan and Johnson, 1971; Harrison et al., 1976; Harrison, 1976). The reason for the instability of pure cultures of methaneoxidizing cultures is not clear. Certainly they have a tendency to enter an encysted or resting phase (Whittenbury et al., 1970). Methane-utilizing organisms are known to be inhibited by amino acids (Ecclestone and Kelly, 1972; Eroshin et al., 1968; Malashenko et al., 1974) and, as has been seen above, there is a tendency in pure cultures for lysis products to accumulate. These may accumulate to toxic levels when cultures are grown at high cell densities and also when growth is perturbed in any way, e.g., by interruption of methane supply. Mixed cultures recover much more readily from perturbations in the culture than do pure cultures (Harrison et al., 1976). Methanol accumulation was shown to be the cause of instability in the culture studied by Wilkinson et al. (1974)but was not the cause of instability in the culture studied by Linton and Buckee (1977). A more rapid recovery of mixed cultures has been the experience of workers using mixed cultures. Mixed cultures might be expected to recover more quickly from adverse perturbations in growth conditions than pure cultures owing to the ability of the heterotrophic symbionts in mixtures to scavenge organic molecules released by the primary substrate utlizer when the culture was subjected to suboptimal growth conditions or during lysis. For any large-scale production of SCP it is most probable that the process water will have to be recycled. Considerable cost savings will accrue if greater than 90% recycle of process water can be achieved. A severe problem posed by water recycle to a continuous culture is that there is likely to be an accumulation of extracellular metabolites which are normally produced at very low concentrations. For instance, consider a culture supplied 20 gm/ liter methanol, in which I% of the methanol carbon is released from the cell as an amino acid which is not reused by the methanol utilizer. Without water recycle the concentration of the amino acid would be about 0.15 gm/liter but with 90% recycle of water this would increase to 1.5 gm/liter. While the lower concentration might be harmless to the culture, the higher concentration might interfere with organism growth. In mixed cultures accumulation of metabolites is prevented by the scavenging action of heterotrophic or-

MIXED CULTURES IN INDUSTRIAL FERMENTATION

155

ganisms. Thus, increased stability can be achieved in mixed cultures, even with water recycle, which enables higher productivities to be attained in methane- and methanol-utilizing cultures than can be reached by pure cultures. 4 . Foam Prevention

Antifoam addition is to be avoided in SCP production for the obvious reason that it creates yet another possible toxic hazard. The cause of foaming in cultures is not altogether well understood but it is generally thought to be due to release of organic molecules from cells rather than the cells themselves. In the three cultures described above, foaming was virtually absent during well-balanced growth of the mixtures. Pure cultures of methanol and methane utilizers, however, were found to foam copiously and were difficult to maintain in steady states without addition of chemical antifoams (Barnes et al., 1976). That the mixed culture did not foam is not surprising, considering the low concentrations of organic matter in the culture supernatent compared with the pure culture. Possibly removal of proteins and peptides released by cell lysis of the primary C, utilizer prevented foaming.

5 . Resistance to Contamination Monocultures grown on methane or methanol, in common with virtually all pure cultures, are susceptible to contamination. Contaminants were commonly gram-negative heterotrophic bacteria which fed on extracellular organic material produced by the primary C1 utilizer. In a well-balanced mixed culture these organic molecules would be already utilized by the heterotrophic moieties of the mixture. Thus, in order to establish itself a contaminant must compete with the heterotrophic constituents for the low levels of nutrients present in the culture. The methanol-utilizing mixed culture described by Harrison et al. (1976) was grown for several thousand hours in continuous culture without sterilization of the media supply. During this time no contaminant established itself in the culture, the only organisms being found at the end of the experiment being those shown in Table 111. Mixed cultures of yeasts have also been found to support less contaminating bacteria than did pure cultures (Kanegafuchi et al., 1972). Resistance to contamination is clearly a significant advantage, for if an unknown organism establishes itself in an SCP culture, the product must be held suspect until the harmlessness of the contaminant is established. In reality any contaminant occurring in significant numbers will probably lead to the rejection of the entire batch of product. 6. Use of Mixed Substrates

The cheapest available substrates for SCP production are not likely to be of the highest purity. Methanol and n-alkanes may be exceptional as being

156

DAVID E. F. HARRISON

readily available carbon sources which are in a reasonably pure state. Certainly methane does not occur in a pure state but as natural gas which contains up to 25%ethane and also butane and propane. Mixed cultures offer the possibility of more stable growth on these (Malashenko et al., 1973). Waste as sources of substrate for SCP will undoubtedly consist of a mixture of substrates. For mixtures of substrates there is a clear advantage in using a tailored mixed culture which can more completely use all the organic material present. Also, the use of cellulose as a basis of biomass production is likely to require the use of mixed populations. Cellulytic molds might be grown in symbiosis with starch- and sugar-utilizing yeasts or bacteria to obtain the most rapid conversions.

7. Vitamin Supply Many microorganisms which are otherwise suited for SCP production require growth factors in order to attain maximum growth rate and efEciency. This would seem to be true of the methylotrophic organisms in the first and second examples above. Kuono et al. (1973)isolated many strains of the methanol-utilizing bacterium Methylomonas methylovora which required high concentrations of thiamine for growth. To supply vitamins in growth media is an added complication and expense in large-scale SCP production SO that the addition of symbiotic species which provide the required growth factors is of definite advantage. This forms the basis of a patent for the production of yeast from n-alkanes.

D. PROBLEMS RELATED TO THE USE OF MIXED CULTURES The use of mixed cultures for SCP production is not necessarily an easy option. A completely uncontrolled and unidentified mixed culture would probably be quite unacceptable as a basis for a product to be sold on a scale of megatons for feeding either animals or humans. IUPAC guidelines have been produced which state that cultures used for SCP production should be clearly identified. This in no way precludes the use of mixed cultures, for as we have seen in the examples described above, these can be as reproducible and stable as monocultures. There is no reason why mixed cultures cannot be exactly defined in terms of the constituent organisms. However, mixed cultures do add another dimension to the identification and characterization of the culture. Apart from the fact that there are simply more organisms to characterise, some of these organisms may be obligate symbionts and thus difficult to grow in pure cultures. New techniques have to be developed to deal with these. Also it has been found that many of the heterotrophic

MIXED CULTURES IN INDUSTRIAL FERMENTATION

157

symbionts present in mixed cultures isolated on methane and methanol do not fit any previously described organism and are species new to microbiology. It seems that there may be a vast number of symbiotic organisms which are not isolated by normal enrichment and isolation techniques and remain to be discovered. For the purposes of defining and, let it be said, patenting commercial processes it is necessary to give a name to microorganisms used. This has often proved difficult for constituents of a symbiotic culture. Although mixed cultures are more resistant to contamination than monocultures it would still be necessary to apply rigorous checks to a commercial process for contaminants. Again this is bound to be made more difficult simply by the greater number of species present. Similarly, product control may be somewhat more complicated by the fact that the product is derived from five rather than one species, although it has been shown that over 90% of the product is likely to consist of one organism. The relative populations of the constituent organisms in a mixture has been demonstrated to be very constant and so there is no reason why the use of mixed cultures should lead to any greater product variability than the use of monocultures. Disadvantages associated with microbiological control can all be overcome but involve a greater amount of microbiological checks. These are certainly not outside our present technical expertise and, with the increasing automation of techniques in this field, will probably not lead to greatly increased costs.

VI. Other Applications of Mixed Cultures A. VITAMIN Blz PRODUCTION Some early papers on the production of vitamin BIZdescribed the use of mixed cultures. Hodge et al. (1952) isolated a mixture which accumulated vitamin BIZconsisting of a Pseudomonas sp., Streptococcus bouis, Proteus vulgaris, and Clostridium pdringens. They went on to isolate the components and found that good yield of B12could be obtained with a mixture of the Pseudomonas sp. and P . vulgaris (0.32 arbitrary units), although pure cultures of those two species gave much reduced yields (0.03 and 0.13 arbitrary units, respectively). Another mixed culture for the production of BIZ was described by Leviton and Hargrove (1952). In this case a mixture was deliberately synthesized &om Lactobacillus casei and Propionibacterium freundenreichii for the production of BIZfrom whey. The Lactobacillus converted the lactose to lactate, which then served as substrate for the Propionibacterium, which produced the vitamin. More recently Tanaka et al. (1974) reported the production of vitamin BIZby methanol assimilation in a

158

DAVID E. F. HARRISON

mixed culture of bacteria. The major methanol-utilizing organism in this mixture was a Protaminobacter ruber. Mixed cultures are presently employed in Hungary for the commercial-scale production of vitamin B12 from methanol (D. Perlman, personal communication). €3. PRODUCTION OF ORGANIC ACIDS

Acetic acid production by fermentation has always been an “unprotected,” i.e., nonaseptic process and acetifiers no doubt sustain a very mixed population. The interrelationship of these and whether they are beneficial or harmful to the process does not seem to have been studied. A most interesting suggested use of a mixed culture is for the conversion of a fumaric acid fermentation to a succinic acid fermentation (Sasaki et al., 1970a). These workers took as their starting point the production of fumarate by Rhizopus chinensis. They screened 17 species of Enterobacteriaciae for the production of succinate from fumarate, adding 1%glucose to promote the reduction step. Finally, by adding Escherichia coli to the Rhizopus culture after 7 days, they could get complete conversion of fumaric acid to succinic acid (Sasakiet al., 1970b). Alternatively, the fumarate could be converted to L-malate by the addition of the yeast Pichia membranaefaciens to the Rhizopus culture in place of E. coli. Processes have also been described for the production of a-ketoglutaric acid using mixed populations (Sanada et al., 1960)and also for production of the amino acids glutamic (Yamatodaniet al., 1967)and alanine (Ozaki et al., 1962).

C. METAL LEACHING The recovery of metals, such as copper and nickel, from low-grade ores by means of dump leaching is certain to rapidly grow in importance as world resources dwindle. That bacteria are important in this process is now well established. The organisms involved in the oxidation of insoluble metal sulfides during leaching all belong to the genus Thiobacillus. In modeling leaching systems in the laboratory, investigators generally assume that the system resembles a pure culture of Thiobacillusfen-oxidans or Thwbacillus thwoxidans (Jones and Kelly, 1976), although the open, uncontrolled nature of leaching dumps makes it virtually inevitable that other organisms will become established (Ehrlich and Fox, 1967). These other species must be dependent upon the Thiobacillus as no other source of energy for heterotrophic growth is present in the dump. Whether these symbionts are harmful or beneficial to the process does not appear to have been shown. However, it is possible that Thwbacillus depends on a symbiotic association for supply of

MIXED CULTURES I N INDUSTRIAL FERMENTATION

159

TABLE XI COMPARISON OF LEACHING OF COPPERAND NICKEL BY PURECULTUREOF Thiobacillus ferrooxidrrns WITH A MIXTUREOF T . fmooridans AND THE NITROGEN-FIXING BACTERIUM Beijerinckia lacticogenes (AFTER TREVIDIAND TSUCHIYA,1975)

A

Percent extraction after 500 hours

Pure culture of T . ferrooxidans Pure culture of B . Zucticogenes Mixed culture

Copper

Nickel

23

75

5" 72

340 100

"Same as chemical control.

fixed nitrogen, although some workers (MacIntosh, 1971) have suggested that Thwbacillus can itself fix nitrogen. Trevidi and Tsuchiya (1975) made mixtures of Thiobacillus ferrooxidans and an acid-tolerant, nitrogen-fixing bacterium, Beijerinkia lacticogenes. They showed that these two organisms would form a stable population based on nutualism, which would grow on a medium of simple salts, with no nitrogen source, to which was added copper or nickel ores. Beijerinkia lacticogenes fixed atmospheric nitrogen and provided nitrogen for the growth of T . fmooxidans, which fixed atmospheric C 0 2 and supplied organic carbon substrates for growth of B. lacticogenes. Trevidi and Tsuchiya (1975) were able to show that the mixed culture was much more efficient at leaching copper or nickel than a pure culture (Table XI). They suggest that the improved leaching rate may not be entirely due to the supply of fixed nitrogen by B . lacticogenes, but that polysaccharides produced by the N2-fixing bacterium may be important in rendering the surface of the ore more amenable to attack by the Thwbacillus. This work obviously opens up the possibility of enhancing ore leaching by deliberate seeding with synthetic mixed cultures.

D. STEROIDTRANSFORMATIONS Steroid oxidation was developed in the 1950s (Perlman, 1977) and it was soon appreciated that several species of organisms might be needed to achieve multistep transformations. McAleem et al. (1959) described the use of mixed cultures to achieve oxygenations of steroids in the 11, 17, and 21

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DAVID E. F. HARRISON

positions and Spuller et al. (1962) described the production of hydrocortisone by a multiple fermentation. These processes involved multistep transformations and were achieved by the sequential addition of different organisms. Shull (1959), however, mixed Curvularia lunata and Mycobacterium phlei to effect a one-step conversion of cortexalone to predinisolone. Later, Lee et al. (1969) reported the one-step conversion of 9afluorohydrocortisone to l-dehydro-16a-hydroxy-9a-fluorohydrocortisone by a mixture of Arthrobacter simplex and Streptomyces roseochromogenes. The Arthobacter carried out a 1-dehydrogenation while the Curvularia was responsible for the Ilp-hydroxylation. In this case the one-step process, using both organisms together, was more successful than a two-step process because, in pure cultures of A. simplex the enzyme 20-ketoreductase is derepressed and all the product and intermediate is reduced at the 20-keto position. In the presence of Curvularia lunata, however, 20-ketoreductase is repressed and the conversion stops at the product l-dehydro-16a-hydroxy-9afluorohydrocortisone (or triamanalone acetonide). Lee et al. (1970) suggested that Curvularia might remove an inducer of 20-ketoreductase which was present in the soybean meal used in the medium. Ryu et al. (1969) tried different combinations of A. simplex with different organisms. Besides Curvularia, Absidia coerula also gave a mixture which could perform the transformation, but Aspergillus ochraceus did not because 1-dehydrogenase formation by the Arthrobacter was repressed. Here, then, we have a fine example of a mixture performing a function which, apparently, could not be carried out by the same organisms acting in sequence.

VII. Future Prospects In the foregoing account we have explored some of the properties and possible applications of mixed culture systems. The fact remains, however, that apart from the often ill-defined cultures used in the more traditional processes, such as dairy fermentations and brewing, there are very few industrial-scale fermentation processes which employ mixed cultures. No doubt there are very good reasons why use of mixed cultures is avoided where pure cultures can perform as well. We have discussed some of the disadvantages of mixed cultures for SCP production and most of these apply equally to other processes: Microbiological control is certainly made more difficult. To this list may be added problems involved in patenting and depositing mixed cultures. For the purpose of patent cover it is certainly advantageous, if not necessary, to be able to clearly identlfy and name the organism in a culture. This is simple enough for a pure culture but more complicated for mixtures. Ideally,

MIXED CULTURES IN INDUSTRIAL FERMENTATION

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all the component species of a mixture should be separately described and deposited but this is not easy where some of the components are obligate symbionts. It may be acceptable to deposit a culture as a mixture but it must still be readily stored and maintained under culture collection conditions. This is fraught with difficulties in the case of complex mixed cultures: Freeze drying and storage in the freeze-dried state often selectively kill one or more component species of a mixture and a mixture adapted to grow at high rates in liquid culture may undergo radical population changes when grown on solid medium. In the author’s experience, storage of mixed cultures is best achieved by freezing the whole culture in liquid nitrogen and storage at temperatures < -80°C. Sequential transfers of complicated mixtures on a solid medium are to be avoided. If possible, it is preferable to maintain separate cultures of all the component organisms. None of the problems is insurmountable but each certainly forms a disincentive to the use of mixed cultures for established monoculture processes. Added to this the in-built prejudice among microbiologists for monoculture systems and it is fair to question whether mixed cultures have any really significant role to play in future processes. If mixed cultures are to be used widely in future fermentation systems then they must be demonstrated to confer definite benefits over monocultures. That this may be so in specific cases is amply illustrated by the examplies discussed above. More important, however, is that these examples illustrate how combinations of organisms can be used to overcome certain limitations of pure cultures. For single-cell protein production from C , compounds the advantages were clear cut and manifold: higher yields, higher productivity rates made possible by increased stability, no foaming, and greater resistance to contamination. Where these advantages can be demonstrated there will most certainly be an application of mixed cultures. A second organism may be used to remove inhibitory substrates that may otherwise cause instability: The inhibitor may be either a product, as in the first example of methaneutilizing mixed cultures given above, or a constituent of the medium. The supply of growth factors, as we have seen, is common in symbiotic relationships. In such cases, the use of a mixture of organisms may circumvent the necessity of using complex media which are required for a pure culture. This may lead not only to direct cost saving but to better control of a fermentation. Trevidi and Tsuchiya (1975) suggested the seeding of nitrogen-fixing organisms to promote the growth of a Thiobacillus in metal leaching. This principle may find wider application to systems which are potentially nitrogen limited. Certainly for waste treatment and SCP production it is a possibility. In many cases the most economical substrate for a fermentation is a mix-

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DAVID E. F. HARRISON

ture of compounds; for instance, natural gas and other higher hydrocarbons are likely to become more important as fermentation substrates and these occur very much as mixtures. Mixed cultures often can more completely exploit all the nutrients present in such mixtures. Another particular use of mixed cultures which has been demonstrated is for achieving multistep conversions where no one organism can be isolated to perform the complete transformation. A few such systems have been described, e. g., steroid transformations and succinic acid production, but there seems to be some potential for further exploratory studies in this area. Two-step fermentations may be combined in such mixed cultures by judicious use of mixtures to give higher rates of conversion by removal of endproduct inhibitions. This may find application in the use of high molecular weight molecules as substrates where feedback inhibition accumulation by breakdown products is often rate limiting. Cellulose activity of molds, for instance, is much increased if cellobiose and glucose are continuously removed. The use of cellulose as a substrate therefore might become more widespread with the judicious use of mixed cultures. Mixed cultures should no longer be thought of as crude, uncontrolled systems. They can be carefully assembled to achieve precise ends. Used thus, mixed cultures are not a soft option: The microbiological research and control required is in order of magnitude more d a c u l t than for pure cultures but modem automated techniques make them quite feasible. N o claim for novelty can be made for mixed cultures: They form the basis of the most ancient fermentation processes. With the exploitation of monocultures having been pushed to its limits it is perhaps time to reappraise the potential of mixed culture systems. They provide a means of combining the genetic properties of species without the expense and dangers inherent in genetic engineering which, in general terms, aims at the same effect. REFERENCES Akaki, M. (1965).J . Fennent. Technol. 43, 365373. Attwood, M. M . , and Harder, W. (1972). Antonie van Leeuwenhoek 38, 369378. Barnes, LJ., Drozd, J. W., Harrison, D. E. F., and Hamer, G. (1976).In “Microbial Producpp. tion and Utilisation of Gases” (H. G. Schlegel, G. Gottschalk, and N. Pfennig, ed~.), 301315. E. Golze K. G., Gottingen, West Germany. Chalin, Y., and Mateles, R. I. (1972). A w l . Mimobiol. !23, 135-140. Chet, I., Fogel, S., and Mitchell, R. (1971).J . Bacteriol. 106, 863-867. Eccleston, M . , and Kelly, D. P. (1972)./. Gen. Microbiol. 71, 5 4 - 5 5 4 . Ehrlich, H. L., and Fox, S. I. (1967). Biotechnol. Bioeng. 9, 471-485. Eroshin, U. K . , Harwood, J. H . , and Pirt, S. J. (1968).I. Appl. Bucteriol. 31, 560567. Galesloot, T. E., Hassing, F., and Veringa, H. A. (1968).Neth. Milk Dairy J . 22, 50-63.

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Goldberg, I. (1977). Process Biochem. 12, 12. Goswell, R. W. (1967). Process Bwchem. 2, 5-11. Haggstrom, L. (1969). Biotechnol. Bioeng. 11, 1043-1054. Hamer, G., Heden, C. G., and Carenberg, C. 0. (1967). Biotechnol. Bioeng. 9, 499-514. Harder, W., and Attwood, M. M. (1975). Antonie oan Leeuwenhoek 41, 421429. Harrison, D. E. F. (1972). J . Appl. Chem.-Biotech. 22, 417440. Harrison, D. E. F. (1973).J . Appl. Bacterial. 35, 301-308. Harrison, D. E. F. (1976). Chem. Technol. 6, 570574. Harrison, D. E. F., and Wren, S. J. (1976). Process Biochem. 11, 3032. Harrison, D. E. F., Topiwala, H. H., and Hamer, G., (1972).Ferment. Technol. Today, Proc. Int. Fennent. Symp., 4th, 1972 pp. 491495. Harrison, D. E. F., Harwood, J. H., and Herbert, B. N. (1974). British Patent 1,469,022. Harrison, D. E. F., Wilkinson, T. G., Wren, S. J., and Harwood, J. H. (1976). In “Continuous Culture 6: Applications and New Fields” (A. C. R. Dean et al., eds.), pp. 122-134. Ellis H a r w d Ltd., Chichester, England. Higgins, I. J., and Quayle, J. R. (1970). Biochem. J . 118, 201-208. Hodge, H. M., Hanson, C. T., and Allgeier, R. J. (1952). Ind. Eng. Chem. 44, 132-135. Jones, C. A,, and Kelly, D. P. (1976). Proc. Int. Ferment. Symp., 5th, 1976 Abstracts, p. 126. Kanegafuchi Kaguku Kogyo Kabushiki Kaisha Co. (1972). British Patent 1,294,810. Kuono, K., Oki, T., Nomura, H., and Ozaki, A. (1973).J . Gen. Appl. Microbiol. 19, 11-21. Lee, B. K., Ryu, D. Y., Thoma, R. W., and Brown, W. E. (1969). J. Gen. Microbiol. 55, 145-153. Lee, B. K., Brown, W. E., Ryu, D. Y., Jacobson, H., and Thoma, R. W. (1970). J. Gen. Microbiol. 61,97-105. Leviton, A,, and Hargrove, R. E. (1952). Ind. Eng. Chem. 44, 2651-2655. Lewis, P. M. (1967). J . Appl. B a c t h l . 30, 406409. Linton, J. D., and Buckee, J. C. (1977).J . Gen. Microbiol. 101, 219-225. McAleer, W., Dulaney, E., and Dulaney, E. L. (1959). U.S. Patent 2,875,132. McCarty, P. L., and Haug, R. T. (1971).In “Microbial Aspects of Pollution” (G. Sykes and F. A. Skinner, eds.), pp. 215-232. Academic Press, New York. MacIntosh, M. E. (1971). J. Gen Microbiol. 66, i-ii. Malashenko, Y. R., and Romanovskaya,V. A. (1973). Bull. Ecol. Res. Commun. (Stockholm) 17, 159-165. Malashenko, Y. R., Klimenko, A. P., Kvasnikov, E. I., Romanovskaya, V. A., and Podgorsky, V. A. (1970). Int. Gus Con$ [Proc.],Ilth, 1970. pp. 1-18. Malashenko, Y. R., Kvasnikov, E. I., Romanovskaya, V. A,, and Boyachenko, V. N. (1971). Mikrobiologiya 70, 724-729. Malashenko, Y. R., Romanovskaya, V. A,, Boyachenko, V. N., Khotyan, L. V., and Voloshin, N. V. (1973). Mikrobiologiya 42, 403-408. Malashenko, Y. R., Romanovskaya, V. A,, Bogachenko, V. N., and Kryshtab, T. P. (1974). Mikrobiologiya 43, 290-294. Mateles, R. I., and Battat, E. (1974). Appl. Microbiol. 28, 901-905. Meers, J. L. (1973). Crit. Reo. Microbiol. 2, 139-184. Miller, I., Martin, H., and Kandler, 0. (1964).Milchwissenschaft 19, 18-25. Miller, T. L., Lie, S., and Johnson, M. J. (1964). Biotechnol. Bioeng. 6, 299307. Murakami, H. (1972). Ferment. Technol. Today, Proc. Znt. Ferment. Symp., 4th, 1972 pp. 639-643. Namsaraev, B. B., and Zavarzin, T. A. (1972). Mikrobiologiya 41, 999-1006. Nurmikko, V. (1956). Experientia 12, 245-249. Okuda, A., and Kobayashi, M. (1963). Mikrobwlogiya 32, 797 and 936-945.

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Ozaki, A,, Kitai, A,, and Oki, K. (1962). Japanese Patent S. 37-3544. Perlman, D. (1977). Chenttech. 7,, 434-443. Pette, J. W., and Lolkema, H. (1950). Neth. Milk D a i q J . 4, 197-208. Pike, E. B., and Curds, C. R. (1971). In “Microbiol Aspects of Pollution” (G. Sykes and F. A. Skinner, eds.), pp. 123-142. Academic Press, New York. Pitone, G. J . , and Rankine, B. C. (1974).Ain. J. E n d . Vitic. 25, 99-107. Pirt, S. J. (1975). “Principles of Microbe and Cell Cultivation.” Blackwell, Oxford. Ryu, D. Y., Lee, B. K., Thoma, R. W., and Brown, W. E. (1969). Biotechnol. Bioeng. 11, 12-55-1276, Sanada, H., Tanaka, M., and Ota, S. (1960). Japanese Patent S. 35-12642. Sasaki, Y., Takao, S., and Hotta, K. (1970a).J. Ferment. Technol. 48, 776-781. Sasaki, Y., Takao, S., and Hotta, K. (1970b).J. Ferment. Technol. 48, 782-786. Sheehan, B. T., and Johnson, M. J. (1971). Appl. Microbiol. 21, 511515. Shull, J. J. (1959). German Patent 1,050,335. Spuller, C., Modelli, R., and Amici, A. M. (1962). U.S. Patent 3,030,278. Tanaka, A., Ohya, Y., Shimizu, S., and Fukui, S. (1974).J. Fennent. Technol. 52, 921-924. Taylor, G. T., Kelly, D. P., and Pirt, S. J. (1975).In “Production and Utilization of Gases” (H. G. Schlegel, G. Gottschalk, and N. Pfennig, eds.), pp. 173-180. Akad. Wiss., Gottingen, West Germany. Topiwala, H. H., and Hamer, G. (1971). Biotechnol. Bioeng. 13, 919-923. Trevidi, N. C., and Tsuchiya, H. M. (1975). Int. J. Miner. Process. 2, 1-14. Tsuchiya, H. M . , Drake, J. F., Jost, J. L., and Fredrickson, A. G. (1972).J . Bacteriol. 110, 1147-1 153. Vary, P. S., and Johnson, M. J. (1967).AppZ. M i c r o b i d 15, 1473-1478. Veldkamp, H. (1976).In “Continuous Culture 6: Applications and New Fields” (A. C. R. Dean et al., eds.), p. 315. Ellis Hanvood Ltd., Chichester, England. Veringa, H. A., Galesloot, T. E., and Davelaar, H. (1968).Neth. Milk Dairy J. 22, 114-120. Watson, T. G. (1972). In “Environmental Control of Cell Synthesis and Function” (A. C. R. Dean, S. J. Pirt, and D. W. Tempest, eds.), pp. 229-244. Academic Press, New York. Whittenbury, R., Phillips, K. C., and Wilkinson, J. F. (1970).J . Gen. Microbial. 61, 205-218. Wilkinson, T. G. (1972). Ph.D. Thesis, University of London. Wilkinson, T. G., and Hamer, G. (1971).J. Appl. Bacteriol. 35, 577588. Wilkinson, T. G., and Harrison, D. E. F. (1973).J. Appl. Bacteriol. 36, 309313. Wilkinson, T. G., Topiwala, H. H., and Hamer, G. (1974). Biotechnol. Bioeng. 16, 41-59. Wolin, M. J. (1975). In “Microbial Production and Utilisation of Gases” (H. 6. Schlegel, G. Gottschalk, and N. Pfennig, eds.), pp. 141-150. Akad. Wiss., Gottingen, West Germany. Wren, S. J. (1978). Ph.D. Thesis, University of London (in preparation). Yamatodani, S., Kakinuma, A,, Suzuki, M., and Abe, M. (1967).Annu. Rep. Tukeda Res. Lab. 26, 107. Yong, F. M., and Wood, J. B. (1974).Ado. Appl. Microbiol. 17, 157-188. Zeikus, J. G. (1977). Bacteriol. Reo. 41, 514-541.

Utilization of Methanol by Yeasts

YOSHIKI TANI,

NOBUOKATO, AND

HIDEAKIYAMADA Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Dissimilation and Assimilation of Methanol in Methylotrophs Other than Yeast.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Dissimilation and Assimilation of Methanol in Yeasts . . . . . . . . A. Enzyme System for the Dissimilation of Methanol . . . . . . B. Assimilation of Methanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cell Yield and the Metabolic Pathway, .................... V. Production of Cells and Metabolites . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 167 170 170 179 180 182 183

1. Introduction Methanol has attracted much attention as a convenient raw material for industrial fermentation. Consequently, investigations of microorganisms which grow on reduced C, compounds, e.g., methane and methanol, as the sole source of carbon and energy have increased. Investigations have been limited to studying the unique metabolic pathway of these compounds. Results of this research have made methanol of practical use in the fermentative production of cells and metabolites. This is a typical pattern in the development of applied microbiology, in which applied and fundamental studies are interrelated. The ability to assimilate reduced C , compounds was first reported by Sohngen (1906),who isolated an aerobic methane-utilizing bacterium Bacillus methanicus. No further information on the utilization of reduced C, compounds was forthcoming for the next 50 years, in spite of the wide occurrence of methane in nature. Microbiologists were interested only in methane-producing microorganisms. Sohngen’s strain was reisolated as a methane and methanol utilizer by Dworkin and Foster (1956) and named Pseudomonas methanica. Their studies showed the physiological peculiarity of methylotrophs. The microbial utilization of methane and methanol has since become the concern of several groups of scientists. Table I is a chronological list of researches on methylotrophs, showing that various kinds of methylotrophs 165 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0026244

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YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

TABLE I MICROBIAL UTILIZATION OF REDUCEDc ,COMPOUNDS First description of a reduced C, compound utilizer, Bacillus methanicus Reisolation of Sohngen's strain, Pseudomonas methanica Isolation of the facultative methylotroph, Pseudomonas PRL-W4, and identification of serine as the first stable intermediate in its metabolic pathway Outlining the serine pathway, Pseudomom AM1 Characterization of primary alcohol dehydrogenase, Pseudomonas M27 Outlining the ribulose monophosphate pathway, Pseudomonas methanica Isolation of a methanol-utilizing yeast, Kloeckera sp. 2201 Systematic characterization of methane-utilizing bacteria Completion of the id+-serine pathway, Pseudomonas MA Crystallization and characterization of alcohol oxidase, Kloeckera sp. 2201 Identification of hexose phosphate as the primary stable intermediate in yeast, Candidu N-16 Isolation of methanol-utilizing fungi, Trichodemna lignorum Identification of Durabino-3-hexulose phosphate, completion of the ribulose monophosphate pathway, Methylococcus capsulatus First International Symposium on Microbial Growth on C, Compounds Isolation of a methanol-utilizing actinomycete, Streptomyces sp. 239 Adoption of Methylomonadaceae in Bergey's Manual of Determinative Bacteriology Finding of the microbody in methanol-utilizing yeasts Second International Symposium on Microbial Growth on C, Compounds

Sohngen (1906) Dworkin and Foster (1956) Kaneda and Roxburgh (1959a,b,c)

Large et al. (1962) Anthony and Zatman (1964) Johnson and Quayle (1965), Kemp and Quayle (1965) Ogata et al. (1969) Whittenbury et aZ. (1970a,b) Bellion and Hersh (1972) Tani et a/. (1972a,b) Fujii and Tonomura (1973) Tye and Willetts (1973) Kemp (1974)

at Tokyo (1974) N. Kato et al. (1974a) Buchanan and Gibbons (1974) Fukui et al. (1975a), van Dijken et al. (1975a), Sahm et a / . (1975) at Moscow (1977)

have been isolated and that mechanisms which produce energy and which synthesize cell materials from reduced C compounds have been extensively investigated. These studies are related to the need for a global food supply, which has been a concern since the late 195Os, and to the consequent international trend to develop single-cell protein production. At present, the practical use of methylotrophs on an industrial scale is limited to cell production in a few countries. The utilization of reduced C1compounds by yeasts began with the investigations of Kloeckera sp. 2201 by Ogata et al. (1969). The history of research on methanol-utilizing yeasts is short in comparison with that on bacterial

UTILIZATION OF METHANOL BY YEASTS

167

methylotrophs. However, after the brief review by Cooney and Levine (1972), research on methanol utilization by yeasts has made rapid progress (Sahm, 1977). In the present review, metabolic features of the methanolutilizing yeasts will be compared with those of other microorganisms. II. Dissimilation and Assimilation of Methanol in Methylotrophs Other than Yeast There are many unique features of the metabolic pathway for methanol, especially in the early steps of the dissimilation and assimilation pathways. A large part of this unique metabolic pathway has been made clear by studies with bacterial methylotrophs (Quayle, 1972). The dissimilation pathway provides electrons for the respiratory chain to produce ATP. Each step in the reaction sequence leading to the complete oxidation of methanol to C 0 2 through formaldehyde and formate has been demonstrated enzymatically. Enzymes which oxidize methanol in bacteria are listed in Table 11. Methane is introduced into the sequence after oxidation to methanol. Phenazine methosulfate-dependent primary alcohol dehydrogenase (EC 1.1.99.8) is characteristic in bacterial methanol oxidation and has been thoroughly investigated. This enzyme also oxidizes formaldehyde, although the K , value for formaldehyde is larger than that of methanol (Sperl et al., 1974). Almost all reported bacterial methylotrophs have this enzyme. Methane-utilizing bacteria are divided into two groups according to the localization of this enzyme in the cell fraction (Pate1and Felix, 1976), which coincides with the arrangement of their intracytoplasmic membranes. The electron acceptor of this enzyme reaction in uivo has been suggested to be cytochrome c (Anthony, 1975; Netrusov et a l . , 1977). The prosthetic group of the enzyme is thought to be a pteridine derivative (Anthony and Zatman, 1967), but the structure of the compound is still unknown. There are only a few examples of methanol oxidation by fungi. Activities of NAD-linked alcohol dehydrogenase and formaldehyde dehydrogenase and of methylene blue-linked methanol dehydrogenase have been detected in cell extracts of Paecilomyces varioti and Gliocladium deliquescens (Sakaguchi et al., 1975). Streptomyces sp. 239, which is the only known actinomycete which utilizes reduced C, compounds, has a different system for methanol oxidation (Kato et a l . , 1975). The oxidation of methanol, formaldehyde, and formate by the cell-free extract requires the presence of phenazine methosulf;?te-2,6-dichlorophenolindophenol or cytochrome c . The incorporation of methanol into cell constituents has been investigated chiefly with bacterial systems. A first oxidation product of methanol, formaldehyde, enters into two different assimilation pathways. One is the ribulose

TABLE I1 OXIDATION OF METHANOL IN BACTERIA Step CH,OH

4

HCHO

Enzymes

Strains

References

(1) Primary alcohol dehydrogenase electron acceptor; phenazine methosulhte (cytochrome c ) :cofactor; pteridine compound, ammonium ion

Pseudomonas M27 Pseudomonas AM1 Methylococcus capsulatus Hyphomicrobium WC Methylosinus sporiurn Pseudomonas C Pseudomonas sp. 2941 Pseudomonas PRL-W4

Anthony and Zatman (1964) Johnson and Quayle (1964) Patel et al. (1972) Sperl et al. (1974) Patel and Felix (1976) Goldberg (1976) Yamanaka and Matsumoto (1977) Kaneda and Roxburgh (1959b)

Pseudomonas methanica

Harrington and Kallio (1960)

Methylococcus capsulatus Methylobacter capsulatus Methylomonas methanica Pseudomonas AM1 Methylococcus capsulatus Pseudomonas methanica Pseudomanas M27 Hyphomicrobium WC Methylosinus sporium Pseudomonas C Pseudomonas sp. 2941 Pseudomonas methanica

Wadzinski and Ribbons (1975) Patel and Felix (1976)

Pseudomonas AM 1

Johnson and Quayle (1964)

Pseudomonas AM1 Methylococcus capsulatus

Johnson and Quayle (1964) Patel and Hoare (1971)

Methanol dehydrogenase electron acceptor; NAD Peroxidase (in the presence of glucose and glucose oxidase) (4) Methanol oxidase present in particulate fraction HCHO 4 HCOOH

HCOOH

-+

COP

(5) Aldehyde dehydrogenase the same enzyme with primary alcohol dehydrogenase

(6) Formaldehyde dehydrogenase electron acceptor; NAD: glutathione dependent (7) Aldehyde dehydrogenase electron acceptor; 2,6-dichlorophenol indophenol (8) Formate dehydrogenase electron acceptor; NAD

Heptinstd and Quayle (1970) Patel and Hoare (1971) Patel et al. (1972) Sperl et al. (1974) Patel and Felix (1976) Goldberg (1976) Yamanaka and Matsumoto (1977) Harrington and Kallio (1960)

169

UTILIZATION OF METHANOL BY YEASTS

monophosphate pathway (or the pentose monophosphate pathway), in which formaldehyde is condensed with ribulose 5-phosphate to form a unique compound, ~urabino-3-hexulose6-phosphate, which undergoes isomerization to fructose 6-phosphate (Strem et al., 1974) (Fig. 1). The second is the serine pathway, in which formaldehyde reacts with glycine to form L-serine by the catalysis of serine transhydroxymethylase (Quayle, 1972) (Fig. 2). Another pathway, which fixes C 0 2instead of formaldehyde, is the ribulose diphosphate pathway. This is usually found in autotrophs (Quayle, 1972). The product of the hexulose phosphate synthase reaction, the first step in the ribulose monophosphate pathway, has recently been identified (Kemp, 1974) and the enzyme has been extensively purified (Ferenci et al., 1974; Sahm et a l . , 1976; Kato et a l . , 1978). Now studies are focusing on metabolic control in the pathway. This may involve cyclic oxidation of formaldehyde through 6-phosphogluconate as a dissimilation pathway (Strpm et al., 1974; Colby and Zatman, 1975a,b; Ben-Bassat and Goldberg, 1977). One undetermined part of the serine pathway was the regeneration system ofglyoxylate and glycine. Bellion and Hersh (1972)showed the presence of isocitrate lyase, malate thiokinase (malate + ATP + CoA 4 malyl-CoA ADP + Pi), and malyl-CoA lyase (malyl-CoA + glyoxylate acetyl-CoA)in Pseudomonas MA. This led to the completion of “the icl +-serine pathway.” However, neither isocitrate lyase nor malate thiokinase activity has been detected in some methylotrophs, including Pseudomonas AM1, a typical strain possessing the id-serine pathway (Salem et al., 1973). Fungi assimilate methanol through the ribulose monophosphate pathway (Tye and Willetts, 1973) or through the serine pathway (Sakaguchi et al., 1975). An actinomycete, Streptomyces sp. 239, is thought to use both pathways. Activities of hydroxpyruvate reductase and hexulose phosphate synthase, respectively the key enzymes of the ribulose monophosphate and serine pathways, have been detected in this strain (Kato et al., 1977a).

