ADVANCES IN
Applied Microbiology VOLUME 5
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ADVANCES IN
Applied Microbiology VOLUME 5
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ADVANCES IN
Applied Microbiology Edited by WAYNE W. UMBREIT Department of Bacteriology Rutgers, The State University New Brunswick, New Jersey
VOLUME 5
@
1963
ACADEMIC PRESS, New York a n d London
COPYHICHT @ 1983, n Y ACADEAIIC PHGS INC. ALL HIGkITS RESEHVED
NO PAHT OF THIS BOOK M A Y BE HEPHODUCED IN ANY FORM BY PHOTOSTAT, MICROFILM, OH ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROhI THE PUBLlSHEH5
ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK 3, N. Y.
United Kingdow Edition
Published by
ACADEMIC PRESS INC. (LONLWN) L ~ D . SQUARE HOUSE,LONDONW. 1 BERKELEY
Library
of
Congress Cutulog Curd Nuinber 59-13823
PHINTEU IN THE VNlTED STATES 01; AhIEHlCA
CONTRIBUTORS Numbcrs in parcnthescs indicate pages on u-hich thc authors’ contributions begin.
ADRIENALBERT,Department of Medical Chemistry, lnstitute of Advanced Studies, Australian National [Jniversity, Canberra, Australia ( 1 )
J. B. Dnvrs, Socony Mobil Oil Compan!y. lnc., Field Research Laboratory, Dallas, Texas (51) G. F. GAUSE,Institute of Antibiotics, Acade3ng of Medical Scieimx, hloscozc, U.S.S.R. (65)
L. INGRAHAM,Department of Bacteriology, Universitv of California, Davis, California (317)
JOHN
GILBERTV. LEVIN,Resource Research, lm., Washington, D. C . ( 9 5 )
w. R. LOCKHART,Department
of Bactcriolog!/, Iowa State [Jnivcr-
sit!/, Ames, lowa (157) STERLING K. LONG,University of Florida Citriis Experiment Slution?, Lake Alfred, Florida (135) ROGERPATRICK,Universit!l of Florida Citrus Experiment Station, Lake Alfred, Florida (135)
FRITZ REUSSER,Researciz Laboratories, Tlie I7pjohn Conzpang, Ka1om(i;oo, Michigan (189) RICHARDT. Ross, Birckman Lalmratories, lnc., Memphis, Tennessee (217) R. W. SQUIRES, Antibiotics hlanufuctiiring and Deoelopment DioiTion, El!/ Lilhl and Coinpan!/, Indianapolis, Indiana ( 157) S E L M . ~A. ~VAKSMAN,Institrrtc of Microbiology, Rutgers, Tlic State Universit!y, New Brrinszcick, Nezc Jersey ( 235)
A. DINSMOOR WEBB, Department of Vitictiltiirc and Enology, University of California, Davis, California (317) V
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PREFACE The present volume, the fifth in the series, again covers a broad range of subjects of interest to the applied microbiologist. These include the action of antimicrobial agents, methods for the search for antitumor agents, microbial determinations using isotopes, the stability and preservation of cultures under a variety of conditions, a review of recent work on actinomyces antibiotics, the perennial problem of aeration, fuse1 oils, 2,3-butylene glycol, even including the microbiology of paint deterioration and the generation of electricity by microbial means. As such, Advances in Applied Microbiology reflects a vigorous and growing field. We hope to have Advances continue to provide a focal point for the varied interests of the field and we would welcome suggestions as to which areas might require more attention than they are now being given. W. W. UMBREIT
Rutgem University May, 1963
vii
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CONTENTS CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V
PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
Correlations between Microbiological Morphology and the Chemistry of Biocides ADRIEN ALBERT
I. Introduction
. . . . . . . .. .. . . . . . . . . . . . . .... . .
1
.........
11. Bacteria . . . . . .. . . . , . . . . . . . . . . .. .. , . . . . . .. 111. Fungi . . . . . . . . . . . . . . . . ......................
28
IV. Viruses . .. . . . . .. .. . . . . ...................... V. Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Appendix on Chelation . . . . . . . . . . . . . . . . . . . . . . . . _. . . . .. . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .....
I. 11. 111.
Generation of Electricity by Microbial Action J. B. DAVIS Introduction ... . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . _ . . . . Redox Potential . . . .. . . .. .. . . . . . . . . . . .. .. _ . . . . . . . _ . .. . Fuel Cells . . . .. .. . . . . .. .. . . . . . . . . . . . . .. .. .. . , .. .. . . . . . . Corrosion Cell . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. .
IV. V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microorganisms and the Molecular Biology
34
38 42 45
51 53
55 62 63 64
of Cancer
G. F. GAUSE I. Introduction
. . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . .. . . . .
11. The Molecular Biology of Cancer . . . . . . . . . . . . . . . _ . . . . . . . HI. Microbial Models of Cancer ... . _.. ... . . . . . . _. _ . .. . . . .. . IV. Microbial Models of Cancer as Sources of Biological Inhibitors
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
65 66 69 87
90 91
Rapid Microbiological Determinations with Radioisotopes GILBERTv. LEVIN I. Classic Microbiological Techniques
. .. .... .. . . . . . . . . . . . . . . .
11. Radioisotope Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References
. .. . . .. .. . . . ... .. . . . . . . . . .. .... . . . . . . . . . . . . . . ix
93 100 132 132
X
CONTENTS
The Present Status of the 2. 3.Butylene Glycol Fermentation STERLING K . LONGAND ROGERPATRICK
. . .
I I1 I11 IV. V. VI . VII .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organisms Producing 2. 3.Butylene Glycol . . . . . . . . . . . . . . . . . . The Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recovery of 2.3.Butylene Glycol ......................... Potential Uses of 2. 3.Butylene Glycol ..................... Probable Future of the 2.3.Butylene Glycol Fermentation .... References .............................................
135 136 138 142 149 150 152 153
Aeration in the Laboratory W . R . LOCKHART AND R . W . SQUIRES
I. I1. I11 IV. V.
.
Introduction ........................................... The Necessity for Aeration .............................. Methods of Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control of Aeration ..................................... Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
157 159 162 169 185 185
Stability and Degeneration of Microbial Cultures on Repeated Transfer FRITZREUSSER
I . Introduction ........................................... 189 I1. Mechanisms Mediating Genetic Recombination in Microorganisms 190
. .
111 Examples of Culture Stability and Degeneration . . . . . . . . . . . . IV. Prevention or Circumvention of Culture Degeneration . . . . . . . . V Discussion and Conclusion ............................... References .............................................
199 210 212 213
Microbiology of Paint Films RICHARD T. Ross I . Introduction ........................................... I1. The Paint Environment .................................. 111. Microflora of Paint Films ................................ IV Microbiological Degradation of Paint Binders . . . . . . . . . . . . . . V The Deteriorative Role of Microorganisms on the Durability of Paint Films ............................................ VI Factors Contributing to Paint Film Deterioration and Their Relationship to Bacterial Degradation ........................ VII . Methods of Microorganism Control in the Paint Industry . . . . . . VIII . Effect of Preservation on Paint Durability . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
.
217 218 219 221 224 227 229 231 234
CONTENTS
xi
The Actinomycetes and Their Antibiotics SELMANA . WAKSMAN I . Introduction
...........................................
I1. The Actinomycetes ......................................
. .
111 The Antibiotics ........................................ IV Mode of Action and Utilization of Antibiotics . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
235 236 259 280 293
Fusel Oil A . DINSMOOR WEBBAND JOHN L . INGRAHAM
. History ................................................ . Characteristics of Fusel Oil Components .................... . Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results of Fusel Oil Analysis ............................. . Biosynthesis of Fusel Oil Components .....................
I I1 111 IV V
References
.............................................
317 318 322 331 338 350
AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
355
SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
379
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Correlations between Microbiological Morphology and the Chemistry of Biocides ADRIENALBERT Department of Medical Chemistry, Institute of Advanced Studies, Australian National University, Canberra
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Bacteria . . . . . . . . . . . . . . . . . . . .................... A. Microstructures of Bacteria . . . . . . . .
1
.......................
28
B. Biocides . . . . . IV. Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
............
....................
1
34
40
I. Introduction Much effort is spent in random screening to discover selectively toxic agents, namely those which can destroy bacteria, fungi, protozoa, and viruses in man, and in the animals and plants that provide his food. The last twenty years have brought an enormous increase in knowledge of the microstructure of microorganisms. Hence it may prove timely to record what correlations exist between the chemistry of such microstructures (“organelles”) and the chemistry of those biocides which act selectively on, or in, a particular microstructure. Such comparisons may provide a more direct approach to the discovery of useful new biocides.
II.
Bacteria
Bacteria will be discussed first, because more information is available for bacteria than for other microorganisms. Fungi, viruses, and protozoa will then be discussed, in that order. 1
2
ADRIEN ALBERT
A. MICROSTRUCTURES OF BACTERIA Bacteria have many structural peculiarities which differentiate them from the cells of higher plants and animals. 1. Cell Wall
Bacteria are under high osmotic pressure: 20 atmospheres is common for gram-positive types and 5 atmospheres for gramnegative types (Mitchell and Moyle, 1956a, 1957). They share this property with fungi, but not with the higher forms of life. Bursting is prevented by a thick, strong cell wall which often constitutes 25% of the total dry weight. This cell wall has large holes and is not a permeability barrier. But when the protoplast (i.e., all portions of the cell inside the cell wall) grows, additional quantities of cell wall must be synthesized. If this synthesis is prevented by a biocide, the cell will burst (see Section 11, B, 1). Chemically the bacterial cell wall is unlike any other kind of cell wall. It consists of a highly polymerized glycopeptide (the so-called “mucopeptide”) which is usually (but not always) loosely linked to a teichoic acid (see below). The glycopeptide has, as a repeating unit, acetylglucosamine linked to acetylmuramic acid which is, in turn, linked to a short polypeptide. Acetylglucosamine polymers are widespread in nature, but muramic acid is confined to bacteria, as is the presence of a peptide in a cell wall. This peptide, moreover, has some amino acids never found in proteins (see below). Acetylmuramic acid ( I ) is acetylglucosamine which is etherified at 0-3’ with lactic acid (Strange and Dark, 1956; Strange and Kent, 1959). Such ether linkages are uncommon in nature. The lactic acid is, in turn, linked to the polypeptide by an amide group. This part of the structure is shown in (11) (where the link has been made to alanine). The other molecule of glucosamine is attached at C-1, leading to a backbone of alternating N-acetylmuramic acid and N-acetylglucosamine residues joined by alter1-+6 ) linkages. The digestion of the cell nating @(1+ 4 ) and @( wall of Micrococcus lysodeikticus with lysozyme hydrolyzes the link between these two sugars; examination of the products gave the earliest clue to the structure of the glycopeptide. Lysozyme readily hydrolyzes the glycopeptides of some other gram-positive bacteria, and of gram-negative bacteria also ( Mandelstam, 1962).
MICROBIOLOGICAL MORPHOLOGY AND BIOCDDES
3
Many streptococci and micrococci have relatively few peptide side chains. A typical polypeptide, that from Staphylococcus aureus, is shown in (111). It should be noted that three of the five amino acids are in the “unnatural” D-configuration (Park, 1952; Park and Strominger, 1957) which has not been found in peptides from any other form of life. The presence of D-forms was discovered by Snell d al. (1955), and further studied by Ikawa and Snell (1960). CH,OH
I
Acetylmuramic acid (anion of) (1)
(11)
(D-lactyl) -L-ala-D-glu-L- lys-D-ala-D-ala
(III) The amino acid sequences in other species have not yet been so clearly defined. Among gram-positive organisms, lysine is confined to the cocci, and its place is taken by diaminopimelic acid in the rods (micrococci may contain either, but not both). Glycine is absent from many species. Serine is found in some staphylococci, and D-aspartic acid in some streptococci (Cummins and Harris, 1956). Diaminopimelic acid occurs as LL-, DL-, and DD-isomers, of which the DL- (i.e., meso-) form is commonest (Hoare and Work, 1957). Apart from bacteria (in which Streptomyces, Nocardiu, Actinomyces, and Mycobacterium are now included as Actinomycetales), this acid is found only in algae (especially in the primitive blue-green algae) (Work, 1957). No true fungi have polypeptides in the cell wall (Cummins and Harris, 1958). In various bacteria, hydrolysis has revealed the presence of other sugars ( mannose, glucose, arabinose, rhamnose, or galactose) presumably present as polymeric anhydrides of the mannan type (Cummins and Harris, 1956). Gram-negative bacteria have only about 3% of glucopeptide in the cell wall, and it consists mainly of
4
ADRIEN ALBERT
muramic acid, diaminopimelic acid, and alanine, in most of the species so far examined ( Mandelstam, 1962). Much polysaccharide is present in the cell walls of gram-negative organisms, but they depend for their rigidity on the glycopeptide, and burst when this is removed. These cell walls, unlike those of gram-positive organisms, cling tenaciously to the lipoprotein that forms the cytoplasmic membrane ( Weidel et al., 1960). Indeed there is some evidence in Escherichia coli for a lipoprotein membrane on both sides of the cell wall (Clarke and Lilly, 1962). For comparative figures of the lipid content of gram-positive and -negative bacteria, also of yeasts, see Salton (1963). The teichoic acids, which are extracted from bacterial cell wall with hot trichloroacetic acid, are believed not to be covalently linked to the polysaccharide backbone described above. These acids are polymers of glycerol phosphate and ribitol phosphate. They make up a substantial proportion (e.g., 40%) of the cell wall of various gram-positive and gram-negative species ( Armstrong et al., 1959; Baddiley and Davison, 1961), although some streptococci (also E. coli) have very little of these acids and Micrococcus Zysodeikticus has none. They all possess labile D-alanine ester linkages, and sugars are frequently attached as glycosides to the glycerol or ribitol. The occurrence of teichoic acids in nature seems to be confined to bacteria (Baddiley, 1962). The teichoic acid from the walls of Bacillus subtilis is a 9 unit polymer of 4-0-( P-D-glucopyranosyl)-D-ribitol5-phosphate, joined through phosphodiester linkages involving positions 1 and 5 in the ribitol. D-Alanine is attached to either the 2- or the 3-hydroxyl groups of each ribitol residue. That from S . aureus H is similarly constituted from 8 units in which the sugar is N-acetylglucosamine, and that from S . albus has a 1,3-glycerol phosphate polymer in which each fourth unit carries a N-acetyl-D-galactosamine residue in (mainly) a-linkage with the 2-hydroxyl group of glycerol. The teichoic acids confer group-antigenic properties on the cell walls. These acids are also found intracellularly, but only in traces, in association with ribonucleic acid (RNA), The capsule surrounding some bacteria is a polysaccharide secretion from the cytoplasmic membrane. Flagella, seen in some other bacteria, consist of pure protein which often contains N-methyllysine, an unusual amino acid (Ambler and Rees, 1959). For more details of the bacterial cell wall, see Salton (1960).
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
5
2. Cytoplasmic Membrane, Mitochondria1 Function, Permeability Immediately inside the cell wall, and containing the cytoplasm, lies the thin cytoplasmic membrane which regulates the permeability of the cell (Mitchell and Moyle, 1956a). It is 60-100 A thick. In some bacteria, this membrane extends a few simple protrusions into the cytoplasm, but there is nothing comparable to the cytoplasmic reticulum of higher organisms (Kellenberger and Ryter, 1958). Its structure seems to be that of a simple lipoprotein sandwich (Hughes, 1962). It forms about 10% of the dry weight of the cell and has a lipid content of about 25%. Sterols are absent from this lipid fraction, and from the whole bacterium, although they are present in fungi, including yeasts (Asselineau and Lederer, 1960). An analysis of the lipid of M . Zysodeikticus is available ( McFarlane, 1961) . About 80% is phospholipid (mainly diphosphatidyl glycerol, also some phosphatidyl inositol) . The protein has all the common, and no uncommon, amino acids (Gilby et al., 1958; Weibull and Bergstrom, 1958; Mitchell and Moyle, 1956b; Work, 1957). The membrane of M . lysodeikticus also has mannosan and a carotenoid pigment (Gilby et al., 1958). For contemporary ideas on a common structure for cytoplasmic membranes, see Section IV, A. The lipid present in bacteria is almost entirely confined to the cytoplasmic membrane which, because of it, stains strongly in electron micrographs. When the cell wall is completely hydrolyzed by lysozyme, the cytoplasmic membrane becomes the outermost layer (see Section 11, A, 1). Because of the small size of bacteria compared to other cells, many of the enzymes that are usually intracellular are incorporated by bacteria into the cell membrane (De Ley and Docky, 1960; Storck and Wachsman, 1957; Mitchell and Moyle, 195613). In particular, the enzymes of the tricarboxylic acid cycle are found there, e.g., more than 90% of the succinic and malic dehydrogenases of S . aureus (Mitchell, 1961). Bacteria contain no mitochondria, and it seems that the protoplasmic membrane takes over their function in the phosphorylative oxidation of carbohydrates. (For the general nature of mitochondria, see Section 111, A, 3.) RNA is found in thoroughly washed preparations of cytoplasmic membranes: it is controversial whether this is an artifact or not (Hughes, 1962). Cytoplasmic membranes of bacteria also contain the enzymes
6
ADRIEN ALBERT
responsible for synthesizing new cell wall (Crathorn and Hunter, 1958). They also contain the permeases, enzymelike substances which control the penetration of sugars and amino acids (Cohen and Monod, 1957 ) . Little is known about the permeability of bacteria to substances which they do not commonly encounter. Mitchell and Moyle (1959) found that the membrane of S . aureus was impermeable, by simple diffusion, to organic molecules with more than four hydrogenbonding groups, and to inorganic ions with more than four molecules of bound water.