+

+

Fructose 1.6-PZ 4 6-P-Gluconate

acetone- P

FIG. 1. The rihulose monophosphate pathway in methylotrophs. @ hexulose phosphate synthase, phosphohexulose isomerase.

@

170

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

@ @

@

FIG. 2. The icl +-serine pathway in rnethylotrophs: serine transhydroxyrnethykse, hydroxypyruvate reductase, malate thiokinase, rnalyl-CoA lyase, iswitrate lyase.

@

0

Ill. Dissimilation and Assimilation of Methanol in Yeasts A number of methanol-utilizing yeasts has been found since the first isolation by Ogata et al. (1969), and a number of type strains of yeasts has also been shown to utilize methanol (Hazeu et al., 1972). These species are limited to several genera which include both ascomycetous and asporogenous yeasts (Table 111).All are facultative methylotrophs. Multipolar budding and a requirement for biotin and/or thiamine are common features of many of these yeasts. These peculiar characteristics of methanol-utilizing yeasts may lead to a new classification system, such as that of Methylomonadaceae for bacterial methylotrophs (Buchanan and Gibbons, 1974). More detailed studies of classification using chemotaxonomy are needed. Candida sp. WY-3, which grows on secondary and tertiary amines, is another type of reduced C, compound-utilizing yeast (Yamada et al., 1976). This yeast may dissimilate the methyl group of the amines in a pathway which affords formaldehyde (Colby and Zatman, 1973). The principal parts of the dissimilation and assimilation pathways of methylotrophs have been shown in studies with bacteria. Knowledge of the enzyme systems in yeasts has accumulated rapidly because of extensive studies with bacteria and because of the usefulness of yeasts in single-cell protein production. THE DISSIMILATION OF METHANOL

A. ENZYME SYSTEMFOR

Each step in the reactions involved in methanol oxidation by yeasts has been investigated at the enzymatic level. Table IV gives the enzymes responsible for the oxidation of methanol to COz through formaldehyde and formate.

TABLE I11 CHARACTERISTICS OF METHANOL-UTILIZING YEASTS Strains Ascomycetous yeasts Hansenula (9)a H . pdymorpha H . ofunaensis Pichia (6) P . methanolica P . lindnerii P. inethanothenno Saccharornyces (2) Asporogenous yeasts Candida (7) C. N-16 C. methanolica C.boidinii Kloeckera (1) K. sp. 2201 Rhodotomla (1) Twulopsis (12) T . methanolooescens T . methanoswbosa T . methanodonnercqii T . nagoyaensis

Budding

Methylotrophism

Growth factor

Multipolar Multipolar

Facultative Facultative

Biotin, thiamine

Multipolar Multipolar Multipolar

Facultative Facultative Facultative

Biotin Biotin, thiamine

Henninger and Windisch (1975) Minaini et al. (1978)

Multipolar Multipolar Mu1tipolar

Facultative Facultative Facultative

Biotin Biotinb Biotin

Tonomura et al. (1972) Oki et al. (1972) Sahm and Wagner (1972)

Bipolar

Facultative

Thiamineb

Ogata et al. (1969, 1970)

Multipokr Multipolar Multipolar Multipolar

Facultative Facultative Facultative Facultative

Biotin, thiamine Biotin, thiamine Biotin, thiamine

Oki et al. (1972) Yokote et al. (1974) Yokote et al. (1974) Asai et al. (1976)

“Number of strains reported in paper. bNot essential for but stimulative to growth.

References

Levine and Cooney (1973) Asai et al. (1976)

K. Kato et al. (1974)

TABLE IV OXIDATION OF METHANOL Step CH,OH

+

HCHO

Enzymes (1) Alcohol oxidase cohctor; FAD

(2) Alcohol dehydrogenase electron acceptor; NAD: glutathione requiring (3) Catalase (peroxidative) HCHO -+ HCOOH

(4) Formaldehyde dehydrogenase electron acceptor; NAD: glutathione dependent (HCHO + HCO-glutathione)

(5) Alcohol oxidase substrate; hydrated formaldehyde

HCOOH + CO,

(6) Catalase (peroxidative) (7) Formate dehydrogenase electron acceptor; NAD (HCO-glutathione -+ CO, or HCO-glutathione + H,O 4 HCOOH + CO,) (8) Catalase (peroxidative)

IN

YEASTS

Strains

Kloeckera sp. 2201 Candida N-16 Candida boidinii Hansenula polymorpha Pichia pinus, Kloeckeru sp. 2201, Candida boidinii Hansenula polymwpha Candida boidinii Candida N-16 Hansenula polymorpha Candida N-16 Kloeckera sp. 2201 Candida boidinii Hansenula polymorpha Candida N-16 Candida boidinii Hansenula polymorpha Kloeckera sp. 2201 Hansenula polymwpha Candida N-16 Candida boidinii Kloeckera sp. 2201 Hansenula polymorpha Hansenula polymorpha

References Tani et al. (1972a,b) Fujii and Tonomura (1972) Sahm and Wagner (1973a) Kato et al. (1976) Mehta (1975a,b) Dudina et al. (1977) Roggenkamp et al. (1974) Fujii and Tonomura (1975b) van Dijken et al. (1972) Fujii and Tonomura (1972) Kato et al. (1972) Sahm and Wagner (1973b), Shiitte et al. (1976) van Dijken et al. (1976a) Fujii and Tonomura (197513) Sahm (1975) Kato et al. (1976) van Dijken et al. (1975b) Fujii and Tonomura (1972) Sahm and Wagner (1973b), Schiitte et al. (1976) N. Kato et al. (197413) van Dijken et al. (1976a) van Dijken et ul. (197513)

UTILIZATION OF METHANOL BY YEASTS

173

1 . Oxidation of Methanol to Formaldehyde

A unique characteristic of the methanol oxidation system of yeasts appears in the first step, methanol to formaldehyde. All yeasts examined have an

alcohol oxidase (EC 1.1.3.13) which catalyzes the following reaction using molecular oxygen as the electron acceptor: CH,OH

+ Oz+

HCHO

+ H202

This type of enzyme has been found only in Basidiomycetes (Farmer et al., 1960; Janssen et al., 1965; Fukuda and Branron, 1971). The electron from methanol in this reaction is transferred to molecular oxygen to form HzOz. In comparison with the bacterial enzyme, this first step of methanol oxidation in yeasts is disadvantageous to ATP regeneration. The enzyme has been purified from the cell-free extract of a methanolutilizing yeast, Kloeckera sp. 2201, by a procedure which includes ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 column chromatographies (Tani et d., 1972a). The crystals obtained had a specific activity about 12-fold that of the crude cell-free extract. The content of the enzyme in the cells was estimated to be about 8% of the total soluble protein. The ease of the preparation makes possible the use of the enzyme in the determination of alcohols (Guilbault, 1970). Physicochemical and enzymological properties of alcohol oxidases from different yeasts are similar (Tani et al., 1972a,b; Kato et al., 1976; Fujii and Tonomura, 1972; Sahm and Wagner, 1973a; van Dijken, 1976) (Table V). The enzyme of Hansenula polymorpha, a thermotolerant yeast, differs in its behavior to temperature. The enzyme is composed of eight subunits, each of which contains one coenzyme, FAD. Each subunit is arranged in an octad aggregate composed of two tetragons face to face based on electron microscopical observations (Kato et al., 1976) (Fig. 3). The enzyme can be induced when the yeast is grown on a methanol medium (Tani et al., 1972a). This enzyme should be called an alcohol oxidase because it is almost equally active for ethanol and methanol: To a lesser extent it is active for several primary alcohols (Tani et al., 1972b). The oxidation of methanol by alcohol oxidase is accompanied by the formation of a toxic compound, HzOz. Catalase is also induced when the yeast is grown on a methanol medium (N. Kato et al., 197413; Fujii and Tonomura, 1975a; Yasuhara et al., 1976). Table VI shows the simultaneous formation of alcohol oxidase and catalase in methanol-utilizing yeasts. Catalase also functions like peroxidase to oxidize methanol (Roggenkamp et al., 1974; Fujii and Tonomura, 1975b; van Dijken et al., 1975b), formaldehyde, and formate (van Dijken et al., 197513) in the presence of excess HzOz.

PROPERTIES OF

TABLE V ALCOHOLOXIDASES

OF YEASTS

Relative activityn for Optimum temperature

K , for methanol (mM)

Molecular weight

Molecular weight of subunit

FAD content (moles/mole)

83,000

8.4

ec,

Ethanol

Kloeckera sp. 2201

35

106

79

0.44

673,000

Candida N-16 Candida boidinii Hansenuh polymorpha DL-1 Hansenulu polymorpha CBS 4732

35

93 75

75 25

210,000

-

6o0,OOo

50

44

2.1 2.0 0.23

669,000

74,000 83,000

7.4

Tani et al. (1972a,b) Kato et al. (1976) Fujii and Tonomura (1972) Sahm and Wagner (1973a) Kato et al. (1976)

78

60.5

1.3

616,000

77,000

8

van Dijken (1976)

Origin of enzyme

30 45

50

n-Propanol

"Relative activity is expressed as 100 for the activity against methanol.

References

UTILIZATION OF METHANOL BY YEASTS

175

FIG. 3. Electron micrograph of alcohol oxidase. The crystalline enzyme of Hansenuh polymorpha was negatively stained with sodium phosphotungstate at pH 7.2.

Subcellular localization of enzymes involved in methanol oxidation is an interesting feature of methanol-utilizing yeasts. A specific organelle called the microbody (Fukui et al., 1975a; Sahm et al., 1975)or the peroxisome (van Dijken et al., 1975a), which is surrounded by a single-unit membrane, has been found in methanol-grown cells. This fine structure also occurs in other methanol-utilizing yeasts (Tsubouchi et al., 1976). The organelle has been isolated using density gradient centrifugation (Roggenkamp et al., 1975; Fukui et a l . , 1975b). Alcohol oxidase and catalase are found in this particle, but formaldehyde dehydrogenase and formate dehydrogenase are not (Fukui et d.,1975b). The localization of alcohol oxidase was confirmed using a cytochemical staining technique (Veenhuis et al., 1976). This organelle may be responsible for the first step in methanol oxidation. Immobilization of the organelle has been studied using photocrosslinkable resins (Tanaka et al., 1977). Immobilized microbodies may be useful as a multifunctional biocatalyst. The presence of an enzyme other than alcohol oxidase in the oxidation of methanol to formaldehyde has been reported. Activity of NAD-dependent alcohol dehydrogenase was observed in extracts of all the methanol-utilizing yeasts tested (N. Kato et al., 1974~).Sahm and Wagner (1973a), in a study

176

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA TABLE VI ACTIVITYOF ALCOHOLOXIDASE AND

CATALASE IN YEASTSa

Activity in methanol-grown

cells

Activity in glucose-grown cells

Strains

Alcohol oxidaseb

Catalaseb

Alcohol oxidaseb

Catalaseb

Kloeckera sp. 2201 Candida rnethanolica Torulopsis pinus Torulopsis methanolovescens Hansenuh capsulata Pichia pinus Pichia trehalophila

0.13 0.40 0.16 0.08 0.07 0.24

12.9 10.7 6.2 9.4 5.7 6.9 3.2

-e -

0.64 0.20 0.18 0.18 0.09 0.35 0.20

0.48

0.002

0.002

aAdapted from N. Kato et al. (1974~). %pecific activity: alcohol oxidase, pmoles of H,Oz/min/mg protein; catalase, AE,Jmin/mg protein. Nondetectable.

using Candidu boidinii, showed that the alcohol dehydrogenase was inactive for methanol, that it was not induced by methanol, and that a mutant which could not grow on methanol had this enzyme activity. This suggests that the enzyme has no physiological significance. Some researchers, however, assume that there is positive participation of the alcohol dehydrogenase in methanol oxidation. Glutathione-dependent dehydrogenation has been shown in cell-free extracts of several yeasts (Mehta, 1975a,b). In this case, cooperative action of contaminating enzymes, such as alcohol oxidase and glutathione-dependent formaldehyde dehydrogenase, may indicate that a dehydrogenase activity reduces NAD. However, methanol oxidation has recently been detected also under anaerobic conditions in which alcohol oxidase should be inactive (Dudina et al., 1977). Therefore, whether methanol dehydrogenase is active in vivo, which would be advantageous for ATP-regeneration and would result in better cell yields, is still debatable. 2 . Oxidation of Formaldehyde to Furmute A glutathione-dependent dehydrogenase which uses NAD as the electron acceptor is known to catalyze formaldehyde oxidation in various organisms, including methanol-utilizing bacteria. Formaldehyde dehydrogenase (EC 1.2.1.1) has also been found in methanol-utilizing yeasts (Fujii and Tonomura, 1972; Kato et al., 1972; Sahm and Wagner, 1973b; Schutte et al., 1976; van Dijken et al., 1976a).

UTILIZATION OF METHANOL BY YEASTS HCHO

177

+ NAD + H,O -+ HCOOH + NADH,

The enzyme can be induced when the yeast is g r o w on a methanol medium. Its content in the cells is about 0.8% as calculated from the specific activity of a highly purified preparation (Schutte et al., 1976). The enzyme is highly specific for formaldehyde. The reaction mechanism is: HCHO -t GSH + H,O + HOCH,-S-G HOCHZ-S-G + NAD + HCO-S-G NADH, GSH HCO-S-G + HZ0 -+ HCOOH

+ +

In this sequence, the oxidation of formaldehyde occurs after the nonenzymatic formation of hemimercaptal from formaldehyde and glutathione. The reaction product, S -formylglutathione, is then hydrolyzed to formate by a hydrolase. Induction of the hydrolase by methanol has also been reported (Schutte et al., 1976). The electron of formaldehyde is introduced into a respiratory chain through the reduction of NAD. It appears that, in contrast to the first step, the second step in methanol oxidation is responsible for energy production. Oxidation offormaldehyde by alcohol oxidase has been found (Sahm, 1975; Fujii and Tonomura, 197513; Kato et al., 1976). This reaction occurs because more than 99% of the formaldehyde is hydrated to form an alcoholic compound in aqueous solution. The apparent K , value of alcohol oxidase for formaldehyde (2.40 mM) is much higher than that of formaldehyde dehydrogenase for formaldehyde (0.29 mM) and that of alcohol oxidase for methanol (0.44 mM) (Katoet al., 1976). A mutant ofCandida boidinii, which lacks alcohol oxidase, oxidizes formaldehyde as well as the parent strain does (Sahm, 1975).These data support the hypothesis that there is no physiological significance in the oxidation of formaldehyde by alcohol oxidase in vim. Formaldehyde oxidation by dehydrogenase is favorable since it provides electrons for the respiratory chain to produce ATP. Another possible oxidation system for formaldehyde is catalase-catalyzed peroxidation (van Dijken et al., 1975b). $3. Oxidation of Formute to C o g

The enzyme which catalyzes the final step of methanol oxidation in yeasts is formate dehydrogenase (EC 1.2.1.2)(Fujii and Tonomura, 1972; Sahm and Wagner, 1973b; N. Kato et al., 197413; Schutte et al., 1976; van Dijken et al., 1976a). It has also been found in bacterial methylotrophs. HCOOH

+ NAD + CO, + NADH,

178

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

The enzyme can be induced when the yeast is grown on a methanol medium. Its content in the cell have been calculated to be about 3% for Kloeckeru sp. 2201 (Kato et al., 1974b) and about 5% for Candida boidinii (Schutte et al., 1976). The enzyme may be useful for determining formate since it is highly specific for that compound. The high K , value of the enzyme for formate, 22 mM for the enzyme of Kloeckeru sp. 2201 (Kato et al., 1974b), has been debated. This places doubt on the physiological significance of the enzyme in an energy-giving system, although it is advantageous for the supply of formaldehyde to the assimilation pathway. Recently, van Dijken et al. (1976a) reported that the true substrate of the enzyme is S -formylglutathione, not formate: HCO-S-G

+ NAD + H,O

--f

COP

+ NADH, + GSH

When S-formylglutathione is used as substrate, the K , value of formate dehydrogenase for it is about 1mM. Direct oxidation of S-formylglutathione to COz, without hydrolysis to formate, is possibly the best way to complete the oxidation of methanol. Induction of S -formylglutathione hydrolase by methanol has been reported (Schutte et al., 1976). Further studies on the final step of methanol oxidation, however, are necessary. Results of studies of the oxidation system of methanol are as follows (Fig. 4): In methanol-utilizing yeasts, methanol is first oxidized to formaldehyde

FIG.4. Schematic representation of the localization and the mechanism of oxidation of nonenzymatic, formaldehyde methanol by yeasts: alcohol oxidase, dehydrogenase, formate dehydrogenase, S-formylglutathione dehydrogenase.

0@

@

@

@

UTILIZATION OF METHANOL BY YEASTS

179

by alcohol oxidase and by catalase in the manner of peroxidase. These reactions occur in a specific organelle, the microbody (peroxisome). Subsequently formaldehyde, which is hydrated in aqueous solution, is oxidized to S-formylglutathione by cytoplasmic formaldehyde dehydrogenase after the nonenzymatic formation of hemimercaptal. Then, S-formylglutathione is oxidized to C 0 2 by formate dehydrogenase alone or in cooperation with hydrolase. In addition, the oxidation of methanol to formate through formaldehyde by the catalysis of alcohol oxidase is also possible, but the physiological significance of this reaction is questionable.

B. ASSIMILATIONOF METHANOL It is essential to determine the pathway that assimilates methanol in order to make use of methanol-utilizing yeasts. Bacterial methylotrophs assimilate reduced C compounds through the ribulose monophosphate or the serine pathway. Therefore, there have been several attempts to determine the pathway in yeasts. However, sufficient data to explain the assimilation pathway of yeasts have not yet been accumulated. The first step, itself, in the incorporation of C, compounds (possiblyformaldehyde and/or formate) is not yet clear. A system different from that in bacteria may occur in yeasts as seen in the oxidation pathway. Sugar phosphates have been postulated to be early intermediates in the assimilation pathway in yeasts. The incorporation of I4C-labeled C, compounds to phosphate esters of hexose, e.g., fructose and glucose, was first reported by Fujii and Tonomura (1973). This suggests the presence of a pathway similar to the ribulose monophosphate pathway and different from the serine pathway. The absence of hydroxypyruvate reductase, a key enzyme in the serine pathway, in cell-free extracts of Kloeckera sp. 2201 (Diel et al., 1974) supports this suggestion. Hexulose phosphate synthase is therefore thought to catalyze the first condensation step as in the bacterial pathway. The special feature in the reaction of yeasts is the ATP requirement (Fujii et a l . , 1974; Fujii and Tonomura, 1974), which is not known for any bacterial system. Measurement of the hexulose phosphate synthase activity is only possible using the radioactivity of a 14C-labeledC, compound incorporated into isolated hexose phosphates. The activity of this enzyme seems inadequate for the first-step reaction of the assimilation pathway (Diel et al., 1974; Fujii and Tonomura, 1974; Sahm and Wagner, 1974; Trotsenko et al., 1976), although the enzyme has been induced during adaptation from a glucose to a methanol medium (Sahm, 1977). No phosphohexulose isomerase activity, which is known to catalyze the isomerization of ~arabino-3-hexulose 6-phosphate to fructose 6-phosphate after the condensation reaction of for-

,

180

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

maldehyde to ribulose 5-phosphate in the bacterial pathway, could be detected in the cell-free extract of Kloeckwu sp. 2201 (Kato et al., 197713). A positive change in enzyme activities related to the ribulose monophosphate pathway has been found when a yeast is grown on a methanol medium (Sahm, 1977). Properties of hexulose phosphate synthase also have been reported (Bykovskaya and Voronkov, 1977; Sahm, 1977). The enzyme responsible for the first reaction in the assimilation pathway of methanol in yeast may still be unknown as studies so far are not definitive. Identifications of the substrate and of the product of “synthase” are the most needed studies, at present, in the research on methanol-utilizing yeasts. IV. Cell Yield and the Metabolic Pathway The first aim of the microbial utilization of methanol has been to produce single-cell protein. Knowledge of methanol metabolism may be sufficient to enable us to produce a theoretical cell yield value. Pathways to dissimilate and assimilate methanol have been summarized in terms of cell yield (Fig. 5) where ATP is the principal compound. The oxidation of methanol to CO, functions as an ATP-producing system. The pathway leading to the cell material described as C4H,0,N, from the C1 compound through 3-phosphoglycerate, a common intermediate in the biosynthesis of cell materials, is represented to be an ATP-consuming system (van Dijken and Harder, 1975). Assuming that the oxidative phosphorylation system is also used in methylotrophs, then the type of electron acceptor should directly determine the amount of ATP formed by the oxidation of methanol. The electron transferred to NAD(P) by the oxidation of one molecule of a C1 compound gives three ATPs but its transfer to molecular oxygen, as in the alcohol oxidase reaction in yeasts, produces no ATP. Phenazine methosulfate-dependent primary alcohol dehydrogenase is distributed in almost all bacterial methylotrophs. The electron acceptor of the enzyme in uiuo is thought to be S e r m pathway or Rtbulose mMophosprate pathway

A

X CH3OH

XHz

Y

YHz

HCHO -HCOOH

Z

ZH2 CO2

FIG. 5. Pathways of dissimilation and assimilation of methanol. Adapted from van Dijken and Harder (1975).

UTILIZATION OF METHANOL BY YEASTS

181

cytochrome c (Anthony, 1975; Netrusov et al., 1977). In this case, one molecule of ATP is available for the oxidation of one molecule of methanol to formaldehyde through the respiratory chain. NAD(P) is effective as the electron acceptor for ATP production but it is usually inactive for the first step of methanol oxidation. This may be because the oxidation-reduction potential in this oxidation is not sufficient to reduce NAD(P). The ATP balance of the assimilation pathways of methanol is shown in Fig. 6. The serine and ribulose monophosphate pathways have several reactions related to the consumption or production of ATP up to the synthesis of a common intermediate, 3-phosphoglycerate. The regeneration system of glyoxylate and glycine in the serine pathway is considered here. The serine pathway consumes four molecules of ATP to form one molecule of 3-phosphoglycerate, while the ribulose monophosphate pathway gives three molecules of ATP. Possibly, the later pathway in yeasts consumes one molecule of ATP in the first step to incorporate formaldehyde. Thus, an organism having the ribulose monophosphate pathway is more advantageous for cell yield than is one having the serine pathway. Cyclic oxidation of formaldehyde through 6-phosphogluconate (Strflm et al., 1974; Colby and Zatman, 1975a,b; Ben-Bassat and Goldberg, 1977) produces a greater amount of ATP for the ribulose monophosphate pathway. Van Dijken and Harder (1975) reported yields of microorganisms grown Y of 10.5. Methanol-utilizing yeasts have values on methanol, based on a , of O,3, and 3, for X, Y and 2, respectively, in the dissimilation pathway (Fig. Serine pathway 2 HCHO COP 4 ATP + 3-phosphoglycerate + 4 ADP + 4 Pi Hydroxypyruvate NADH,+glycerate NAD Glycerate ATP -+ 3-phosphoglycerate ADP + Pi Malate ATP CoA + acetyl-CoA + glyoxylate + ADP + Pi Succinate flavoprotein 4 malate flavoprotein.H2 Malate NAD + oxaloacetate NADH,

+

+

+ + + + +

+

+ +

+

Ribulose monophosphate pathway 3 HCHO 3 ADP 3 Pi + 3-phosphoglycerate + 3 ATP (3 HCHO + 3-phosphoglycerate) Fructose 6-phosphate ATP+fructose 1,g-diphosphate ADP Pi Dihydroxyacetone phosphate + NAD + ADP + Pi + 3-phosphoglycerate NADH, ATP (HCHO pentose phosphate ATP + hexose phosphate + ADP + Pi)

+

+

+

+

+

+ +

+

+

FIG. 6. Overall reactions leading to the synthesis of 3-phosphoglyceratefrom methanol. The reactions within parentheses may be possible in yeasts.

182

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

5) and possibly have the ribulose monophosphate pathway. Obligate methanol-utilizing bacteria have values of 1, 3, and 3 for X, Y, and 2 and possess the ribulose monophosphate pathway. Calculations based on these values showed a higher cell yield for the methanol-utilizing bacteria (0.63 gm per gram methanol) than for the methanol-utilizing yeasts (0.54 gm per gram methanol). Van Dijken et al. (1976b) also reported a low yield for a yeast (0.38 gm per gram methanol) due to alcohol oxidase. Primary alcohol dehydrogenase also has been reported to be responsible for the oxidation of formaldehyde (Heptinstall and Quayle, 1970). Th’IS enzyme may transfer the electron of formaldehyde to cytochromec . Therefore, the cell yield of the bacterium should be reduced. However, methanol oxidation by a dehydrogenase in yeasts may increase cell yield.

V. Production of Cells and Metabolites Calculations of cell yields are based only on enzymatic aspects of methanol metabolism. To obtain actual yield data, various factors besides the metabolic pathway must be considered (Cooney and Levine, 1975). One specific drawback to the use of methanol as the sole source of carbon and energy is its toxicity to the growth of the microorganism. Semicontinuous methods of culture (fed-batch culture) have been carried out using methanol-utilizing yeasts and bacteria. In contrast to the usual continuous culture, the fed-batch method keeps the methanol concentration low so that maximum specific growth rate can be obtained during cultivation. Reuss et al. (1975) reported on a fed-batch culture of Candidu boidinii in which the methanol concentration was controlled by measuring the amount of methanol in the exhaust gas. Several methods have been developed using methanol-utilizingbacteria. Yamane et al. (1976) have designed a fed-batch culture which maintains exponential growth with a feed rate programmer. Nishio et al. (1977a)have reported on a culture controlled by pH, in which a methanol-ammonia mixture was fed in response to a direct signal of pH change. Shimizu et al. (1977d) have obtained about 85 gmAiter of Protaminobacter ruber with a fed-batch culture using dissolved oxygen tension as the control indicator. Fermentor design has been improved and allows the use of methanol for the industrial process of single-cell protein. Using methanol-utilizing yeasts, Kuraishi et al. (1977)have cultivated Pichia aganobii in an air-lift fermentor, made as a pilot plant fermentor to obtain high oxygen transfer and miscibility (Kuraishi et. al., 1975). A high cell density culture at a normal dilution rate could be made. Several efforts to obtain metabolites of methylotrophs have been also reported. Accumulation of L-glutamate (6.8 mg/ml) was first reported with

UTILIZATION OF METHANOL BY YEASTS

183

Methanomonas methylouora (Oki et al., 1973). Other metabolites, e.g., pyruvate, a-ketoglutarate, and p l y saccharide, have also been detected in the culture filtrate. Methylomonas aminofaciens has been isolated as a producer of branched chain amino acids (Ogata et al., 1977). A valine hydroxamate-resistant mutant of the strain accumulated 2.2 mg/ml of Lvaline and 0.8 mg/ml of L-leucine (Izumi et al., 1977). A mutant of Methylomonas methanolophila, resistant to aromatic amino acid analogs, accumulated L-phenylalanine (4 mg/ml), L-tyrosine (1.1 mdml), and L-tryptophan (0.2 mglml) (Suzuki et al., 1977). Methanol-utilizing bacteria also produce vitamin B,, (Tanakaet al., 1974; Nishio et al., 1975a,b, 1977a,b; Toraya et al., 1975; Sato et al., 1977) and polysaccharide (Hagstrom, 1977; Kodama et al., 1977). The production of L-serine by methylotrophs is one use of their unique metabolic pathway, the serine pathway. Serine transhydroxymethylase, which fixes formaldehyde to glycine to form L-serine, is a key enzyme in this pathway. Keune et al. (1976) reported the accumulation of 4.7 mg/ml of L-serine in the culture filtrate of Pseudomonas 3ab. Arthrobacter globtjbrmis, a gram-positive methylotroph, is another L-serine producer. A methionine-requiring mutant of the strain can produce 5.2 mg of L-serine per milliliter (Tani et al., 1978). Methanol-utilizing yeasts have an alcohol oxidase catalyzing the first step of methanol oxidation. The content of the enzyme is 8% of the intracellular soluble protein, and the enzyme has eight molecules of FAD in each molecule (Kato et al., 1976). The increased amount of FAD in the cell and the derepression of FAD pyrophosphorylase, the last enzyme in FAD biosynthesis, have been observed when yeasts are grown on a methanol medium (Shimizu et al., 1977a,b; Eggeling et al., 1977). The induction of FAD biosynthesis by methanol has led to a study of the production of FAD by methanol-utilizing yeasts. Riboflavin or F M N added to a yeast culture on a methanol medium was converted to FAD in a good yield (45.4 pg/ml) (Shimizu et a l . , 1977~). The amounts of metabolites produced by methylotrophs are still low, but the advantage of using methanol as a raw material should promote its utilization not only for single-cell protein production but also for metabolite production. To further develop this field, details of methanol metabolism need to be determined. ACKNOWLEDGMENTS We wish to express our thanks to the late Professor Koichi Ogata, Kyoto University, who was the first to report on a methanol-utilizing yeast at 1969. Due to his leadership and encouragement in the study of rnethylotrophs, this review has been possible.

184

YOSHIIU TANI, NOBUO KATO, AND HIDEAIU YAMADA

REFERENCES Anthony, C. (1975). Biochem. J. 146, 286. Anthony, C., and Zatman, L. J. (1964). Biochem. J. 92, 614. Anthony, C., and Zatman, L. J. (1967). Biochem. J. 104,960. Asai, Y., Makiguchi, N.,Shimada, M., and Kurimura, Y. (1976).J. Gen. Appl. Microbiol. 22, 197. Bellion, E., and Hersh, L. B. (1972). Arch. Biochem. Biophys. 153, 368. Ben-Bassat, A., and Goldberg, I. (1977). Biochim. Biophys. Acta 497, 586. Buchanan, R. E., and Gibbons, N. E., eds. (1974). “Bergey’s Manual of Determinative Bacteriology,” 8th ed. Williams & Wilkins Co., Baltimore, Maryland. Bykovskaya, S. V., and Voronkov, V. V. (1977). Mikrobwlogiya 46, 46. Colby, J., and Zatman, L. J. (1973). Biochem. J. 132, 101. Colby, J., and Zatman, L. J. (1975a). Biochem. J. 148, 505. Colby, J., and Zatman, L. J. (1975b). Biochem. J. 148, 513. Cooney, C. L., and Levine, D. W. (1972).A h . Appl. Microbiol. 15, 337. Cooney, C. L., and Levine, D. W. (1975).In “Single-Cell Protein 11” (S. R. Tannenbaum and D. I. C. Wang, eds.), pp. 402423. MlT Press, Cambridge, Massachusetts. Diel, F., Held, W., Schlanderer, G., and Dellweg, H. (1974). FEBS Lett. 38, 274. Dudina, L. P., Sukovatova, L. V., and Eroshin, V. K. (1977). Abstr., Znt. Symp. Microb. Growth C,-Conipounds, 2nr1, 1977 pp. 89-90. Dworkin, M., and Foster, J. W. (1956).I . Bacteriol. 72, 646. Eggeling, L., Sahm, H., and Wagner, F. (1977). FEMS Microbiol. Lett. 1, 205. anner, V. C., Henderson, M. E. K., and Russell, J. D. (1960). Biochein. J. 74, 257. r’erenci, T., StrBm, T., and Quayle, J. R. (1974). Biochem. J. 144, 477. Fujii, T., and Tonomura, K. (1972). Agric. Biol. Chem. 36, 2297. Fujii, T., and Tonomura, K. (1973). A@. Biol. Chem. 37, 447. Fujii, T., and Tonomura, K. (1974). Agric. Biol. Chem. 38, 1763. Fujii, T., andTonomura, K. (1975a). A@. Bwl. Chem. 39, 1891. Fujii, T., and Tonomura, K. (1975b). Agric. Biol. Chem. 39, 2325. Fujii, T., Asada, Y., and Tonomura, K. (1974). Agric. Biol. Chem. 38, 1121. Fukuda, D. S., and Branron, D. R. (1971). Appl. Microbiol. 21, 550. Fukui, S., Tanaka, A,, Kawamoto, S., Yasuhara, S., Teranishi, Y., and Osumi, M. (1975a). J . Bacteriol. 123, 317. Fukui, S., Kawamoto, S., Yasuhara, S., Tanaka, A,, Osumi, M., and Imaizumi, F. (1975b).Eur. J. Biochem. 59, 561. Goldberg, I. (1976). Eur. J. Biochem. 63, 233. Guilbault, G. G. (1970). “Enzymatic Methods of Analysis.” Pergamon, Oxford. Haggstrom, L. (1977). Appl. Enuiron. Microbiol. 33, 567. Harrington, A. A. and Kallio, R. E. (1960).Can. J. Microbiol. 6, 1. Hazeu, W., de Bruyn, J. C., and Box, P. (1972). Arch. Mikrobiol. 87, 185. Henninger, W., and Windisch, S. (1975).Arch. Microbiol. 105, 47 (in German). Heptinstall, J., and Quayle, J. R. (1970). Biochem. J. 117, 563. Izumi, Y., Asano, Y., Tani, Y., and Ogata, K. (1977).J. Ferment. Technol. 55, 452. Janssen, F. W., Kenvin, R. M., and Ruelius, H. W. (1965).Bwchem. Biophys. Res. Commun. 30,630. Johnson, P. A,, and Quayle, J. R. (1964). Biochem. J. 93, 281. Johnson, P. A,, and Quayle, J. R. (1965). Biochem. J. 95, 859. Kaneda, T., and Roxburgh, J. M. (1959a). Can. J. Microbiol. 5 , 87. Kaneda, T., and Roxburgh, J. M. (1959b). Can. J. Microbiol. 5, 187. Kaneda, T., and Roxburgh, J. M . (1959~).Biochim. Biophys. Acta 33, 106.

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Kato, K., Kurimura, Y., Makiguchi, N., and Asai, Y. (1974).J . Gen. Appl. Microbiol. 20, 123. Kato, N., Tamoki, T., Tani, Y., and Ogata, K. (1972). Agric. Biol. Chem. 36, 2411. Kato, N., Tsuji, K., Tani, Y., and Ogata, K. (1974a). J. Ferment. Technol. 52, 917. Kato, N., Kano, M., Tani, Y., and Ogata, K. (1974b). Agric. Biol. Chem. 38, 111. Kato, N., Tani, Y.,and Ogata, K. (1974~).Agric. Biol. Chem. 38, 675. Kato, N., Tsuji, K., Tani, Y.,and Ogata, K. (1975). In “Microbial Growth on C,-Compounds” (The Organizing Committee, ed.), pp. 91-98. SOC.Ferment. Technol., Osaka, Japan. Kato, N., Omori, Y., Tani, Y., and Ogata, K. (1976). Eur. J. Biochem. 64, 341. Kato, N., Tsuji, K., Ohashi, H., Tani, Y., and Ogata, K. (1977a). Agric. Biol. Chem. 41, 29. Kato, N., Ohashi, H., Hori, N., Tani, Y., and Ogata, K. (197%). Agric. Biol. Chem. 41, 1133. Kato, N., Ohashi, H., Tani, Y., and Ogata, K. (1978). Biochim. Biophys. Actu 523, 236. Kemp, M. B. (1974). Biochem. J. 139, 129. Kemp, M. B., and Quayle, J. R. (1965).Biochim. Biophys. Actu 107, 174. Keune, H., Sahm, H., and Wagner, F. (1976). Eur. I . Appl. Microbiol. 2, 175. Kodama, T., Nakahara, T., Ohmori, T., Binh, N. T., Hoshino, K., and Minoda, Y. (1977). Abstr., Znt. Stytnp. Microb. Growth C,-Cotnpounrls, 2 n d 1977 pp. 211-213. Kuraishi, M., Matsuda, N., Term, I., Kamibayashi, A., Tonomura, K., and Fujii, T. (1975).In “Microbial Growth on C,-Compounds” (The Organizing Committee, ed.), pp. 231-240. SOC.Ferment. Technol., Osaka, Japan. Kuraishi, M., Ohkouchi, H., Matsuda, N., and Terao, I. (1977). Abstr. Znt. Symp. Mkrob. Growth C,-Cotnpounrls, 2nd, 1977 pp. 180-181. Large, P. J., Peel, D., and Quayle, J. R. (1962). Biochem. J. 82, 483. Levine, D. W., and Cooney, C. L. (1973). Appl. Microbiol. 26, 982. Mehta, R. J. (1975a). Erperienth 31, 407. Mehta, R. J. (1975b).J . Bacteriol. 124, 1165. Minami, K.. Yamamura, M., Shimizu, S., Ogawa, K., and Sekine, N. (1978). J. Fennent. Technol 56, 1. Netrusov, A. I., Rodionov, Y. V., and Kondratieva, E. N. (1977). FEBS Lett. 76, 56. Nishio, N., Yano, T., and Kamikubo, T. (1975a). Agric. Biol. Chem. 39, 21. Nishio, N., Yano, T., and Kamikubo, T. (1975b). A@. Biol. Chem. 39, 207. Nishio, N., Tsuchiya, Y., Hayashi, M., and Nagai, S. (1977a).J. Ferment. Technol. 55, 151. Nishio, T., Tanaka, M., Matsuno, R., and Kamikubo, T. (197%). J. Fennent. Technol. 55, 200. Ogata, K., Nishikawa, H., and Ohsugi, M. (1969). Agric. Biol. Chem. 33, 1519. Ogata, K., Nishikawa, H., Ohsugi, M., and Tochikura, T. (1970).J. Fennent. Technol. 48,389 (in Japanese). Ogata, K., Izumi, Y., Kawamori, Y., Asano, Y., and Tani, Y. (1977).J . Ferment. Technol. 55,

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Recent Chemical Studies on Peptide Antibiotics from the Genus Bacillus JUN’ICHI

SHOJI

Shwnogi Research kboratory, Shionogi 61 C o . , Ltd., Fukushinuz-ku, Osaka, 553 Japan I. Introduction, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

........................................ A. Bacilysin, Linear Gramicidins, and Edeines . . . B. Cerexins A, B, C, and D ............................ C. Tridecaptin Group of Antibiotics ...................... 111. Cyclic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gramicidin S, Tyrocidins, and Bacitracin . . . . . . . . . B. Mycobacillin ....................................... C. Iturin A, Mycosubtilin, and Bacillomycin L . . . . . . . . . . . . D. Octapeptin Group of Antibiotics ...................... E. New Polymyxins ................. IV. Peptide Lactones.. .................... A. Esperin and Surfactin ................. B. Brevistin . . . . . . . . . . . 11. Linear Peptides

V. Concluding Remarks. . References . . . . . . .

187 188 188 189 192 194 194 195 196 199 203 208

. . . . . . . . . . . . . . . . . 212 . . . . . . . . . . . . . . . . . 213

1. Introduction Studies of the genus Bacillus have not yet shown this group to be a good source for antibiotic substances. While 168 antibiotics had been reported from the genus Bacillus (Berdy, 1974), many of them have been reported as crude or partially purified preparations. New interest was generated by the isolation of the amino glycoside antibiotics, the butyrosins. The antibiotics isolated in recent years are chemically well characterized and their descriptions usually accompanied by structural studies (Table I). It is almost impossible to estimate the exact number of antibiotics which has been isolated from cultures of the genus even if the antibiotics which have not been well characterized are eliminated from count. Some antibiotics given different names are presumably identical, and in some cases several named antibiotics are likely to be a single entity of a complex. However, a conservative review suggests that the number of antibiotics from Bacillus is of the order of 117, of which 80 members are peptides. Those for which structures have been proposed are 64, and 47 of these are peptides. A recent review of the chemistry and biogenesis of the peptide antibiotics of Bacillus has been published by Katz and Demain (1977). The present author has been engaged in a series of studies on antibiotics from the genus 187 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in m y form reserved. ISBN 0-12-002624-4

188

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SHOJI

TABLE I THE NUMBEROF ANTIBIOTICSFROM THE GENUSBacillus &PORTED IN THE PASTFOURDECADES

Era

Number of reported antibiotics"

up to 1950 1951-1960 1961-1970 1971-1977

30 (10) 28 (16) 13 (1) 58

OThis is the total number; that of not well-characterized antibiotics is in parenthesis. An antibiotic reported as a complex in the first report and separated later is counted as a single one.

Bacillus and has now reported the isolation of 21 new antibiotics. Sixteen of these are peptides and structure elucidation has been completed on 12. In the present paper chemical studies on peptide antibiotics from the genus Bacillus which have been reported in the past several years are reviewed. These peptide antibiotics can be conveniently divided into three large groups: linear peptides, cyclic peptides, and peptide lactones. Brief descriptions of the biological activities of newly isolated antibiotics also are included and there are some briefly cited references to previously described antibiotics.

II. Linear Peptides A. BACILYSIN,Lrmm GFLMICIDINS,AND EDEINES

Bacilysin is a dipeptide with antibiotic activity produced by B . subtilis. The structure (Fig. 1) was determined in 1970 by Walker and Abraham. Tetain, produced by B . pumilus, was proved to be identical with bacilysin (Kaminski and Sokolowska, 1973).

CH3 FH2 I NH,-CH-CO-NH-CH-COOH ILI

ILI

FIG. 1. Bacilysin.

189

RECENT CHEMICAL STUDIES ON PEF'TIDE ANTIBIOTICS

IL)

(L)

H C O * X - G l y * Ala ILI

Trp

-

ID1

IL)

-

ILI

ID1

ID1

ID)

IL)

Leu * Ala - V a l - V a l * V a l * ILI

ID)

ILI

ID1

Leu * Y * Leu * Trp * Leu * T r p - N H C Y C Y O H

Valine-gramicidin A Iroleucine-gramidin A Valine-gramicidin B koleucine-gramicidin B Valine-gramicidin C Iroleucin-gramicidin C

X

Y

Val Ile Val Ile Val Ile

Trp Trp Phe h e Tyr Tyr

FIG. 2. Linear gramicidins.

Tyrothricin, an antibiotic complex produced by B. brevis, was separated into the linear pentadecapeptides, gramicidins, and the cyclic decapeptides, tyrocidins. The linear gramicidins (Fig. 2) have been deduced to be composed of six structurally related variants (Sarges and Witkop, 1965). Edeine (Fig. 3), produced by B. brevis Vm 4, was separated into four active components, edeines A,, B,, C, and D. These antibiotics are particularly of interest because of the fact that they contain unusual amino acids (P-tyrosine or P-phenyl-P-alanine, isoserine, diaminopropionic acid, 2.6diamino-7-hydroxyazelaic acid), glycine, and an amine, spermidine or guanylspermidine (Hettinger and Craig, 1970; Wojciechowska et al. , 1972). B. CEREXINS A, B, C,

AND

D

In 1975, Shoji et al. (1975a)reported the isolation of cerexins A and B from the culture broths of B. cereus 60-6 and B. cereus Gp-3, respectively. These antibiotics are amphoteric in nature and soluble only in dimethyl sulfoxide, R, ~

~

~

~

H

~

-

COI HO

N :YNY ~ -

C

-

~

-

I

(FYh CH-N%

I

CH-OH

R2-NH-(CH,),-NH-(CH,b-NHCO-CH,-NHCO-C%

Rl

R2

EdeineAl

-OH

-H

Edeine Bl

-OH

-C-NY

-H

NH -H

EdeineD

I

FIG.3. Edeines A,, B,, and D.

I

C

~

-

~

~

~

~

190

JUN'ICHI SHOJI

dimethylformamide, and alkaline water. Their antimicrobial activities in uitro are not so strong against gram-positive bacteria: MIC (pgml) S. aureus, 50; S . pyogenes, 6.25;S . pnemoniue, 3.13; however, appreciable activities in uiuo are shown: EDmo'(mg/kgX 2) S. aureus, 4.7; S . pyogenes, 4.4; S . pnemoniue, 3.9. The acute toxicity of cerexin A to mice is relatively low: LDs0 (mg/kg) 100-200 (i.p.), 50-100 (i.v.), >500 (s.c.). By acid hydrolysis cerexin A released Asp (2D, l ~ )D-aThr , (l),L-Ser (l), D - V ~(2), D-dle (l),D-Trp (l), L-threo -7-hydroxylysine (abbreviated as ~-yHyl)(l),and ammonia (3) (Shoji and Hinoo, 1975). By 1-hour hydrolysis from the acid hydrolyzate, a Eatty acid was isolated. It was separated as a methyl ester by gas chromatography and was identified with P-hydroxy isoundecanoic acid (abbreviated as i-Cllh3) by MS and nuclear magnetic resonance (NMR). When the hydrolysis time was prolonged, three additional peaks were given in the gas chromatogram. One was identified with that of an a#-unsaturated acid formed by a dehydration reaction during the acid hydrolysis; another one was supposed to be that of a y-lactone probably formed through a P, y-unsaturated acid. Such complex phenomena commonly are observed in the acid hydrolysis of acylpeptides whose acyl groups are P-hydroxy acids, e.g., other cerexins, octapeptin group antibiotics, and some members of tridecaptins, as will be described later. The N-terminal end of cerexin A was indicated to be FA + Val (FA means fatty acyl groups) by isolation of this fragment from a partial acid hydrolyzate. The C-terminal end was accidentally indicated to be Trp 4 aIle, as the cleavage reaction with N-bromosuccinimide released aZZo-isoleucine itself. Absence of lactone or ester linkage was suggested by the infrared (IR) spectrum and the presence of one each of amino and carboxyl function was indicated by titration. Thus, a linear structure of acylated decapeptide was assumed for cerexin A; furthermore, the 3 moles of aspartic acid found in the hydrolyzate were considered to be present as amide forms in the intact molecule. A molecular formula Cs3HlmN15019 anticipated from the above coincided well with the elemental analytical data. Treatment of cerexin A with concentrated hydrochloric acid at low temperature selectively cleaved it at the C-terminal side of the yHyl residue. The C-terminal peptide fragment obtained by the cleavage reaction was indicated to be aThr + Ser + Trp + aIle by successive Edman degradation reaction. An enzyme preparation, polymyxin acylase, from the cell of Pseudomonas sp. M-6-3 has been reported to be able to deacylate polymyxin E. This enzyme acted on cerexin A to afford deacyl cerexin A. The successive Edman degradation reaction on deacyl cerexin A proceeded only up to the third step, indicating a sequence, Asn + Val + Val. From these results it has been concluded that the C-terminal side of cerexin A is yHyl + aThr + Ser + Trp +aIle and the N-terminal side is FA + Asn 4Val + Val, and the

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

191

remaining two residues, i.e., Asn, Asn, must be present between the two sequences. For the reason the Edman degradation reaction stopped at the fourth step, several possibilities were assumed. To clarlfy this problem, a dehydrationreduction procedure was applied to cerexin A and deacyl cerexin A. By the procedure, an asparaginyl residue (a-carboxyl peptide linkage) gave rise to 2,4-diaminobutyricacid, while an isoasparaginyl residue (P-carboxyl linkage) gave rise to p-alanine. Consequently, it was clarified that the three asparagine in intact cerexin A existed really as asparaginyl residues, and it was suggested that some bond migration of an asparaginyl residue, to produce largely an isoaspartyl residue, might occur during the deacylation procedure. The three asparagine residues in the sequence of cerexin A were isolated separately as DNP-Asp, whose CD curves were measurable. Thus, the total structure of cerexin A was concluded (Shoji et al., 1976d). The constituent amino acids of cerexin B were found to be Asp (2D, l ~ ) , D-aThr (l),Gly (l),D - V (l), ~ D-aIle (I),D-Phe (I), D-Trp (1)and L-yHyle (1). This indicated replacement of Ser and one Val residue in cerexin A by Gly and Phe in cerexin B. The constituent fatty acids were elucidated to be P-hydroxyisodecanoic acid (i-C1,,h3), P-hydroxydecanoic acid (n-Cl,,h3), P-hydroxyisoundecanoic acid (i-Cllh3), and P-hydroxyanteisoundecanoic acid (a-Cl,h3)by procedures similar to those used for cerexin A. The relative abundance of these fatty acids was approximately estimated as i-C1,,h3(66%), n-C1,,h3(12%),i-C,,h3 (8%),and a-Cllh3 (14%), although it varied somewhat in preparations from different fermentation media. Therefore, cerexin B was considered to be a complex of four acyl peptides, whose peptide parts were common. The amino acid sequence of cerexin B was determined by essentially the same methods as in cerexin A (Shoji and Kato, 197613). During an attempt to increase the antibiotic production of the Bacillus strains 60-6 and Gp-3, it was found that both the strains produced an additional antibiotic component in an improved medium. These new antibiotic components were isolated and named cerexins C and D, respectively. The physicochemical properties of cerexins C and D are closely similar to those of cerexins A and B. Also, both the antibiotics exhibit the same antimicrobial spectra as cerexin A, although they are somewhat less active (Shoji et al., 1976e). By acid hydrolysis and amino acid analyses, it was found that cerexins C and D contained lysine but not y-hydroxylysine. All the amino acids other than y-hydroxylysine in cerexins A and B were found in cerexins C and D, respectively. Similarly the constituent fatty acids of cerexins C and D were found to be essentially the same as those of cerexins A and B respectively. Thus, it was concluded that cerexins C and D were the acyl peptides analo-

192

JUN’ICHI SHOJI FA - A m - V a l (Dl

ID1

-

X -Am (Dl

IL)

- Am - Y - aThr - Z - Trp(0)

(L1

(D)

FA Gxexin A

wco

alle (01

ID)

X

Y

Z

Val

y-Hyl

Ser

Phe

y-Hyl

Gly

Val

Lys

Ser (L)

(L)

OH

B

* C o

-CO OH

c

W

C

OH

O OH

y-Hyl:

y-Hydroxylysine

FIG. 4. Cerexins A, B, C , and D.

gous to cerexins A and B, respectively, in which the y-hydroxylysine residue was replaced by a lysine residue. The four peptide variants, cerexins A, B, C, and D, isolated from the strains of B. cmew are presented in Fig. 4. They differ from each other in the nature of the htty acid component and in variations at the third, sixth, and eighth position of their sequences.

C. TIUDECAPTINGROUPOF ANTIBIOTICS Very recently Shoji et al. (1978) reported the isolation of tridecaptins A, B, and C from the culture broths ofB. polyrnyxa AR-110, B-2, and E-23. These antibiotics were characterized as acyl tridecapeptide and named the tridecaptin group of antibiotics. Tridecaptin A is active against gram-negative and gram-positive bacteria in uitro and in uiuo: MIC (pg/ml) E. coli EC-14, 3.13; K . pneumoniue, 6.25; P . aeruginosa, 50; S . aureus, 50: ED, (mgkg x 2) E. coli, 0.41; K . pneumoniae, 5.5. The LD, value by the i.p. route is 25 mgkg. The hydrochloric acid salt of tridecaptin A is a colorless amorphous powder. The molecular N 1 7 0 2 0 * HC1.H20. 3 The infrared abformula was indicated to be C73H115 sorption spectrum indicated the presence of peptide bonds and carboxyl functions. Amino acid analysis of the acid hydrolyzate clarified the amino acid composition of tridecaptin A and the constituent amino acids were isolated from the hydrolyzate. By their measured optical activities, they were determined

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

Tridecaptin A

*co

Val

Phe

alle

193

Ala

OH

b

c

o

Ser

Dab: 2,4-Diaminobutyric acid

FIG.5. Tridecaptins A, B, and C.

to be Dab (ZD, l ~ ) Ser , ( l ~l ,~ Glu ) , (IL),Gly (I),Ala (IL),Val (ID, 1L), aIle (ID), Phe (IL), and Trp (ID). When the ethereal extract of the hydrolyzate was examined with gas chromatography (GC), the presence of p-hydroxyanteisononanoicacid (a-Cgb3)was indicated. The fatty acid methyl ester was isolated by thin layer chromatography (TLC) and the identity was confirmed by MS and NMR. This fatty acid already had been found as the constituent of octapeptin C as described later. This peptide antibiotic was selectively cleaved at the N-terminal side of the Trp residue with N-bromosuccinimide and the sequence of the C-terminal peptide fragment was determined by Edman degradation reaction as Ser + Dab -+ Dab -+ Phe + Glu -+ Val 4aIle -+ Ala. By partial acid hydrolysis, two further peptide fragments, F A 4Val -+ Dab + Gly and Ser -+ Trp* (whose Trp residue was partially degenerated during acid hydrolysis), were obtained. The sequence of the former tripeptide was determined by further partial hydrolysis. Thus, the amino acid sequence of Tridecaptin A was clarified. The remaining problem was elucidation of the chiralities of the individual residues: the three Dab residues (2D, l ~ ) two , Ser residues ( l ~ l, ~ )and , two Val residues (l~1 , ~ These ). residues were individually isolated from appropriate peptide fragments and their optical activities were measured. The total structure of tridecaptin A was thus determined (Kato and Shoji, 1978a). Tridecaptins B and C were obtained as complexes of acyl peptides. Their structures were deduced in a way similar to those above (Kato and Shoji, 197813). The structures of tridecaptin group of antibiotics clarified to date are presented in Fig. 5.

194

JUN'ICHI SHOJI

111. Cyclic Peptides A. GRAMICIDIN S, WOCIDINS,AND BACITRACIN

Gramicidin S (Fig. 6), produced by strains ofB. breuis, is a cyclic decapeptide (Battersby and Craig, 1951). Tyrocidins A, B, and C (Ruttenberg et d . , 1965)are also cyclic decapeptides (Fig. 7), produced by a certain strain of B. breuis (Dubos) which simultaneously elaborates linear gramicidins. Gramicidin S and the tyrocidins are closely similar to each other, as shown by the fact that the half parts of these peptides are identical. Two other tyrocidins (D and E) have been elaborated by biosynthetic techniques (Ruttenberg and Mach, 1966; Fujikawa et al., 1968). Izumiya and his co-workers synthesized approximately 50 cyclic decapeptides analogous to gramicidin S, most of which showed antibiotic activity (Izumiya, 1976). Bacitracin is one of the oldest known antibiotics produced by B . Zichenifmis. The structural determination of this antibiotic had been rather complicated. Structural studies carried out by researchers in two separate laboratories in agreement allowed two possible amino acid sequences for this peptide antibiotic. The most probable structure (Swallow and Abraham, 1959; Soffel and Craig, 1961), which contains a cyclic hexapeptide moiety and a thiazoline ring, is believed to be the structure of bacitracin A. However, it should be more commonly known that Ressler and Kashelikar (1966) reduced the possibilities to one conclusive one by a dehydration-reduction procedure, which they devised for rapid identification of asparaginyl and

c

ILI

ILI

Val - O m Pro-PheID1

ILI

- - - 1 ILI

ID1

ILI

Leu

Phe

Pro

Leu-Om-Val ILI

ILI

ILI

FIG. 6. Gramicidin S. IL)

ILI

ILI

ID1

ILI

Val -Om -Leu -Phe - P

L,?ILI

Gln- Asn

- -

IL)

ILI

Y

X

ID1

ILI

X

Y

Z

Tyrocidin A

Phe

B C D E

Trp Trp Trp

Phe Phe Trp Trp

Tyr Tyr Tyr Trp

Phe

Phe

Phe

FIG. 7. Tyrocidins A, B, C, D, and E

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

~H-cH21LI

CO-

ID1

ILI

Leu-Glu-Ile-

ILI

(Dl

ILI

195

ID1

~ys-Orn-Ile-Phe,

FIG.8. Bacitracin A.

glutaminyl residues in peptides. By the procedure, they proved the presence of a L-asparagine residue inside the peptide ring and the absence of a D-isoasparagine residue outside the ring, indicating the structure (Fig. 8) which contains a cyclic heptapeptide moiety as the strucure of bacitracin A. Very recently this structure was confirmed by total chemical synthesis (Hirotsu et al., 1978). B. MYCOBACILLIN Mycobacillin, an antifbngal antibiotic produced by B . subtilis, is particularly interesting, because it is an example of the presence of unusual peptide linkages (non-a-peptide linkages) in the peptide antibiotics from the genus Bacillus. Therefore, the structural studies are outlined here, although they were reported in 1971. Mycobacillin is a cyclic peptide with 13 amino acid residues [ D - A s ~(4), L - A s ~(l),D-G~u(2), L-Tyr (2), L-Ser (l), L-Ala (l), L-Leu (l),L-Pro (l)] whose sequence had been determined in 1960 by Majumdar and Bose. Sengupta et al. (1971) reported their final studies in 1971 and proved the presence of y-linked glutamic acid residues in the peptide antibiotic. When mycobacillin was reacted with N-bromosuccinimide in succinimide solution the yield was 2 moles of carbon dioxide per mole of the antibiotic. It was previously proved that only a-carboxyl groups were responsible to this reaction. Therefore, this indicated the presence of two non-a-peptide linkages in the peptide antibiotic. Then mycobacillin was oxidized with sodium hypobromite, followed by hydrolysis. Succinic acid was detected in the hydrolyzate by paper chromatography. This meant that at least one glutamic acid residue was y-linked in the molecule. However, it was not possible to determine the exact number of y-glutamyl residues, so the technique of hydrazinolysis was adopted. By this method, mycobacillin gave the a-hydrazide of aspartic acid and y-hydrazide of glutamic acid. This indicated that the five aspartic acid residues were all a-linked, whereas the two glutamic acid residues were y-linked.

196

JUN’ICHI SHOJI

ILI

ILI

ILI

Tyr -Asp * Tyr

HO-Glu-AspID1

ID1

-

ILI

ID1

ILI

Pro- Asp- Ala-Asp ILI

ID1

Ser -Asp *Leu * GIu-OH

ID1

ILI

-ly

ID1

FIG. 9. Mycobacillin.

This conclusionwas further confirmed as follows. The free carboxyl groups of mycobacillin were esterified with methanol and methanolic HC1. The methyl ester of mycobacillin thus formed was reduced with lithium borohydride. When the product was hydrolyzed, a-amino-y-hydroxybutyric acid and y-amino-6-hydroxyvalericacid were released. P- and y-esters of aspartic acid and glutamic acid should give a-amino-y-hydroxybutyric acid and a-amino-6-hydroxyvaleric acid, and a-esters should give P-amino-y-hydroxybutyric acid and y-amino-6-hydroxyvalericacid. Therefore, this result conclusivelyproved that all aspartic acid residues were a-linked and glutamic acid residues were y-linked in mycobacillin. The fact that the glutamyl linkages are more susceptible to hydrolysis also supports this conclusion. From these studies, they concluded the structure of mycobacillin to be as shown in Fig. 9.

C. ITURINA,

MYCOSUBTILIN, AND

BACILLOMYCIN L

In recent years, Peypoux, Delcambe, and their group have studied a group of antibiotics, such as iturin, bacillomycin B, bacillomycin K, eumycin, mycosubtilin, bacillomycin of Landy et al., and the antibiotic of Kaubitschek, which have been known for a long time to be crude or partially purified preparations isolated from cultures of Bacillus subtilis, primarily active against fungi. The active entity of iturin was isolated and named iturin A, and the active principles of bacillomycin B, bacillomycin R, and eumycin were proved to be identical with iturin A (Besson et al., 1976). It was also proved that all these antibiotics had common structural characteristics, containing p-amino acids with 14-17 carbon atoms. They have reported elucidation of the structure of iturin A, mycosubtilin, and bacillomycin of Landy et al. (later they called it bacillomycin L). The antibiotic of Kanbitschek is now under their investigation (personal communication). An outline of their structural studies on these antibiotics is described here. By total acid hydrolysis, iturin A gave a mixture of lipophilic amino acids in addition to water-soluble amino acids. The lipophilic amino acids were extracted with ethyl ether or chloroform. Gas chromatography combined with MS of its derivatives (N-trifluoroacetyl butyl ester) allowed separating of several components (XI +XJ: The two most abundant components were X 1

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

197

(34%) and X , (47%).In the mass spectra of the N-acetyl methyl esters of X, and X 2 , several common fragment ion peaks which indicated p-amino acid structure were observed: 102 (H,N+ = CH-CH,-COOCH,), 144 (H3C . CO - HN, = CH-CH,-COCH,), etc. A similar mass spectrum was obtained with the synthetic P-aminopentadecanoic acid. The is0 structure [CH3-CH (CH3)-l for XI and the anteiso structure [CH,-CH,-CH(CH,)-] for X 2 were suggested by the MS data and confirmed by formation of acetone from X and methylethylketone from X by chromic acid oxidation. From these results, X was concluded to be 3-amino-12-methyltridecanoic acid and X was 3-amino-12-methyltetradecanoicacid. The components X , X , and X were also found in the hydrolyzate of mycosubtilin. Similar experiments determinedx to be 3-amino-14-methylpentadecanoicacid; X 4, 3-amino-hexadecanoicacid; and X 5 , 3-amino-14-methylhexadecanoicacid. Quantitative analysis of the water-soluble amino acid gave the molar ratio: Asp,,,, Glu,, Proo.,, Ser,,,, Tyro.,, and their configurations were determined by enzymatic methods using D-amino acid oxidase, t-glutamate oxidase, and L-glutamate-oxalacetate transaminase. As iturin A showed no mobility on paper electrophoresis, the presence of asparagine and glutamine was suggested. When iturin A was tested by the dehydration-reduction procedure developed by Ressler and Kashelikar (1966), the hydrolyzate of the product showed the presence of diaminobutyric acid and ornithine. Thus, the amino acid composition of iturin A was concluded to be: D-Am,, L-Asn,, L-Gln,, L-Pro,, L-Ser,, D-Tyr,. Dinitrophenylation of iturin A gave only 0-DNP-tyrosine. The IR spectrum of the antibiotic did not show any characteristic absorption of an ester function and titration indicated the absence of a free carboxyl group. These facts can only be explained on the basis of a cyclic peptide structure for iturin A. When iturin A was hydrolyzed with 6 N HC1 at 105°C for 16 hours, an another lipophilic substance was liberated besides the C14,C15 p-amino acids (abbreviated as PNCl4,CI5hereafter). It was further hydrolyzed to give Ser and PNC,,,C,,. The presence of Ser in the N-terminal side was confirmed by dinitrophenylation, Edman degradation, and mass spectrometry. The lipophilic substance was concluded to be Ser +. PNCl4,CI5.Iturin A was partially hydrolyzed under various conditions. From the hydrolyzate obtained at conditions of 10N HC1, 80"C, and 3 hours, four lipophilic peptide fragments, A,, A,, B,, and B,, were isolated. Further, four hydrophilic peptide fragments, PI, P,, P,, and P,, were isolated from the hydrolyzates at 3 N HC1, 105"C, 4 hours and 0.1 N HC1, 105"C, 6 hours. End group analysis by dinitrophenylation (Sanger method) or by dansylation combined with Edman degradation and hydrazinolysis clarified the sequences of these fragments as follows:

,

,

,

198

A, A, B, B, P, P, P3 P,

JUN’ICHI SHOJI

L-Ser + PNCl4,CI5+ L-ASP+ D-Tyr L-Ser -+ PNC14, C15 + L - A s ~+ D-Tyr + D-AsP L-Ser + PNC14,C15 L-Ser + PNC14,C15+ L - A s ~ D - A s -+ ~ L-G~u L-G~u + L-Pro + D - A s ~ L-G~u -+ L-Pro D-Tyr 4 D - A s ~

From these sequences, the total amino acid sequence of iturin A was deduced (Fig. 10). This structure for iturin A was confirmed by mass spectrometry of the permethylated and perdeuteriomethylated derivatives. Molecular ion peaks were observed at m/e 1280 and 1294 with the permethylated derivative and at m/e 1331 and 1345 with the perdeuteriomethylated derivative. This is compatible with the formulas C48H74N12014 and C46H76N12014 for two homologs of intact iturin A containing PNC15 or PNC14.The interpretation of the fragment ion peaks also supported the structure of iturin A concluded from the chemical degradation (Peypouxet al., 1973, 1978; Delcambe et al., 1977). The structure of mycosubtilin was determined by the same techniques as used in the case of iturin A. It contains eight a-amino acids: Asp (2D, 2 ~ )Ser , ( l ~Glu ) , ( l ~Pro ) , ( l ~ )Tyr , ( l ~ )and , onep-amino acid; 3-amino-14-methylacid, or 3-aminohexapentadecanoic acid, 3-amino-14-methylhexadecanoic decanoic acid. A cyclic structure was determined as shown in Fig. 11 (Peyboux et al., 1976). Besson et al. (1977) published a report on structural elucidation of bacillomycin of Landy et al. In the report, they used the name bacillomycin L instead of bacillomycin of Landy et al. The procedures used for the structural elucidation were essentially the same as those for iturin A. Bacillomycin L contains seven a-amino acids: Asp ( l ~l,~ )Glu , ( l ~Ser ) , ( l ~l ~ , Thr ) , (l~), (LI

(DI

ID1

C y - C O -Asn -Tyr -Am I

R-yH NH-

ILI

ID1

ILI

ILI

1

Ser- Am- Pro- Gln

R: CHs-CH-(CY),-

I

CH3 CH,-CH,-CH-(CH& I

cH3

FIG. 10. Iturin A.

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

(Ll

(Ll

199

(DI

(Ll

CHz-CO -Asn -Gln -Pro -Tyr

I R-CH 1

ILI

(DI

(Dl

NH- Am- Ser- Asn-

(DI

Am

1

R: CH,-(CH?),z-

CH,-CH-(CH,),OI CH3 CH,-CH,-CH-(CHz),oI CH3

FIG. 11. Mycosubtilin.

Tyr ( l ~ )and , one p-amino acid: 3-amino-12-methyltridecanoic acid, 3-amino-12-methyltetradecanoicacid, or 3-amino-14-methylpentadecanoic acid. The cyclic structure shown in Fig. 12 was determined by sequential analysis of the peptide hagments obtained by partial acid hydrolysis.

D. OCTAPEPTINGROUPOF ANTIBIOTICS 1 . E M 4 9 (Octapeptins

A1,2,3

and

B1,2,3)

In 1973, Meyers and his colleagues (1973a) reported the isolation of an antibiotic complex EM49 &om the culture broth of a strain of B. circulans. It is a basic substance and the hydrochloride is freely soluble in water and methanol. By analysis of the acid hydrolyzate, it was indicated to be a complex of acyl peptides and was later separated into four components on C M cellulose column. EM49 possesses activity against gram-positive and gram-negative bacteria as well as against yeasts, fungi, and protozoa in uitro. The higher order of activity against gram-negative than against gram-positive bacteria is shown. The antibiotic is not cross-resistant with a number of other antibiotics representing different chemical types. The absence of cross-resistance was shown ILI

ID1

(DI

C y - C O -Asp -1yr -Asp I

1

R-CH N H - Thr- Ser- Gln- Ser ILI

(DI

ILI

ILI

R: CH,-CH-(CY),-

kH3 CH,-CH~-CH-(CHZ),& I

FIG. 12. Bacillomycin L.

200

JUN’ICHI SHOJI

even between this antibiotic and polymyxin B. The action of EM49 is bacteriocidal and not bacteriostatic. There is no essential difference between the activities of the separated four components of EM49. EM49 is also active in uiuo. A therapeutic effect against experimental infection of S . pyogenes or Escherichia coli was shown when it was given subcutaneously (Meyerset al., 197313). The primary site of action of this antibiotic was shown to be in disturbing membrane permeability in the experiment using E. coli (Rosenthd et a[. , 1976). Amino acid composition of the EM49 complex was determined to be Leu, Phe, and 2,4-diaminobutyric acid (abbreviated as Dab) in a molar ratio of 2.55 : 0.44 : 5.01. These amino acids were isolated and measured for [ M I D values, from which the Phe was indicated to be in the L configuration and the Dab and Leu were mixtures of D and L forms. A mixture of P-hydroxy fatty acids was liberated by acid hydrolysis of brief hydrolysis time. They were separated by preparative gas chromatography of the methyl esters into three components. By mass spectrometry, elemental analysis, NMR, and [ M I D values in chloroform and in methanol, the major one (ca. 65%) was deduced to be 8(S)-methyl-3(R)-hydroxydecanoic acid (C1lHzzO3); the others were 8-methyl-3(R)-hydroxynonanoicacid and 3(R)hydroxydecanoic acid (C10H2003). The EM49 complex was separated into four components (a,/3, y , and 6) by column chromatography on CM cellulose. Amino acid analysis of the hydrolyzates showed that the a and P fractions contained five Dab and three Leu residues, whereas y and 6 contained one Phe, five Dab, and two Leu residues. Examination of the lipid portions of the hydrolyzates showed that the more abundant fractions, p and 6, released the Cl1acid, whereas the smaller fractions, a and y, released the C,, acid and two other minor lipid components. These data suggested that EM49 complex was a complicated mixture of acyl octapeptides, which were constructed from combinations of two peptide parts and three or more fatty acyl residues. Sequential analysis was made on the EM49 complex, not on the separated components. After the four amino groups of EM49 were blocked with 2,4dinitrophenyl (DNP) groups or with benzyloxycarbonyl (Z) groups, the P-hydroxy group of the fatty acyl residue was oxidized to a ketone with dimethyl sulfoxide-dicyclohexylcarbodiimide (DMSO-DCC). Then the P-ketoacyl group was cleaved from the peptide with hydroxylamine to give derivatives of deacyl EM49. Further dinitrophenylation and then hydrolysis of tetru(DMP)-deacyl EM49 gave bis(DNP)-Dab, which was proved to be in the D configuration by its optical rotation. This N-terminal amino acid was removed by Edman degradation reaction. Dinitrophenylation and then hydrolysis on the remaining peptide gave a-DNP-Dab. This established the

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

201

structure of a cyclic heptapeptide with a branched amino acid residue. The Edman degradation reaction, up to the final step, succeeded with tetrabenzyloxycarbonyl deacyl EM49, and the N-terminal amino acid of the remaining peptide at each step was identified as its DNP derivative (the Sanger method). The second step of this degradation reaction opened the peptide ring. The remaining peptide of the fourth step, a pentapeptide, was indicated to be not homogeneous by TLC and it released both DNP-Leu and DNP-Phe by the end group analysis. Each of the residues in the sequence was isolated as its DNP derivative and the optical rotation was measured. The N-terminal Dab and the Leu at the fourth position were indicated to be in the D configuration. Thus, the structure of EM49 complex was shown to be as in Fig. 13 (Parker and Rathnum, 1973, 1975).

2. 333-25 (Octapeptin C , ) In 1976, Shoji and his co-workers (1976b) reported the isolation of an antibiotic from the culture broth of a strain belonging to B. circulans. The antibiotic is closely related to EM49 but differentiated in mobility on TLC and in analysis of fitty acids by GC. Its physicochemical properties and antimicrobial activity are closely similar to those of EM49. Amino acid analysis on the acid hydrolyzate of 333-25 revealed its amino acid composition. Isolation of the constituent amino acids and measurement , ), of their optical rotations clarified their chiralities as follows: Dab ( l ~4 ~ Leu ( 2 ~ )and , Phe (ID). The fatty acid constituent was determined to be P-hydroxyanteisononanoicacid (u-Ch3)by GC, NMR, and MS. Deacylation by the action of polymyxin acylase was unsuccessful, probably because the antibiotic was insoluble in the buffer solution used for the enzyme reaction. Therefore, the chemical reaction used for deacylating EM49 was applied to

,

*

L-Dab+D-Leu-(L-Le.u,

X - D-Dab-L-Dab a

x=

L-&)

1

“y\ L:Leu-l-Dab+L-Dab

+ HO H O

Dab: 2,4-Diaminobutyric acid

FIG. 13. Structure of EM49.

202

JUN’ICHI SHOJI

the dinitrophenyl derivative of 333-25, and tetra(DNP)-deacyl 333-25 was afforded. Successive Edman degradation reaction on this derivative was carried out with some modification in order to diminish the loss of remaining peptide during the reaction. The reaction proceeded well up to the final step, and the total sequence was elucidated (Fig. 14) (Shoji et al., 1976c; Kato and Shoji, 1976).

3. Nomenclature In 1976, the researchers of two laboratories (Squibb Institute and Shionogi Laboratory) agreed in the following nomenclature proposal (Meyers et al., 1976). EM49 and 333-25 and possibly some other antibiotics make a new group of antibiotics. Octapeptin is proposed as the group name, with provisions for identlfying individual members. Each of the peptide variants is designated by a different capital letter in order of discovery, and the fatty acid residues are differentiated by subscript numbers following the letter. Consequently, each member of EM49 complex is named as octapeptins A,,2,3 and B 1,2,3, and 333-25 is named as octapeptin C 1. Now, the general structure of the octapeptin antibiotics can be presented and their individual members can be distinguished as in Fig. 14. FA

-

0-Dab

-

L-Dab

-

L-Dab

t~-Leu

- X

- L-Dab

Y

L-Dab

Name

Synonym

X

Y

FA

Octapeptin A,

EM4p

0-leu

L-Leu

a-Cllh3

0-bU

L-Leu

i-clohJ

0-LCU

L-Leu

n-cl&3

0-leu

L-Phe

a-Cllh3

O-LCU

L - ~ C

i-C10h3

EM49y

0-Leu

L-Phe

r~-cl&~

33-25

0-Phe

L - ~ U a-@

Octapeptin El Octapptin

4

Octapeptin Bj

EM4%

}

Octapaptin CI

a-C&

:

i-Cloh3 :

dCo Dab: 2.4-Diaminobufyyric acid

I

d

c

o

FIG. 14. Structure of octapeptin antibiotics.