3. Nuclear Material ancl Ribosomes Instead of a nucleus, such as is found in all higher forms of life, bacteria have “chromatin bodies” which are strands of nuclear material unprotected by the usual nuclear membrane. Phase-contrast microscopy shows that the chromatin bodies lie in the central region of the protoplasm, and each divides directly (often at the time the cell divides) without visible spindle formation. When a cell has more than one chromatin body, they seem to be equivalent, There is some evidence that the genetic information in the chromatin body is contained in a single chromosome. The principal nucleic acid present is deoxyribonucleic acid (DNA), but the protein with which this is paired differs from that of higher organisms in not giving the usual tests for a basic protein (Mason and Powelson, 1956). Close to the DNA, a strand of RNA has been located, at least in some species (Gale, 1959). The gross composition of bacterial RNA is not strikingly different from that of higher organisms. However, fractionation has revealed that the messenger RNA of bacteria has a base composition which mirrors that of the DNA from which it was formed. Only one kind of DNA seems to be present, but the ratio of bases in this differs greatly from one bacterial species to another. This is evident from Table I, where the last column gives the sum of the two amphoteric bases divided by the two monofunctional bases (Belozersky and Spirin, 1958). This figure ranges from 0.45 to 2.80, whereas in higher animaIs it has so far been found to vary only between 0.6 and 0.9, a range to which higher plants are also restricted although these have one-quarter of the cytosine replaced by methylcytosine. Ribosomes are abundantly present in bacterial protoplasm. They consist of particles 10-20 mp in diameter, i.e., the same size as in
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
7
mammalian cells. About 60% of the ribosome consists of RNA and 40% of protein (they lack lipids). The ultracentrifuge can separate these ribosomes into two smaller particles which must be united before protein synthesis can recur. TABLE I BASE RATIOS IN DNA OF BACTERIA Guanine + Cytosine Species Clostridium perfringens Staphylococcus aureus Pasteurella tularensis Proteus vulgaris Escherichiu coli Shigella dysenteriue Salmonella typhosa Corynebacterium diphtheriae Azotobacter spp. Bmcella abortus Pseudomonos aeruginosa Mycobacterium tuberculosis Actinomyces spp.
Adenine + Thymine 0.45 0.53 0.53 0.68 1.09 1.14 1.14 1.20 1.28 1.37 2.03 2.08 2.80
A discussion of the cooperation between DNA and ribosomes in protein synthesis and cell division will be found under Section 11, B, 3a. 4. Other Components Linear inorganic polymers, consisting only of orthophosphoric acid units, are common in bacteria, fungi, algae, and insects, but are not found in higher animals or plants. They may be phosphagens (energy accumulators), or merely waste products. Poly-p-hydroxybutyrate is widely distributed among bacteria but seems to be uncommon elsewhere in nature. It is believed to be used for energy storage (Doudoroff and Stanier, 1959; Bickel et al., 1960). Polyamines are also common, but more abundant in gramnegative than in gram-positive bacteria (Herbst et ul., 1958). All bacteria have iron-containing growth factors ( sideramines ). One of the best known is the ferrichrome consisting of the ferric complex of a cyclic hexapeptide containing three residues of glycine and three of a hydroxamic acid ( acetyl-N-hydroxyornithine ) (Emery and Neilands, 1961). Another iron-binding substance, 2,3dihydroxybenzoylglycine, has been isolated from Bacillus subtilk
8
ADRIEN ALBERT
(Ito and Neilands, 1958). The function of these substances is unknown, and the iron is tightly bound. For possibly analogous substances in fungi, see Section 111, A, 4. For an account of some unusual substances in tubercle organisms, see Asselineau and Lederer (19f30). Bacterial spores, e.g., those of Bacillus cereus, are surrounded by a protein rich in disuEde bonds. Behind this layer is the muramic acid and calcium dipicolinate material ( Gould and Hitchins, 1963).
B. BIOCIDES Many structural features of bacteria, as enumerated above, are so different from those of higher organisms that a whole system of selective toxicity could be based on this. To some extent this has taken place, largely accidentally, as the following account will show. 1. Acting on the Cell Wall Many substances are known which block the synthesis of new cell wall. This inhibition does not harm the bacterium until it begins to grow, whereupon the high osmotic pressure ruptures the unprotected cytoplasmic membrane. This catastrophe can, however, be averted if the organism is placed in a concentrated solution of sucrose (e.g., 0.3 M ) , which lessens the difference in pressure on both sides of the membrane (sucrose penetrates the membrane only very slowly, see Section 11, A, 2 ) . Inhibition of wall synthesis, as a cause of death was first established for penicillin (Lederberg, 1957),l but is now known to be the mode of action of several antibiotics, such as oxamycin, novobiocin, and bacitracin. None of these affects protein synthesis. At the end of the following discussion on these substances, beginning with oxamycin, an account will be given of other substances that are injurious to cell wall, but are not so specific as penicillin. a. Oxamycin, Penicillin, and Other Specifics. The mode of action of the antibiotic oxamycin (formerly called cycloserine) is known in considerable detail. Oxamycin ( ~-4-amino-3-isoxazolidone)( IV ) is a structural analog of ~-alanine( V ) and can displace this amino acid from two enzymes, in true competition. The first enzyme inhibited is the one that racemizes L-alanine, and the second is the enzyme which synthesizes D-alanyl-D-alanine from D-alanine ( Strominger et al., 1959). This dipeptide is the terminal feature of the 1
E . cold was used in this historic experiment.
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
9
wall peptide (111) of staphylococci, and presumably of some other organisms. This antibiotic causes S . aweus to accumulate a nucleotide in which uridine diphosphate is linked to C-1 of acetylmuramic acid ( I ) which is, in turn, linked through 0-7' to the tripeptide L-alanyl-D-glutamyl-L-lysine. If D-alanyl-D-alanine is supplied, this is rapidly attached to L-lysine, an event which shows that oxamycin does not interfere with incorporation of the dipeptide. H,C-CNH,
H2c-!i!~~,
I I 9 ,c=o
H
I
,c=o
0 H
N H
Benzylpenicillin (anion of)
HNKN
~ o ~ 3 - o p o ~ o P o 2 - o -
n O
0 (VII 1
It is noteworthy that D-oxamycin (the natural isomer) does not present incorporation of L-alanine, and that L-oxamycin does not prevent the biosynthesis of D-alanyl-D-alanine ( Strominger et al., 1959, 1960). Oxamycin is used for treating refractory infections of the human urinary tract ( E . coli) (Fairbrother and Garrett, 1960). Doses larger than 0.75 gm. daily causes distressing mental symptoms. Penicillin, like oxamycin, prevents the laying down of new cell wall and hence, as soon as the bacterium begins to grow, it ruptures and dies. Although the end result is the same as with
10
ADRIEN ALBERT
oxamycin, the biochemistry is different and less well understood. However, in both gram-positive and -negative bacteria, the site of attack is the same, namely, the muramic acid-containing glucopeptide (Rogers and Mandelstam, 1962). Subjection of S. auras to sublethal amounts of benzylpenicillina (VI) causes accumulation of a pentapeptide (Park, 1952). This substance, acetylmuramic acid ( I ) , is linked to uridine through C-1, and to ~-alanyl-~-glutamyl-~-lysyl-~-alanyl-~-alanine through 0 - 3 (Park, 1952). This, in short, is a typical piece of the cell wall glycopeptide in which uridine (VII) is in the place that acetylglucosamine will occupy later in the biosynthesis. It is evident that a standard transglycosylation reaction of the type UDP - X
+ ROH +UDP + ROX
has been interrupted by the penicillin. It is not known exactly on what chemical groups penicillin acts. Penicillin is an acylating agent, because the 4-membered lactam ring readily opens between C-8 and N-1’. Hence it is commonly supposed that penicillin irreversibly acylates some enzyme playing a key role in cell wall synthesis. (For an account of drugs which act by acylation, see Albert, 1960.) Experiments with S35 penicillin show that resistant strains of staphylococci, even those that produce no penicillinase, take up no penicillin from solution, but susceptible strains of various species combine with from 200 to 750 molecules per cell. This amount is held tightly, cannot be washed away, and does not exchange with nonradioactive penicillin (Rowley et al., 1950; Cooper, 1956; Maass and Johnson, 1949). Hence it has been concluded that penicillin combines covalently with a receptor in susceptible bacteria, and that this receptor is a group of atoms playing a key role in the biosynthesis of cell wall. It is known that penicillin combines most vigorously with highly nucleophilic groups (e.g., mercaptan anions), and much less vigorously with less nucleophilic groups such as amines, and the anions of alcohols, and water. This knowledge should assist in locating the group concerned. Meanwhile an explanation has come to hand as to why penicillin should injure bacteria only, whereas mammalian cells are freely permeable to it. Collins and Richmond (1962) have shown by 2 From 1942 onward, this has been the standard form of penicillin, against which other penicillins may be compared.
hlICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
11
atomic models that three hydrogen-bonding atoms in penicillin, viz. 0-9’,N-l’, and 0-11’occupy positions almost identical to those occupied by three hydrogen-bonding atoms in acetylmuramic acid, viz. 0-lo’, 0-2, and 0-7,respectively. A comparison of ( I ) and (VI ) will make this relation somewhat clearer, but only models ( o r the authors’ photographs of these ) can properly represent these highly 3-dimensional molecules. These authors suggest that penicillin is taken up, in the place of acetylmuramic acid, by the enzyme responsible for transglycosylation of the uridine-muramic acid-pentapeptide. They further suggest that the acylating carbonyl group (in the 4-membered ring), which is seen in the model to be pointing away from the plane containing 0-9’, N-l’, 0-ll’,is in this way brought near to a highly nucleophilic group, which is then acylated, and so the enzyme is irreversibly inactivated. This nucleophilic group may be the free amino group of a terminal cysteine residue, because penicillin has been shown to have a high specificity for this, in oitro ( Wintersteiner et al., 1949; Cavallito, 1946). So far, this hypothesis (of similar spacings of three hydrogenbonding groups) has proved compatible with all that is known of active and inactive analogs of penicillin. The cephalosporins, which have the sulfur atom in a 6-membered ring but otherwise resemble penicillin, contain these same three hydrogen-bonding groups in almost identical conformations. Penicillin causes S . UUTCUS to accumulate also a cytidine derivative of ribitol, apparently a precursor of a teichoic acid (Saukkonen, 1961). Those penicillins ( e.g., metliacillin and oxacillin) which are resistant to hydrolysis by penicillinase have been found to inactivate this enzyme (Gourevitch et ~ l . 1962). , Penicillin is still the most generally useful of all the antibiotics used in daily medical practice. Some other substances are known to inhibit cell wall synthesis specifically. These include the antibiotics vancomycin, novobiocin (Strominger and Threnn, 1959), and bacitracin (Abraham, 1957), und a synthetic substance, crystal violet. Novobiocin, a complcx thiazolidine peptide, is ineffective against penicillin-resistant bacteria. b. Less SpwiJic Biocitlcs. It is convenicnt now to mention tlirec chemically unrelated classes of substances wliicli disintegrate preformed cell walls. Experiments on cytoplasts, in hypertonic media,
12
ADRIEN ALBERT
indicate that these substances can disintegrate cytoplasmic membranes as well. They are, respectively, the phenols, the quaternary amines, and the polypeptide antibiotics (members of the first two classes are synthetic). Electron microscopy shows that they rapidly
FIG. 1. Cytolytic damage ( S . aureus) after a few minutes in 0.01% CTAB.
MXCROBIOLOGICAL MORPHOLOGY AND BIOCIDES
13
cause quite large areas of the cell wall to dissolve away. The appearance of S. uureus after a few minutes in 0.01% cetyltrimethylammonium bromide (VIII ), a typical quaternary amine, is shown in Fig. 1 (Salton et ul., 1951). Similar results were obtained for various phenols and polypeptides.
CTAB
(vm) Hexylresorcinol, as shown in Fig. 2, liberates almost the whole cell contents as fast as it is bound by the cell, a process which is often almost complete in 2 minutes (Beckett et ul., 1959). Similarly fast action has been recorded in this way for the quaternary amines. Opticol density ot exudote (260mp)
0
0
20 40 Contoct (minutes)
60 Contoct (minutes)
FIG.2. Hexylresorcinol (350 pg./ml.) and E . culi (3 x 1@/d.)at 25°C.
All three classes of substances are surface-active. The lipophilic side chains (nonanoyl in polymycin, cetyl in CTAB, hexyl plus benzene-ring in hexylresorcinol) all confer surface-active properties because they are situated in juxtaposition to a strongly hydrophilic
14
ADRIEN ALBERT
group (as usual, embodying nitrogen or oxygen atoms). T ~ Uthe S molecule of grarnicidin S has been found to be a pleated sheet with the lipophilic groups (leucyl and valyl) all on one side, and the hydrophilic group (ornithyl) on the opposite side ( Schwyzer, 1958). The high surface activity of these three substances does not explain why they cause damage but only explains how they become concentrated on the bacterial surface. The damage probably arises from their loosening the structure by breaking ionic bonds (“salt linkages”), possibly between lysine and a teichoic acid. 2. Acting on the Cytoplasmic Membrane So far, no substance has been discovered that specifically destroys the bacterial membrane (contrast with fungal membrane, Section 111, B, 2). However the situation of many catabolic enzymes in this membrane is so exposed that they can be assaulted with a freedom impossible in any other form of life. Many of these enzymes have metabolically active mercapto ( “thiol,” “sulfhydryl”) groups, and the inhibition of bacteria by mercurials probably proceeds at this structural level. Thus, succinic dehydrogenase is known to be situated in the bacterial membrane ( Mitchell and Moyle, 1956b), to be easily inhibited by inorganic and organic mercurials, and to have a key position in carboxylic acid metabolism.
3. Acting on Nuclear Material, or on Microsoms
The lack of protective membranes leaves both the nuclear material (“chromatin”) and the ribosomes very exposed. Many biocides with a specific action on these organelles are known, and several distinct points of attack are now recognized. The therapeutic benefits to be derived from “sequential blocking,” i.e., attacking a related series of biochemical processes at two or more points, makes it desirable to study these substances in detail (Albert, 1960). This discussion will be prefaced with a few remarks on the general function of nucleus and ribosomes in handing on genetic information by effecting and controlling the synthesis of nucleic acid and protein. These remarks apply to bacteria if “chromatin body” is read for “nucleus.” a. The Nature of Nucleic Acids and Protein Synthesis (Hoagland, 1960). Deoxyribonucleic acid ( DNA) is usually confined to the nucleus of cells. It is widely believed to have all of the cell’s genetic information recorded by means of the order of purine and
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
15
pyrimidine bases. It consists of two strands arranged in a spiral. Each strand has a deoxyribose-phosphoric acid backbone on which the bases are inserted. The two spirals are kept together by hydrogen bonding between each pair of bases (Watson and Crick,
P
-
Phosphoric ocid; ’5- Deoxyribose. 1-Thymine
A IAdenine.
G. Guanine;
GCytosine.
FIG. 3. Diagrammatic representation of DNA “spiral-strands” on which purine and pyrimidine bases are inserted.
I
FIG.4.
,
Structural details of a single turn of the DNA spiral.
1953). Figure 3 shows this spiral diagrammaticalIy, and Fig. 4 shows structural details of a single turn of the spiral. The DNA acts as a template for the synthesis of a special RNA known as “messenger RNA,” which travels to the ribosomes. The latter already contain other (unorganized) RNA and a nonbasic protein. The “messenger RNA” is highly organized in that it bears information for the synthesis of specific proteins, information built
16
ADFUEN ALBERT
into it by the DNA (Brenner d al., 1961; Volkin and Astrachan, 1956). Amino acids in the cytoplasm become esterified to a third type of RNA (“transfer RNA,” “soluble RNA”) which is of relatively low molecular weight. There are more than 20 different varieties of this RNA, each of them specific for one amino acid. Esterification seems to be confined to the 3-OH group of a terminal adenylic acid (IX). These esters settle on the “messenger RNA” in the ribosomes
1. DNA makes Messengrr-RNA in crll’s nucleus.Thir RNA trawls to ribosomes.
2. Meanwhik. obout 24 kinds of Transfer-RNA rstrrify (activated) amino acids.
3. These rsteri tit Inlo Mesunqer-RNA on ribosomes.
=TT 2
4. Amino acids are linkrd togdher to m form protein. Tronsfrr-RNA is rruud.
FIG. 5. Diagrammatic representation of Transfer- and Messenger-RNA engaged in protein synthesis.
(Hoagland et al., 1957). The order of bases in the “messenger RNA” determines the order in which the esters are attracted, and hence the order of amino acids in the protein synthesized. The code which connects the order of bases with the order of amino acids is not known, but some promising clues have been found (Speyer d al., 1962; Martin d aZ., 1962). Once the esters are in position on the ribosomes, enzymes unite the amino acids to form a protein, and at the same time hydrolyze off the “transfer RNA” residues which are used to make a new supply of esters. These operations are shown diagrammatically in Fig. 5. “Transfer R N A is now known to be a twin spiral, like DNA. Unlike the latter, however, it is formed of a single strand twisted
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
17
around itself. At the point where the direction of the chain is reversed, three nucleotides are unpaired and may be code-determining (Spencer et al., 1962).