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

203

4 . Other Related Antibiotics Some other antibiotics which seem to be related to the octapeptin group have been reported. Bu-1880 (Japanese Patent, 1974)produced by B. circulans contains Phe (l),Leu (2), Dab (5), and 3-hydroxy-8-methyldecanoicacid (U-C,,h3). It seems to be identical with octapeptin B except for ambiguity in the chiralities of the amino acid residues. TM-743 (Japanese Patent, 1976a) was reported to be produced by B. circulans and was found to be constructed from Dab (S),Leu (2), Phe (l), and two kinds of P-hydroxy acid with C8and C9. It therefore seems to be closely related to octapeptin C,. Y-8495 (Japanese Patent, 1975), produced by B. bungoensis, and AB-1 (Japanese Patent, 1976b), by B. subtilis, have been reported constructed from Phe, Leu, Dab, and hydroxy acids, although the amino acid composition ratios have been reported different from those of the octapeptin group antibiotics.

,

E. NEW POLYMYXINS It seems likely that the producing organisms of the polymyxin group of antibiotics are distributed very widely in nature. In the course of screening studies for new antibiotics from the genus Bacillus carried out by the present author and his group, a lot of strains which produce this group of antibiotics have been found. They have isolated the strains producing almost all members of this group of antibiotics, i.e., polymyxins A (or M), B, C (or P). D, E, and circulin from soil samples collected only in Japan (unpublished data). They have isolated two strains which produce new members of this group, as judged from amino acid composition. Polymyxins Sl and TI have been isolated &om the culture broths of the strains RS-6 and E-12 which were identified as B. polymyxa (Shoji et al., 1977a). Polymyxins S, and T, are strong basic substances; their hydrochloric acid salts are soluble in water and methanol. Polymyxin S1 is active only against gram-negative bacteria in uitro and in uiuo as same as hitherto-known polymyxin group antibiotics: MIC (pg/ml) E . coli, 0.78; K . pnemoniae, 0.78; P . aeruginosa, 6.25; S. aureus, >50: ED, (mgkg X 2) E. coli, 0.22; K . pneunwniae, 0.19. Polymyxin T1is less active against gram-negative bacteria than other members are but it is also active against gram-positive bacteria: MIC (pg/ml) E. coli, 3.13; K . pneumoniae, 3.13; P . aeruginosa, 6.25; S . aureus, 12.5: ED, (mgkg X 2) E. coli, 1.00. Acute toxicities of polymyxins S1 and T1 seem to be at the same level as other members: LD, (i.p., mgkg) 2 5 5 0 and 32.4, respectively. The hydrochloric acid salts of polymyxins S1 and TI were colorless amorphous powders, whose molecular formulas were indicated to be HC1.2 H,O, respectively. C53H91N15015*4 HC1. H 2 0 and C58H1D2N16012-5

204

JUN’ICHI SHOJI

The infrared absorption spectra of both the antibiotics indicated the presence of a peptide bond and the absence of lactone and carboxyl function. Automatic amino acid analyses carried out on the acid hydrolyzates of both the antibiotics revealed the amino acid composition of polymyxin S, to be Dab (5), Thr (3), Ser (l),and Phe (l), and that of polymyxin T1 to be Dab (6), Thr (l),Leu (2), and Phe (1).These amino acids were isolated from the hydrolyzates by preparative paper chromatography and the use of a porous polymer Amberlite XAD-2 column. From their measured [MID values and ORD curves it was concluded that only Ser and Phe in polymyxin Sl and Phe in polymyxin T1were present in the D configuration and that all other amino acids were present in the L configuration. The fatty acids liberated in the acid hydrolyzates of polymyxins S, and TI were extracted with ether, methylated, and analyzed with gas chromatography. A main peak of retention time identical to that of methyl anteisononanoate was observed with both the specimens, and the identification was confirmed with gas chromatographymass spectrometry. By the action of polymyxin acylase, deacyl polymyxin S and deacyl polymyxin T were readily prepared. In an attempt to use the successive Edman degradation reaction on deacyl polymyxins S and T, some modifications in the procedure were required. During the reaction, the uncovered Dab residues are converted to yPTC-Dab residues; consequently the peptides become considerably lipophylic, which causes a great loss of the remaining peptides. Therefore, the Edman degradation procedure was modified as follows: The solvents, benzene and ethylene chloride, used for extraction of an excess phenylisothiocyanate and phenylthiocarbamylamino acid, were replaced by cyclohexane and ethyl ether, respectively. Furthermore, as an opening of a peptide ring by the degradation reaction was anticipated at the fourth step, the PTC-peptide in the fourth step was heated for a prolonged period in tduoroacetic acid to complete the formation of the thiazolinone derivative of a linear heptapeptide. The Edman degradation reaction with the above modifications carried out on deacyl polymyxin S proceeded up to the final step, revealing the presumable amino acid sequence of the antibiotic as shown in Fig. 15. Further evidence for the sequence and the branching mode of the peptide ring with a branched chain was obtained by partial acid hydrolysis. Tetru(DNP)FA

-

-

L-Thr

- o-Ser - L-Dab

-

- L-Thr - L-Dab J hco L-Dab

*

L-Dab

-

D-he

Y L ~ - T h r L-Dab

FA:

Dab: 2.4-Diarninobutyric acid

FIG. 15. Structure of polymyxin S,.

RECENT CHEMICAL STUDIES ON PEF'TIDE ANTIBIOTICS

205

DNP I Dab Phe

-

DNP

Ha-Dab

- I

Dab

Phe

L T h r

DNP

DNP

I FA + D a b

Thr

- Ser - Dab - DabI - Phe L-Thr

Dqb

- ThrJ Dpb

DNP DNP

FIG. 16. Peptide fragments obtained from partial hydrolysis of tetra (DNP) polymyxin S,.

polymyxin S, was prepared and was partially hydrolyzed with a mixture of formic acid and concentrated hydrochloric acid at 37°C. Several DNPpeptide fragments were isolated by TLC, and their sequences were determined to be as shown in Fig. 16. From these results, the structure of polymyxin S, was concluded (Shoji et ul., 1977b). Successive Edman degradation reactions on deacyl polymyxin T were carried out as on polymyxin S,. However, the loss of remaining peptide was larger in this case, because this peptide contained more residues of lipophilic amino acid. Therefore, the result of this reaction did not clarify the sequence beyond the seventh step, but the sequence Dab --+ Thr 4 Dab +-Dab + Dab .+ Phe + Leu was identified. It was noteworthy for structure elucidation of this antibiotic that this peptide contained only one Thr residue which was indicated to be present at the second position from the N terminus. Pentu(DNP)-polymyxin TI was selectively cleaved at the Thr residue by the chemical cleavage method developed by DiBellow et ul. Consequently, a DNP-octapeptide formed by cleavage at the C-terminal side of the Thr residue of pentu(DNP)-polymyxin T, was obtained. Edman degradation on this DNP-octapeptide was carried out with the same modification. This reaction proceeded well, and the result clearly indicated the sequence of this octapeptide to be as shown in Fig. 17. Furthermore, when the remaining peptide of the first step of the Edman degradation reaction, a cyclic heptapeptide, was dinitrophenylated and hydDNP

DNP

I

I

H-Dab -Dab -Dab -Phe tLeu

-

Leu_l - DabDab I I D N P DNP

FIG. 17. Amino acid sequence of DNP-octapeptide.

206

JUN’ICHI SHOJI

FA

-

L-Dab * L-Thr

FA:

-

L-Dab

-

-

-

-- J

L-Dab L-Dab D - h e L-LU y L ~ - L e u L-Dab L-Dab

-

hco

Dab: 2,4-Diaminobutyric acid

FIG. 18. Structure of polymyxin TI.

rolyzed, approximately 1 mole of a-DNP-Dab was obtained. This provided the conclusive evidence for the branching mode in the peptide ring. Thus, the structure of polymyxin TI was concluded to be as shown in Fig. 18 (Shoji et al., 197713). Before the isolation of the new polymyxins, about 15 members of the polymyxin group of antibiotics had been known in the literature (Table II). Most of them had been known since the earlier period, and much effort was devoted to structural studies. A review on the chemistry of polymyxin antibiotics has been published by Vogler and Studer (1966). Wilkinson and Lous, in a short communication (I=), proposed a structure for polymyxin A, which contains one D-Dab and five L-Dab residues, without any experimental data. They also pointed out a possible identity between polymyxins A and M. Silaev and co-workers continued structural studies on polymyxin M and accomplished it in 1975. They showed the structure of polymyxin M, in which all six Dab residues are in the L configuration, and gave plenty of experimental data, i.e., the [aIDvalue, the CD curve, and the behavior to D-amino acid oxidase. It might be reasonable to suppose polymyxins A and M to be identical, although direct comparison between them has not been made. Polymyxins C and P have the same kind of amino acid residues. At present it is impossible to know the exact chemical nature of polymyxin C, but it may be rational to suppose a possible identity between polymyxins C and P from a survey of all the members of polymyxin group antibiotics. Recently J. Shoji and his co-workers isolated an antibiotic which was judged to be identical with polymyxin C or P from the amino acid composition, and they determined the amino acid sequence to be as shown in Fig. 19 (unpublished data). From these considerations, the substantial number of polymyxin group antibiotics hitherto isolated can be reduced and the structures that are shown in Fig. 19. As seen in the structures of the polymyxin group antibiotics, there are four replaceable positions with respect to amino acid residues (W, X, Y,Z in Fig. 19). Before the isolation of polymyxin TI, only three replaceable positions (W, X, Y) had been known. It should be noted that polymyxin TI is unique in this respect and it has somewhat different antimicrobial properties from other members of this group. Another new member of the polymyxin group of antibiotics, polymyxin F (Parker et al., 1977), has been recently isolated;

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

207

TABLE I1

REP~RTEDMEMBEFS

OF THE

POLYMYXIN CROUP OF ANTIBIOTICS

Amino acid"

Antibiotic

Dab

Thr

Polymyxin A * Polymyxin A, Polymyxin M Polymyxin K Polymyxin B, Polymyxin B, Polymyxin C Polymyxin PI Polymyxin P, Polymyxin D, Polymyxin D, Polymyxin E l (colistin A) Polymyxin E, (colistin B) Circulin A Circulin B Polymyxin S, Polymyxin T, Polymyxin F

SL, ID

3L

SL, 1D

3L 3L 3L 2L

6~ 6~ 6~ 6~

+

Leu

Phe

Ser

Ile

Fatty acida a-C, i-C, a-C, +c

a-C, i-C,

2L

+

+

6 6

3 3

5L

3L

ID

a-C, i-C, a-C,

5L 6~

3L 2L

1D

i-C,

6~

2L

6~ 6~

2L

5L

3L

6~ 5

1L

1L, 1D

1~ IL

2L

1

1D

1

1

Reference Wilkinson and Lous (1966) Silaev et al. (1975) Kimura (1971) Suzuki et al. (1964b) Wilkinson and Lous (1964) Jones (1949) Kimura et al. (1969) Hayashi et al. (1966)

a-C,

Suzuki et al. (1963)

i-C,

Suzuki et al. (1964a)

a-C, i-C, a-C, a-C, a-C, i-C, n-C,

Fujikawa et a2. (1965) Hayashi et al. (1968) Shoji et al. (1977b) Shoji et al. (1977~) Parker et al. (1977)

+

'The figures give the number of amino acid residues per molecule. The signs express the presence of the amino acids or unidentified fatty acids. Dab, 2,4-diaminobutyric acid; a-C,, anteisononanoic acid; i-C,, isooctanoic acid. *Identity with polymyxin M has been suspected. CThepresence of hydroxy fatty acids was stated.

its amino acid composition is Dab (5),Ser (l),Ile (l), Leu (2), with only one residue of Thr as in polymyxin TI. It should be noted here that there is close similarity between the structures of the polymyxin group and the octapeptin group antibiotics. Both the groups of antibiotics have a fatty acyl residue and a cyclic heptapeptide moiety, although polymyxin group antibiotics are decapeptides and octapeptin group antibiotics are octapeptides. Moreover, further close similarity is shown in the replaceable positions in both the cyclic heptapeptide moieties (see Figs. 14 and 19). It is of interest to note that the cyclic heptapeptide moieties of octapeptin C, and polymyxin T, are quite identical. Similarity is also shown in their modes of action. Disturbance of membrane permeability

JUN’ICHI SHOJI

208 FA

dab' -L-Th?

-W3 -L-Dav

-

L-DabS -x6

t Z I o - L-D&-

-

Y7

L-Dabe

w

x

Y

Z

tirculin A, B

L-Dab

D-kU

L-lle

L-Thr

Polymyxin El,2 (Colirtin A, 6)

L-Dab

o-Leu

L - L ~ u L-Thr

Polymyxin M (A)1,2

L-Dab

0-Leu

L-Thr

Polymyxin Bl,2

L-Dab

o-Phe

L - L ~ u L-Thr

Polymyxin C (P)l,2

Dab

Phe

Thr

Thr

Polymyxin D1,2

o-Ser

o-Leu

L-Thr

L-Thr

Polymyxin Sl

o-Ser

o-Phe

L-Thr

L-Thr

Polymyxin TI

L-Dab

D-Phe

L-Leu

L-Leu

L-Thr

FA: Anteisononanoyl or irooctanoyl Dab: 2,4-Diaminobufyric acid

FIG. 19. Structures of polymyxin group of antibiotics.

has been observed with octapeptin antibiotics, as is well known in the polymyxin group of antibiotics. The action mechanism of these antibiotics was discussed in detail in a recent review titled “Polymyxin and Related Peptide Antibiotics” published by Storn et al. (1977).

IV. Peptide Lactones A. ESPERINAND

SURFACTIN

Esperin is an antibiotic produced by B. mesentericus with hemolytic activity. The structure was finally determined in 1969 (Fig. 20) (Thomas and Ito, 1969). The structure is of interest because of a lactone linkage between the hydroxy group of a P-hydroxy acid and the P-carboxyl group of an Asp residue. Surfactin, produced by B. subtilis, is a potent clotting inhibitor in the thrombin-fibrinogen reaction. A closely similar structure was shown (Fig. 21) (Kakinuma et al., 1969). It should be noted that the essential difference R-CHCb-CO

*

Glu * Leu

-b R:

- Leu - Val *Asp - Leu - Leu(Val)OH

c12&

(45%)

cIlb3

(35%)

CIOYI (2%)

FIG.20. Esperin.

RECENT CHEMICAL STUDIES O N PEPTIDE ANTIBIOTICS

CH3, ,CH-(CH&-CH-CH,-CO CH3

A

-

G l u * Leu

-

ID1

Leu -Val -Asp

-

209

(Dl

Leu-Leu

FIG. 21. Surfactin.

between both the antibiotics is only the position of the amino acid residue involved in a lactone linkage. B. BREVISTIN In 1976, Shoji et al. (1976a) reported the isolation of an antibiotic, named brevistin, from the culture broth of a strain of B. breuis. Brevistin is an amphoteric substance, soluble in acid and alkaline water but not in water at neutral pH. The hydrochloric acid salt is soluble in methanol. The antibiotic is active against gram-positive bacteria in uitro and in uiuo: MIC (pglml) S . aureus, 3.13; S . pyogenes, 3.13; S . pneumoniae, 6.25: ED, (mgkg X 2) S. aureus, 0.72; S . pyogenes, 1.23; S . pneumoniae, 3.54. Amino acid analysis on the acid hydrolyzate of brevistin revealed the constituent amino acids. These amino acids were isolated by preparative paper chromatography or chromatography on a column of porous polymer XAD-2. From their optical rotatory activities, these were deduced to be: Asp (2D, l ~ Thr ) , ( l ~ Gly, ) , sum of L-Val and L-Ile (l),Trp ( l ~and ) , Dab ( l ~1, ~ ) . From its amino acid composition, brevistin is thought to be a complex of undecapeptides, consisting of a major peptide (ca. 80%)containing Ile and a minor peptide (ca. 20%) containing Val: Ile or Val is present in the same position of'the sequence. When an ethereal extract of the acid hydrolyzate was methylated and analyzed by gas chromatography, a peak of retention time identical to that of methyl anteisononanoate was observed, and evidence for the identification was provided by a GC-MS experiment. Thus, all the constituents of brevistin were determined. A molecular formula, Cs3H,,N 150 calculated for the isoleucine peptide, is in agreement with that from elemental analytical data. Titration with sodium hydroxide in aqueous dimethylsulfoxide indicated the presence of two carboxyl and two amino groups in the brevistin molecule. The presence of a lactone linkage was suggested by an absorption at 17401 cm. In most peptide lactone antibiotics, the carbonyl stretching bands of lactone linkages are located in the neighborhood of 1740lcm. Treatment with dilute sodium hydroxide converted brevistin to a product caused by lactone ring opening, named brevistinic acid. When brevistin was reduced with sodium borohydride and the product was hydrolyzed and analyzed with an amino acid analyzer, the reduced amino acid was indicated to be Phe.

210

JUN'ICHI SHOJI

Chromic acid oxidation did not destroy the Thr residue of the intact antibiotic, whereas it destroyed that of brevistinic acid. Thus, the presence of a lactone linkage between the carboxyl group of the Phe and the hydroxy group of the Thr was proved. A deacylation reaction with an enzyme preparation polymyxin acylase was applied with success to deacylating brevistinic acid. Consequently, deacyl brevistinic acid was obtained, and an Edman degradation reaction revealed its amino acid sequence up to the Trp residue to be Thr 4 Dab + Asp + Gly + Asn + Asp + Gly + Trp. Oxidation with N-bromosuccinimide cleaved bis(DNP)-brevistinic acid into two peptide fragments at the Trp residue. As the fragment of the C-terminal side, a mixture of tripeptides, composed of Ile (or Val), y-DNP-Dab, and Phe, was isolated. Edman degradation carried out with this mixture clarified the sequence to be Ile (or Val) + y-DNP-Dab + Phe. Thus, the sequence of all the amino acid residues of brevistin was determined. Elucidation of the chiralities of individual residues of three Asp and two Dab residues remained to clarlfy completely the total structure of brevistin. The nonapeptide with Asp3 at the N terminus was prepared by a two-step process of Edman degradation from deacyl brevistinic acid. Dinitrophenylation and hydrolysis afforded DNP-Asp3, which was isolated by TLC. Similarly, the heptapeptide with Asn5 at the N terminus was prepared. Dinitrophenylation and hydrolysis gave DNP-Asp and non-dinitrophenylated Asp6, which were easily separable. From CD curves measured with these DNP-Asp specimens, Asp3 was deduced to be in the L form, and Asn5 and Asp' were deduced to be in the D form. Cleavage reaction with N-bromosuccinimide on bis(DNP)-brevistinic acid gave two peptide fragments. One, the N-terminal side, contained Dab2, whereas the other, the C-terminal side, contained Dab lo. From each of the fragments, Dab2 and Dab lo were isolated as their bis-dinitrophenyl derivatives. Their CD curves clarified that Dab2 is in the D form and Dab l o in the L form. From all the results, the total structure of brevistin was concluded to be as shown in Fig. 22 (Shoji and Kato, 1976a).

ILI

FA * Thr

b

-

IDI

b h e ILI

FA:

(LI

D a b - Asp

- -

- Dab ILI

Gly

(DI

ID1

Asn -Asp

Ile(val) * Trp ILI

(Ll

-

7

Gly

LCO

Dab: 2.4-Diaminobutyric acid

FIG. 22. Brevistin.

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

211

C. TL-119 AND A-3302-A In 1975, Shoji et al. (197513) reported the isolation of an antibiotic TL-119 from the culture broth of a strain of B. subtilis. The antibiotic is preferentially active against Staphylococcus aureus (MIC, 1.56 pglml). It has low toxicity to mice (LDo>50 mgkg, i.p.), but no therapeutic effect is shown to infected mice by subcutaneous administration. TL-119 is obtained as a colorless amorphous powder. It is a neutral substance soluble in dimethylsulfoxide, dimethylformamide, and a mixture of methanol and chloroform. A molecular formula of C,,H,,N,O, (803) was indicated by elemental analysis and mass spectral molecular ion peak. Amino acid analysis on the acid hydrolyzate revealed the presence of Thr (l), Ala (l), Val (l),Leu (l),and Phe (2). The presence of a-ketobutyric acid in the acid hydrolyzate was confirmed by a GC-MS experiment, and a-aminobutyric acid in an equimolar ratio was estimated in the hydrolyzate of the hydrogenated antibiotic. Thus, the presence of 1mole of a-aminodehydrobutyric acid (AAbu)in TL-119 was proved, which had previously been found in a peptide antibiotic stendomycin produced by a Streptomyces. When TL-119 was examined by mass spectrometry, the arrangement of the above residues and an acetyl group at the N-terminal end was directly indicated to be as shown in Fig. 23. This was also supported by the mass spectrum of the permethylated product of the antibiotic. The presence of a lactone linkage between the C-terminal amino acid, a-aminodehydrobutync acid, and Thr was suggested by an absorption at 1723hm in IR. The slight shift to lower wavenumbers is explicable by a conjugated lactone linkage. TL-119 was converted to an acid by treatment with a dilute alkaline solution. Chromic acid oxidation destroyed the Thr residue of the acid, whereas it did not have any effect on the Thr residue of the intact antibiotic. This provided the evidence that the Thr residue was involved in the lactone linkage. The structure of TL-119, without the stereochemistry of the amino acid

1 ./I

Ac

Phe

Phe

Leu

(fir)

IH'IH] Va I

Ala

Mh,

0 0 0 0 0 0 0 0 I H H CHS-C N-CH-C N-CH-C N-CH-C N-C3H,-C N-CH-C N-CH-C NH-C-C iH2 C,H,

{CH H2H[H

#2 C6H5

/ \

CH3 /k \HCH,

&

1

cy 0 A

cn, CH3

43

190

303

450

33

632

FIG. 23. Fragmentation pattern of TL-119.

703

8a3(M;Fj

212

JUN’ICHI SHOJI

0 Mbu: a-Aminodchydrotutyric acid

FIG.24. Structure of TL-119.

residues, was determined to be as shown in Fig. 24 (Nakagawaet al., 1975). In 1976, Ogawa et al. (1976) reported structural studies on the antibiotics A-3302-A and -B produced by a strain of B . subtilis. The structural difference between the components A and B was found to be the presence of a propionyl group in A in the position of an acetyl group in B. The same amino acid composition and arrangement as in TL-119 were proved in a similar manner. By partial acid hydrolysis of A-3302-B, two peptide fragments, D-Phe + D-Leu -+ L-Phe and L-Thr -+ L - V + ~ L-Ala, were obtained. From these experiments, Ogawa et al. proposed the structures of A-3302-A and B, whose amino acid sequences were identical with TL-119, including the stereochemistry of the constituent amino acids. A-3302-B can be reasonably thought identical to TL-119.

V. Concluding Remarks As already pointed by Berdy (1974),the nomenclature of antibiotics is in a chaotic situation. There are nearly a thousand superfluous antibiotic names in current use. In the case of antibiotics from the genus Bacillus, we have somewhat fewer than 200 names but the real number may be assumed to be around 117, The number of not well-characterized antibiotics is relatively large. If possible, some of these antibiotics should be reexamined using modern chemical technology. Some examples of the results of this type of reexamination are given in this review, e.g., Iturin-related antibiotics and the polymyxin group of antibiotics. Some of the confusion in the nomenclature of peptide antibiotics might be eliminated if we adopted a policy of giving a group name to a family of related antibiotics and of distinguishing the individual members by appropriate suffixes. This was attempted in our studies on the octapeptin group of antibiotics which were examined in two independent laboratories. One remaining problem in studying peptide antibiotics is that of further fine resolution of complexes which cannot be separated by present techniques. At present the chemistry of certain peptide antibiotics is established only by studying a complex (a mixture) of presumably related peptide variants. However, this problem may be mostly solved by introducing a new separation technique, high performance liquid chromatography. Very recently complete resolution of such complexes, i. e., cerexins, tridecaptin

RECENT CHEMICAL STUDIES ON PEPTIDE ANTIBIOTICS

213

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The CBS Fungus Collection J. A.

VON

Am

AND

M. A. A. SCHIPPER Centraalbureau vow Schirnmelcultures, Baarn, The Netherlands I. Introduction and History . . . . . . . . . .............. 11. The Collection of Fungi and Actino Baarn. . . . . . . . A. Maintenance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Identification and Applied Mycology Service . . . . . . C. The Division of Medical Mycology .................... D. Scientific Research and Other Activities 111. The Collection of Yeasts in Delft A. Maintenance.. . . . . . . B. Identi6cation Service ............................ C. Scientific Research. . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 218 219 226

235

1. Introduction and History The Centraalbureau uour Schirnmelcultures (CBS) collection includes only yeast and filamentous fungal cultures. Bacteria and all other microorganisms, with the exception of Actinomycetes, are excluded, a specialization which resulted from the historical development of the CBS. The collection has often been compared with a botanical garden of higher plants, and the CBS referred to as a “fungus garden” or even a “herbarium of living specimens.” Toward the end of the last century techniques were developed for obtaining pure cultures of bacteria and fungi on semisolid media. The need arose for the maintenance of such cultures as reference material for later research workers. It was especially important to keep cultures used in the description of new taxa. At a meeting of the Association lnternationale des Botanistes in 1903, it was proposed that a central culture collection of fungi be established. The following year at the First Botanical Congress in Vienna, the foundation of such a collection was proposed by Prof. F. A. F. C. Went of Utrecht, and he agreed to take up the task. Prior to his appointment in Utrecht he had worked as a phytopathologist in Bogor (Java) and isolated quite a large number of fungal strains which were to form the basis of the new collection. In 1906 the establishment of the CBS was announced in the Botanisches Centrulblatt volume 101 and the first “List of Cultures,” containing approximately 80 cultures, was given on an unnumbered page. Raper (1957) published a reproduction of this page in an article marking the fiftieth anniversary of the CBS. 215 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O - l Z - O L l E Z 4 4

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In 1907 the care of the collection was assigned to a young biologist, Dr. Johanna Westerdijk, who had been appointed shortly before as director of the Phytopathological Laboratory Willie Commelin Scholten in Amsterdam. Johanna Westerdijk remained in charge of the collection for more than 50 years until she retired in 1959. As a phytopathologist she was especially interested in fungi which caused plant diseases, but she was always a keen collector of cultures of all types of fungi. The first researchers to send cultures to the collection in 1907/1908 included M. W. Beyerinck, B. Namyslowski, C. A. J. A. Oudemans, C. Wehmer, and H. W. Wollenweber. Type cultures of Mucordes, described and sent to the collection by 0. Hagem and A. Lendner in 1908, are still kept in the collection as well-sporulating strains which are important as authentic reference material. That the collection continued to grow and even survived such difficult periods as the two World Wars was due to the constant devotion of Johanna Westerdijk. There were always financial difficulties because all the costs of maintaining the collection had to be derived from the sale of cultures, identification fees, and private donations. In 1920 Johanna Westerdijk, the Phytopathological Laboratory, and the associated CBS moved 35 km from Amsterdam to Baarn, a village peacefully situated in a wooded area. Although the number of cultures increased, the number of stafF members was forced to remain limited due to a continual shortage of funds. The few members of the CBS thus had little time for research work. Mr. F. H. van Beyma thoe Kingma was the only mycologist who was able to attain some recognition from publications appearing between the World Wars. He was a staffmember of the CBS from 1925 to 1944 and his main task was the identification of cultures, the results ofwhich were reflected in approximately 30 publications mainly concerned with the taxonomy of Hyphomycetes (e.g., Penicillium and Phialophora) and Ascomycetes. The daily management of the collection was in the hands of Dr. G. E. Bunschoten from 1933 to 1967, who was responsible for new accessions to the collection and also for distribution of cultures in the early years. In addition Dr. Bunschoten did much bibliographic work and was involved in the compilation of numerous editions of the regularly published “List of Cultures.’’ A. C. Stolk was a member of the CBS from 1948 to 1975, and became a renowned specialist in the taxonomy of Aspergillus and Penicillium and the connected ascigerous states, on which she published a number of important papers. A. L. van Bevenvijk was at the CBS from 1948 and was director from 1959 until her death in 1963. Her main research work was concerned with Fusarium but she became better known for her publications on fresh-water Hyphomycetes. A number of other mycologists worked at the

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CBS for longer or shorter periods, e.g., Dr. M. B. Schol-Schwarz in a part time function from 1949 to 1969. Until 1964 the CBS (Fig. 1)was closely associated with the Phytopathological Laboratory Willie Commelin Scholten; they were housed in the same building, they used the same laboratory facilities and the same library and, furthermore, Prof. Westerdijk was director of both laboratories. A large donation made by Mr. 0. van Vloten enabled the CBS to erect its own laboratory building in 1964. Prior to this period the CBS had already received financial support from the Royal Netherlands Academy of Arts and Sciences. In 1968 it became a research institution of this Academy, which is supported by the Dutch government. In 1922, a collection of approximately 60 strains of yeasts was taken over by Prof. A. J. Kluyver of the Microbiological Laboratory of the Technical University in Delft (in exchange for a number of test tubes and conical flasks). The maintenance of the yeast collection was supervised and stimulated by Prof. Kluyver until his death in 1956. Mrs. N. M. Stelling-Dekker worked in Delft for her doctorate and her thesis was entitled “Die Hefesarnmlung des ‘Centraalbureau vow Schimmlcultures,’ Ted 1 : Die sporogenen Hefen” (1931). From 1932 to 1938 Dr. J. Lodder worked as a mycologist in the department and her position was subsequently filled by the following workers: Dr. T. Hof (1938-1944), Dr. N. J. W. Kreger-van Rij (1946-1964), and W. C. Slooff (1951-1971). Dr. Lodder published the second part of “Die anascosporogenen Hefen” in coauthorship with Dr. H. A. Diddens (1934, 1942). The monograph “The yeasts, a taxonomic study” appearing in 1952 was drawn from the work of J. Lodder and N . J. W. Kregervan Rij. They later cooperated with Miss Slooff on the second edition of “The Yeasts,” which appeared in 1970. Other contributors to this standard work,

FIG. 1. The CBS-laboratory building in B a r n .

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such as Dr. H. J. PhafTand Dr. J. P. van der Walt, had earlier worked under the guidance of Prof. Kluyver at the yeast division in Delft. The CBS in Baarn gained a division of Medical and Veterinary Mycology in 1952. One mycologist and two technical assistants today make up this division. Since 1952 the number of CBS workers has also been increased. In 1977 the staB included 16 professional members (two in Delft), 16 technical (two in Delft), and four clerical (one in Delft).

II. The Collection of Fungi and Actinomycetes in Baarn At the close of 1976, there were approximately 19,300 cultures in the collection. These include a number of Myxomycetes, Acrasiomycetes, approximately 70 Chytridiomycetes, 460 Oomycetes (especially Pythiurn, Phytophthoru, and Achlyu species), over 800 Zygomycetes (especially Mucorales but also some Entomophthorales), and about 2600 cultures of Basidiomycetes. The remaining strains are isolates of Ascomycetes and Deuteromycetes. In addition the collection has 945 cultures of Actinomycetes (Fig. 2).

800. 700-

600500.

.WO

200 100

1907

1917

1927

n

1947

FIG. 2. Histogram of the number of strains in the present collection in Baarn in relation to the year of accession.

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The collection also includes rather numerous “restricted cultures,” maintained on request by certain customers. Such cultures are usually involved in some process for which patent applications are being considered. They are handled as requested by the depositor. The “List of Cultures” (catalog) only includes those strains determined to the species level, strains with only genus names being excluded. In some cases numerous strains of one species may be maintained in the collection (e.g., single-spore isolates) but not all are listed. Fungi mentioned in the “List of Cultures” may be ordered at set prices or obtained in exchange for strains of other species which are either not yet in the collection or of which the CBS only has one representative strain. Cultures not listed may also be obtained upon request.

A. MAINTENANCE Strains of the collection are maintained by three methods: (a) active on agar slants, (b) lyophilized, and (c) under mineral oil.

1 . The Active Collection Natural nutrient media are preferred, e.g., malt, cherry decoction, oatmeal, cornmeal, potato-dextrose, potato-carrot, and soil extract agars, lupine stems, carrot pieces, or Acer twigs. These media are easily prepared, inexpensive, and effective. Each strain is simultaneously cultured on two different media. At each transfer the media are interchanged such that any beneficial effects lacking in one medium can be supplemented by the other. Prof. Westerdijk developed this method from the observation that in nature luxurious growth occurs when food is abundant but poor conditions often lead to fructification. Illumination may enhance or even be essential for sporulation. The light sources used include daylight on north- or south-facing window-sills, blue light or “black light,” and incubation at 17 or 25°C. In the active collection the cultures are arranged alphabetically. Cultures which have been allowed to develop at optimum temperatures are stored in the collection room at 16-17°C and a humidity of 60-70%, or in refrigerators at 5°C (Fig. 3). The 5°C cultures are transferred annually onto new media. Some species respond favorably to prolonged storage at low temperatures, rather than to oft-repeated transfers, less subculturing also saving extra labor. However, not every species survives a 1-year storage at 5°C and, further, the capacity of the refrigerators is limited. The initial choice is based on the principle that fungi which would naturally survive a west European winter would also be likely to respond favorably to storage at 5°C. All the strains of such a genus

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FIG. 3. Part of the active collection

are then refrigerated. If after 1year too many difficulties arise with reinoculation, then the whole genus is transferred to 16°C (this with a view to efficiency). At present the refrigerated genera include: Absidia, Achaetomium, Actinomyces, Alternark, Aspergillus, Ceratocystis, Cercospora, Cercosporella, Chaetomium, Cladosporium, Clathrosphaerina, Colletotrichum, Coniochaeta, Gymnoascus, Mastigosporium, Mortierella, Muc w (with a few exceptions), Nectria, Penicillium, Podospora, Pestalotia, Streptomyces (with isolated exceptions), Streptosporangium, Streptoverticillium, and Syncephalis. Cultures kept at 16°C are usually transferred onto new media every 6 months. Some strains need to be transferred more frequently, that is, either once per month or every 2-3 months, e.g., the genera Allomyces, Achlya, lsoachlya, Phytophthora, Pythium, and Saprolegnia, and the basidiomycete genera Boletus, Coprinus, Corticium, Cortinarius, and Mycena, and also some smaller groups. The technical st& members handling the transfers of the cultures are each responsible for a particular group in the collection so that they can familiarize themselves with these fungi. The assistants learn to recognize the cultural habits and sporulation patterns and to deal with any special growth requirements. Transfers are usually made by spore smears but in some cases pieces of the substrate must be placed on the new medium, e.g., aging Mucorales. As a rule strains are reinoculated onto fresh media, except in the case of some osmophilic species for which drier, more concentrated media are advantageous. Immediately following inoculation the cotton plugs of the test tubes are ringed with a mercuric chloride solution to prevent mite infections. These slants are subsequently allowed to develop under their optimal conditions of temperature and light. The technical staff supervise their own subcultures after 2 4 weeks if these have been incubated at room temperature. Thermophilic strains are controlled earlier to prevent drying of the substrate, as are cultures under “black

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light.” Unsuccessful or contaminated subcultures are treated as special problems. Bacterial infections can easily be detected by growing the strains in liquid malt peptone which turns milky in the presence of bacteria. Most bacterial infections can be removed by means of culturing the infected strain on a medium containing penicillin and streptomycin or other antibiotics. Where cultures suddenly show poor development it is often advantageous to activate an older culture by one of the following methods: (a) sterile water or liquid malt peptone solution is poured onto the old slant, which is then allowed to soften for a few days prior to a new transfer; (b)small pieces of carrot or lupine stem and a little liquid are added to the tube; or (c)warm agar is slowly allowed to spread over part of the old agar surface. (It is, of course, always easier to employ lyophilized or oil cultures if these are available.) The same methods are followed in cases of contamination. Alternatively, cultures may be purified by making single-spore isolations or by using the different reactions of the strain and contaminant to temperature and/or media, etc. Final control of the reinoculated new collection is by the technician in charge or by mycologists who control the particular groups which they study. The collection always consists of two controlled complete series of all the strains. On renewal of cultures the oldest series is autoclaved and discarded. 2. Lyophilized Collection

Freeze drying as a means of prolonged preservation of (sporulating) fungal strains has been used at the CBS since 1958. Since 1960 all incoming sporulating fungal strains have been lyophilized immediately to conserve the original characteristics of these strains. The older strains (pre-1960) which still sporulate are now being lyophilized on an intermittent basis. A suspension of spores in skimmed milk is rapidly frozen at -25 to -30°C. The milk forms a protective layer around each spore. The deep-frozen suspension is then dried under high vacuum by sublimation of the ice, while the temperature rises slowly. The tubes are subsequently sealed so that the vacuum is retained in the tube. The whole procedure takes approximately 3 hours when 4-mm diameter ampules and 0.2 ml of liquid are used at a pressure of O.02-O.04mm mercury. The procedure followed at the CBS includes the following preparatory work. A narrow slip of paper indicating the “lyophile” number is placed in each (Hysil) ampule which is plugged with a loose piece of cotton wool. The ampules are then sterilized for 2 hours at 160°C. Glass pasteur pipettes are plugged with cotton wool and sterilized in a tall preserve bottle with cotton wool at its base to prevent damage of the pipette points. Sterile test tubes are filled with 2 3 ml of a suspension of skimmed milk in 100 ml ofwater and sterilized on three consecutive days for 30 min at 100°C in an autoclave.

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The alcohol bath of the freeze dryer is cooled some time before use. A spore suspension is made by pouring the skimmed milk (mentioned above) from one test tube over the culture, after which the spores are loosened with a long flat inoculation needle. The required number of ampules is filled with the spore suspension by means of a sterile pipette which is then discarded. The cotton plug is flamed and pushed into the ampule until it nearly reaches the slip of paper. The working table is cleaned with 70% alcohol before the next series is prepared. A number of unnumbered sterile ampules and pipettes is always kept in reserve. If there is even a slight doubt as to sterility the material is discarded. Finally the filled tubes are attached to the lyophilizing apparatus so that the spore suspension is plunged entirely into the cooled alcohol, and the vacuum pump is switched on. When the temperature of the alcohol bath has risen to zero and the spore suspension is “dry,” the alcohol bath is either removed or emptied and the pellets are allowed to air dry for approximately half an hour. The tubes are then sealed while still attached to the apparatus and while the pump is still working. (If the vacuum during drying is insufficient and if the suspensions show any ice particles when the temperature in the baths has risen to approximately -5“C, then the temperature must again be lowered and the period of drying increased.) Normally six tubes are made for each strain (the apparatus has 72 connections). After sealing the vacuum in the tubes is checked by means of a vacuum tester. One tube of each set of six is then opened and the contents are suspended in sterile water and spread over a suitable nutrient medium in a petri dish, to test the viability and/or possible contamination of the culture. The remaining lyophile tubes are stored in sealed plastic bags with labels indicating their lyophile numbers, CBS numbers, data of lyophilization, nutrient media, and any special light or temperature requirements. The plastic bags are stored in metal drawers in a room with a rather low and constant temperature. When only two tubes remain for a strain (due to outside orders or revival of poor cultures), one tube is used to make a new series of six. The advantages of this method include longer viability and preservation of original characters, provided the drying process is withstood. In 1976 a survey of 100 of the oldest (17-18 year-old tubes) gave very satisfactory results (Schipper and Bekker-Holtman, 1976). Cultures which give inoculation problems, such as Neurospora species or Phycornyces species, or cultures which hardly withstand storage on agar, such as some Streptomycete species, can successfully and more easily be stored in the lyophilized state. In tests which are spread over a long period of time lyophile cultures will insure that the material retains its original characters. Cultures transported

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in a lyophilized state are more likely to survive adverse conditions than cultures on agar slants. A lyophile collection takes up less space and the sealed tubes prevent any contamination. One of the disadvantages of the method is the unavailability of material for quick comparison, such as when a culture is needed for the identification of a fungus. Each tube can only be used once and nonsporulating strains cannot be stored by this lyophilization routine. A few more points need to be mentioned. The spores of some genera, such as Mucur, Mortierella, Rhizopus, and Streptomyces, appear to lose their ability to germinate rather rapidly with age and, therefore, only young cultures can successfully be lyophilized. Species with large and/or intricate spores and delicate cell walls, such as Helicodendron species, seldom survive the drying process.

3. Oil Collection When living cultures are covered with liquid paraffin, the metabolic processes of the fungus are retarded. The data available from literature indicate that this retardation is approximately by a factor of ten. The CBS oil collection was initiated in 1956/1957 and consists of one tube for each strain. Purified paraffin oil of viscosity 68-81 cp dry sterilized at 170°C for 2 hours is poured onto young slant cultures. (After steam sterilization the oil is turbid due to floating water droplets which later form one large drop at the base of the tube.) As the heavy oil can cause light spores to disperse and adhere to the remaining oil not being poured into the tube, it is separately sterilized and oil from only one tube is used for each culture. Any oil remaining in the first tube is stored next to the slant culture tube for 1 week and may be used to fill the latter if the oil level in the second tube has dropped due to air loss from the mycelium. After 10 years the oil-sealed fungus is inoculated onto a fresh medium. When the new culture has developed sufficiently it is again immersed in paraffin oil. Long inoculating needles are used here to prevent adherence of spores to the needlehandle connection. Reinoculation is done onto agar slants to allow the adherent oil to slide away from the point of inoculation. On a horizontal petri dish much of the inoculated area would remain under oil. It has become apparent that subcultures from oil tubes onto X-agar (cherry + peptone-glucose-saccharose + oatmeal agar) or cherry agar are often poor and hence these media are avoided. About 85% of all the cultures have survived a 10-year storage under oil, but marked differences may be seen between the genera. There were many failures among the Oomycetes and the Agaricales, groups which also require special care in the active collection.

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The advantage of oil sealing as compared to lyophilization is that nonspomlating strains can be preserved as well as sporulating ones and, further, that more than one subculture can be made from one oil-sealed culture. A disadvantage, however, is that after 10 years the subcultures from an oil-sealed strain are often poorer than the original. The oil collection at the CBS is regarded as a reserve collection, that is, a safeguard, and is stored in a subterranean room away from the main building.

4 . New Accessions and Documentation New cultures sent to the collection are first studied by mycologists specializing in the fungi concerned and camera lucida drawings are prepared. Subcultures are made onto at least five different media. If a culture spomlates poorly it is transferred onto media with low sugar contents or such natural substrates as lupine stems and incubated at different conditions of light and temperature. Usually four final slant cultures are prepared on two different (optimal)media: two for the active collection, one for lyophilization, and one for the oil collection. Finally the strain is given an accession number. Information about each accessioned strain is recorded on two identical cards which give all the relevant data and which are stored separately. The accession numbers include two parts: the number assigned in the calendar year starting with 101, followed by the actual year, e.g., the number 102.77 indicates the second strain accessioned in 1977. This is followed by the Latin name of the fungus and the authors’ names; the strains in the collection are arranged alphabetically by Latin name. Subsequently any synonyms and/or additional names of imperfect or perfect states, and the name of the authority who has sent in the strain (if he or she has identified it correctly) are all given. In addition any data concerning the strain are listed; especially the substrate, locality, and date of isolation. Type cultures are indicated as such and numbers given to the same strain in other collections are listed. Information on biochemical and other properties, in particular ploidy and compatibility groups, is added. Finally the appropriate nutrient media, conditions of temperature and light, date of lyophilization, and, possibly, the number of herbarium specimens made from petri dish cultures are indicated. If a strain has been discarded from the collection, its card is stored in a separate file. In another file the cards are arranged according to the accession numbers, here only the names of the fungi being given. Any literature concerning the strains is recorded on yet another set of cards. Finally, new strains are included in a card system which is to be used for the next “List of Cultures.” From 1977 on these data have been copied onto punch cards with auxiliary numbers that allow insertions, corrections, and deletions at any time in the

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computer tape. FORTRAN programs have been developed for sorting the stock and printing the “List of Cultures” at the Academic Computer Centre of the University of Utrecht. AND APPLIED MYCOLOGY SERVICE B. IDENTIFICATION

The identification service offered includes the classification of pure cultures of filamentous fungi and Actinomycetes. Only occasionally are fungi on the natural substrates identified; these are mainly fungi on diseased plants and insects, which do not grow in pure culture (rusts, downy and powdery mildews). However, the identification of killed herbarium specimens is not a part of our service. If necessary the cultures are purified before identification. On arrival a preliminary examination of the cultures is made by one of the mycologists. The cultures are then distributed among the specialists in the groups concerned. There are specialists in Mucorales, Ascomycetes, Basidiomycetes, Coelomycetes, Hyphomycetes, Oomycetes, the PenicilliumAspergillus-Paecilomyces group, Fusarium, soil fungi, entomogenous fungi, and “black yeasts.” Most of the strains are grown on different media in tubes and petri dishes. Fungi with no or insufficient sporulation are cultured on potato+arrot or oatmeal agar with lupine stems and incubated under “black light.” Strains which are sterile or only form microconidia or sporangia may represent mating partners or heterothallic species of Mucorales, Oomycetes, Ascomycetes, or Basidiomycetes. Such strains can often only be identified by mating with known collection strains. In general the culture collection is an indispensable aid in the identification work because many sporulating or sterile strains can only be identified by comparison with authentic cultures. The optimal temperature for growth and sporulation often has to be determined. A fungus may be psychrophilic, mesophilic, thermotolerant, or thermophilic. Osmophilic fungi have to be grown on media with a high sugar concentration; keratinophilic fungi on media containing forms of keratin (e.g., hairs of humans or horses). Out of the strains sent in for identification, those of interest are incorporated in the CBS collection with permission of the sender; all others are destroyed after 6 months. As a result of the identification service, the CBS collection is enriched by many interesting cultures. Moreover many strains sent in for identification become the subjects of scientific papers. Undescribed taxa are described by either the sender alone, by CBS specialists, or by both in a joint communication. In addition to the identification of pure cultures, for which there is usually a set fee, some research work is done at the CBS on request. This work

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mainly falls in the field of applied mycology, for example on the biodegradation of materials, textiles, plastics, leather; wood attacking fungi; toxic or other fungi; contaminations in food, feed or fodder, or drinks; fungi which cause plant diseases or fruit rot; and fungi parasitic on insects, the latter in connection with some ecological and phytopathological problems. Fungi from agricultural soils are isolated and classified.

c. THE DIVISIONOF MEDICAL MYCOLOGY The division of Medical Mycology maintains a fairly large collection of human and animal pathogens. This collection includes the three dermatophyte genera Epidermophyton (two species, 18 strains), Microsporum (14 species, 64 strains), and Trichophyton (47 species, 193 strains) and their respective perfect states, which are classified as Arthrodermu and Nannizzia species. Other pathogenic fungi are Madurella (three species, nine strains), Glenospora metamericana (one strain), Glenosporella loboi (one strain), Blastomyces dermutitidis (13 strains), Cladosporium trichoides (five strains), Coccidioides (2 species, nine strains), Histoplasma (three species, 17 strains), and Paracoccidwides brasiliensis (nine strains). The seven last-mentioned species and genera are only transferred in a safety cabinet. Subculturing takes place every 6 months. Madurella strains are transferred every 3 months. Sabouraud agar with dextrose or maltose, potatodextrose agar and malt agar are the media most often used for cultures. The temperature for incubation is 25°C. Some of the dimorphic species, such as Histoplasma capsulatum and Ajellomyces dermutitidis, are also cultured, at least as long as possible, in their yeastlike form. This requires a rich medium, such as brain heart infusion agar or blood agar, and a temperature of 37°C. The dermatophytes are maintained on malt agar and Sabouraud maltose instead of Sabouraud glucose agar, because dextrose favors pleomorphism. They are subcultured every 4 months. For identification of dermatophytes other media are used including Littman Oxgail agar, cornmeal agar, Czapek agar with human hair, etc. The incubation is at 24°C until optimal cultural development. The increasing numbers of opportunistic fungi that cause disease in patients who for some reason became predisposed to fungal disease are all incorporated in the main collection of the CBS. The Division also takes care of a collection of actinomycetes comprised of 945 strains distributed over 28 genera. Streptomyces is the best represented in this collection, having 668 strains including a great number of type and neotype cultures of the international Streptomyces Project and several strains deposited in connection with patent applications. In addition 140 Streptomyces strains are only maintained as lyophilized cultures. For the maintenance of the actinomycetes the same

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three methods are used as for the fungi. Oatmeal, cornmeal, and malt-yeast extract agar are the most appropriate media. The microaerophilic Actinomyces israelii is only preserved in a lyophilized state. An important part of the program is the identification of cultures and the investigation of clinical materials. In a study on the conidial states ofPetrieZla and Petriellidium, which are often isolated from clinical material, the value for species delimitation of several morphological and physiological characters is investigated. Since 1952 papers have been published on various subjects, e. g., medical mycological methodology, maduromycosis, Aspergilhs fumigatus and actinomycetes in air, keratinophilic fungi, tinea capitis in chimpanzees, dermatophilosis in a horse, the variability of Trichophyton rubrum, Lobo’s disease in dolphins, Trichophyton equinum var. equinum in horses, and zygomycosis. For a study of keratinophilic fungi and actinomycetes soil samples are collected two or three times a year in the new polders S. and E. Flevoland. They are analyzed by the hair bait method. The E. Flevoland soil was negative for keratinophilic fungi until 1971, when both Trichophyton ajelloi and T . terrestre were isolated from a sample collected at the periphery of a meadow area. This appeared to be the second recorded observation of keratinophilic fungi in this polder, as Arthroderma curreyi already had been isolated in 1969. In S. Flevoland the first keratinophilic species was isolated in 1976, 7-8 years after the polder was completely drained. The fungus was identified as Ctenomyces serratus, a species known to occur on bird feathers in neutral to slightly alkaline soils. In E. Flevoland, which fell dry in 1957, eight keratinophilic species were recorded up to 1977.

D. SCIENTIFICRESEARCH AND OTHERACTIVITIES In the classical taxonomy of fungi, the delimitation of taxa was based on morphological characters visible under the light microscope. Fresh collections or usually dead dried specimens were studied. For many fungi it was supposed that a species was limited to a single or a restricted number of closely related hosts. A fungus collected on a new host was described as a new species without prior comparison with similar fungi described &om other hosts. The study of killed specimens is static, often practically no observations of the life cycle, the variability, and the influence of growing conditions being possible. When working with material on the natural substrate, especially fungi growing on plants, a reliable delimitation of species is usually impossible. Pure cultures have the great advantage over dead specimens that they can be subcultured on various media, propagated to any desired extent, and

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treated to show all the differentiations of the fungus in the most typical way. A fungus in pure culture can be studied over a prolonged period under defined conditions of medium, temperature, humidity, and light. The life cycle can be elucidated, e.g., the connection between sexual and asexual states. Cultures should be grown under optimal conditions, which can be realized by the choice of medium, temperature, and light. Fungi isolated from living or killed plants are preferentially grown on media containing starch and/or cellulose and little or no sugars, e.g., cornmeal agar. For other fungi at the CBS a diversity of media is available, some being enriched with yeast extract. For a comparative taxonomic study as many strains as possible should be available. One of the subjects studied at the CBS is the generic and specific delimitation in Mucor and allied genera. Mating experiments play a major role in species delimitation (Schipper, 1973, 1975, 1976). Some Mucor species, e. g., Mucor hiernalis and M . circinelloides, can occasionally only be recognized with difficulty by their cultural and sporangial characters; in such cases mating experiments are very helpful. In the latter species the zygospores are formed in matings on whey agar at 25°C and are reddish brown; in the former species they develop in matings on beerwort agar at 15°C and are blackish brown (Schipper, 1969) (Fig. 4). In Oomycetes, sterile or only sporangia-forming cultures may represent mating partners of heterothallic species. Such cultures can often only be identified by mating experiments. Pythium sylvaticum, P . heterothallicum, or P . intermedium, the most common soil-inhabiting Oomycetes, are heterothallic and the oogonial states have only recently been discovered. In a rather large number of other mycelial Oomycetes the sexual states have not

FIG. 4. Zygospores of (a) Mucw ciscinelloides and (b)M . hiemalis

(SOOX).

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yet been found and these isolates may represent undescribed species (van der Plaats-Niterink, 1975). Some Ascomycetes, mainly belonging to the Gymnoascaceae and Sordariaceae, also develop their ascomata in mating experiments. The mating partners usually produce conidial or spermatial states; these of the Gymnoascaceae are generally arthric (form genera Chrysospwium and Malbranchea), these of the Sordariaceae phialidic or blastic (e.g., form genera Phialophwa, Cladorrhinum, and Myceliophthwa; van Oorschot, 1978). Some thermophilic Ascomycetes, in particular, include Myceliophthora conidial states and have been found to be heterothallic. Many other Ascomycetes are studied in connection with their conidial states. They are often isolated from soil or litter. Species of Eupenicillium and Talaromyces (ascigerous states of Penicillium), for example, are commonly isolated from soil in warmer regions and have been studied by Stolk and Samson (1972) and Scott (1968). Other genera studied at the CBS are Thielavia, Microascus, and Petriellidium (von Arx, 1973, 1975a,b). A morphological-biochemical study showed that the species classified in the genus Ceratocystis represented two unrelated phylogenetic groups. The genus Ceratocystis is therefore restricted to species with phialidic conidial states of the genus Chulara. Ceratocystis may be related to Chaetosphaeriu with similar phialidic conidial states which are treated as Chloridium. Species formerly placed in Ceratocystis with Sporothrir, Graphium, or similar conidial states forming blastic conidia, have been classified in a separate genus for which the name Ophiostoma is now available (de Hoog, 1974; Weijman and de Hoog, 1975). The genus Ophiostoma has a rather isolated position within the Ascomycetes and it may be related to the Microascaceae. A large number of hngi studied in pure culture are Fungi imperfecti, mainly Hyphomycetes. Many of these are very common and widespread in nature and often are of economic importance. Species of the generaAspergi1lus and Penicillium may be usehl or harmful in producing antibiotics and toxins, may cause biodegradation in a desired or undesired sense, may be used in the production of food and chemicals (organic acids), but may also cause diseases in humans, animals, and plants. Recent studies carried out at CBS showed that the existing species delimitation in Penicillium had been partly based on inadequate, variable, and hardly reproducible characters. A modified classification, particularly for the most common species, has therefore been proposed and the number of accepted species considerably reduced (Samson et al., 1976). Some other large genera including some important species have been monographed by CBS co-workers. Gams (1971) classified most of the hngi hitherto known as Cephalosporium in Acremonium. He distinguished over 80 species and more have since been described (Gams, 1975). Ascigerous states of Acremonium mainly belong to the genus Emericellopsis. The conidial states of the ascomycete genus Nies-

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slia were described in Monocillium, while other Cephulosporium-like fungi were classified in Verticillium and some additional genera. The genus Paecilomyces was monographed by Samson (1974). About 30 species were accepted and some were transferred to other genera, such as Mariannaea, and Nomuraea. Nomuraea rileyi is an important entomogenous species used in pest control. Chloridium and some other phialidic, dematiaceous Hyphomycetes which commonly grow on decaying wood have been treated by Gams and HolubovA-Jechova (1976). Most of the species proved to represent conidial states of Chaetosphaeria species (Sphaeriaceae). Other species of this genus include Gonytrichum conidial states, which can be distinguished from Chloridium by having phialides which are partly arranged in false whorls. The so-called “black yeasts,” a previously imperfectly known group of fungi partly pathogenic to humans and animals and partly growing on living and dead plants, have been revised by de Hoog (1977) and HermanidesNijhof (1977) in a joint study. The genera Aureobasidiurn and Rhinocladiella have only been accepted in a restricted sense and many species have been transferred to other or new genera, such as Leptodontium (eight species), Ramichloridium (15 species or varieties), and Exophiala (eight species, including the human pathogens E. jeanselmei and E . munsonii). Black yeasts without septate hyphae and only forming blastoconidia have been classified in a new genus Phueococcus. Fungi causing leaf spots on higher plants, hitherto classified as Kabatiella, Microstroma, and other genera, have been transferred to Aureobasidium. The conidial states of several Dothioraceae and Botryosphaeriaceae are treated in a separate, morphologically distinct genus, Hormonemu. In the Coelomycetes (Sphaeropsidales and Melanconiales)much attention has been paid to the genera PhyUosticta, Phomu, and Colletotrichum, in which several thousand often morphologically indistinguishable species have been described. Many of these cause leaf spots, necroses, anthracnoses, or fruit rots. In Colletotrichum the number of species was reduced by von Arx (1957) from approximately 800 to 12 after a comparison of numerous cultures. It was shown that the Colletotrichum conidial state of Glomerella cingulata had been previously described under a few hundred names, mostly in Gloeosporium and Colletotrichum. Similar comparative studies based on cultures would show, for example, that in Pestalotia (including Pestalotiopsis‘, Truncatella, and probably also Monochuetia), Conwthyrium (sensu Saccardo), or the Dothiorella 43otryodiplodia relationship, the number of distinguishable species could be greatly reduced. The genus Phyllosticta has been monographed by van der Aa (1973) and includes some pycnidial states of the ascomycete genus Guignurdia, about 70 species being recognized. As yet unpublished data show that hundreds of species de-

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scribed as Phyllosticta and Phomu belong to a limited number of Phomu and Phompsis species, e.g., P b m u exigua, P. mucrostomu, P. herbarum, and P . glomerata. These and other species can usually be easily recognized by their cultural characters. Basidiomycetes in general are studied from freshly collected specimens (or from herbarium specimens). In pure culture they usually only develop hyphae and sometimes conidia but normally no basidiomata or basidia. Wood decaying species are often isolated, however, from various substrates and for their identification only the vegetative (cultural)characters are available. Stalpers (1978) prepared keys based on the structures of the hyphae (clamp connections),presence or absence and kind of conidia, colony characters, and enzyme activities. Much attention has been paid to entomogenous fungi, especially on species known to be parasitic on insects of economic importance. Genera such as Paecilomyces, Nomuraea, Beauueriu, and Tritirachium have been treated by Samson (1974) and de Hoog (1972). In some taxonomic studies carried out at the CBS microscopic characters are used in conjugation with chemical characters for the delimitation of taxa. Approximately 10 years ago a chemical department was formed at CBS to deal with this side of the taxonomy. Investigations have since been carried out on the presence of anthraquinone in Curuularia and related fungi (Van Eijk and Roeymans, 1977), naphthaquinones in dermatophytes, carotenoids in Mucorales, triterpenoids in Aschersonia species, or sesquiterpenoids in several basidiomycetes. Most emphasis is placed on the carbohydrate composition of cell walls or intact cells, especially in the case of certain yeasts, “black yeasts,” and yeastlike ascomycetes, such as Dipodascus and Ascoidea (e.g., Weijman and de Hoog, 1975; Weijman, 1977; de Hoog, 1977). Cell wall analysis, the GC content of the DNA, and submicroscopic details have proved to be useful in delimiting the Endomycetes (ascomycetous yeasts), the Ustomycetes (ustilaginaceous yeasts), the Basidiomycetes, and the true Ascomycetes (von Arx et al., 1977). Chemical and submicroscopical characters will undoubtedly become indispensible in future taxonomic work on fungi. For the isolation, purification, and characterization of fungal metabolites, several physicochemical techniques can be applied, e.g., thin layer (TLC), liquid (LC), and gas-liquid (GLC)chromatography (van Eijk and Roeymans, 1976) and infrared (IR) and ultraviolet-visible spectrophotometry (MS). When dealing with certain taxonomic problems, good results are also obtained by the application of pyrolysis gas-liquid chromatography (Py-GLC) and pyrolysis mass spectrometry (Py-MS) (Weijman, 1977). Scanning electron microscope studies (Fig. 5) on spore ornamentation and, in particular, conidiogenesis of various Hyphomycetes and other fungi,

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J. A. VON ARX AND M. A. A. SCHIPPER

FIG. 5. T h m a s c u s thennophilus, conidiophores and plyphialides of the Polypaecilum conidial state (~OOOX).

were previously carried out in collaboration with the Laboratory of Electromicroscopy at the University of Amsterdam. In 1976 the CBS acquired its own Leitz AMR scanning electron microscope. This technique gives instructive and aesthetic pictures of structures visible with the light microscope and reveals previously unobserved details. Two 4-week courses are annually given and attended by students in mycology, research workers, etc., from different parts of the world. In March a practical and theoretical course on medical and veterinary mycology is given in order to promote the extension and intensification of scientific research on mycoses in the Netherlands. A second course in September1 October deals with general mycology and involves theoretical as well as practical work with field excursions. A handbook entitled “CBS Course of Mycology” (Gams et al., 1975)is offered to participants. Set fees are charged for attendance of both courses. Guest workers from various countries occasionally use the CBS facilities for longer or shorter periods. For more voluminous papers in taxonomic mycology, the CBS has

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published a series “Studies in Mycology,” of which 15 numbers have appeared since 1972.

111. The Collection of Yeasts in Delft A. MAINTENANCE

The CBS Delft collection includes approximately 4000 cultures representing 59 genera and 485 species as classified at present. Most of the strains are maintained on agar slants in tubes plugged with cotton wool which until recently were stored at room temperature (16-25°C) and subcultured every 5-6 months. Psychrophilic yeasts, such as Leucosporidium species, are stored at 3” C. Some strains, such as those of Pityrosporum species and Saccharomyces telluris, require subculturing every month and some, such as Dekkera species, every 2 months. The cultures are today stored in refrigerators at 5°C. Most strains are cultivated on GPY agar (glucose 40 gm; peptone, 5 gm; agar, 20 gm; yeast water, 500 ml; demineralized water, 500 ml), some (mainly ustilaginous yeasts) on 2%malt agar, and a few on YM agar (Difco) or V8 agar. The two strains of Cyniclomyces guttulatus present in the collection are maintained at 37°C in an acid medium consisting of 10 gm yeast autolyzate, 1 gm proteose peptone, 2 gm glucose, and 100 ml demineralized water and the pH is adjusted to 3 . 5 4 . 0 with HC1 before sterilization. These strains are subcultured every 2 weeks. In addition as many strains as possible are maintained in the lyophilized state, although this has not always proved possible. In cases such as Candida m u s c m m and C. javanica, none of the strains live for long, while in others survival depends very much on the strain, some strains of a given species reacting well, others poorly or not at all. Of 557 cultures stored at room temperature for 25 years, 394 were still viable. For lyophilization the cultures are grown for 3 days in 20 ml of liquid GPY medium or malt extract on a rotary shaker; the cells are harvested, suspended in the lyophilization medium (inositol, 7.5%; sodium glutamate, 2%; dextran, 5%; pH 7.0-7.5), and then cooled to -30°C in the chamber of the freeze dryer. The cultures are dried in high vacuum overnight; then the temperature is allowed to rise to approximately 20°C and the ampules are stoppered, removed from the apparatus, and flame sealed. One tube is opened to check viability within a few days of drying. Newly accessioned strains are lyophilized in six ampules as soon as possible after entry into the collection. The ampules are stored in filing cabinets in small envelopes on which are recorded the date of lyophilization and the date on which an ampule is opened. Each strain has a file card on which are entered the name and CBS

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number, numbers of the strain in other collections, previous identifications, designation under which the strain has been received, donor, source and date of isolation, date received, medium and any special conditions required for cultivation and storage, any special properties, uses, and literature.

B. IDENTIFICATION SERVICE Morphological, physiological, and genetic characters are used in the identification of yeasts. In the morphology emphasis is laid on the manner of vegetative reproduction (unipolar, bipolar, or multilateral budding), the formation of pseudo- and true mycelium, chlamydospores, ballistospores, and vegetative endospores. The most important physiological characteristics include the fermentation and assimilation patterns of carbon and nitrogen sources. Six carbon sources, viz. D-glucose, D-galactose, sucrose, maltose, lactose, and raffinose, are generally tested in the fermentation. In the assimilation test 32 carbon sources are used but this number can be increased (Lodder, 1970). In standard identifications the assimilation of nitrate as a nitrogen source is determined. Some additional physiological characters include growth in a vitamin-free medium, growth on 50 and 60% glucose-yeast extract agar, growth at 37"C, acid production, resistance to actidion, and assimilation of ethylamine-HC1. In the genus delimitation of ascosporic yeasts attention is paid to the manner of ascus formation and also the nu'mber, size, shape, and ornamentation of the ascospores (e.g., round, hat-shaped, kidney-shaped, smooth, or warty). In the ustilaginaceus type of yeasts note is made of the shape of the resting spores (angular or round) which may in some cases be formed after conjugation. C. SCIENTIFICRESEARCH

The group of yeasts related to the Ustilaginales, including the genera Rhodotorula, Rhodosporidium, Sporobolomyces, Sporidiobolus, Cryptococcus, Leucosporidium, and Bullera, is investigated. Efforts are made to construct the life cycles of these yeasts. Numerous mating experiments are done to identlfy strains of opposite sex of the same species (mating types). All sorts of media are used, for instance: malt extract agar, cornmeal agar, hay infusion agar, vegetable juice agar, and water agar with additional salts. If septate hyphae with clamp connections and chlamydospores are produced, then attempts are made to induce chlamydospore germination. Mating types have been found in Cryptococcus macmans, Sporobolomyces odorus, S p . roseus, S p . salmonicolor, and S p . hispanicus. The apiculate yeast genera, Hanseniaspora and Kloeckera, have been

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revised and their morphological and physiological properties examined (von Am, 1976). A joint investigation by Sally A. Meyer and M. Th. Smith into the DNA base composition of various strains of both genera and into the relationships between the perfect and imperfect states was carried out at the American Type Culture Collection. At present six species in the genus Hanseniaspora and six imperfect counterparts can be recognized. Detailed data will be published in a joint publication. A taxonomic study of the genera Brettanomyces and its perfect counterpart Dekkera has been initiated. Morphological and physiological characters and the DNA base composition of various strains present in the CBS yeast collection will be examined. The genus Sacchuromyces, sensu J. P. van der Walt in Lodder et al. (1970), is being revised and it has been proposed that the genera Zygosaccharomyces and Torulaspora be restored. At present six species can be recognized in the genus Sacchuromyces, namely S. cerevisiae, S. dairensis, S . exiguus, S . kluyveri, S . seroazzii, and S. unisporus. All other species will have to be excluded. A number of species assigned to the genera Zygosaccharomyces and Torulaspora were reduced to synonymy on the basis of their variable characteristics, namely, S. amurcae with Z . cidri; T. rosei, T . fermentati, S . florenzanii, T . vafer, and T. nilssonoii with T . delbrueckii; and Z . mellis with 2. rouxii. The DNA base compositions reported by Yarrow and Nakase (1975), and those since determined, do not contradict this classification and the results of DNNDNA hybridizations (Bicknell and Douglas, 1970; Groot et al., 1975; PhaE, 1977)confirm some of these proposals.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to their colleagues for supplying information, to Dr. W. Gams for reading the manuscript, to Miss C. A. N. van Oorschot for correcting the English text, and to Mrs. E. J. Hilhorst-Timmer for typing the final draft.

REFERENCES Bicknell, J. N . , and Douglas, H. C. (1970). J . Bacterial. 101, 505512. de Hoog, G . S. (1972). Stud. Mycol. 1. de Hoog, G . S . (1974). Stud. MycoZ. 7. de Hoog, G . S. (1977). Stud. Mycol. 15, 1-140. Gams, W. (1971). “Cephalosporium-artige Schimmelpilw (Hyphornycetes).” Fischer, Stuttgart. Gams, W. (1975). Trans. Br. Mycol. SOC.64,389404. G m s , W., and Holubov&J~hova,V. (1976). Stud. M ~ c o Z . 13.

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Cams, W., van der Aa, H. A,, van der Plaats-Niterink, A. J., Samson, R. A., and Sdpers, J. A. (1975). “CBS Course of Mycology,” B a r n . Groot, G. S. P., Flavell, B. A,, and Sanders, J. P. M. (1975). Biochim. Biophys. Acta 378, 186-194. Hermanides-Nijhof, E. J. (1977). Stud. Mycol. 15, 141-177. Lodder, J., and Diddens, H. A. (1942). “Die anascosporogenen Hefen,” Part 2, NorthHolland Publ., Amsterdam. Lodder, J., and Kreger-van Rij, N. J. W. (1952). “The Yeasts, a Taxonomic Study.” NorthHolland Publ., Amsterdam. Lodder, J . (1970). “The Yeasts,” 2nd ed. North-Holland Publ., Amsterdam. PhafT, H. J. (1977). Abstr., Int. Mycol. Congr., 2nd, p. 519. Raper, K. B. (1957). Mycologia 49, 884-892. Samson, R. A. (1974). Stud. Mycol. 6. Samson, R. A,, and Evans, H. C. (1977). Pt-oc. K. Ned. Akad. Wet., Ser. C , 80, 128-134. Samson, R. A., Stolk, A. C., and Hadlok, R. (1976). Stud. Mycol. 11. Schipper, M. A. A. (1969). Antonie uan Leeuwenhoek 35, 189-208. Schipper, M. A. A. (1973). Stud. Mycol. 4. Schipper, M. A. A. (1975). Stud. Mycol. 10. Schipper, M. A. A. (1976). Stud. Mycol. 12. Schipper, M. A. A. and Bekker-Holtman, J. (1976). Antonie oan Leeuwenhoek 42, 325-328. Scott, De B. (1968). “The genus Eupenicillium.” C.S.I.R., Pretoria, South Africa. Stdpers, J. A. J. M. (1978). Stud. M!ycoZ. 16. Stelling-Dekker, N. M. (1931).Verh. Kon. Akad. Wet. Anlsterdani, aft/. Natuurk., Sect. 2 , 28. Stolk, A. C., and Samson, R. A. (1972). Stud. Mycol. 2. van der Aa, H. A. (1973). Stud. Mycol. 5. van der Plaats-Niterink, A. J. (1975). Neth. I . Pl. Path. 81, 2237. van Eijk, G. W., and Roeymans, H. J. (1976).j. Chromatog. 134, 6648. van Eijk, G. W., and Roeymans, H. J. (1977). Erperientia 33, 1283. van Oorschot, C. A. N . (1978). Persoonia 9, 401408. von Arx, J. A. (1957). Phytopothol. Z. 29, 413468. von Am, J. A. (1973). Persoonia 7, 367-375. von Arx, J. A. (1975a). Persoonia 8, 191-197. von Arx, J. A. (1975b). Stud. Mycol. 8. von Arx, J. A. (1976). Verh. K. Ned. Akad. Wet., Afd. Natuurkd. 11, 67. von Am, J. A., Rodrigues de Miranda, L., Smith, M. T. and Yarrow, D. (1977). Stud. Mycol. 14.

Weijman, A. C. M . (1977).In “Analytical Pyrolysis” (C. E. R. Jones and C. A. Cramers, eds.), p. 225. Elsevier, Amsterdam. Weijman, A. C. M. (1977). Antonie oan Leeuwenhoek 43, 323331. Weijman, A. C. M . , and de Hoog, 6 . S. (1975). Antonie oan Leeuwenhoek 41, 353-360. Yarrow, D., and Nakase, T. (1975). Antonie uan Leeuwenhoek 45, 8 1 4 8 .

Microbiology and Biochemistry of Oil-Palm Wine NDUKAOKAFOR Department of Microbiology, University of Nigeria, Nsukka, Nigeria I. Introduction ........................... B. Tapping the Palm for S a p . . .......................... C. Composition of the Sap. . . . . . . . . . . . . . .......... 11. Microorganisms in Palm Wine ...........................

111.

IV. V.

VI.

B. Bactena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Succession of Microorganisms ........................ D. Source of the Microorganisms . . . . Biochemical Changes in Palm W i n e . . ..................... A. Changes in the Composi Wine with Storage. . . . . B. Changes in the Composi Increasing Period of Tapping ......................... Preservation of Palm Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Survival of Bacterial Pathogens.. ......................... Conclusion ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 237 239 240 241 241 241 242 244 245 245 250 252 254 254 255

I. Introduction A. NATURE OF PALMWINE

Palm wine is the collective name for a group of alcoholic beverages produced by the fermentation of the sap of palms (tribe Cocoineae, Family Palmae, Anonymous, 1966).It is drunk in various parts of the tropical world (Adriens, 1952; Ahmad et al., 1954; Bassir, 1962, 1968; Bois, 1937; Busson, 1965; dAymeric, 1921; Irvine, 1961; Miracle, 1967; Munier, 1965; Van Pee and Swings, 1973), including South America, Asia, and Africa, and is often referred to as “toddy” (Blatter, 1926; Comer, 1966). In Africa, the production of palm wine appears to have been known for several centuries, but the earliest record of it comes from the chronicles of European travellers recorded as early as 1591(Pigatetta and Lopes, 1591).Other early chronicles of the beverage are to be found in Capelle (1641), de Rome (1648), and Da Firenze (1820). The type of palm whose sap is fermented varies from one part of the world to another and includes the coconut palm, the oil palm, the date palm, and the raphia palm. Table I illustrates the world distribution of palms tapped for palm wine. Palm wine, no matter the origin of the sap, is usually a whitish liquid which is effervescent because the microorganisms causing the fermentation are alive when it is consumed. In this respect it is similar to some of the 237 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-00262&1

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TABLE I PALMSFROM WHICH PALM WINE IS OBTAINED~ Name of palm Acromia oinilfera Oerst Arenga pinnata (Wurmb.) Merr. (Syn. A. sacoharifera Labill.) Attaka speciosa Mart. Borassus aethiopum Mart. Broassus flabelifer Linn. Caryota wens Linn. Cocos nucilfera Linn. Corypha umbraculifera L. Elaesis guineensis Jacq. Hyospathe elegans Mart. Hyphaenae guineensis Thonn. Jubaea chiknsis (Molina) Baillon Mauritiella aculeata (H.B. and K.) Burret (Syn. Lepidococcus acukatus H. Wendl and Drude) Murenia montana (Humb. and Bonpl.) Burret (Syn. Kunthia montana Humb. and Bonpl.) Nypa fiuticans Wurmb. Orbignya cohune (Mart.) Dahlgreen ex Standley (Syn. Attaka cohune Mart.) Phoenix dactylifera Linn. Phoenix reclinata Jacq. (Syn. Phoenix spinosa Schum. and Thonn.) Phoenix syluestris (L) Roxb. Raphia hookeri Mann and Wendl. Raphia sudanica A. Chev. Raphia oinifera Beauv. Scheeka princeps (Mart.) Karsten (Syn. Attaka princeps Mart.)