Ester of tyrosine with "transfer W A R (it is connected, at the asterisked oxygen atom, by a phosphoryl linkage to the next nucleotide)
(MI
N
I
Puromycin
(X1
b. Purornycin (X). This antibiotic prevents the synthesis of proteins by ribosomes. In place of proteins, the ribosomes of rabbit reticulocytes release short, puromycin-N-terminated peptides (Allen and Zamecnik, 1962), but E. coli is said not to form any peptides in the presence of puromycin (Nathans and Lipman, 1961). Yarmolinsky and de la Haba (1959) pointed out the striking re-
18
ADRIEN ALBERT
semblance of puromycin to the esters that amino acids form with “transfer RNA.” This is particularly striking if the amino acid in question is tyrosine (IX). Three hydrogens in (IX) are replaced by methyl groups in (X), but the most important difference between these two formulas is marked by a dagger ( t ) ; here the ester group is replaced by an amide group in puromycin. All available evidence suggests that puromycin, possibly after phosphorylation at the site marked by an asterisk ( ” ) in (X), is taken up by the ribosome on the receptors designed to receive the amino acid “transfer RNA” esters, and that lengthening of the peptide is thereby prevented. Puromycin inhibits protein synthesis in all living cells. Neither it nor any analogous substance, many of which have been synthesized, has found much clinical application. c. Ch2o~ampFRnicoZ ( X I ) . Like puromycin, chloramphenicol specifically inhibits protein synthesis in ribosomes (Gale and Folkes, 1953a,b; Hancock and Park, 1958). It acts much more powerfully against bacteria than against mammalian cells. Although the biochemical reason for this selectivity is unknown, it enjoys wide use in the clinic, especially in the prompt management of dysenteries. Chloramphenicol is D-(-)-threo-Z-dichloroacetamido-l-p-nitrophenylpropane-l,3-diol. The L-eythro isomer has no effect on protein synthesis, but inhibits the formation of D-glutamyl polypeptide in B. subtilis, although chloramphenicol does not do so (Hahn et al., 1954). The remaining two stereoisomers affect neither process. Excellent photos of models of chloramphenicol are available (Collins et al., 1952,). X-ray crystallography shows the two liydroxy groups close together (the amide group points away from these), and the whole aliphatic portion is roughly in a plane at right angles to the benzene ring (Dunitz, 1952). Although it has been said that the terminal-CH20H group is not essential for the action of chloramphenicol, this should be confirmed (Buu-Hoi et al., 1950). Other workers have found that such very small changes in the aliphatic portion lead to almost complete inactivation, e.g., the substitution of any hydrogen atom by a methyl group, or of one halogen by another (Feitelson et al., 1951; Collins et al., 1952). However, in the benzene ring, the nitro group can be replaced by several other electron-attracting groups with a less drastic loss in activity, e.g., chloro-, bromo-, and iodo-
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
19
substitution leave 10, 20, and 3% of the activity respectively (BuuHoi’ ef a2., 1950; Dann et al., 1950). Chloramphenicol inhibits the uptake by ribosomes of amino acids from the esters with “transfer RNA,” e.g., (IX) (Lacks and Gros, 1960). This is at or near the site of action of puromycin and hence it is not surprising that the two molecules have been seen to have some features in common (Hopkins, 1959). Indeed, Hopkins saw chloramphenicol as an analog of uridine (XII), which has been supposed by some to play a special role in the coding process (Speyer et al., 1962). Whereas puromycin makes an unnatural analog of transfer RNA, chloramphenicol interferes with the functioning of messenger RNA (Hahn and Wolfe, 1961). In comparing the formulas of chloramphenicol and uridine, it is important to realize that the electronic effect of the nitrophenyl group in (XI) would be similar in polarity, if not so intense, as the dioxopyrimidyl group in (XII). This is illustrated by the following pK values which show uracil-6-carboxylic acid to be about 100 times stronger than benzoic acid (Brown, 1962): benzoic acid, 4.18; n-nitrobenzoic acid, 3.49; uracil-6-carboxylic acid, 2.21. 0
Chloramphenicol
(XI)
Ur idine
(xn1
The asterisked primary alcohol groups present features in coinmon, and that of chloramphenicol may be phosphorylated (as in uridylic acid). The amide group, marked with a dagger ( t ) in (XI), is similarly placed to one in puromycin; but it could have a parallel significance only if uridine (or cytidine) were known to form esters with amino acids. Thus, it is too early to say whether these similarities are relevant to the action of chloramphenicol. d. Metabolite Analogs of Bases. Some analogs of the pyrimidine
20
ADRIEN ALBERT
and purine bases of nucleic acids have antibacterial properties. Such analogs have been made either by replacing a hydrogen by a halogen atom, or by replacing a -CH= group by -N=. These analogs are, in many cases, incorporated into the nucleic acids by biosynthesis. Thus in E. coli, each gram of DNA incorporates at least 6 mg. of 5-bromouracil (XIII) (Wacker et al., 1960), and, in some strains, the whole of the thymidylic acid of RNA is replaced by bromouridylic acid. 5-Chloro-, 5-iodouracil ( Dunn and Smith, 1957), and 5-fluorouracil (Cohen et al., 1958) are incorporated similarly. 5-Fluorouracil, and the more potent 5-fluorouracil deoxyriboside, are converted by E . coli to 5-fluorouracil deoxyribotide which inhibits the enzyme that synthesizes thymidylic acid from thymidine. As a result, the organism dies rapidly because no new thymine is incorporated into the DNA (Cohen et d.,1958). 8-Azaguanine (XIV) is incorporated into both DNA and, especially, the RNA of Bacillus cereus, and almost completely stops protein synthesis (Otaka et al., 1961). It also blocks the incorporation of uridine (Mandel, 1961). The RNA units containing azaguanine are smaller than normal (Mandel and Markham, 1958). It is also known to be incorporated into the RNA of various other bacteria (Matthews and Smith, 1956; Heinrich et al., 1952). 0
Most of these analogs have been evolved for antitumor work in mammals, and some of them have been found to display very striking effects in viruses (see Section IV, B ) . So far they have not provided useful antibacterials because of their profound effects on the cells of higher organisms, Nevertheless they have afforded the biochemist much interesting information about the synthesis and incorporation of nucleotides in bacteria. e. Pteridines, Sulfonamides, and Other Antifolic Acid Agents (Albert, 1960). Pteridines, of the folic acid type, play a key role
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
21
in the biosynthesis of nucleic acids. They are responsible, as coenzymes, for inserting the carbon atoms into both the 2- and 8position of purines, and the methyl group into thymine. They are also concerned in the biosynthesis of methionine, and in equilibrating serine with glycine. Hence it is less surprising that antifolic drugs should be so deleterious to bacteria, than that they should be so safe for man. The explanation is that, broadly speaking, mammals cannot synthesize folic acid but must take it, preformed, in the food; pathogenic bacteria, however, cannot absorb preformed folic acid, but must synthesize it. Hence any substance that can interfere with the synthesis of folic acid is of potential value as a biocide selective against pathogenic bacteria. (There are a few nonpathogens that can absorb folic acid but not synthesize it; these are useful for the assay of folic acid.) Pteroylglutamic acid (XV) is the fundamental unit of the folic acid family of coenzymes, and is what is usually intended when
Pteroylglutamic acid ("Folic acid")
(XV)
the term "folic a c i d is used. Reading from left to right, it is seen to consist of three components, covalently linked, namely: glutamic acid, p-aminobenzoic acid, and a simple pteridine. The COenzymes are all 5-, 6-, 7-, 8-tetrahydro-derivatives which bear, in position 5, a one-carbon substituent which eventually becomes part of the molecule undergoing synthesis. The same molecule of tetrahydrofolic acid is then recharged with this substituent, and functions repeatedly in this way. The substituent may be -CHO, -CH20H, or -CH3, depending on the required product. The biosynthesis of the nucleic acid bases seems to be more sensitive to antifolic agents than are the reactions involving amino acids. The first antifolic drugs were the sulfonamides. The discovery
22
ADRIEN ALBERT
of their mode of action was not made until some time after they had been successfully introduced into medical practice. Briefly, they prevent p-aminobenzoic acid being converted into folic acid by blocking the enzyme which inserts it. This is inferential, because such an enzyme has not yet been isolated. However p-aminobenzoic acid can competitively reverse the bacteriostatic action of ally sulfonamide drug, and folic acid inhibits the action of these drugs noncompetitively on such bacteria as can absorb this acid. There is little evidence, if any, that the sulfonamides become built into folic acidlike molecules. But an antitubercular drug, p-aminosalicylic acid, is converted by some enterobacteria into a pseudofolic acid, which is nonfunctional and hence injurious to the bacteria (Wacker et al., 1954). Metabolite analogs of the pteridine portion of folic acid have also been made, but mainly for antitumor work, or as antimalarials. Thus, if the 0x0 group in the 4-position is changed to an amino group, a drug useful in treating leukemia is obtained. This substance, aminopterin, not surprisingly, has been found to block the synthesis of DNA (Simon, 1961).
Pyrimethamine (XVI f
Excellent antimalarial drugs, e.g., pyrimethamine (XVI ), have been obtained by making analogs of the pyrimidine ring of the pteridine nucleus in (XV). To secure a high concentration in the red blood cells, where the protozoon is located, lipophilic groups (-C1, and -CaHn) were added ( Hitchings, 1952). These antimalarials were found to be true competitors of folic acid, and have since been discovered to block the hydrogenation stage described above. Antibacterial action has been found in the pteridine series, especially among the 2,4-diaminopteridines, and a little more work should yield clinically useful ones. The most characteristic property of pteridines, the tendency to saturate one double-
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
23
bond by adding water covalently across it, profoundly affects their properties (Albert et al., 1962). f. Acrulines (XVZl). The aminoacridines, introduced by Browning as local antiseptics in 1913, and used in severe wounds during the two world wars, have an excellent record for sterilizing infected tissues which then heal rapidly. As they are highly active against penicillin-resistant staphylococci, they deserve detailed study. The antibacterial action of aminoacridines is annulled if DNA is added to the medium in which the bacteria grow, but it is unaffected by serum proteins, Under the fluorescence microscope, aminoacridines can be seen to concentrate in the nucleus of living mammalian cells (De Bruyn et al., 1950), and presumably they do likewise in bacterial “chromatin.” There is some evidence that the aminoacridines act on the outside of bacteria (Albert et al., 1945), and in this connection the possible occurrence of nucleic acids in the cytoplasmic membrane (see Section 11, A, 3) is relevant. Certainly proflavine injures E . coli without penetration to the cytoplasm. That the aminoacridines can act on DNA is shown by the mutagenic action of euflavinej on yeast (Ephrussi et al., 1949), and to the prevention by acridine orange* of the union, in phage, between DNA and protein, at very low concentrations of the acridine, and of the synthesis of new DN,4 by slightly higher concentrations ( D6nes and PolgAr, 1980). In &TO, proflavine inhibits both RNA-polymerase and DNApolymerase, particularly the latter. Proflavine concentrations ( 30 pM) which inhibit DNA synthesis by 85% do not inhibit RNA synthesis by more than 30% (Hurwitz et al., 1!362). These inhibitions must play a vital part in all chemotherapy with aminoacridines. Physical studies of the interaction of aminoacridines with DNA have shown that proflavine is bound by two mechanisms, ( a ) a first-order reaction that reaches equilibrium at one proflavine molecule per 4 or 5 nucleotides, and ( b ) a weaker, higher-order process that leads to the fixation of one proflavine molecule per nucleotide (Peacocke and Skerrett, 1956). The latter process is probably simply the adsorption of acridine molecules on to those 3
Neutral acriflavine, the methochloride of proflavine.