Location Nicaragua, Panama, Costa Rica Far East Brazil, Guyana Tropical Africa India, Cambodia, Java India India, Sri Lanka, Africa Sri Lanka Africa Brazil, Guyana West Africa Chili Brazil, Venezuela

Brazil Sri Lanka, Bay of Bengal, Philippines, Carolines, Salmomon Islands Honduras, Mexico, Guatemala

North Africa, Middle East Central Africa India Africa Africa Africa Brazil, Bolivia

“From Swings and de Ley (1977).

indigeneous cereal beverages drunk in various parts of Africa where the fermenting organisms are alive when the brew is consumed. Such beverages include burukutu (Okafor, 1966; Faparusi et al., 1973; Faparusi, 1970), pito (Ekundayo, 1969; Okafor, 1966), and K&r beer (Novellie, 1960; Schwartz, 1956; Van der Walt, 1956). These beverages contrast sharply with grape wines, barley beers and sake where the organisms participating in the fermentation are removed before the beverages are drunk (Rose, 1977). Palm wine is usually sweetish and has a variable alcohol content of 0.57.1% (Bassir, 1962; Van Pee and Swings, 1971) by volume depending on a

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number of factors, including the source of the sap and the length of the fermentation (Faparusi and Bassir, 1972a,b). At consumption, the most favored palm wine has a pH of 3-5 (see below). In West Afi-ica the two palms most commonly tapped are the oil palm (Elais guineensis Jacq. and the raphia palm Raphia hookeri (Minn. et Windl.), Raphia raphia, and Raphia vin$wa Beav. (Okdor, 1975a). Much of this review centers on wine from the oil palm Elaeis, as a lot less seems to be known about other palm wines. It is sometimes distilled to produce a gin whose names vary according to the locality4ai-kai or ogururo in Nigeria (Akinrele, 1968)or akpeteshie in Ghana (Ayernor and Matthews, 1972). Accurate figures of the quantity of palm wine produced are not available, but in Nigeria, with a population of some 80 million, a rough estimate of 450 million gallons per annum has been given (Akinrele, 1968). Bassir (1968) suggests that some four million people consume palm wine daily in Nigeria alone and that these people derive some nutritional benefits from the approximately 300 calories (from sugar and alcohol), 0.5-2.0 gm protein, and large amounts of vitamins present in each liter.

B. TAPPINGTHE PALM

FOR SAP

The procedure for obtaining (or tapping) the fresh unfermented sap from a palm tree varies not only according to the palm tree being considered but, even for the same palm it depends on the locality. In some Asian countries, such as India, Indonesia, Phillipines, Sri Lanka, and Malaysia, sap is obtained from the coconut palm Cocos nucqera L. by first pulverizing the male inflorescence. Sap is then later collected from the injured flowers (Swings and de Ley, 1977). A slit made at the base of the male inflorescence of this palm has also been successfully used at the Nigerian Institute for Oil Palm Research, Benin, Nigeria (Obashola, private communication, 1974). The stage at which the Raphia palm (especially R. hookeria) is tapped is critical. Raphia palms are monocarpic, dying once they have flowered and fruited, after a period of vegetative growth lasting from 7 to 10 years. The palm must be tapped at the proper time, which is at the inception of the inflorescence. Tapping is done by cutting the terminal bud and collecting the sap issuing from it (Tuley, 1965b). This method is used in Nigeria as well as in Zaire (Swings and de Ley, 1977). The Cambodian method of tapping Burassus Jlabillifer L. has been described by Martin (1950). Several methods for tapping the oil palm Elaeis guineensis L. have been described (Tuley, 1965a). In the first method the tree is felled and the

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terminal bud is cut. The wine from this method, the so called “down wine” is not highly favored in Nigeria partly because it may result in the elimination of the palm population and also because of the unusually high sugar (and hence alcohol)content of the wine therefrom. This method appears to be the preferred method in Ghana, which requires that a palm seedling be planted for every tree felled (Ayernor and Matthews, 1972). The second method consists of tapping the stem of the standing tree, just below the apical growing point. In the third method a triangular incision is made in the base of the immature male inflorescence. The incision is widened twice daily. This method is the most acceptable, as it not only spares the tree but produces a wine which commands a high price-the so-called “up-wine” in Nigeria. It is widely used in West Afi-ica, including Nigeria, Benin, Dahomey, and the Ivory coast. The yield of sap is highly variable. Sometimes a slit in the trunk or the male inflorescence yields no sap at all. Simonart and Laudelout (1951), however, report that a felled oil palm yielded some 150 liters of sap in about a month. Swings (Swings and de Ley, 1977) and this author have collected about 3 liters daily in Zaire and Nigeria, respectively. Raphia palms generally yield more than oil palms and about 10 liters or more per day up to a month or more are not unusual. C. COMPOSITION OF

THE

SAP

The sap of the oil palm tapped from the immature inflorescence is a clear sugary syrup. Because of the exposure of the sap to microbial contamination and consequent alteration in the sap’s composition, Bassir (1962) collected the sap in a flask immersed in a freezing mixture of salt and ice. Okafor (1972b, 1975b)collected sap by using sterile tapping equipment, including a sterile knife for widening the slit in the base for the inflorescence, a funnel made of sterile plastic. and a sterile shield and glass tube, etc. Sap (about 100 ml) was collected in about I hour and Sietz filtered immediately. Using both of these methods sap believed to be only slightly altered in composition was obtained. Chinarasa (1968) collected what can be described as unfermented sap (“fresh sweet wine”; alcohol content, nil) in a vessel sterilized with 0.1% of sodium metabisulfite. Chromatographic analysis of the sap showed it to contain sucrose, fructose, glucose, and some r a n o s e (Bassir, 1962, 1968; Faparusi, 1966; N. Okafor, unpublished 1965). Quatitatively the amounts of the various sugars vary tremendously (See Table 11) probably because of the natural variations of the sap composition, the method of analysis adopted, the changes taking place in the sap before it

2,441

MICROBIOLOGY AND BIOCHEMISTRY OF OIL-PALM WINE

TABLE I1 COMPOSITION OF PALMSAP

Bassir (1962) Sucrose (%) Glucose (%) Fructose (%) R d n o s e (%) Protein (%) Ammonia (%) Vitamin C (mgl100 ml) Vitamin B,, (pglml)

4.29-1.4 3.31-0.95

Chinerara (1968) 13.00 -

-

N. Okafor, unpublished 11.2 0.95 1.0

Fapanusi and Bassir (1972a) 3.00 1.20 1.20 0.80

0.36

0.38-0.015

-

-

-

10.00

18.6 160.00

-

is analyzed, the time of the year it has been tapped, the variety of palm tapped, and a vast range of other factors (Faparusi, 1966). What is certain is that the total sugar content usually has an upper limit of about 12%.

II. Microorganisms in Palm Wine

A. YEASTS The yeasts which have been reported in palm wine belong largely to the genus Saccharomyces. Other genera include Kloeckera, Pichia, Candida, and Endomycopsis (see Table 111). Okafor (1972a) isolated and unidentified yeasts from palm wine in various parts of Nigeria. Most of the yeasts were Saccharomyces and Candida, even though the sources of the isolations were often hundreds of miles apart. He concluded that the distribution of yeasts was fortuitious and did not seem to have been dictated either by the type of palm from which the sap was obtained or the locality in which the palm grew.

B. BACTERIA

A wide range of bacteria has been encountered in palm wine, including the following genera: Lactobacillus, Acetobacter, Sarcina, Streptococcus, Leuconostoc, Bacillus, Zymomonsa, Brevibacterium, Micrococcus, Serratia, Corynebacterium, Pediococcus, and Klebsiella (Bassir, 1962; Faparusi, 1966; Faparusi and Bassir, 1971, 1972a,b; Simonart and Laudelot, 1951; Okafor, 1975a; Roelofsen, 1941). The type of bacteria would appear to depend on several factors but most importantly on the stage of the fermentation and the composition of the sap.

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NDUKA OKAFOR

TABLE 111

YEASTSIDENTIFIED IN PALMWINES Type of Wine

Yeast Saccharomyces pastorianus Saccharomyces ellipsoides Saccharomyces cerevisiae Saccharomyces cerevisiae

Oil palm Oil palm Arenga palm Oil palm

Saccharomyces chevalieri Pichia sp. Schizosaccharomyces pombe

Oil palm Oil palm Oil palm Oil palm Palmyra palm (Brosassusfibelliter) Oil palm Oil palm Oil palm Oil palm

Saccharomyces vafer Endomycopsis sp. Saccharomyces markii Kloekera apiculata Saccharomyces florentius Saccharomyces chevalieri Saccharomyces rosei Candida spp. Saccharomycoides ludwigii

Oil palm Palmyra palm (Brosassusfibellifir) Raphia Oil palm and Raphia Oil palm Palymra palm

Reference Van Pee and Swings (1971) Van Pee and Swings (1971) Bassir (1962); Faparusi and Bassir (1972a); Simonart and Laudelot (1951);VanPee and Swings (1971); Ayernor and Matthews (1972) Guilliermond (1914) Bassir (1962) Faparusi and Bassir (1971) Faparusi and Bassir (1972b);Ahmad et al. (1954); Saito and Otani (1936)

Okafor (1972a) Okafor (1972a) Okafor (1972a) Van Pee and Swings (1971); Simonart and Laudelot (1951) Okafor (1972a) Van Pee and Swings (1971) Okafor (1972a) Faparusi and Bassir (1971); Okafor (1972a) Saito and Otani (1936) Ahmad et aZ. (1954)

C. SUCCESSION OF MICROORGANISMS Faparusi and Bassir (1971) reported the presence of Leuconostoc and Lactobacilbs early in the fermentation of their palm wine sample. Okafor (1975b)found that while lactic acid bacteria as a group were important, there was no consistent pattern of the distribution of the various lactics; all four genera of lactic acid bacteria were not found in any one sample. In one sample, for example, Streptococcus was found throughout the 7-day period, whereas lactobacilli (Fig. 1) remained only during the first 3 days of fermentation. In contrast, the only lactic acid bacteria observed in another sample was Leuconostoc and even then only after 2 days of incubation. In the same vein, Streptococcus was important in some wine samples but not in others. Okafor (197213, 1975b) reported the presence of Enterobacteriaceae, such as Serratiu and Klebsiella, early in the fermentation. These quickly disappeared and he suggested that the enterics (as well as the lactics) proba-

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243

19

Period (days)

FIG. 1. Succession of microorganisms in, and pH of, wine produced from three trees of the same age, 8-15 yards apart. 0, yeasts; 0 , Lactobacillus spp.; V, Acetobacter spp.; 0 , Micrococcus spp.; A, Serratia spp.; B, Leuconostoc spp.; X , Aerobacter spp.; A---A, pH (Okafor, 1975a).

bly also contributed to the drop in the pH fi-om the neutral of the fresh unfermented sap to about pH 4 in the first 24 hours. Gram-negative bacteria belonging to the Enterobacteraceae have not been generally reported in palm wine. Probably they are missed since they disappear within 24 hours of the fermentation. That they were observed in this particular experiment was

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probably because sap was collected in sterile containers where the organisms developing in the sap could grow without undue competition from organisms already present in the collecting vessel. Yeasts and Micrococcus seem to occur consistently in many samples of palm wine (Bassir, 1962; Faparusi and Bassir, 1971; Okafor, 1975b). Workers who have studied the succession of microorganisms in palm wine consistently report the development of Acetobacter after about 3 days, at which time alcohol should be present in reasonable quantities (Faparusi and Bassir, 1971; Okafor, 1975b). One of the most striking features of the study of the microbiology of palm wine is the variability in the nature of the lactics encountered. In Fig. 1 the trees tapped were only 8-15 feet apart and were tapped by the same tapper who followed the same asceptic procedure. Yet the nature of the microorganisms differed tremendously, even though they were collected in sterile tlasks. Bassir (1962) introduced into palm sap unidentified palm wine yeasts and bacteria either alone or as mixtures of bacteria and yeasts. Activity of the organisms was determined by COz release and the pH of the medium. From his observations, Bassir (1962) concluded that the conversion of fresh sap to wine followed the two-stage fermentation described in cassava by Collard and Levi (1959). The first stage according to Bassir (1962) consists of the production of organic acids by bacteria. The second stage, which is triggered off at pH 6.8 and ends at pH 4.40, is the alcohol and organic acid stage. The major difficulty with the theory is that it places all the inversion of sucrose on the yeasts, whereas it is well known that several bacteria can bring about this phenomenon. It is possible that those bacteria employed in the Bassir (1962) experiment simply did not do this. Swings and de Ley (1977) claim that the bacterium Zymomnnas can produce reasonably large amounts of alcohol under anerobic conditions. D.

SOURCE OF THE

MICROORGANISMS

As palm wine is currently produced it ferments spontaneously by the activity of microorganisms which find their way into the palm sap. The source of the microorganisms, however, is of interest since a known reservoir could be an asset in isolating microorganisms for future attempts at standardizing the quality of the beverage through artificial microbial inoculations. Okafor (1972b), in attempting to discover the abinitio source of the organisms, carried out an experiment which eliminated four of these sources: (a) the tapper’s knife, (b) the bamboo funnel through which sap drips, (c) the gourd in which wine is collected, and (d) the air. The fifth-the bark of the male inflorescencrwas identified as the source. The microorganisms which had been found on scrapings of the bark were also found in wine, ex-

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245

cept Pseudomonas, which was assumed to have found the environment of the wine inclement. Faparusi (1973) came to a similar conclusion by examining a wide range of the aerial portions of the oil palm. His conclusion that the xylem stream may be a source of yeasts because these organisms are consistently found at the tapping sites (where presumably the tapping has been done with unsterile equipment) is open to further experimentation.

111. Biochemical Changes in Palm Wine A. CHANGES IN THE COMPOSITION AND PROPERTIES OF PALM WINE WITH STORAGE Palm wine is not considered acceptable after about 48 hours of fermentation from the time it is brought down from the tree even by the most hardened connoisseurs. Some studies (e.g., Faparusi and Bassir, 197.213; Okafor, 1975a,b) have followed changes in palm wine for between 4 and 7 days. Such studies have more relevance for such processes as the distillation of gin from fermented wine. Changes in this review will therefore not be pursued beyond 72 hours.

1 . Changes in Sugar Content of the Wine The sugar content of palm wine is very important from the organoleptic point of view. Sugar rapidly diminishes with storage as shown in Table IV. The table also shows that sucrose is the most important sugar in oil palm wine, its presence conferring on the wine a sweet taste. The table also shows that at least in some wine samples there is a preferential usage of various sugars. It remains for further experimentation to decide how the taste is affected by such sugars as fructose, glucose, and r a n o s e which would appear to form only a small percentage of oil-palm wine sugars and whose content seems to change little with progressive storage and fermentation. 2 . Changes in Alcohol and Other Votatile Compound

Contents Alcohol content of fermenting palm wine increased in all the studies available up to the first 7 days, when there was usually a decline. According to Faparusi and Bassir (1972a) this decline is attributable to Acetobacter spp., although Okafor (197513)has observed the presence of these bacteria as early as 24 hours from the time fermentation is observed. The alcohol content at the time palm wine is drunk (i.e., after 2448 hours offermentation) is in the range of 1.0-7.0% (Table V). It appears that the variety of the palm is important in determining the yield of alcohol. Thus Bassir (1968)states that

T A B L E IV CHANGES IN THE SUGAR CONTENTAND TASTEOF WINE WITH STORAGE Bassir (1962) Time (hours)

0 3 6 12 24 48 72

Sucrose (%)

4.29-1.4 0.0124.2 -

Chinarasa (1968)

Glucose (%)

Sucrose (%)

3.3-0.95 0.05-0.07 -

13.0 6.8 4.16 1.48 0.58 -

-

Taste

Sucrose

Fructose

Glucose

Raffinose

-

2.8 1.6 1.2 1.0

1.2 1.1 1.0 0.9

1.1 1.0 1.1 0.8

0.8 0.6 0.5 0.5

FresMsweet Sweet Slightly/sour Very sour

-

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TABLE V CHANGESIN ALCOHOLCONTENTIN PALMWINE WITH TIME

Bassir (1962, 1968)

Chinarasa (1968)

Faparusi and Bassir (1972b)

3.78 4.84 6.32 6.70 -

-

48

1.5-2.1 -

72

-

-

Days

0 3 6 12 24

1.5 4.8 5.5

Ayenor and Matthews (1972)

3.76 4.58 (4 hours) 5.24 7.22 7.80 8.22

the alcohol content of ripe wine is highest in the duara variety and lowest in the tenera. In the same publication Bassir (1968) also points out that palm wine is higher in alcohol content in the rainy than in the dry season, a surprising fact in view of the possibility of dilution of the wine in rain water and the lower temperatures of the rainy as compared with the dry season. Chinarasa (1968),for example, states that it is not surprising that the rate of palm wine fermentation is faster on a sunny day than on a cloudy or rainy one. Figure 2 shows a gas chromatographic study of slightly fermented sap and the wine fermented subsequently therefrom (N. Okafor, unpublished, 1976). It can be seen that isoamyl alcohol, active isoamyl alcohol, isobutanol, 1-propanol and ethyl acetate originally absent in the sap now appear in the wine. One can assume that these include the congeners which confer on palm wine its distinctive flavor. Ayernor and Matthews (1972)also identified methanol, isopropanol, n-propanol, and n-butanol by gas chromatography.

3. Changes in Total Acidity and p H One remarkable property of palm wine is its acidity, which quickly develops as microorganisms break down the sugar in the original sap. Changes in total acidity and pH in fermenting palm wine are given in Table VI. Palm wine sap usually begins with a pH value of neutrality (Bassir, 1962, 1968; Okafor, 197513; Faparusi and Bassir, 1971, 1972b).The pH tends to fall within 24 hours to 4.5-5.0 and at 48 hours to 4.0 or below. The figures of Ayernor and Matthews (1972) are different from these (Table VI) and it is possible that wine from felled trees which is used in Ghana (in contrast to the inflorescence wine of Nigeria and elsewhere) has a lower pH. That the total acid increases with increasing storage is expected. Much of this acidity is due to tartaric, lactic, and acetic acids. Bassir (1968)states that a 24-hour sample of wine contained 11.7-36.0, 32.156.7, and 18.6-28.6

248

NDUKA OKAFOR

5

Ethyl alcohol

-

Ethyl acetate

lsoamyl

alcohol I/

lsobutanol

Actctlve lsoarnyl f alcohol

%

FIG. 2. Chromotographs of partially fermented palm sap (bottom)and fully fermented palm wine (top). (Okafor, 1976, unpublished.)

(mg/100 ml), respectively, of these acids. Van Pee and Swings (1971), however, did not observe lactic acid in fresh sap as did Bassir (1968).In addition to the above acids Van Pee and Swings (1971) also observed malic acid, pyruvic acid, succinic acid, and citric acid. All the acids except tartaric and citric acids were either absent or occurred only as traces in palm sap.

4 . Changes in Protein and Amino Acid and Total Nitrogen Content The total nitrogen content of palm wine is a crude measure of the microorganisms therein and their nitrogenous metabolites. It is therefore not surprising that Faparusi and Bassir (1972b)observed a steady increase with the fermentation of the wine from 120 mg/100 ml total nitrogen on the first day to 200 mg/100 ml on the seventh. Bassir (1968)states that the amount of protein in palm wine is related not

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TABLE VI CHANGES IN TOTALACIDITYAND pH IN FERMENTINGPALM WINE Ayernor and Matthews (1972)

Faparusi and Bassir (1972b)

Hours of fermentation

Titratable acidity (mlO.1 N NaOH)

pH

0 4 6 12 24 48 72

5.3 6.4 7.1 7.2 7.8 9.2

3.8 3.6 3.6 3.6 3.6 3.6 3.6

Okafor (1975b) PH

Titratable acidity (mlO.1 N NaOH)

pH

2.5 5.5

5.5 3.8

7.0 -

-

4.0 4.5

only to the variety of oil palm, but also to the season. The variety Pisferu produced about twice as much protein nitrogen and three times as much amino nitrogen as the Duru variety. In the dry season these constituents approximated 32 mg/100 ml and 104 mg/100 ml, and rose to about 70 mg/100 ml and 137 mg/100 ml, respectively, in the wet season. No explanation has been given for this unexpected observation. Bassir (1968) names thirteen amino acids which are usually found in ripe palm wine. Van Pee and Swings (1971), however, found a total of 27 amino acids as well as four unidentified ones. These two authors group the amino acids of palm wine into five groups: a. Those which are always present in both palm sap and palm wine; aspartic acid, glutamic acid, serine, asparagine, citrulline, alanine, tyrosine, aminobutyric acid, valine, methionine, typtophane, leucine, isoleucine, and proline b. Amino acids absent in palm sap but appearing in palm wine after 12, 24, 36, 48, and 72 hours: cystine, cysteic acid, histidine, lysine, and arginine c. Amino acids present after 0, 12, and 24 hours and absent thereafter: threonine, ornithine, and histamine d. /3-Alanine, which is completely absent e. Those amino acids which fluctuate during fermentation: glycine and pheny lalanine

5. Changes in the Vitamin Components The vitamins which have been examined in palm wine are vitamins B,, B2, B,, B,,, and C.

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NDUICA OKAFOR

Bassir (1968) reports the presence of 25 pg/liter of vitamin B, in fresh palm wine. This amount increased to 150 pg/liter after an unspecified period of storage. Vitamin B2increased from 35 p g i t e r to 50 p g i t e r over the same period. The same author collaborating with Faparusi (Faparusi and Bassir, 1972a) later reported much lower amounts of these two vitamins and also much less change in amounts with storage of the wine: figures of approximately 8.0-10.0 pg/liter were found over a period of 7 days. Figures for vitamin B6 were much higher and ranged from 30 pg/liter to 35 pg/liter over the same period. Vitamin C, according to the authors, dropped from about 11mg/100 ml in fresh palm sap to 9.0 mg in 2.4 hours. Thereafter it remained approximately constant for the next 6 days. Similar figures (10.6 mg/100 ml dropping to 7.4 mg/100 ml in 24 hours) were found by N. Okafor (unpublished, 1976). Vitamin BIZ,in contrast, tended to increase with storage as shown by the observations given in Table VII. IN B. CHANGES

THE

COMPOSITION OF PALMWINE TAPPING

WITH INCREASING PERIOD OF

As palm sap drips from the tree in the traditional method of tapping it is immediately attacked by microorganisms which are naturally present on the tapping materials and utensils, especially the collecting gourds. The process of palm sap fermentation may therefore be seen as a modified continuous culture, which differs from the system as it is usually known because there is no simultaneous outflow of sap (Okafor, 1975b). For this reason it is imprecise to refer to palm wine as “fresh (except in terms of taste) since the term usually applies only when the wine has been brought from.the tree. Since oil-palm wine is collected once or twice a day a “24-hour” sample of potable wine may have indeed been fermenting over a total period of 36 or 48 hours. Because the microorganisms in palm wine are alive, the biochemical properties of the wine-and with them the organoleptic properties-change rapidly. A number of workers have studied these changes. It appears from TABLE VII CHANGES IN VITAMINB,, CONTENT OF PALMWINEWITH STORAGE (pg/ml) Storage (hours)

Van Pee and Swings (1971)

N. Okafor, unpublished (1977)

0 12 24

17-180 140-190 190-280

150 500

MICROBIOLOGY AND BIOCHEMISTRY OF OIL-PALM WINE

TABLE VIII CHANGES OCCURRING IN VARIOUS COMPONENTS OF PALM WINE LENGTH ENS^ Days Sucrose Fructose 1 2 3 4 5 6 7

2.7 2.0 1.4 1.0 1.0 0.8 0.8

1.3 1.6 1.8 1.8 1.8 1.6 1.2

1.2 1.5 1.7 1.6 1.8 1.2 1.1

Glucose

0.3 0.4

7.1

Raffinose Titratable

-

110 0.9 0.5

4.04 6.2 1.0 120 2.0 5.0 5.8 2.0 1.0

0.5 6.5 1.6 0.5 0.6 0.9 0.6 10.0

4.2 6.5 4.0 39

3.0 170 4.2 3.00 4.5 190 4.6 190

AS THE

pH 7.1 1.8 3.2 3.4 170 3.22 3.4

251

TAPPINGPERIOD

Ethyl Total Vitamins 10.0 6.0 2.5 2.6 1.4 7.2 2.6

9.0 6.2 6.3

8.6

6.1

8.2 8.0

88

'Approximate figures compiled from graphs in Faparusi and Bassir (1972a).

the work of Faparusi and Bassir (1972a) that a major factor affecting the quality of palm wine is the length of the time a tree has yielded wine. This factor should be clearly distinguished from the further changes which occur as wine from each particular day is stored. As indicated in Table VI certain properties or components, such as sucrose, pH, and vitamin C, decrease while others such as ehtyl alcohol, titratable acidity, total nitrogen, and vitamins B1 and B2, increase with the lengthening period of tapping. Fructose, glucose, and vitamins B6 and C remain virtually the same (Table VIII). The import of this observation emerges fully when it is noted that from the observations of Faparusi and Bassir (1972a) that wine obtained from a tree which has yielded sap for only 1 day contains no alcohol at all within the usual time limit of fermentation (say 8-12 hours). That this cleary is a result of the buildup of the microorganisms responsible for the changes is illustrated in Table IX, which shows that a plateau begins to develop in microbial TABLE IX CHANGES IN

THE VIABLECOUNTSOF

YEASTS AND BACTERIAIN PALM WINE INCREASINGPERIODSOF TAPPING"

WITH

Days of tapping

Yeast cells ( x 106)

Bacteria ( x 102)

1

0.007 2.50 8.00 24.00 48.00 68.50 80.50

1.10 7.00 18.50 26.00 34.00 41.00 43.20

2 3 4

5 6 7

"Faparusi and Bassir (1972a).

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NDUKA OKAFOR

counts of palm wine between the second and the fourth day of tapping. This is the period sap is allowed to flow in Nigerian practice before the ensuing wine is considered suitable for consumption. For a full history of any sample to be given (especially for experimental purposes), therefore, the length of the tapping period should, where possible, be stated.

IV. Preservation of Palm Wine Because the organisms in palm wine are alive they soon metabolize away the sugar, producing acid among other metabolites (see Tables 11-VI). The result of this is that the wine soon loses its sweetish, pleasant taste for a sour, harsh one. Owing to the popularity of palm wine among all classes of West Africans a number of attempts have been made to increase its shelf life from 2436 hours to much longer. Theoretically this could be achieved in a number of ways: (a) by slowing down the metabolism of the palm wine organisms through refrigeration; (b) by inhibiting or totally destroying the microorganisms by the use of chemicals; (c) by destroying some or all of them by heat; or (d) by the physical removal of the microorganisms. The first method is not, at the present time, practicable in the economically developing countries where palm wine is produced and drunk. This is because the infrastructure of constant electric power supply, refrigerated transportation, sales facilities, etc., which would make this method economical is not available. The attempts at preserving palm wine have therefore been limited to the other three methods. Levi and Oruche (1957)rejected pasteurization because, according to the authors, this method “changes the character of the beverage.” Instead they used the chemicals sulfur dioxide and benzoic acid contained in two commercial preparations (from Bush and Co., Ltd., Hackney, London E. 8): a. “Bush’s Fermenticide Powder containing sulphur-dioxide.” b. “Bush’s Preservative Powder containing benzoic acid.” The authors considered pure potassium or sodium bisulfite and sodium benzoate, but most surprisingly rejected their use in favor of the above proprietary products which they considered “as the most suitable for Nigerian producers who will want to be sure of obtaining regular supplies of chemicals which can be relied upon for strength and purity.” (sic)The authors recommended the addition of 5.5 gm of these materials to 2 gallons of wine, which should be bottled and crowned immediately the chemicals dissolved. Wine so treated would keep for many months and could be transported without explosion. Adverse effects on the flavor could be restored by pro-

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prietary flavors. However, the fact that this method has been discarded at the very research institute where it was developed (Chinarasa, 1968; Akinrele, private communication, 1975) would appear to belie this claim. One major drawback to this method is that benzoate is not used as a food additive in some countries. With respect to sulfite, it appears (Faparusi, 1969) that at pH 4-5 when the wine is preferred the amount of sulfite required would be too high. Levi and Oruche (1957) took into account neither the wine pH nor the acceptable level of sulphite (Okafor, 1975a). The bark of the tree Saccoglotis gabonensis Urban (family Humiriaceae) is added to palm wine by the local people in parts of Nigeria (Ogan, 1971). Attention was therefore turned to its use for possible prolongation of the shelf life of palm wine. Faparusi (1972) showed that three of five bark constituents inhibited unnamed palm wine bacteria and yeasts. Okafor (1975a), however, found no inhibition. The differing results could be due to several factors. First is the question of the proper identity of the tree whose bark was used in the two experiments. Second is the treatment which the bark received: whether it is oven dried and at what temperature. The third factor is the method of extraction; Faparusi (1972) used several solvents, including light petroleum, ethanol, ethyl acetate, and methanol, whereas Okafor (1975a) used water. The antimicrobial activity of the bark of Saccoglottis would appear to deserve further study as a possible antimicrobial food additive, since it is already being used in palm wine. Heat has been applied for the preservation of palm wine. Chinarasa (1968) experimented with temperatures ranging from 50°C to 90°C for different durations and found 68°C for 30 min to be most suitable for preserving the wine as well as for maintaining its flavor. Wine so preserved kept for 6 months, according to the authors, without loss of flavor. The darkening of the sediment which formed was arrested with 70 ppm SO, added before pasteurization. Okafor (1975a) studied a combination of various dilutions of sodium metabisulfite, diethyl pyrocarbonate (DEPC), and sorbic acid with heat treatment. DEPC, although successfully used in other alcoholic beverages (Amerine and Kunkee, 1968), left a pungent smell in palm wine. Metabisulfite, although the most suitable of the three as a preservative, was not acceptable because the effective dose in the wine would be too high for human consumption based on the maximum requirement of 0.35 mg/kg of body weight given by Mossel (1971). Pasteurization at 70°C for % hour and subsequent treatment with sorbic acid was therefore chosen (Okafor, 1975a). The removal of the microorganisms of palm wine by centrifugation made it possible for Okafor (1977) to use lower temperatures and chemical concentrations than hitherto. Samples of wine which were virtually sterile could be produced with this method (see Table X). The loss of the whitish appearance

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NDUKA OKAFOR

TABLE X COUNTS OF BACTERIA ON WHOLE PALM WINE AND ON SUPERNATANT OF CENTRIFUGED PALM WINE AFTER VANOUS TREATMENTS’ Number of bacteria

Treatment Whole palm wine Untreated 0.05%potassium sorbate

0.10% 0.151 0 . 1 5 1 sodium metabisulfite 0.10% 0.15% Supernatant after centrifuging Untreated Pasteurized at 60°C for 1/2 hour Pasteurization plus 0.05%potassium sorbate Pasteurization plus 0.05%sodium metabisulfite

2.80 x 2.96 x 2.32 x 2.24 x 1.40 x 1.20 x 4.80 x

10” 109 109

109 109 108 105

4.00 x 104 2.00 x 102 2.00 x 102 0.4 x lo2

Percent survival

100.00 1.0571 0.8286 0.8OoO

0.5oOo 0.4285 0.00017 O.oooO1 14285 0.0000007

0.00000007 Virtually sterile

~

‘From Okafor (1977).

consequent on the removal of microorganisms was restored by using a cloudifier. With this method wine was kept in a “fresh” and acceptable form for between 6 and 9 or more months without refrigeration. Furthermore a sediment which usually turns an unpleasant brown color with storage is formed in wines preserved by all the previous methods. The sediment does not form in wine preserved in part by centrifugation.

V. Survival of Bacterial Pathogens Because water of dubious sanitary quality is sometimes used by unscrupulous middlemen to dilute palm wine it was decided to study the survival of Salmonella and Shigella in palm wine. Neither of these two survived (Okafor, 1974), probably because of the acidity of the beverage.

VI. Conclusion Palm wine can be regarded as a suspension of bacteria and yeast in palm sap whose fermentation they bring about. Its study could be seen as a branch of microbial ecology. A fuller understanding of its nature has permitted a method of its preservation to be developed. Much of the work on palm wine has been carried out on wine from the oil palm. Virtually nothing seems to be known of the other palm wines, especially Raphia wine.

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REFERENCES Adriens, L. (1952). Bull. A@. Congo Belge 43, 556. Ahmad, M . , Chaudhury, A. R., and Ahmad, K. U. (1954). Mycologia 46, 708 (translated from Italian by W. Bal, Nauwelaerts, Louvan and Pans, 1965). Akinrele, I. A. (1968). Proc. Agric. SOC. Nigeriu 5, 16-18. Amerine, M. A., and Kunkee, R. E. (1968).Annu. Reu. Microbiol. 22, 323. Anonymous. (1966). “The Oil Palm in Malaya Ruala Lumpur.” Ministry ofAgriculture, Malysia. Ayernor, G. K. S., and Matthews, J. S. (1972). Trop. Sci. 13, 71433. Bassir, 0. (1962). West Aj?. 1. B i d . Appl. Chem. 6, 21-25. Bassir, 0. (1968). West A$. 1. Biol. Appl. C k m . 10, 4 1 4 5 . Blatter, E. (1926). “Palms of British India and Ceylon.” Oxford Univ. Press, London and New York. Bois, D. (1937). “Les Plantes alimentaires,” 4th ed. Lechavalier, Paris. Busson, F. (1965). “Plantes alimentaires de L’Quest Africain.” Les Palmiers. Capelle, F. (1641). Bull. Inst. Hist. Belge Rome 37, 221 (quoted in Swings and de Ley, 1977). Chinarasa, E. (1968). Research Rep. No. 38. Federal Ministry of Industries, Lagos. Collard, P., and Levi, S. (1959).Nature (London) 183, 620-621. Comer (1966). “The Natural History of Palms.” Weidenfeld & Nicholson, London. Da Firenze, Z. (1820). “Etudes Histoire Africaine, Vol. I. Kinshasha, Zaire (quoted in Swings and de Ley, 1977). dAymeric, M. (1921).J. Phann. Chem. 23,272. de Rome, J. -F. (1648). Trasktion of Italian by F. Bontnic, Nauwelaerts, Louvain and Paris, 1964 (quoted in Swings and de Ley, 1977). Ekunday, J. A. (1969).J . Food Technol. 4, 217-22.5. Faparusi, S. I. (1966). Ph.D. Thesis, University of Ibadan. Faparusi, S. I. (1969). Appl. Microbid. 18, 122-123. Faparusi, S. I, (1970).1. Sci. Food Agric. 21, 79-81. Faparusi, S. I. (1972).Appl. Microbiol. 24, 853456. Faparusi, S. I. (1973).1. Appl. Bacteriol. 36, 559-565. Faparusi, S. I., and Bassir, 0. (1971).J. Food Sci. Technol. 206. Faparusi, S. I., and Bassir, 0. (1972a). West A@. J . B i d . Appl. Chem. 15, 24-28. Faparusi, S. I . , and Bassir, 0. (1972b). West Af;. J. B i d . Appl. Chem. 15, 24-28. Faparusi, S. I . , Olorunboba, M. O., and Ekundayo, J. A. (1973). 2. Allg. Mikrobiol. 13, 563568. Guilliermond, A. (1914).Ann. Sci. Nat., Bot. Biol. Veg. [91 19. Irvine, F. R. (1961). ‘‘Woody Plants of Ghana.” Oxford Univ. Press, London. Levi, S. S., and Oruche, C. B. (1957).Res. Rep. N o . 1 . Federal Ministry of Industries, Lagos. Martin, F. (1950).2nd. Agric. Aliment. 67, 237-245. Miracle, M. P. (1967). “Agriculture in the Congo Bassin.” Univ. of Wisconsin Press, Madison. Mossel, D. A. A. (1971). Symp. SOC. Gen. Miwobiol. 21, 177. Munier, P. (1965). Fruits 20, 577. Novellie, L. (1960).J. Sci. Food Agric. 11, 408421. Ogan, A. U. (1971).Phytochemistry 10, 2832-2833. Okafor, N . (1966). West Afr. J . Appl. Biol. Chein. 4. Okafor, N. (1972a). J . Sci. Food Agri. 23, 1399-1407. Okafor, N. (1972b). Symp. Proc. Nigerian SOC. Microbiol. 1, 102-106. Okafor, N. (1974). “Symposium Committee on Food Microbiology and Hygiene of the IAMS.” Kiel.

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Okafor, N. (1975a).J. Appl. Bacteriol. 38, 1-7. Okafor, N . (1975b).J. Appl. Bacteriol. 38, 81-88. Okafor, N. (1977).J. Appl. Bacteriol. 43, 159-161. Pigatetta, F.,and Lopes, D. (1951).Translated from Italian by W. Bal, Nauwelaerts, Louvain and Paris, 1965 (quoted in Swings and de Ley, 1977). Roelofsen, P. A. (1941).Natuurwet. Tijdschr. Ned. Zndie 101, 374. Rose, A. H.,ed. (1977).“Alcoholic Beverages,” p. 33. Academic Press, New York. Saito, K., and Otani, Y. (1936).Jpn. J. Bot. 8, 23. Schwartz, H.M. (1956).J . Sci. Food A@. 7 , 105-113. Simonart, P., and Laudelot, H. (1951).Znst. R. Colon. Belge, Bull. Seances 22, 38541. Swings, J., and de Ley, J. (1977).Bacteriol. Reu. 41, 143. Tuley, P. (1965a).Niger. Field 30, 28-36. Tuley, P. (1965b).Niger. Field 30, 120-131. Van der Walt, J. P. (1956).J . Sci. Food A@. 7, 105-113. Van Pee, W.,and Swings, J. G. (1971).East Af;. Agric. For. J. 36, 311314.