4
3,6-Bisdimethylaminoacridine.
24
ADRIEN ALBERT
already attached to the DNA, as demonstrated for acridine orange by Stone and Bradley (1961). But the strong attachment of proflavine to DNA is best explained, on all evidence available at present, as follows. The proflavine seems to be intercalated between the layers of (pairs of) nucleotide bases (see Fig. 4 ) . As these layers are normally almost touching, the DNA helix must become extended (e.g., by partial unwinding of the whole, without any rupture of hydrogen bonds). In this arrangement, the aminoacridine molecules would be, like the nucleotide bases, perpendicular to the phosphate-deoxyribose backbone, and no more -~ than every second gap between bases can receive an aminoacridine molecuIe.
a m
NHZ
NHZ
1
’
4/
I
‘
d
10
Ac r idine
(xvn)
Am inacrine
(xvIn1
4 - Aminoquinoline
(-1
This picture has emerged from measurements of viscosity and sedimentation of the complex in solution, of X-ray diffraction patterns (Lerman, 19Sl), and of small-angle X-ray scattering measurements (Luzzati et al., 1961). It was found that the meridional spacing of 3.4 A was retained, but the rods which had adsorbed the proflavine had a diminished mass per unit length. Calculation of the intrinsic viscosity from the intercalation hypothesis gave a figure in good agreement with experiment. Further confirmation was obtained from measurement of flow dichroism, and polarization of fluorescence ( Lerman, 1963). Both RNA- and DNA-polymerases (the enzymes which synthesize RNA and DNA) are strongly inhibited by aminoacridines (Hurwitz et al., 196.2). Examination of a series of 106 acridine-,, against a range of bacterial species, showed that only those acridines which were highly ionized (as cations) under the conditions of the test were highly antibacterial (Albert ct al., 1945). This is conveniently illustrated in Table 11. The five isomeric monoaminoacridines differ widely in basic strength thanks to a basestrengthening resonance that is possible only in the 3- and 9positions. It is evident from this table that those isomers which are more highly ionized (under the conditions of the test) have much
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
2.5
greater antibacterial action. This is seen to be true also for diaminoacridines. Indeed, throughout the whole series of 106 acridines, it was found that antibacterial activity was independent on the type of substituent present except in so far as that influenced ionization. One interesting outcome of these ionization studies was the adoption of aminacrine, i.e., of 9-aminoacridine (XVIII) (“5aminoacridine,” in the older numbering), by the British Pharmacopoeia because of its high antibacterial activity, low mammalian toxicity, and freedom from staining. TABLE I1 AMINOACRIDINES: DEPENDENCE OF BACTERIOSTASIS ON ABUNDANCE OF CATIONS (pH 7.3; 37°C.) Per cent ionized
Minimal bacteriostatic concentration (24 hours) Streptococcus pyogenes
4-Amino%Aminol-Amino3-Amino9-Amino- ( Aminacrine) (XVIII)
< 1 2 2 73 100
1 in: 5,000 10,000 10,000 80,000 160,000
4,5-Diamino2,7-Diamino3,g-Diamino3,7-Diamino3,6-Diamino- (Proflavine)
< 1 3 100 76 99
Acridine (XVII)
< 5,000 20,000 160,000 160,000 160,000
Most nonacridine cations are not antibacterial, or if antibacterial they are inactivated by protein. These facts led to a search for the source of the good properties of the acridine nucleus. It was soon found that removing a ring, to give 4-aminoquinoline (XIX), entirely abolished the high activity of 9-aminoacridine. This abolition occurred also if one of the outside rings of 9-aminoacridine (XVIII) was reduced, giving 1,2,3,4-tetrahydro-9-aminoacridine. Because the molecules of (XVIII) and (XIX) are quite flat, whereas hydrogenation creates a three-dimensionally bulky ring, it was evident that a minimal area of flatness is necessary for antibacterial activity in these cations. This critical area is about 40 square A. This knowledge made it possible, by extending the flat area of 4-aminoquinoline ( XIX ) as in 2-styryl-4-aminoquinoline, to restore the lost antibacterial activity (Albert et al., 1949). It should be noted that all the substances mentioned in this paragraph are 100% ionized under the conditions of test. These requirements of high ionization and flatness become un-
26
ADRIEN ALBERT
derstandable in the light of Lerman’s model, discussed above. Nonheterocyclic antibacterials, e.g., 2-guanidinoanthracene7 have been prepared from knowledge of these requirements, and have a typical acridinelike activity against bacteria (Albert et al., 1949). g. Miscellaneous. The tetracyclines ( e.g., Aureomycin ) play a highly important part in mammalian chemotherapy. Little is known of their mode of action, except that they inhibit protein synthesis in cells ( like chloromycetin) , and polypeptide formation in cell walls (like penicillin). No clues to the biochemical reason for their high tolerance by mammals and yeasts are available. They are strong chelators of heavy metals (see Section VI) (Albert and Rees, 1956) . Structure-action relationships in the tetracyclines are constantly being studied, but the difficult chemistry involved in preparing new examples retards progress. For a table of structureaction relationships, see Hlavka et al. (1962). Elimination of both methyl and hydroxy groups from the 6-position increases potency, and high activity has been found in a 1,8,9,10-tetraoxyanthracene ( Shemyakin and Kolosov, 1962). Erythromycin, a macrolide ring with two unusual sugars attached as glycosides, blocks protein synthesis in bacteria. Actinomycin C and D, which have a phenoxazine nucleus (flat) and t w o polypeptide side chains, inhibit RNA synthesis in bacteria, viruses, fungi, and mammalian (normal and tumor) cells. Actinomycin D seems to act by occupying the surface, on DNA, upon which “messenger RNA” is synthesized (Harbers and Miiller, 1962; Reich et al., 1961a; Kersten et al., 1960; Hurwitz et al., 1962). Biosynthesis of DNA continues in its presence. It is of more interest as a biochemical tool than in therapy. Actinomycin D penetrates into B. subtilis and inhibits RNA synthesis in the cytoplasm but does not penetrate into E. coli. Antimycin, a mixture of homologues from Streptoniyces kitazuwaensis, is based on a m-aminosalicylamido-substituted di-lactone with a 9-membered ring. It is used by biochemists specifically to inhibit synthesis of RNA. Mitomycins A, B, and C are violet antibiotics from Streptoirqces caespitosis (Webb et al., 1962). They inhibit synthesis of DNA, as a result of which much thymine is liberated. The principal use, so far, is as a biochemical reagent (Reich, 1961b; Kersten and Rauen, 1961). Azaserine ( O-diazoacetyl-L-swine) , and DON ( 6-diazo-5-oxo-
MICROBIOLOGICAL hlORPHOLOGT AND BIOCIDES
27
L-norleucine), obtained from species of Streptomyces, inhibit growth of bacteria by preventing the amidination of formylglycinamide ribotide by glutamine. This results in a blockade of the de novo synthesis of purines (Tomisek ef aE., 1956; Levenberg et al., 1957). Streptomycin, is still much used in human antibacterial chemotherapy, especially in tuberculosis, although attention has to be given to its toxic hazards. It is initially accumulated by the cell wall of bacteria, and then transferred to the cytoplasmic membrane (Anand and Davis, 1960). The site and nature of the lethal injury is unknown in spite of an embarrassingly large number of clues (e.g., Spotts and Stanier, 1961; Erdos and Ullmann, 1959). Alkylating agents, including the “nitrogen mustards,” combine with the most nucleophilic nitrogen atom in each purine or pyrimidine base. They are mutagenic or, in excess, lethal (Reiner and Zamenhof, 1957; Lawley, 1957; Brookes and Lawley, 1!360). Their chief application, however, is in cancer therapy. Nitrous acid deaminates cytosine to uracil, giving a mutation that is often viable (Schuster and Schram, 1958). Culture in highly acidic media removes some purine bases, another mutagenic effect ( C . Tamm et al., 1952). Glyoxals react with guanine (Staehlin, 1958). Hydrazine breaks, and removes, large portions of the pyrimidine rings, but purines are unharmed (Takemura, 1959; Baron and Brown, 1955). Hydroxylamine converts cytosine to the 4-hydroxylamino analog (Brown and Schell, 1961), and is mutagenic. 4. Other Types of Action
The most important of the antibacterial agents not yet discussed are the chelators. Because these are also of much interest for fungi and viruses, they are dealt with in a separate section (see Section VI). Of the remaining antibacterial agents, only brief mention need be made of the oxidizing agents (e.g., potassium permanganate), halogens (of which iodine acts in the form of elementary iodine whereas chlorine acts as hypochlorous acid above pH 2 ) , and formaldehyde and ethylene oxide which are presumably alkylating agents (see also 3 g, above). Iodine, formaldehyde, and ethylene oxide are much relied upon to destroy bacterial spores. For this purpose, formaldehyde requires a much higher relative humidity than ethylene oxide. Sideromycins are iron-containing antibiotics, found usually in
28
ADRIEN ALBERT
the Actinomycetales (see Section 11, A, 1). They specifically antagonize the iron-containing growth factors ( sideramines ) of common bacteria (see Section 11, A, 4). Common examples of sideromycins are albomycin, and grisein (Kuehl et al., 1951).