Bacteriala-Amylases M. B. INGLE AND

R. J. EFUCKSON Research and Development, Industrial Products Group. Miles Laboratories, Inc., Elkhart, Indiana Introduction . . . . ........................ Thermophilic Am ........................ Alkaline Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acidic Amylases.. ................................. Biosynthetic Aspects of Amylase Production. A. Extracellular Enzyme Synthesis and the Culture Growth Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. RNA Metabolism and Amylase Biosynthesis . . . . . . . . . . . . C. Induction and Catabolite Repression . . . . . . . . . . . . . . . . . . D. Genetic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Selection of the Amylase for Development . . . . . . . . . . . . . VI. Conclusion . . . .. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 11. 111. IV. V.

257 258 258 260 260 260 264 267 271 273 275 276

1. Introduction Starch hydrolyzing enzymes, analogous to catalysts used in the petrochemical industries, are manufactured in small amounts when compared to the amount of material produced by their reaction. The bacterial a-amylases are used for liquefaction of starch to allow the efficient production of dextrose by sacchrifying enzymes. These a-amylases are endoamylases capable of randomly hydrolyzing the w ~ 1-4)( glucosidic linkages of starch. The a-amylases are used with continuous starch cooking by direct steam contact. This process is called “jet cooking” in commercial terms. This type of process provides improved process control which produces a consistent level of saccharides. As the use of bacterial a-amylases increased, it became important to isolate new high-producing strains (Ingle and Boyer, 1976) and enzymes with characteristics more amenable to industrial use. Through these studies it became apparent that the characteristics of a-amylase varied as widely as the organism which produced them. In addition, the efficient production of these enzymes has produced a need for a more complete understanding of the regulatory events which control synthesis and secretion. This review describes the characteristics of some new a-amylases and factors regulating the biosynthesis of these enzymes. 257 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press,Inc. All rights of reproduction in any form reserved. ISBN 0-12-002624-4

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II. Thermophilic Amylases Although most bacterial a-amylases are used in commercial practice at relatively high temperatures (80-90°C) there has been interest in improving this characteristic by isolation of alternate enzymes from a variety of microorganisms. In addition, the continuing interest in the molecular basis of enzyme thermal stability has provided more definitive information in this area (Singleton and Amelunxen, 1973). A summary of these organisms, and characteristics of the respective enzymes, is presented in Table I. The initial description of an a-amylase from B. coagulase demonstrated increased heat stability (Campbell, 1954, 1955). Later studies concentrated on the a-amylase of B. stearothemophilus (Campbell and Cleveland, 1961; Campbell and Manning, 1961; Manning and Campbell, 1961; Manning et al., 1961). Although some confusion exists on the exact amount of heat stability and the molecular characteristics of this a-amylase (Pfueller and Elliott, 1969) the existence of a thermostable enzyme is well documented (Singleton and Amelunxen, 1973). Although it appears that thermophilic bacteria are likely to produce thermostable a-amylases, it is apparent that there is no obligatory relationship between the thermophilic character of a particular microbe and its extracellular a-amylase. For example, Thermonorpora curuata produces an amylase with a temperature optimum of 65°C and B. acidocaldarius 104-A one with an optimum of 6043°C (Table I), while Bacillus lichen$brmus (not generally considered a thermophile) amylase has an optimum of 76°C (Saito, 1973). Although there certainly is a trend, there are enough exceptions to prevent any conclusions based on the nature of extracellular enzymes from an organism that occupies a particular ecological niche. 111. Alkaline Amylases

The search for enzymes which operate in an alkaline pH range was initiated during the major interest in the use of enzymes in detergents. Subsequently, a number of patents and publications appeared. Robyt and Ackerman (1971) described an amylase from Psuedomonas stutzeri with an optimum pH of 8.0. Several Bacillus species were reported to produce alkaline amylases which had pH optima 10 or higher (Rikagaku Kenkyusho, 1970; Ajinomoto Co., Inc., 1971). Boyer and Ingle (1972) described and characterized an alkaline amylase from B. alkalophilic subsp. halodurans (Boyer et. al., 1973). The amylase had an optimum temperature of 50°C and a pH optimum of 9.2. The enzyme was an endo-amylase and was not sensitive to chelating agents. The lack of

TABLE I a-AMYLASES CHARACTERISTICS OF BACTERIAL Strain

pH optimum

Temperature optimum (“C)

Acidic a-amylases B. acidocaldarius 104-1A B. acidocaldarius Agnano 101 Clostridium acetobutylicum Thermostable a-amylases Thermomonospora cumata

4.0

6043 75 50

5.5-6.0

65

Themoactinonyces vulgaris

5.9-7.0

Thermophile V-2

6.0-7.2

B. stearothermophilus

5.0-6.0

B. licheniformis

5.0-8.0

60°C optimum, activity unstable above 70°C Optimum of 70°C but stable at 80°C for 2 hours 70°C optimum activity; varies according to growth temperature and enzyme isolate 76°C optimum activity; 50% of maximal activity at 90°C

Alkaline a-amylases Bacillus alkalophilus Bacillus No. A-40-2 Pseudomonas stutzeri

4.5 3.5

9.2 10.5 8.0

51 50-55 Inactivated above 40°C

Type

endo-Amylase endo-Amylase Not characterized Produces maltotetraose and maltopentaose multiple forms

Not clear hut produces no glucose or maltose in early stages @-AmylaseI and I1 with different temperature optimum Unusually high production of G5. Early reaction products predominate in high molecular weight oligosacchrides endo-Amylase, products with p configuration Insensitive to EDTA Sacchanfying, insensitive to EDTA

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sensitivity to chelating agents was considered to be a majro advantge for use in detergents containing sodium tripolyphosphate. Yamamoto et al. (1972) have confirmed that there is a wide range of a-amylase that can be found in alkalophilic bacteria. Eight strains were selected from 300 isolates. The pH ranges for optimum amylase activity were found to be as low as pH 4.0 and as high as pH 11.0, Unlike the thermophilic enzyme and organisms, the alkalophilic bacteria all produce alkalophilic extracellular enzymes.

IV. Acidic Amylases Since portions of the industrial processes concerned with production of dextrose are carried out in a pH range of 4.05.0,an advantage can be obtained with an acidophilic liquefying amylase. Although little is known about these types of enzymes, their existence indicates potential improvement in starch processing. The observation of starch hydrolysis on solid media by B. acidocaldarius isolate 104-1A led to studies on the characteristics of the a-amylase. The enzyme was isolated from solid culture and characterized. Buonocore et al. (1976) studied on a different isolate designated Agnano 101. There are differences between the enzymes produced by the two isolates with regard to pH and temperature optimum (Table I). Both enzymes are endo-amylase but maltotetrose is the main product of the amylase from Agnano, 101 while maltose is the predominant product from strain 104-1A. Since there are some differences between the enzymes, it should be noted that the growth temperature ranges are 5840°C and 4070°C for Agnano 101 and 104-1A, respectively.

V. Biosynthetic Aspects of Amylase Production A. EXTRACELLULAR ENZYMESYNTHESIS AND CULTURE GROWTHCYCLE

THE

The process of amylase biosynthesis has usually been investigated under experimental conditions that include high aeration, an efficiently metabolizable carbon source, a complex nitrogen source, and an optimal temperature. Since most of the studies on bacterial amylase production also involve the genus Bacillus, the relationship between the specific processes of amylase biosynthesis and the differentiation of the vegetative cell into a spore becomes obscure and confusing. As pointed out by Schaeffer (1969), the existence of amy mutants that sporulate normally indicates that the enzyme does not play a direct role in sporulation. However, as our conceptualization of

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the total process of sporulation becomes more complex, it is more difficult to unequivocally demonstrate independence of all aspects of the two processes. For example, alterations in the ability of RNA polymerase to select different promotors may play a direct role in both sporulation and amylase biosynthesis. A close relationship can also be justified on teleologic grounds. As a cell depletes the immediate carbon source in the environment, it must use the remaining energy source either to form a spore to preserve its genetic information or to secrete degradative enzymes to allow further growth and the generation of more genome copies. The Bacillus cells have evolved to carry out both activities simultaneously. Two postulates can be advanced for this situation. First, sporulation is an energy intensive process and it would be more economical for the cell to elaborate the degradative enzymes to find alternative carbon sources. Second, the presence of the enzymes (e.g., amylases, proteases, RNase, glucanases, cellulases) might act to insure the completion of the sporulation process by scavenging all remaining energy sources. A theoretical understanding of this interrelationship could provide a rational basis for a process or strain improvement program. This will be illustrated in the following discussion. The growth cycle of bacteria that produce amylases (and all other secondary products as described by Drew and Demain, 1977) can be divided into three important phases: the trophophase, in which the potentially productive biomass is produced; the idiophase, in which the amylase is produced; and the transition phase that separates the first two phases. This delineation is not rigorous and amylase biosynthesis is occasionally observed in growing cells but usually not under the conditions described above. It is, of course, during the idiophase that the processes of sporulation are operative. Since both amylase synthesis and sporulation are inhibited during the trophophase, one can ask whether there exist common regulatory steps. At present it appears that regulation takes place at the transcriptional level. It has been known for quite some time that new classes of mRNA appear in the idiophase in Bacillus (Aronson, 1965; DiCioccio and Strauss, 1973) and that both vegetative and sporulation genes appear to be transcribed simultaneously (Sumida-Yasumoto and Doi, 1974), although there is evidence that certain vegetative genes are not transcribed during the later stages of sporulation (Linn and Losick, 1976). Net synthesis of RNA ceases during the transition phase (Doi, 1969) and there is a rapid turnover of preexisting molecules into new RNA (Balassa, 1972). The ribosomal RNA genes do not appear to be transcribed during the idiophase (Hussey et al., 1971) and the level of mRNA in Bacillus amyloliquefaciens increases from 3 to 6% of the total cellular RNA (Brown and Coleman, 1975).Based upon the differential synthesis of extracellular and cellular protein, Brown and Cole-

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M. B. INGLE AND R. J. ERICKSON

man (1975) estimated that content of mRNA specific for extracellular proteins increased %-fold during the switch from trophophase to idiophase. Bacterial sporulation has been utilized as a model system for cellular differentiation and a general mechanism to explain the activation of a large number of genes has been sought. For the present discussion two hypotheses are briefly summarized and their applicability to amylase synthesis is considered. Two recent in-depth reviews are available and the reader interested in additional information should refer to these. The first hypothesis to be considered is that structural alterations in the complex RNA polymerase molecule influence and selection of the operons to be transcribed. The initial work in this area was, of course, prompted by the work done with the sigma factor of Escherichia coli RNA polymerase. The core RNA polymerase molecule contains four subunits, two a , p, and PI. When this RNA polymerase was added to T4 phage DNA, RNA synthesis was initiated at random; however, the addition of sigma factor, which was normally part of the polymerase complex of uninfected cells, restricted transcription to operons that were expressed early during the infection process (Bautz et al., 1969). As infection proceeds, the sigma factor shows a decreased affinity for the core polymerase (Bautz and Dunn, 1969) and four new polypeptides are found to be associated with core RNA polymerase (Stevens, 1972). The appearance of these modifications has been correlated to changes in the transcription pattern observed during T4 phage infection. Proof that such RNA polymerase modifications are associated with the transcriptional control of sporulation and extracellular enzyme biosynthesis would require the demonstration of both the occurrence of such alterations and the dependence of the specific function on that alteration. The Bacillus subtilis RNA polymerase has a sigma factor associated with the core enzyme during the trophophase of the growth cycle (Avila et al., 1971) which is replaced by two other polypeptides during sporulation (Fukuda and Doi, 1977; Linn et a l . , 1975). Although there exist some discrepancies in the data of the two reports cited, the more recent work of Fukuda and Doi (1977) has demonstrated the appearance of an enzyme factor designed 6l at stage I11 of sporulation and another factor, ij2,at stage IV. The same investigators also showed that the vegetative RNA polymerase, carrying the sigma factor, could be found in sporulating cells. Regardless of the correct interpretation of the results to date, it appears that a modified RNA polymerase enzyme can be found in B . subtilis cells that are producing amylases. Is there a correlation, then, between extracellular enzyme synthesis and these modifications? Unfortunately, an unequivocal answer cannot be given. Most of the data on this subject are more qualitative than quantitative in nature and when an effort is made to be quantitative, the results are ambiguous. An example is the work of Sonenshein et al. (1974) on their class I1

BACTERIAL ff -AMYLASES

263

sporulation-defective, rifampin-resistant mutants. These mutants demonstrate drastic reductions in sporulation, an altered RNA polymerase core enzyme, and only minor changes in serine protease and esterase production. Mutants M027 and M O B , for instance, demonstrate a 104-foldreduction in sporulation efficiency and no change in either enzyme. A further degree of ambiguity is introduced when one considers that the mutations examined to date modify the core enzyme and not the associated factors. Conformational alterations of this type need affect not only the attachment of the subunit factors but, in addition, other parameters influencing template specificity. Thus, in contrast to the models and conclusions of others (Coleman et al., 1975; Priest, 1977) the relationship between RNA polymerase modification and extracellular enzyme synthesis, including the amylases, remains obscure. A second alternative is related to the modulation of RNA polymerase activity by highly phosphorylated nucleotides. Table I1 lists some of these compounds with their common designations. MS I was found to accumulate in stringent (rel+) strains of E. coli in response to amino acid starvation (Cashel, 1969). As MS I accumulates, the synthesis of RNA is preferentially inhibited (Reiness et al., 1975; van Doyen et al., 1975)and ribosomes ofrel+ cells synthesize both MS I and MS I1 in cell-free systems (Haseltine et al., 1972). It was later shown that MS I could alter the preference ofE. Cali RNA polymerase (the holoenzyme) for different promotors (Travers, 1976). An analogous situation also appears to exist in B. subtilis (Fortnagel and Bergman, 1974) except for the interesting observation that ribosomes from idiophase, sporulating cells produce HPN I and HPN 11, while ribosomes from trophophase cells produce MS I and MS I1 in an in uitro system (Rhaese and Goscurth, 1974). In the fermentation vessel, HPN I11 is found to accumulate intracellularly and HPN IV accumulates in the medium (Rhaese et d.,1976) and conditions that repress sporulation and extracellular enzyme TABLE I1 HIGHLYPHOSPHORYLATED NUCLEOTIDES FOUNDIN Bctccillus CELLS Chemical abbreviation

Designation

PPGPP PPPGPP PPAPP PPPAPP PPZPUP PPPAPPP

MS I MS I1 HPN 1 HPN 11 HPN 111 HPN IV

264

M. B . INGLE AND R. J. ERICKSON

synthesis (e.g., high glucose levels) reduce the levels of HPN I11 and HPN

IV.Conversely, lack of a carbon source or phosphate stimulates the production of HPN I11 and HPN IV. The definitive experiments regarding the importance of these highly phosphorylated nucleotides on promotor selection in amylase synthesis have not been reported but an interesting experiment carried out with B. licheni,formis may have some relevance to this point. Cells (of varying age) were washed and resuspended in sterile growth medium that had been conditioned by previous growth of the same organism (Saito and Yamamoto, 1975). When trophophase cells were resuspended in medium taken from idiophase cultures, maximum stimulation of a-amylase production was observed. The reverse experiment greatly repressed enzyme synthesis. Although one could argue that this could be due to a more trivial factor, such as pH, one might also propose the HPN IV could be acting to stimulate the formation of extracellular enzymes (Priest, 1977). A hypothetical mechanism would be that HPN IV induced the synthesis of the intracellular HPN 111 which acted to direct the RNA polymerase to recognize the promotors for extracellular enzymes and sporulation related genes. Although no definite conclusions have been deduced from the data presented on the modulation of RNA polymerase activity, a hypothetical example can be considered for the potential application of such basic knowledge. If one assumed that the a-amylase gene was transcribed by the holoenzyme (aa p p' 6) in the presence of HPN I11 one could, with ingenuity, construct a mutant with constitutively produced HPN I11 at a certain temperature that was defective in the formation of the sporulation associated forms of RNA polymerase (i.e., a a p p' 6l and a a p p' P).This genetic background would maximize the available RNA polymerase for transcription of the amylase gene which then could be directed to the amylase gene by the HPN I11 as a function of temperature.

B. RNA METABOLISMAND AMYLASEBIOSYNTHESIS There are two unusual characteristics of RNA metabolism in cells that are capable of producing excessive amounts of exocellular enzymes, the existence of a large pool of RNA precursors and an apparently long half-life of exoenzyme mRNA. A large ribonucleotide pool has been demonstrated in both B. amyloZiquefaciens (Stormonth and Coleman, 1974) and B. Zichenijx-mis(Leitzmann and Bernlohr, 1965)and Coleman et al. (1975)have implicated this observation into a general mechanism of exoenzyme biosynthesis. In their hypothesis, the RNA precursor pool is channeled into three pathways; nontranslatable RNA (rRNA and tRNA), cell protein mRNA, and exoprotein mRNA. During the trophophase of the fermentation the exopro-

BACTERIAL CY -AMYLASES

265

tein mRNA synthetic system (i.e., specific RNA polymerase, modifying molecules, promotors, etc.) is at a competitive disadvantage and the other two types of RNA predominate. In idiophase the situation changes. Synthesis of rRNA is significantly inhibited and there is a rapid turnover in this class of RNA (Hussey et al., 1971). This could be related to the accumlation of ppGpp in idiophase cells (Rhaese and Goscurth, 1974). This increases the RNA precursor pool size and allows the transcriptional system for exoenzyme mRNA to compete favorably with the cellular transcriptional system. Hence, in this model, the primary control factor is the precursor pool size and one would predict mutations affecting this pool (e.g., a decrease in rRNA turnover or a release of the feedback controls of nucleotide biosynthesis) to alter exoenzyme production in a hypothetically consistent manner. Such data could not be located in the literature. The studies regarding the apparent half-life of exoenzyme mRNA are controversial and intellectually intriguing and may offer insights into the basis of cellular differentiation. The studies have been reviewed in detail (Priest, 1977; Glenn, 1976; Erickson, 1976) and the present discussion briefly reviews the data regarding the a-amylase. The original observations implicating an abnormality of mRNA metabolism were based upon the insensitivity of penicillinase (Pollock, 1963) and RNase (Coleman and Elliott, 1965) biosynthesis to low levels of actinomycin D. The latter report indicated that this observation did not apply to amylase production but subsequent work demonstrated that the synthesis of this enzyme, in idiophase cells, was insensitive to both antinomycin D and rifampicin (Gould et al., 1973). A short description of the experimental protocol will aid in understanding the interpretation of the results. Cells are grown to the idiophase, washed, and resuspended in a production medium. Two phases of amylase production are observed. The initial phase, which is of 30-90 min in duration, is insensitive to antinomycin D and rifampicin, as described above, is not subject to glucose repression (Priest, 1975), but can be blocked by chloramphenicol (Gould et al., 1973). From these data, the extrapolation from the data gathered with protease biosynthesis (Both et al., 1972), one would predict that the rate of amylase synthesis was controlled by translational mechanisms and that the amylase mRNA was transcribed prior to the washing step. After this initial phase of production, amylase synthesis ceases for a short time and then a second phase of biosynthesis is initiated. This production phase is normal in that it is sensitive to inhibition of RNA synthesis and glucose repression (Priest, 1975). Three hypotheses could be advanced to explain these observations. Portions of these hypotheses have been previously discussed in this paper and details are found in those sections. The first hypothesis to be advanced was

266

M. B. INGLE AND R. J. ERICKSON

that a large pool of exoenzyme mRNA accumulated in idiophase cells, perhaps molecules in transit to membrane sites, and that synthesis would be sustained simply due to the massive pool of mRNA (Both et al., 1972). This model would explain the observation that uracil-requiring mutants, when treated as described above, will produce amylase in the absence of uracil (Kinoshitaet al., 1968)as well as the data with the inhibitors of RNA synthesis. This hypothesis appears to be inconsistent with data accumulated by Coleman and Brown (Brown and Coleman, 1975; Coleman et al., 1975). They showed that although the total amount of mRNA increased two-fold in B. amyloliquefaciens and exoenzyme mRNA might have increased ten-fold, this increase paralleled enzyme secretion. Hence, if one assumes that the efficiency of translation of the exoprotein mRNA is not extremely unusual, then the increased level of this mRNA simply reflects increased translation and not an extraordinary pool size of the mRNA. In addition, when trophophase cells are washed and resuspended in fresh medium at four times their original cell density, exoenzyme synthesis is derepressed even in the presence of the drug rifampicin. Since these cells would not have accumulated a large pool of exoenzyme mRNA, the data can be used as an argument against the existence of such a mRNA pool. The model advanced by Coleman et al. (1975), based on a changing RNA precursor pool size and a modified exoenzyme RNA polymerase, may account for the transition to the idiophase biosynthetic pattern, but it does not account for the continued synthesis of amylase by a uracil. The data of Kinoshita et a2. (1968) indicate that at a time of complete cessation of net RNA synthesis and no alkaline phosphatase synthesis, amylase synthesis continues at a significant rate. If these data are of general significance, then it appears that the only valid explanation is the existence of a mRNA species with an extremely long half-life. This introduces the last explanation, that the amylase mRNA has a relatively long half-life and that even under starvation conditions, exoenzymes continue to be excreted. Lampen has discussed the unusual structure of the 5’ end of the penicillinase mRNA (Yamamoto and Lampen, 197613) and the structure at this end of the RNA molecule is known to significantly increase mRNA half-life. For example, reovirus mRNA that has a blocked (i.e., terminates in GpppG) or capped (i.e., terminated in m7GpppGm)is much more stable than unmodified mRNA when injected into Xenopus laevis oocytes (Furuchi et al., 1977). In addition, since the structure proposed by Lampen would have a tendency to associate with membranes, the molecules might show unusual efficiency and stability characteristics. In conclusion, RNA metabolism in bacterial strains that produce unusually

BACTERIAL ff -AMYLASES

267

high levels of exoenzyme demonstrates several unusual characteristics. The mechanisms responsible for the observations remain unknown but their elucidation may provide important insights into the processes of enzyme excretion and cell differentiation. The application of techniques for mRNA purification and cell-&ee protein-synthesizing systems. C. INDUCTION AND CATABOLITE REPRESSION There is strong evidence that the rate of bacterial amylase biosynthesis is controlled by both substrate induction and catabolite repression. The evidence for substrate induction (e.g., starch or a-1,Clinked oligosaccharides) has been obtained in studies with B. stearothermophilus (Welker and Campbell, 196313) and B . lichenijbrmis (Saito and Yamamoto, 1975). The studies with B. stearothermophilus demonstrated that although the strain was partially constitutive, there was an instantaneous increase in the rate of amylase synthesis after the addition of a series of oligosaccharides (e.g., maltose through maltohexaose). Maltotetraose was the most efficient inducer and the addition of M increased the rate of synthesis three-fold in long phase cells. The studies with B . lichenqormis used idiophase cells that were grown in the presence of sorbitol (a noninducer) and then washed and exposed to a variety of sugars, oligosaccharides, and polysaccharides. The yield was then assessed after 2A hours’ additional incubation. Some of the carbohydrates produced no measurable enzyme activity while maltotetraose proved to allow the highest yield of enzyme. Thus, final yields were significantly altered by the presence of the potential substrate inducers. There have been several reports that certain strains of Bacillus species, such as B . amyloliquefaciens (Coleman, 1967), B . lichen$ormis (Meers, 1972), and B. subtilis (Sekiguchi and Okada, 1972), are constitutive with respect to a-amylase synthesis. In light of the partial induction noted with the oligosaccharides, however, this issue must be resolved with wellcontrolled experimentation. The experimental evidence regarding catabolite repression is less equivocal and can be demonstrated in all species studied to date. Studies with B . subtilis (Sekiguchiand Okada, 1972),B . stearothermophilus (Welker and Campbell, 1963a,b), B . amyloliquefaciens (Coleman and Grant, 1966), and B . licheni$ormis (Meers, 1972) have all indicated that carbohydrate sources or concentrations that tend to increase the growth rate will inhibit the formation of amylase. Most studies indicate that the Bacillus amylase biosynthetic system is under extreme catabolite repression and that even glyceral and acetate at a level of 0.5% can completely repress amylase synthesis (Saito and Yamamoto, 1975). In addition to the observed reduced rate

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of amylase production caused by conventional catabolite repression, amylase biosynthesis in Bacillus also demonstrates transient repression (Ingle and Boyer, 1976). The mechanism of catabolite repression in Bacillus remains obscure. Our conception of the molecular mechanism of catabolite repression is based on studies on the lac operon in E . coli. In this system, the addition of a rapidly metabolized carbohydrate (e.g., glucose) decreased the intracellular level of cyclic AMP which caused a reduction in the binding of the cyclic AMP receptor protein to the lac promotor (Pastan and Perlman, 1968). Th.is reduced binding to the promotor region results in a decrease in mRNA synthesis (Zubay et al., 1970) thus demonstrating that such control operates at the transcriptional level. Work on catabolite repression of amylase synthesis indicates that although it appears to operate upon transcription of mRNA, cyclic AMP may not be involved. As we have stated previously, idiophase cells appear to have either a large pool of mRNA or an increased stability of the mRNA for exocellular enzymes. Thus, idiophase cells should demonstrate a temporary resistance to catabolite repression and this has been demonstrated in both B. amyloliquefaciens (Ingle and Boyer, 1976) and B. subtiZis (Priest, 1975). The involvement of cyclic AMP, however, can be questioned for two major reasons. First, cyclic AMP could not be detected in B. lichenijormis with cyclic GMP being the only detectable cyclic nucleotide (Bernlohr et al., 1974). Second, although there exists a reported stimulation of amylase synthesis by cyclic AMP (Saito and Yamamoto, 1975), the nucleotides AMP and ATP also stimulate amylase synthesis (Priest, 1975) and it is suggested that the noted effect may be a nutritional response. Hence, there is no doubt that amylase synthesis is under catabolite repression; it appears to be controlled by a system that is not directly analogous to the lac operon in E. coli. Numerous models of exoenzyme secretion have been proposed (Both et a l . , 1972; Coleman et a l . , 1975) and are quite similar in that they are based primarily on data accumulated on secretory eucaryotic cells and the B . lichenijbrmis penicillinase. This discussion first describes the general model of exoenzyme secretion and then presents pertinent supporting observations. Unlike normal cellular protein biosynthesis, a simultaneous transcription and translation probably does not occur in the case of amylase. The transcribed message remains untranslated in the cytoplasm and, by an unknown mechanism, becomes associated with membrane-bound 50s ribosomal subunits. This inert cytoplasmic behavior of the mRNA is most likely due to a unique configuration of the RNA molecule that resists translation by free ribosomes and may direct the mRNA to the membrane. At the membrane site the translation process is initiated and a polar, phospholipoprotein seg-

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ment of the enzyme is synthesized and becomes firmly attached to the membrane. As the remainder of the molecule is formed, it is passed into the membrane and assumes a configuration in which the hydrophilicity of the enzyme is masked. The enzyme transverses the membrane and assumes a stable, hydrophilic configuration as it enters the external environment. A specific protease cleaves the polar NH,-terminal tail of the enzyme and the enzyme is now released from the cell and must now pass through the cell wall. The first issue raised by this model is the characteristic that differentiates the exoenzyme mRNA from normal cell protein mRNA. One possibility suggested by Yamamoto and Lampen (1976a) is that 5' end of the molecule may be attracted to the membrane and aid in the selection of ribosomes at this site. As is described below, the membrane-bound form of penicillinase has a unique phospholipopeptide at the NH, terminus. A mRNA structure can be proposed, based upon the amino acid sequence of the fragment, that is composed of 80% purine bases. The polynucleotide would be 75 nucleotides in length, behave like poly (A) segment, and be located at the 5' end of the mRNA molecule. Since poly (A) is known to have an affinity for membrane (Milcarek and Penman, 1974, Lande et al., 1975) a mRNA molecule with such a structure might be directed toward the cell membrane. Alternatively, the mRNA might associate with the 30s ribosomal subunit which would recognize membrane-associated 50s subunits. It has been proposed that the specificity of cistron selection is a property of certain protein components of the 30s ribosomal subunit (Held et al., 1974; Sprague et al., 1977) and such an interaction might also increase mRNA stability. The data linking membrane-associated ribosomes and exocellular enzyme synthesis and secretion remains equivocal and has been reviewed by both Priest (1977) and Glenn (1976). Membrane-associated ribosomes have been observed in many species of bacilli (Coleman, 1969; Pfister and Lundgren, 1964; Fitz-James, 1964; van Dijk-Salkinoja et al., 1970) and appear to be structurally different from free ribosomes (Brouwer and Planta, 1975). In light of the attractive model for amylase secretion in the pancreas of higher organisms, which involves the polysomes attached to the endoplasmic reticulum (Redman et al., 1966; Palade, 1975), it is tempting to bestow a similar property of the microbial ribosomes. In the eucaryotic amylase synthesis process, it has been proposed that the nascent amylase enzyme passes through a tunnel in the 50s ribosomal subunit and is discharged into the intraluminal compartment of the endoplasmic reticulum. Data to support such a mechanism in bacteria are based on two observations: Synthesis of exoenzymes is more sensitive to inhibition by fusidic acid and pactamycin than general intracellular protein synthesis (Both et a l . , 1972); and cell-free preparations employing membrane-associated ribosomes produce five times

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the amount of amylase as a system employing soluble ribosomes. Obviously, more data are needed to validate this aspect of the model. As translation is initiated, the nascent enzyme is extruded into the plasma membrane. With regard to the amylase enzyme, cell-associated activity can be separated into three fractions by chromatography on Sephadex G-75 (Nagata et ul., 1974). The fraction with the lowest molecular weight (i.e., 55,000) appears to be identical to the exocellular amylase and the two larger components appear to be precursors that are in transit through the membrane. Membrane-associated forms of amylase have also been observed in B. amyloliquefaciens and exhibit higher sedimentation velocities and altered elctrophoretic properties in comparison to the exocellular enzyme (Fernandez-Rivera Rio and Arroyo-Bendich, 1975). These larger molecules have not been analyzed to date but may be related to the membrane-associated penicillinase that has been thoroughly characterized by Yamamoto and Lampen (1976a,b). In this instance the membrane-associated enzyme had a molecular weight of 33,000,in contrast to 29,000 for the exoenzyme (i.e.,the largest membrane-associated amylase had a molecular weight of approximately 70,000). The added peptide was attached to the NH, terminus of the precursor polypeptide and had a very unusual structure. The NH,-terminal residue was found to be phosphatidylserine and contributed the hydrophobic character to the phospholipopeptide. This was attached via an Asn link to a high polar peptide made of 25 amino acid residues. The interesting aspect of this peptide was that it appeared to have arisen from a tetrapeptide structure (i.e., Asp-GluSer-Gly) that was repeated seven times. The modifications could all be accounted for by postulating the occurence of several single base changes, six deletions, and a duplication of a Glu residue. This added polypeptide may play some role in the passage of the penicillinase through the membrane and also in anchoring the enzyme to the cell surface, a role that would be of importance to penicillinase but not an amylase. There have been reports of other presumed phospholipoproteinsin plasma membranes of BaciZlus strains (Aiyappa and Lampen, 1976) and the synthesis of the enzyme levansucrase is very sensitive to the action of cerulenin, an inhibitor of Ltty acid biosynthesis (Caulfield et al., 1976); however, the general importance of such structures remains speculative. The membrane-associated enzyme is converted to this exoenzyme by the action of a single penicillinase-releasing protease that appears to reside in the periplasmic space (Aiyappa et al. , 1977). The specificity of this protease is toward the peptide bond involving the carboxyl group of serine or threonine. with no specificity toward the amino acid residue donating the amino function to the peptide bond (Aiyappa and Lampen, 1977). Since the added phospholipoplypeptide described above is joined to the exoenzyme through

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a Ser-Lys bond, the specificity explains the enzyme-releasing activity. The level of the penicillinase-releasing protease is not dependent upon penicillinase induction and it has been found in B . subtilis and B . amyloliquefaciens (Aiyappaet al., 1977; J. 0. Lampen, unpublished). Thus, this protease may have more general significance in exoenzyme secretion. As the enzyme emerges from the membrane, it appears to undergo a conformational change to assume the hydrophilic, protease-resistant exocellular structure. This hypothesis is, again, primarily based upon work with the B . lichenijbrmis penicillinase (Bettinger and Lampen, 1975). The studies involved the secretion of the enzyme by protoplasts. In the presence of trypsin or chymotrypsin penicillinase activity did not accumulate in the medium but peptide fragments of penicillinase origin could be identified. Similar studies with the B. amyloliquefaciens have shown the same to be true for the a-amylase produced by this species. In addition, and of particular importance in processes based upon amylase production by Bacillus species, the emerging enzyme was also sensitive to purified B . amyloliquefaciens protease. The occurrence of such an inactivation in uiuo could seriously alter the efficiency of the process.

D. GENETICASPECTS The initial genetic studies of amylase biosynthesis were reported by Green and Colarusso (1964) in their genetic transformation analysis in B . subtilis. The selected recipient and donor strains possessed amylases that differed in several respects including heat stability and sensitivity to catabolite repression. Recombinant strains were isolated that appeared to have incorporated the genetic information for both the structural gene and the catabolite control of the donor DNA. Subsequent transformation studies employed both B . natto (Yamaguchi et al., 1974a) and B subtilis var. amylosacchariticus (Yoneda et al., 1974) as the source of donor DNA and B . subtilis as the recipient. In these cases, one of the identifying characteristics of the donor amylase was the rate of synthesis and both studies indicated that the regulatory D N A segment (i.e., the gene controlling rate of synthesis) could be recombined independently of the amylase structural gene. The amylase structural gene was designated amyE and the control locus amyR. The B . subtilis regulatory gene was labeled amyRl and the two donor regulatory loci that conferred the high rate of synthesis were designated amyR2 (B. natto) and amyR3 ( B . subtilis var. amylosacchariticus). The fine structure of the amylase genetic region was studied by Yamaguchi et al. (197413). They isolated 28 amylase mutants of B. subtilis and found the mutations linked in the amyE locus. Various classes of mutations were isolated, including suppressor-sensitive strains, variants that produced

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temperature-sensitive amylases, amylases with reduced activity, and amylase-negative cells that produced immunologically cross-reacting material. The amyR gene was found to adjoin the terminal region of the structural gene. The amylase gene was found to be linked to a mutant locus labeled arollb by Yuki and Veda (1968) but it took several years to place these two markers of the B . subtilis chromosome. The arollb mutation was found to be in the aroZ region and three-fictor genetic crosses have proved the map order to be: lin-amyR-amyE-aroZlzarBdal-purB [Yuki, 1975; also see Young and Wilson (1975) for the complete B. subtilis chromosomal map and marker designations]. The percentage of cotransfer between aroZ and amyE is approximately 2040% which facilitates the isolation of amy transformants. One can simply use aroZ as the selected marker and screen these recombinants for amy cotransformants. In addition to the amyE and amyR genes, several other genes have been found to affect the biosynthesis of amylase. Yoneda et al. (1973) isolated mutants of B . subtilis that demonstrated increased production of protease and amylase but not RNase. This gene was found not to be linked to amyE and was originally labeled pap. It subsequently has been demonstrated that pap mutations are equivalent to the sacU locus studied by Lepesant et al. (1972) and are linked to u w and gtaB on the B . subtilis chromosome (Steinmetz et al., 1976). This mutation appears to be a cell surface alteration and is remarkably pleiotropic in nature. Some of the affected functions include loss of competence for transformation, lack of flagella, reduced autolytic activity (Ayusawaet al., 1975), overproduction of levansucrase, and ability to sporulate in a rich medium (Kunstet. al., 1974).ThesacU andamyR2 were found to act synerpistically in B . subtilis (Yoneda et al., 1974). Another interesting pleiotropic mutation is that of tunicamycin resistance in B . subtilis (Sasaki et. al., 1976). Tunicamycin is a nucleosidecontaining antibiotic (Takatsukiet al., 1977)that is active against gram-positive bacteria, yeast, and hngi and acts by interfering with the formation of lipid-linked intermediates in the synthesis of complex cell wall carbohydrates (Tkacz and Lampen, 1975; Bettinger and Young, 1975). Bacillus subtilis variants resistant to the drug produced five times the amount of amylase as the parent but normal amounts of protease and RNase. The mutant phenotype could be transformation, a result indicative of a single mutated locus. Amylase synthesis still appeared to be under catabolite repression and the increased yield was due to an increased rate of synthesis. Thus it appears that certain cell surface alterations can significantly increase the production of specific exocellular enzymes and the activity of other surface-directed antibiotics should be investigated. Other mutations that would affect amylase biosynthesis would be catabo-

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lite repression resistance and those leading to constitutive enzyme production. One would expect, in most cases, that such mutations would be in the vicinity of amyR and amyE. We have already stated that many strains are partially constitutive and, since starch is an inexpensive medium constituent and acts as a source of inducer molecules during the fermentation, such mutants may be of limited value. Mutants resistant to catabolite repression, however, could prove to be quite important. Such mutants would allow the use of an inexpensive, easily metabolizable carbon source, rapid achievement of maximum biomass, and a greatly increased rate of biosynthesis. Saito and Yamamoto (1975) isolated several such mutants of B. lichentjbrrnis by simply treating the cells on agar-based starch medium in the presence of high levels of glucose (e.g., 2%). Colonies with large halos were easily selected.

E.

SELECTION OF THE AMYLASE FOR

DEVELOPMENT

The initial step in developing an a-amylase fermentation process is the identification of the boundary limits under which the enzyme may be applied. This may appear to be rather obvious, but one’s economic rewards will ultimately depend on this first decision. Amylases are one of the largest volume industrial enzymes on the market (Aunstrup, 1977) and are utilized in numerous and varied processes (Fogarty et al., 1974). Special requirements for the starch and sugar syrup industry may vary considerably from those of the brewing, textile, or paper industry. Such enzyme characteristics as temperature and pH optima, cation or other cofactor requirements, as well as the level of additional enzymes in the product (e.g., p-glucanase, protease, pullulanase, @amylase) may significantly alter the attractiveness of the product for different applications. The subsequent step is the selection of the microorganism that appears to possess the potential for meeting the requirements set in the first step. The initial screening tests could employ isolates from natural sources or established culture collections. An indication of the variability encountered in such a screening program is observed in the work of Ingle and Boyer (1976). Strains of B. Zichen$mis produced significant amounts of alkaline protease but little amylase. Strains of B. amyloliquefaciens, in contrast, produced massive amounts of amylase and smaller amounts of neutral and alkaline protease. Strains of B. natto and B. subtilis that were screened produced low amounts of all enzymes examined. After selection of the potential production strain, a program is initiated to maximize the genetic capability of the strain to produce the specific amylase. Two approaches will be considered in this discussion, conventional mutation and selection and the utilization of genetic engineering technologies. The

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techniques of mutation and selection have been employed almost exclusively in the past in industry with a great deal of success. The results of such strain improvement programs are seldom reported in the literature and those results that do appear may be of limited value. The major reason for this latter statement is that many such studies select a very low-producing strain as the parent. Thus, although the mutants produce large amounts of amylase relative to the parent, the final yields are insignificant on an industrial scale. The genetically well-characterized B. subtilis 168, for example, produces 500600 units of amylase activity while randomly chosen strains of B. umyloliquefuciens produce from 1O0,OOO to 500,OOO units of activity (Ingle and Boyer, 1976). Obviously, a 20-fold increase in productivity in B. subtilis 168 would be insignificant relative to the closely related B. umyloliquefuciens. Bailey and Markkanen (1975)described a strain improvement program in which they used sequential exposure to different mutagens to increase amylase yields in B. subtilis. The agents, in order of use, were ultraviolet (UV) irradiation, ethylene imine, and a combination of ethylene imine and UV irradiation of N-methyl-N'-nitro-N-nitrosoguanidine.Final yields were increased only twofold but the authors claimed that the parent strain was initially a good amylase producer. The utilization of a combination of mutagens in sequence was thought to be required since they observed a plateau in yield improvement with repeated use of a given mutagen. They suggested that this loss of response represented the saturation of mutable sites that would elicit a positive response with respect to amylase yield. Although this may be true for some mutagens, it may not apply to the potent alkylating agents which appear to attack nucleic acids with little specificity (Singer, 1976). Continued mutagen exposure, in contrast, could result in an increased resistance of the strain toward the mutagen (e.g., altered uptake or inactivation). Random mutation and massive screening programs, although highly successful in the past, are time consuming, laborious, and often not amenable to good quantitative selection methodology. The techniques of genetic engineering in strain improvement programs should provide new avenues of approach to this problem. These new methodologies have the potential not only of increasing yields but of designing strains that can make novel enzyme products, unique mixtures of enzymes, that are able to grow in very inexpensive media and are extremely safe and efficient to utilize. The application of recombinant DNA technology in Bacillus species is in the developmental stages. Plasmids, a potential source of vectors for information transfer, have been characterized in numerous species (Lovett, 1973; Tanaka and Koshikawa, 1976) but, at present, lack good markers for direct selection of transformed cells. It appears, however, that they can be transferred between

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-AMYLASES

species and carried in a stable manner (Lovett et a l . , 1976). An alternative methodology has been to isolate antibiotic resistance plasmids from Staphylococcus aureus and introduce them into B . subtilis by transformation (Ehrlich, 1977). These small plasmids are maintained in B . subtilis and the antibiotic resistance genes (i.e., tetracycline and chloramphenicol) are expressed in the new host. Lysogenic bacteriophages are obvious alternatives as cloning vectors. Three phages that attack B. subtilis have been considered; 43T, 4105,and SP02. Bacteriophase 43T is of significance in this regard since it contains an easily selectable marker, thyP+ (thymidylate synthetase) (Tucker, 1969), and has already been employed in recombinant DNA experiments (Duncan et al., 1977). In these experiments the @T genome was cleaved with restricted endonuclease EcoRI and ligated to the E . coli plasmid pMB9 that had been treated with the same enzyme. A chimeric thyP+ plasmid was cloned in E . coli and shown to express the Bacillus gene for thymidylate synthetase. The chimeric plasmid, designated pCD1, was then isolated and was used to transform B. subtilis. The efficiency of transformation of thy quite high but the plasmid was not maintained in the extrachromosomalstate. Linkage data indicate that the thyP gene from pCDl is integrated into the chomosomal thyA locus (Williams and Young, 1977). With regard to amylases, one can predict numerous applications of these technologies toward strain design programs. Genes for amylases from any source can be isolated and treated as the thyP gene described above. Multiple gene copies can be inserted into a given production strain. An amylase gene can be isolated, modified by attaching new control elements (e.g., induction by lactose), and inserted. Genes for specific RNA polymerase, ribosomes, or other cellular components can be added to a cell and controlled by altering the environmental and fermentation conditions. The possibilities appear almost unlimited and should make studies involving strain improvement and design exciting, challenging, and of critical importance to the fermentation industry.

+

VI. Conclusion The bacterial a-amylases provide a unique example of new information, developed in response to a commercial and scientific need. These studies have led to the identification of new alkalophilic and acidophilic enzymes and bacteria. This provides models for describing and understanding the role of molecular confirmation and hydrolysis of oligosaccharides. In addition, it is obvious that the presence of these organisms in their ecological niche and the capability of extracellular enzymes to operate in these specific environments provide a wide and diverse possibility for new and novel enzymes.

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The ease of measurement of a-amylase synthesis, the knowledge of Bacillus genetics, and the fact that the enzymes are secreted could also provide excellent models for further study on the regulation of the synthesis of the secretion of these enzymes. Thus, the bacterial a-amylases provide an area of study where basic and applied information both contribute to an understanding of microbiological and biochemical processes. REFERENCES Aiyappa, P. S., and Lampen, J. 0. (1976). Biochim. Biophys. Actu 448, 401410. Aiyappa, P. S., and Lampen, J. 0. (1977). J . Biol. Chem. 252, 1745-1747. Aiyappa, P. S., Trdcante, L. J., and Lampen, J. 0. (1977). J . Bacteriol. 129, 191-197. Ajinomoto Co., Inc. (1971). Netherlands Patent Appl. 70-13,390. Aronson, A. I. (1965).J . Mol. Biol. 11, 576-585. Aunstrop, K. (1977). In “Biotechnology and Fungal Differentiation” 0. Meyrath and J. D. Bu’lock, eds.), pp. 157-171. Academic Press, New York. Avila, J., Hermoso, Vinuela, E., and Saks, M. (1971). Eur. J . Biochem. 21, 526-535. Ayusawa, C., Yoneda, Y., Yamane, K., and Maruo, B. (1975). J . Bucteriol. 124, 459469. Bailey, M. J., and Markkanen, P. H. (1975). J . Appl. Chem. Biotechnol. 25, 73-79. Balassa, G. (1972). Curr. Top. Microbwl. lmmunol. 59, 99-182. Bautz, E. K. F., and Dunn, J. J. (1969). Biochem. Biophys. Res. Commun. 34, 230-237. Bautz, E. K. F., Bautz, F. A., and Dunn, J. J. (1969). Nature (London) 223, 1022-1024. Bernlohr, R. W., Haddox, M. K., and Goldberg, N. D. (1974).]. Biol. Chem. 249,4329-4331. Bettinger, G. E., and Lampen, J. 0. (1975). J . Bacteriol. 121, 83-90, Bettinger, G. E., and Young, F. E. (1975). Bwchem. Biophys. Res. Commun. 67, 16-21. Both, G. W., Mclnnes, J. L., Hanlon, J. E., May, B. K., and Elliott, W. H. (1972).]. Mol. Biol. 67, 199-207. Boyer, E. W., and Ingle, M. B. (1972). J . Bacteriol. 110, 992-1000. Boyer, E. W., Ingle, M. B., and Mercer, G. D. (1973). Znt. J . Syst. Bacteriol. 23, 238-242. Brouwer, J., and Planta, R. J. (1975). Biochem. Biophys. Res. Commun. 65, 3363.14. Brown, S., and Coleman, G. (1975).J. Mol. Biol. 96, 345352. Buonocore, V., Caporale, C., DeRosa, M., and Gambacorta, A. (1976). J . Bacteriol. 128, 515421.

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Priest, F. G. (1977). Bmteriol. Reo. 41, 711-753. Redman, C. M.,Siekevitz, P., and Palade, G. E. (1966).J . B i d . Chem. 241, 1150-1158. Reiness, G., Yang, H.-L., Zubay, G., and Cashel, M.(1975). Proc. Natl. Acad. Sci. U.S.A. 72, 2881-2885. Rhaese, H. J., and Goscurth, R. (1974). FEBS Lett. 44,87-93. Rhaese, H. J., Grade, R., and Dichtelmuller, H. (1976). Eur. J . Biochem. 64, 205-213. Rikagaku Kenkyusho (1970). Netherlands Patent Appl. 70-13,396. Robyt, J., and Ackerman, R. J. (1971). Arch. Bwchem. Biophys. 145, 105-114. Saito, N. (1973). Arch. Bwchem. Biophys. 155, 290-298. Saito, N., and Yamamoto, K. (1975). 1. Bmteriol. 121, 84843%. Sasaki, T., Yamasaki, M., Maruo,B., Yoneda, Y., Yamane, K., Takatsuki, A., andTamura, G. (1976). Biochem. Biophys. Res. C o r r u n . 70, 125-131. Schaeffer, P. (1969). Bmtetiol. Reo. 33, 4-71. Sekiguchi, J., and Okada, H. (1972). J . Fament. Technol. 50, 801-809. Singer, B. (1976). Nature (London) 264,333439. Singleton, R., Jr., and Amelunxen, R. E. (1973). Bacteriol. Reu. 37, 320342. Sonenshein, A. L., Cami, B., Brevet, J., and Cote, R. (1974). J . Bacteriol. 120, 253-265. Sprague, K. U., Steitz, J. A., Grenley, R. M.,and Stocking, C. F. (1977). Nature (London)267, 462-465. Steinmetz, M., Kunst, F., and Dedonder, R. (1976). Mol. Gen. Genet. 148, 281-285. Stevens, A. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 603-607. Stormonth, D. A., and Coleman, G. (1974).J . Appl. Bacteriol. 37, 225-237. Sumida-Yasumoto, C., and Doi, R. H. (1974). J. Bacteriol. 117, 775-782. Takatsuki, A , , Kawamura, K., Okina, M., Kodama, Y., Ito, T., and Tamura, G. (1977). Agric. B w l . Chem. 41,2307-2309. Tanaka, T., and Koshikawa, T. (1977).J . Bmteriol. 131, 699-706. Tkacz, J. S., and Lampen, J. 0. (1975). Biochen. Biophys. Res. Commun. 65, 248-2.57. Tomoyeda, J., Hiroyuki, H., and Kameyama, M. (1962). Gifu Daigaku Nogakubu Kenkyu Hokoku 16, 151-154. Travers, A. (1976). Mol. Gen. Genet. 147, 225-232. Tucker, R. G. (1969). /. Gen. Virol. 4, 479504. van Dijk-Salkinoja, M. S., Stoof, T. J . , and Planta, R. J. (1970). Eur. J. Biochem. 12, 474-

482. van Ooyen, A. J. J., de Boer, A. H., Ab, G., and Gruher, M. (1975). Nature (London) 254,530531. Welker, N. E., and Campbell, L. L. (1963a). J . Bacteriol. 86, 681486. Welker, N. E., and Campbell, L. L. (1963b). /. Bucteriol. 86, 687-691. Williams, M. T., and Young, F. E. (1977). Abstr.. Annu. Meet. Am. Sac. Microbw1.p. 325. Yamaguchi, K., Nagata, Y., and Maruo, B. (1974a). J . Bacteriol. 119, 410415. Yamaguchi, K., Nagata, Y., and Maruo, B. (1974b).J . Bacteriol. 119, 416424. Yamamoto, M.,Tanaka, Y., and Horikoshi, K. (1972). Agric. Bid. Chem. 36, 1819-1823. Yamamoto, S., and Lampen, J. 0. (1976a). J . Biol. Chem. 251, 40954101. Yamamoto, S., and Lampen, J. 0 . (1976b). Proc. Nut/. Acad. Sci. U.S.A. 73, 1457-1461. Yoneda. Y.,Yamane, K., and Maruo, B. (1973). Bwchem. Bwphys. Res. Cqmmun. 50,765-770. Yoneda, Y., Yamane, K., Yamaguchi, K., Nagata, Y., and Maruo, B. (1974)./. Bacteriol. 120, 1144-1150. Young, F. E., and Wilson, G. A. (1975). Spores 6, 596-614. Yuki, S. (1975).Jpn. /. Genet. 50, 155-157. Yuki, S., and Veda, Y. (1968).Jpn. /. Genet. 43, 121-128. Yutani, K. (1973).J . Biochem. (Tokyo) 74, 581586. Yutani, K., Sasaki, I., and Ogasahara, K. (1973).J . Biochem. (Tokyo) 74, 573579. Zubay, G., Schwartz, D., and Beckwith, J. (1970). Proc. Natl. Acad. Sci. U . S . A . 66, 104-110.

Subject Index A

C

Activated sludge bulking problem, 86-93 operation, 86 process variables, 87-89 setding curve, 89-90 solids loss, 91-93 filamentous organisms, 95-114 actinomycetes, 113 bacteria, 95-113 blue-green algae, 113-114 fungi, 114 relationship to bulking floc form, 93-94 a-Amylases. bacterial acidic, sources, 260 alkaline, sources, 258-259 biosynthetic aspects culture selection, 273-275 genetic aspects, 271-273 induction and catabolite repression,

Centraalbureau voor Schimmelcultures history, 215-219 identification services, 225, 234 methods of maintenance, 219-223,233 Cerexins, 189,190-192

D Dextransucrase distribution, 63-64 preparation and purification, 64-75 Streptococcus mutans, 5556,80-82 dextran biosynthesis, 58, 59,61 chemistry and physical properties,

5658 E

Edeines, 189 Esperin, 208

267-271 relation to growth cycle, 260-263 relation to RNA metabolism. 264-265

G

uses, 257

thermophilic types, sources, 258-259 Antibiotics

Gramicidin S, 194 Gramicidins, 189

33-25,201-202 A-3302-B, 212 AB-1, 203 BU 1880, 203 EM-49, 199-202 TL-119,211-212 TM-743,203 V-8495,203

I

Iturin A. 196-198 L

Lyophilization for preservation of microorganisms, 15-26 equipment, 16-17 factors affecting survival, 17-29 cell concentration, 19-20 extent of drying, 23-24 method of reconstitution, 25-26 physiological age, 18-19

B

Bacillomycin L, 199 Bacilysin, 188 Bacitracin A, 194-195 Brevistin, u)s-210

279

280

SUBJECT INDEX

Lyophilization, continued rate of freezing, 22, 23 storage atmosphere, 24, 25 suspending medium, 20-22 temperature of storage, 25 type of organisms, 17-18 ,

M Methanol cell yield, 180-181 dissimilation by organisms other than yeasts, 167-169 by yeasts enzyme systems for dissimilation, 170-172 alcohol oxidase, 174-175 assimilation of methanol, 179-180 oxidation to formaldehyde, 173-175 oxidation of formaldehyde to formate, 176-178 historical, 165-167 production of cells, 182-183 Microorganisms, preservation, 1-53 Mixed culture fermentation enrichment techniques, 135 practice, 140-141 theory, 138-140 turbidostat, 136 two-stage chemostat, 136-137 industrial uses, 129, 130-132 brewing, 130 koji, 130 yoghurt, 131 production of metal leaching system, 158-159 of organic acids, 158 of vitamin B,,, 157-158 single cell protein, 141 advantages, 152-156 methane as substrate, 142-145, 147 methanol as substrate, 145-152 problems, 156-157 steroid oxidation by, 159-160 types of microbial action amensalism, 133 commensalism and mutualism, 134 competition, 132 neutralism, 133

parasitism, 133 predation, 132-133 Mycobacillin, 195, 196 Mycosubtilin, 199 0

Octapeptins, 202

P Palm oil wine, 237 biochemical changes, 245-250 composition of sap, 240-241 composition of wine, 250-251 microbial population, 24-245 origins, 238-2.39 Peptide antibiotics, from Bacillus species, 187-188 Polymyxins, 207-208 Polymyxin S,, 203-205, 207-208 Polymyxin TI, 203-208 Preservation of microorganisms criteria, 2 5 ability to reproduce, 2 3 functional properties, 3-4 genetic complement maintenance, 4 5 in industrial laboratories, 3134 methods, 5-29 dehydrated, 14-15 direct transfer, 5 frozen, 6-14 lyophilized, 15-29 under oil, 543 in water, 6 special groups of microorganisms algae., 34-35 bacteria, 35-41 fungi, yeast and actinomycetes, 41-43 phages, 42-47 S Streptococcus mutans, 77-80

Sufactin, 208-209

T Tridecapeptins, 192-193 Tyrocidins, 194

CONTENTS OF PREVIOUS VOLUMES Volume 1

Volume 2

Protected Fermentation Milos Herold and Jan Necusek

Newer Aspects of Waste Treatment Nandw Porges

The Mechanism of Penicillin Biosynthesis Arnold L. Demin

Aerosol Samplers Harold W. Batchelor

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

A Commentary on Microbiological Assaying F. Kauanagh

The State of Antibiotics in Plant Disease Control David P r a m Microbial Synthesis of Cobamides D. Perlman Factors Affecting the Antimicrobial Activity of Phenols E. 0. Bennett Germ-Free Animal Techniques and Their Applications Arthur W. Phillips and James E . Smith Insect Microbiology S. R. Dutky The Production of Amino Acids by Fermentation Processes Shukuo Kinoshita Continuous Industrial Fermentations Philip Gerhardt and M. C. Bartlett

The Large-Scale Growth of Higher Fungi Radcliffe F. Robinson and R. S. Dauid.son AUTHOR INDEX-SUBJECT

INDEX

Application of Membrane Filters Richard Ehrlich Microbial Control Methods in the Brewery Gwhard J . Hass Newer Development in Vinegar Manufactures Rudolph J. Allgeier and Frank M. Hildebrandt The Microbiological Transformation of Steroids T.H. Stoudt Biological Transformation of Solar Energy William J. Oswald and Clarence 6. Golueke ENGINEERING ADVANCES IN FERMENTATION PRACTICE

SYMPOSIUM O N

Rheological Properties of Fermentation Broths Fred H. Deindoe$er and John M. West Fluid Mixing in Fermentation Process 1. Y. Oldshue Scale-Up of Submerged Fermentations W. H. Bartholemew 281

282

CONTENTS OF PREVIOUS VOLUMES The Metabolism of Cardiac Lactones by Microorganisms

Air Sterilization

Arthur E . Humphrey

Elwood Titus

Sterilization of Media for Biochemical Processes

Intermediary Metabolism and Antibiotic Synthesis

Uoyd L. Kempe

1. D. Bu’Lock Fermentation Kinetics and Model Processes

Fred H . Deindoe7fer

Methods for the Determination of Organic Acids

Continuous Fermentation

A . C. Hulme

W. D. Maxon AUTHOR INDEX-SUBJECT

Control Applications in Fermentation

INDEX

George]. Fuld AUTHOR INDEX-SUBJECT

Volume 4

INDEX

Induced Mutagenesis in the Selection of Microorganisms

Volume 3

S. I. Alikhanian

Preservation of Bacteria by Lyophilization

The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry

Robert]. Heckly Sphuerotilus, Its Nature and Economic Sig-

F . J. Babel

nificance

Norman C . Dondero

Applied Microbiology in Animal Nutrition

Harlow H . Hall Large-Scale Use of Animal Cell Cultures Donald J . Merchant and C . Richard Eidam Protection against Infection in the Microbiological Laboratory: Devices and Procedures

Mark A . Chatigny

Biological Aspects of Continuous Cultivation of Microorganisms

T. H o l m Maintenance and Loss in Tissue Culture of Specific Cell Characteristics

Charles C . Morris Oxidation of Aromatic Compounds by Bacteria Martin H . Rogoff Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, j r . . and Robert F . Pitti110 The C~ssificationof Actinomycetes in ~ tion to Their Antibiotic Activity

Eli0 Baldacci

Submerged Growth of Plant Cells

L. G. Nickell INDEX

Volume 5

~ Correlations 1 ~ . between Microbiological Morphology and the Chemistry of Biocides

Adrian Albert

CONTENTS OF PREVIOUS VOLUMES

283

Generation of Electricity by Microbial Action J . B. Davis

Nonmedical uses of Antibiotics Herbert S . Goldberg

Microorganisms and the Molecular Biology of Cancer G . F . Gause

Microbial Aspects of Water Pollution Control K. Wuhrmann

Rapid MicrobiologicalDeterininations with Radioisotopes Gilbert V. Levin The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K. Long and Roger Patrick Aeration i n the Laboratory W . R. Lockhart und R. W. S p i r e s Stability and Degeneration of Microbial Cultures on Repeated Transfer Fritz Reirsser

Microbial Formation and Degradation of Minerds Melvin P. Siluennan and Henry L. Ehrlich Enzymes and Their Applications Irwin w. Sizer A Discussion of the Training of Applied Microbiologists B. W . Koft and Wuyne W . Umbreit AUTHOR INDEX-SUBJECT

INDEX

Volume 7 Microbiology of Paint Films Richard T . Ross The Actinotnvcetes and Their Antibiotics Selman A . bVaksman Fuse1 Oil A. Dinsmoor Webb andJohn L. Ingraham AUTHOR INDEX-SUBJECT

INDEX

Volume 6

Microbial Carotenogenesis Alex Ciegler Biodegradation: Problems of' Molecular Recdcitrance and Microbial Fallibility M . Alexander Cold Sterilization Techniques John B. Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes D . Perlman

Global Impacts of Applied Microbiology: An Appraisal Carl-Gb;ron Heddn and Mortimer P. Starr Microbial Processes for Preparation of Radioactive Compounds D. Perlmun, Aris P. Bayan, and Nancy A. Giuffie Secondary Factors in Fermentation Processes P. Margalith

Development of Coding Schemes for Microbid Taxonomy S. T . Cowan Effects of Microbes on Germfree Animals Thomas D. Luckey Uses and Products of Yeasts and Yeast-Like Fungi Walter]. Nickerson and Robert G. Brown

2a4

CONTENTS OF PREVIOUS VOLUMES

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

INDEX

Volume 8 Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain Genetics in Applied Microbiology S . 6 . Bradley Microbial Ecology and Applied Microbiology Thomas D. Brock The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes

Mycotoxins in Feeds and Foods Emanuel Borker, Nino F. Insahta, Colette P. k o i , and John S . Witzeman AUTHOR INDEX-SUBJECT

INDEX

Volume 9 The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. Russell, June Jenkins, and 1. H. Harrison Antiserum Production in Experimental Animals Richard H . Hyde Microbial Models of Tumor Metabolism G . F. Gause Cellulose and Cellulolysis Brigitta Norkruns Microbiological Aspects of the Formation and Degradation of Cellulose Fibers L. Juraiek, J . Ross Colvin, and D. R. Whitaker

Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers und Norman A . Clarke

The Biotransformation of Lignin to HumusFacts and Postulates R. T. Oglesby, R. F . Chrbtman, and C . H . Drioer

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

Bulking of Activated Sludge Wesley 0. Pipes

Oral Microbiology Heiner Hoffman

Malo-Lactic Fermentation Ralph E . Kunkee AUTHOR INDEX-SUBJECT

Media and Methods for Isolation and Enumeration of the Enterococci Paul A. Hartman, George W . Reinbold, and Devi S. Saraswat Crystal-Forming Bacteria as Insect Pathogens Martin H . Rogoff

INDEX

Volume 10 Detection of Life in Soil on Earth and Other Planets, Introductory Remarks Robert L. Starkey

285

CONTENTS OF PREVIOUS VOLUMES For What Shall We Search? Allan H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets 6. Stotzky Experiments and Instrumentation for Extraterrestrial Life Detection Gilbert V . Leoin

Structure-Activity Relationships of Semisynthetic Penicillins K . E . Price Resistance to Antimicrobial Agents J . S . Kiser, 6. 0. Gale, and G. A . Kemp

Micromonospora Taxonomy George Luedemann

Halophilic Bacteria D. J . Kushner

Dental Caries and Periodontal Disease Considered as Infectious Diseases William Gold

Applied Significance of Polyvalent Bacteriophages S. G . Bradley

The Recovery and Purification of Biochemicals Victor H . Edwards

Proteins and Enzymes as Taxonomic Tools Edward D. Garber and John W . Rippon

Ergot Alkaloid Fermentations William J . Kelleher

Mycotoxins Alex Ckgler and Eivind B. Lillehoj

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

Transformation of Organic Compounds by Fungal Spores Claude Vizina, S. N . Sehgal, and Kamar Singh

Training for the Biochemical Industries 1. L. Hepner

Microbial Interactions in Continuous Culture Henry R. Bungay, 111 and Mary Lou Bun@Y Chemical Sterilizers (Chemosterilizers) Paul M . Btdck htibiotics in the Control of Plant Pathogens M . /. Thirumalaehar AUTHOR INDEX-SUBJECT

INDEX

AUTHOR INDEX-SUBJECT

INDEX

Volume 12 History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester Thomas Kennedy Walker Fermentation Processes Employed in Vitamin C Synthesis Milo.? Kulhanek

and Microorganisms CUMULATIVE AUTHOR I N D E X ~ U M U L A T I VFlavor E P. Margalith and Y. Schwartz TITLEINDEX

Volume 11

Mechanisms of Thermal Injury in Nonsporulating Bacteria M . C. Alltoood and A . D. Russell

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

Collection of Microbial Cells Daniel 1. C . Wang and Anthony J . Sinskey

286

CONTENTS OF PREVIOUS VOLUMES

Fermentor Design R. Steel and T . L. Miller

Metabolism of Acylanilide Herbicides Richard Bartha and David P r a m

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

Therapeutic Dentifrices J. K. Peterson

Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. Perlman and G. P. Peruzzotti AUTHOR INDEX-SUBJECT

Some Contributions of the U.S. Department of Agriculture to the Fermentation Industry George E . Ward Microbiological Patents in International Litigation John V . Whittenburg

INDEX

Volume 13 Chemotaxonomic Relationships Among the Basidiomyce tes Robert G. Benedict

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

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

Mathematical Models for Fermentation Processes A. G. Frederickson, R. D. Megee, ZIZ, and H . M . Tsuchija

Large-Scale Cultivation of Mammalian Cells R. C . Telling and P. 1. Radlett

AUTHOR INDEX-SUBJECT

Large-Scale Bacteriophage Production K . Sargent

Volume 14

Microorganisms as Potential Sources of Food Jnanendra K . Bhattacharjee Structure-Activity Relationships Semisynthetic Cephalosporins M . L. Sassiver and Arthur Lewis

among

INDEX

Development of the Fermentation Industries in Great Britain John J . H . Hastings Chemical Composition as a Criterion in the Classification of Actinomycetes H . A. Lechevalier, Mary P. Lahevalier, and Nancy N. Gerber

Structure-Activity Relationships in the Tetracycline Series Robert K . Blackwood and Arthur R. English

Prevalence and Distribution of AntibioticProducing Actinomycetes ]ohn N . Porter

Microbial Production of Phenazines J . M . Zngram and A. C . Blackwood

Biochemical Activities of Nocardia R. L. Raymond and V . W . lamison

The Gibberellin Fermentation E . G . Jeffieys

Microbial Transformations of Antibiotics Oldrich K . Sebek and D. Perlman

287

CONTENTS OF PREVIOUS VOLUMES

I n Vim Evaluation of Antibacterial Chemotherapeutic Substances

Microbial Utilization of Methanol Charles L. Cooney and David W . Levine

A. Kathrine Miller Modification of Lincomycin Barney J . Magerlein Fermentation Equipment G. L. Solomons The Extracellular Accumulation of Metabolic Products by Hydrocarbon-Degrading Microorganisms Bernard J . Abbott and William E . Gledhill

Modeling of Growth Processes with Two Liquid Phases: A Review of Drop Phenomena, Mixing, and Growth P. S . Shah, L. T . Fan, I . C . Kao, and L. R. Erickson Microbiology and Fermentations in the Prairie Regional Laboratory of the National Research Council of Canada 1946-1971 R . H . Haskins AUTHOR INDEX-SUBJECT

AUTHOR INDEX-SUBJECT

INDEX

INDEX

Volume 16

Volume 15 Medical Applications of Microbial Enzymes Zrwin W . Sizer

Public Health Significance of Feeding Low Levels of Antibiotics to Animals Thomas H. Jukes

Immobilized Enzymes K . L. Smiley and G. W . Strandberg

Intestinal Microbial Flora of the Pig R . Kenworthy

Microbial Rennets Joseph L. Sardinas Volatile Aroma Components of Wines and Other Fermented Beverages A . Dinsmoor Webb and Carlos J . Muller Correlative Microbiological Assays Ladislav J . Halika Insert Tissue Culture W . F . Hink Metabolites from Animal and Plant Cell Culture Zrving S. Johnson and George B . Boder Structure-Activity Relationships in Coumermycins John C . God$-ey and Kenneth E . Price Chloramphenicol Vedpal S . Malik

Antimycin A, a Piscicidal Antibiotic Robert E . Lennon and Claude Vizina Ochratoxin s Kenneth L. Applegate andlohn R. Chipley Cultivation of Animal Cells in Chemically Defined Media, A Review Kiyoshi Higuchi Genetic and Phenetic Classification of Bacteria R. R. Colwell Mutation and the Production of Secondary Metabolites Arnold L. Demain Structure-Activity Relationships in the Actinomvcins Johannes Meienhofer and Eric Atherton

288

CONTENTS OF PREVIOUS VOLUMES

Development of Applied Microbiology at the University of Wisconsin William B . Sarles

Microbial Penicillin Acylases E . J . Vandamme and J . P. Voets SUBJECT INDEX

AUTHOR INDEX-SUBJECT

INDEX

Volume 18

Volume 17 Education and Training in Applied MicrobiolOgY

Microbial Formation of Environmental Pollutants Martin Alexander

Wayne W . L’mbreit Antimetabolites from Microorganisms David L. Pruess and James P. Scannell Lipid Composition as a Guide to the Classification of Bacteria Norman Shaw Fungal Sterols and the Mode of Action of the Polyene Antibiotics J . M. T. Hamilton-Miller Methods of Numerical Taxonomy for Various Genera of Yeasts 1. Campbell Microbiology and Biochemistry of Soy Sauce Fermentation F. M . Young and B . J. B . Wood Contemporary Thoughts on Aspects of Applied Microbiology P. S. S. Dawson and K . L. Phillips Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast G. 6. Stewart Linear Alkylbenzene Sulfonate: Biodegradation and Aquatic Interactions William E. Gledhill The Story of the American Type Culture Collection-Its History and Development (1899-1973) William A. Clark and Dorothy H . Geary

Microbial Transformation of Pesticides Jean-Marc Bollag Taxonomic Criteria for M ycobacteria and Nocardiae S . 6. Bradley and J . S . Bond Effect of Structural Modifications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine Kenneth E . Price, John C . Godfrey, and Hiroshi Kawaguchi Recent Developments of Antibiotic Researcb and Classification of Antibiotics According to Chemical Structure Janos Berdy SUBJECT INDEX

Volume 19 Culture Collections and Patent Depositions T . G. Pridham and C . W . Hesseltine Production of the Same Antibiotics by Members of Different Genera of Microorganisms Hubert A. Lechevalier Antibiotic-Producing Fungi: Current Status of Nomenclature C . W . Hesseltine and J. J. Ellis

CONTENTS OF PREVIOUS VOLUMES

Significance: of Nucleic Acid Hybridization to Systematics of Actinomycetes S. 6. Bradley Current Status of Nomenclature Antibiotic-Producing Bacteria Erwin F. Lessel

of

Microorganisms in Patent Disclosures Iruing Murcus Microbiological Control of Plant Pathogens Y . Henis and 1. Chet Microbiology of Municipal Solid Waste Composting Melvin S. Finstein and Merry L. Morris Nitrification and Dentrification Processes Related to Waste Water Treatment D. D. Focht and A. C. Chung The Fermentation Pilot Plant and Its Aims D. J . D. Hockenhull

289

Role of the Genetics and Physiology of Bordetellu pertussis in the Production of Vaccine and the Study of Host-Party Relationships in Pertussis Charlotte Parker Problems Associated with the Development and Clinical Testing of an Improved Pertussis Vaccine George R. Anderson Problems Associated with the Control Testing of Pertussis Vaccine Jack Cameron Vinegar: Its History and Development Hubert A. Conner and Rudolph J. Allgeier Microbial Rennets M . Sternberg Biosynthesis of Cephalosporins Toshihiko Kanzaki and Yukio Fujisawa

The Microbial Production of Nucleic AcidRelated Compounds Koichi Ogata

Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells Bernard J . Abhott

Synthesis of 1,-Tyrosine-Related Amino Acids by P-Tyrosinasr Hideaki Yamadu and Hidchiko Kumagai

Cytotoxic and Antitumor Antibiotics Produced by Microorganisms J. Fuska and B . Proksa

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

SUBJECT INDEX

SUBJECT INDEX

Volume 20

Volume 21 Production of Polyene Macrolide Antibiotics Juan F. Martin und Lloyd E . McDuniel

The Current Status of Pertussis Vaccine: An Overview Charles R. Manclark

Use of Antibiotics in Agriculture Tomomasa Misuto, Keido KO, and Isamu Yamuguchi

Biologically Active Components and Properties of Bordetella pertussis Stephen 1. Morse

Enzymes Involved in P-Lactam Antibiotic Biosynthesis E. 1. Vandumme

290

CONTENTS OF PREVIOUS VOLUMES

Information Control in Fermentation Development

D. J . D. Hockenhull Single-Cell Protein Production by Photosynthetic Bacteria R. H. Shipman, L. T. Fan, and 1. C . Kao Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals

Acid Ionophores Produced by Streptomyces J . W . Westley The Microbiology of Aquatic Oil Spills

R. Bartha and R. M . Atlas Comparative Technical and Economic Aspects of Single-Cell Protein Processes

John H . Litchfield

Jitendra Sarena and Philip H . Howard SUBJECT INDEX

Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mammalian Cells

Volume 23

Prasanta K . Ray Pharmacologically Active Compounds from Microbial Origin

Hewitt W . Matthews and Barbara Fritche Wade

Biology of Bacillus popilliae Lee a. B u l k , Jr., Ralph N . Costilow, and Eugene S . Sharpe Production of Microbial Polysaccharides

M . E . Slodki and M . C . Cadmus SUBJECT INDEX

Effects of Cadmium on the Biota: Influence of Environmental Factors

Volume 22 Transformations of Organic Compounds by Immobilized Microbial Cells

H . Babich and G . Stotzky Microbial Utilization of Straw (A Review) Youn W . Hun

Ichiro Chibata and Tetsuya Tosa Microbial Cleavage of Sterol Side Chains Christoph K . A. Martin Zearalenone and Some Derivatives: Production and Biological Activities P. H . Hidy, R. S. Baldwin, R. L. Greasham, C . L. Keith, and]. R. McMul-

Zen Mode of Action of Mycotoxins and Related Compounds F . S . Chu Some Aspects of the Microbial Production of Biotin

Yoshikazu lzumi and Koichi Ogata Polyether Antibiotics: Versatile Carboxylic

The Slow-Growing Pigmented Water Bacteria: Problems and Sources Lloyd G . Herman The Biodegration of Polyethylene Glycols Donald P. Cox Introduction to Injury and Repair of Microbial Cells

F. F . Busta Injury and Recovery of Yeasts and Mold K . E. Stevenson and T . R. Graumlich Injury and Repair of Gram-Negative Bacteria, with Special Consideration of the Involvement of the Cytoplasmic Membrane

L. R. Beuchat

CONTENTS OF PREVIOUS VOLUMES

Heat Injury of Bacterial Spores Daniel M . Adurns

M . D. Pierson, R. F. Gomez, und S . E . Martin SUBJECT INDEX

The Involvement of Nucleic Acids in Bacterial Injury

A B C D E F 6 n 1

8 9 O 1 2 3 4

J 5

291

CONTENTS OF PREVIOUS VOLUMES

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