Ill. Fungi Compared with bacteria, the most typical fungi are complex, uni- or multi-cellular organisms with a bewildering variety of shapes. It is therefore surprising that fungi seem to have a common, and characteristic, chemistry and morphology (the Actinomycetales are now classed, on chemical grounds, as bacteria, see Section 11, A, 1). A. MICROSTRUCTURES OF FUNGI The cells of fungi resemble those of higher organisms very closely but they share one feature with bacteria, namely the high internal pressure and the need for a stout cell wall to prevent bursting (Nickerson et al., 1961). 1. Cell Wall This consists entirely of carbohydrate in all species studied so far (Cummins and Harris, 1958). In mycelial fungi the main carbohydrate is chitin (polymerized N-acetylglucosamine), whereas yeast cell walls contain no amino sugars, but mannan (polymeric mannose), and some glucan attached to proteins ( Nickerson et al., 1961). Lipids have also been found, but these may come from a closely adhering cytoplasmic membrane. At intervals a disulfidase in the cytoplasm sometimes attacks a portion of the wall, and a finger of protoplast is extended through the hole. This finger soon becomes covered with new cell wall, thus forming a yeast “ b u d which later becomes a new organism (Nickerson et al., 1961).
2. Cytoplasmic Membrane. Permeability Fungi have a permeability-regulating membrane, thin and fragile, inside the cell wall. It seems to consist of the usual lipoprotein mosaic (see Section IV, A). It contains inositol as a phospholipid (Shatkin and Tatum, 1961).GThe complex systems of Electron micrographs reveal this phospholipid in aZZ membranes of Neuuros p o ~ acrassu, in an inositol-less mutant of which all the membranes are degenerated. Inositol is essential for balanced growth in yeast, and analogs of inositol antagonize grojvth ( Shatkin and Tatum, 1961).
29
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
enzymes found in bacterial cytoplasmic membranes is absent, but adenosine triphosphatase has been found in the membrane of yeast, and it appears to assist in the uptake of amino acids (Post et al., 1960). Endoplasmic reticulum, an extensive network of membrane filling the cytoplasm, is abundant in fungi just as in higher organisms, It is commonly supposed to consist of invaginations of the cytoplasmic membrane. Little is known of the permeability properties of fungi, except that the membrane seems to be unusually easily penetrated by lipophilic substances. It is noteworthy that few specifically antifungal substances are known, whereas many substances are specifically antibacterial. A great many antifungal substances in common use are general protein precipitants, and they inactivate a wide range of enzymes in both plants and animals. However, they usually have lipophilic groups which assist selective concentration in the fungus. Thus, of three agents widely used against fungus on crops, chloranil (XX) depends on the four halogen atoms, glyodin (XXI) on the long alkyl side chain, and captan (XXII) on both the 6-membered hydrocarbon ring, and the trichloromethylthio group. The walls of spores are freely permeable, provided that the surrounding air is humid.
c1 c*c; 0 Chloranil
H
..
0 Glyodin
Captan
3. Mitochondria, Nuclei, Ribosomes
Fungi have mitochondria, nuclei, and ribosomes in the cytoplasm (the presence of mitochondria in yeast is, however, dependent upon aerobic culture) (Linnane et al., 1962). A few words on mitochondria may be appropriate here. In higher organisms, they form somewhat spherical bodies. The
30
M R I E N ALBERT
peripheral lipoprotein double membrane, about 180 A thick, extends as internal convolutions, called cristae, into the interior so that the entire structure of the mitochondrion visible under the electron microscope seems to be filled with membrane. About one quarter of the protein part of the cristae consists of “respiratory assemblies,” i.e., ordered arrangements of nicotinamide-adeninedinucleotide, riboflavine, cytochromes b, c, and a (in that sequence) together with their specific proteins, and adenosine diphosphate. Mitochondria are the site of (1) the cell’s tricarboxylic acid cycle which transforms (to carbon dioxide, water, and energy) the acetyl Co-A which is produced by the metabolism of both carbohydrates and fatty acids; ( 2 ) the enzymes that convert fatty acids to acetyl Co-A; (3) those enzymes which transmit, to atmospheric oxygen, the electrons removed from all the various metabolic substrates, and store part of the energy, obtained in this way, as adenosine triphosphate (Ball and Joel, 1962). The more soluble enzymes of the Meyerhof carbohydrate degradation sequence are believed to be in the sap of the mitochondria. No important differences between the mitochondria, nuclei, and ribosomes of fungi and those of higher organisms have yet been described. 4. Other Components Fungi contain many substances not found elsewhere (Birkinshaw, 1956; Stickings and Raistrick, 1956). Some of these may be accumulated waste products. However, many of them are chelating agents, e.g., pyrones (such a kojic acid), and the l-hydroxyanthraquinones; these may act as storage for those traces of heavy metals (such as zinc and copper) which seem so necessary for fungal growth. Some fungal enzymes show marked individuality. Thus crystalline aldolase from yeast requires iron (Warburg and Christian, 1943), whereas that from mammals does not (Taylor d a]., 1948). Again, rabbits injected with the glyceraldehyde-3phosphate dehydrogenase of yeast, produced a serum which inhibited the activity of this enzyme but had no effect on the corresponding enzyme from rabbit muscle (Krebs and Najjar, 1948). A similar result was found when the hexokinases of yeast and brain were compared (Miller et aZ., 1948).
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
31
B. BIOCIDES Of the few biocides that have high specificity for fungi, there are several that act specifically on the cytoplasmic membrane (a type of attack not yet achieved on bacteria), but few act on the cell wall and apparently none on the nuclei, mitochondria, or ribosomes. In medicine, there is a need for antifungal drugs to suppress yeasts whose excessive growth in the bowel after tetracycline therapy causes so much discomfort. Better drugs for treating the mycoses are also required. 1. Acting on the Cell Wall Cycloheximide (actidione) (XXV) is a fairly simple antibiotic from Strepfomyces grimus (Kornfeld et aE., 1949). It causes the yeast Saccharomyces pastorianus to form large cells ( apparently through a derangement of cell wall synthesis) and growth becomes arrested (Gundersen and Wadstein, 19SZ). Cycloheximide is highly toxic to yeasts and phytopathogenic fungi, but has much less toxicity for bacteria and for fungi pathogenic to mammals. It is used in culture media to enrich certain species, and also as an agricultural spray against e.g., cherry leaf spot, and fungal infestation of turf (Ford et al., 1958). Unfortunately, it is moderately toxic to plant cells also. Griseofulvin (XXIII) is a highly specific fungicide, which is successfully used in medical practice, to eliminate fungal diseases of the skin, hair, and nails after prolonged oral administration. Because all microorganisms inhibited by griseofulvin contain chitin, it was thought that the antibiotic inhibited the enzyme that polymerizes acetylglucosamine to chitin in laying down new cell wall. However, some ( chitin-containing ) fungi are unaffected by griseofulvin (Abbot and Grove, 1959). Only one of the four isomeric forms of griseofulvin shows biological activity. The configuration of this isomer, determined by MacMillan (1959), has something in common with the repeating unit of chitin; and in both molecules the bulky substituents are equatorial. Whatever the mechanism, the site of action of griseofulvin is confined to the growing tips of the mycelium. Chitinase, an enzyme from snails and almonds, cleanly strips fungi of their cell wall. The quaternary amines also attack fungal cell wall, though not very specifically (see Section 11, B, 1, b ) .
32
ADRIEN ALBERT
Penicillin has no effect on fungal cell walls, and griseofulvin is not antibacterial.
I
c1
I
H Griseofulvin
Cycloheximide
2,. Acting on the Cgtoplasmic Membrane Polyene antibiotics make the plasma membrane swell (and often burst) in 15 seconds. This has been shown for both Neurospora crussu, a mycelial fungus (Kinsky, 1962a, b ) and yeast (Marini et ul., 1961). No damage occurs to the celI wall. These antibiotics act on resting, as well as growing, fungi but they have no action on bacteria.
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
33
When the cell wall of Neurospora is removed with chitinase, in 20% sucrose solution to prevent bursting, stable spherical protoplasts are obtained. These have proved useful for studying the effects of the polyene antibiotics, which cause a nonspecific release of plasma constituents into the medium at about 20 pg. per ml. (1 in 50,000) (Kinsky, 1962a,b). Each polyene antibiotic consists of a large lactone ring which contains several conjugated double bonds and is united by a glycosidic linkage to one (or more) unusual sugar. Thus filipin, C3,HB2OI2from Streptomyces fizipinensis, has the pentaene lactone ring (25-membered) shown in (XXIV), which still leaves about 10 carbon atoms to be accounted for (Berkoz and Djerassi, 1959). The most successful polyene antibiotic in human medicine is amphoteracin B ( “Fungizone”). It is given orally for treating mycoses. The full constitution of this antibiotic is not yet known, but a heptane lactone ring is present.
3. Other Types of Action Chelating agents, much used as fungicides, are dealt with in Section VI. Organic mercurials and inorganic copper compounds are widely used as fungicides in agriculture, but their sites of action have not been located, although SH-containing enzymes are suspected. It has been shown that the leaves of plants secrete acids which solubilize copper carbonate (the residue after spraying with Bordeaux Mixture): the product is absorbed only by the fungus (Arman and Wain, 1958). Bisdithiocarbamates ( e.g., Dithane and Nabam) become degraded, on vegetable matter, to bisthiocyanates which combine with SH-containing enzymes. Tributyl tin is a much used fungicide that interferes with oxidative phosphorylation ( Aldridge, 1958) . Sulfur, which is still much employed in agriculture, interferes with dehydrogenation processes. All the substances mentioned in the last paragraph presumably act on mitochondria. The following substances, which form covalent bonds, presumably act on proteins and possibly nucleic acids as well: quinones, e.g., (XX), formaldehyde, ethylene oxide, halogens. Protein synthesis is interfered with by m- and p-fluorophenylalanine, which compete, reversibly, with phenylalanine (van Andel, 1962). The antibacterials chloromycetin and puromycin do not inhibit fungi.
34
ADRIEN ALBERT
Weak acids (e.g., benzoic acid, sulfurous acid) are weakly fungicidal and are most active at pH values where they are least ionized (Albert, 1960). They probably act by denaturing protein. Phenols, including chlorinated phenols, probably belong to this class, but salicylic acid has a strong chelating action as well.
IV. Viruses Advances in techniques, in recent years, have brought a wealth of new knowledge about viruses. These are structurally simpler than bacteria, and vary in size from particles as small as 200 A, to others that form long rods (10,000 x 100 A ) or spheroids (3000 A ) . Viruses have no cell walls. Rickettsias and organisms of the psittacosis-lymphogranuloma group, although obligate intracellular parasites, have cell walls containing muramic acid. They are parasitic bacteria, not viruses (Perkins and Allison, 1963). A. k~lCHOSTRUC1TJRESOF VIRUSES The complete infective virus, such as exists extracellularly, consists of a core of either DNA or KNA (but not both) surrounded by a protective coat of protein. These two components are arranged in a highly ordered fashion that is characteristic of the virus. The DNA is double stranded in some species, but a single strand in others. The larger viruses have, in addition, lipids which may help to regulate permeability. Cell membranes from Rous sarcoma virus appear to have the same structure as those of chicken liver cells and human erythrocytes, and this pattern may prove to be widely distributed in nature (Dourmashkin et al., 1962). In viruses, no devices for producing energy are present. Pliage viruses contain aliphatic diamines such as putrescine [H,N. (CH,), *NH2] and spermidine [H,N. (CH,),NH( CHZ),NH,], in quantities sufficient to neutralize from 3030% of the DNA present. They seem to be stabilizers of the folded state of DNA (Mahler et al., 1961). But they are nonspecific as they can be replaced by an excess of magnesium ions without disturbing the functioning (Ames and Dubin, 1960). Many other viruses, e.g., poliovirus and tobacco mosaic virus, lack these bases. Myxoviruses are unique in having neuraminase as an integral part of the virus particle, although the presence of enzymes is suspected in some other viruses. Copper is found associated with the viruses of
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
35
the pox group. 5-Hydroxymethylcytosine is found in place of cytosine in the DNA of some coliphages (Wyatt and Cohen, 1953). When a viriis particle infects a cell, the nucleic acid (which is the sole infectious part) passes into the host and not only organizes the host’s supply of intermediates to produce fresh virus, but can also command the synthesis of new intermediates which it requires. Phage, the type of virus which infects bacteria, has a special shape, consisting of a head and a tail. The head of T2 coliphage, for example, contains a single molecule of DNA (M.W. = lo*), which weighs 2 x l O - ’ O pg. and contains 2 x lo6 nucleotide pairs. This molecule is neatly folded into an approximately spherical mass, and surrounded by neatly packed (nongenetic) protein molecules constitntes the “head.” To this is attached a “tail” built of a series of five structures: (1) the outer sheath consisting of a contractile protein with about 110 molecules of adenosine triphosphate; (2) a solid core; ( 3 ) a tip consisting of a spiked plate; ( 4 ) a few molecules of endolysine, a lysozymelike enzyme; (5) a series of fibers which are wound around the distal end. The invasion, by the phage, of the bacterial host is thought to begin with electrostatic attraction between a thiol ester in the tail tip and zinc cations in the bacterial cell wall, followed by a hydrolysis of the ester in which chelation has a possible role (Kozloff et al., 1957). The removal of the tip unmasks the endolysine which depolymerizes the glycopeptides of the bacterial cell wall. The myosinlike sheath then contracts, and the solid core pierces the bacterial membrane, and finally the viral DNA is injected into the cytoplasm (Lwoff, 1961; Hershey and Chase, 1952).
R. BIOCIDES During the short period of its life cycle when virus is extracellular, it is susceptible to chemicals, even to dilute soap solution. However, no useful attack can be made on virus diseases unless the viruses can be killed in the parasitized cell without harm to uninfected host cells. Until quite recently this had seemed an impossible goal. Attention was first concentrated on those agents which interfere with virus reproduction, even when they were not very specific. The effect of aminoacridines, mentioned in Section 11, B, 3, f, on nucleic acids is observable at great dilution, but has not yet
36
ADRIEN ALBERT
led to a usable drug. The mutagenic action of aminoacridines on phages has been much studied ( DeMars, 1953; Crick et al., 1961). The analogs of purine and pyrimidine bases, whose antibacterial properties were mentioned in Section 11, B, 3, d, have shed much light on viral metabolism. Thus, 2-thiouracil (XXVI) becomes built into the RNA of tobacco mosaic virus (TMV), and interferes with reproduction (Jeener and Rosseels, 1953; Matthews, 1956). 5-Chloro-(and bromo-)uracil (XIII) are incorporated into the DNA of phage, and hinder multiplication (Dunn and Smith, 1957). 5-Fluorouracil is incorporated into the RNA of tobacco mosaic virus. 8-Azaguanine (XIV) replaces guanine in the RNA of TMV (Matthews, 1954) which then becomes incapable of reproduction; it is also taken into the RNA of turnip yellow mosaic virus ( Matthews, 1955). 0
Thiouracil (XXVI 1
The first clinically useful analog was 5-iodo-deoxyuridine, which is effective in curing a very painful and long-lasting disease of the eyes, namely infection with the virus of herpes simplex (Kaufman, 1962). This virus attacks the corneal epithelium, an unusually favorable site for drug therapy, because these cells have little metabolism ( Cogan, 1962). Intradermal vaccinia infection can be prevented by giving this drug intravenously (Calabresi et al., 1963). The cytidine analog, and the 5-fluoro-analog,were also highly active in animal experiments ( Perkins et al., 1962). Biochemically this deoxyriboside of 5-iodouracil has been found to inhibit the synthesis of DNA; in addition it is incorporated as the 5' phosphate into DNA, in the place of thymidine-S-phosphate (Prusoff, 1960). Chloramphenicol (at 1 in 10,000) suppresses all protein synthesis, within one minute, in T2 coliphage, but the effect is lost upon dilution (Hershey and Melechen, 1957). The most versatile antiviral agents, so far discovered, are the thiosemicarbazones. These were first observed to be active against
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
37
neurovaccinia infection in mice (HamrB et al., 1950). Later isatin thiosemicarbazone (XXVII, R = X = H ) was prepared, and found to be by far the most active example (Thompson et aE., 1953). Two doses ( 125 mg./kg.), given orally to mice, some hours after intercerebral infection of mice with a thousand LDZoof vaccinia virus, saved all lives. The infection proceeded just sufficiently to create immunity (Bauer, 1955, 1960). Most of the members of the variola-vaccinia subgroup of the pox viruses are sensitive to this drug, in the following order of decreasing susceptibility: variola, rabbit pox, neurovaccinia, white cowpox, cowpox, ectromelia. In some cases, higher homologs of the drug are more effective. Thus when variola major virus, the causative organism of the most serious type of smallpox infection, was inoculated ( a thousand LD50) intracerebrally into mice, the lives were all saved by a dose of 25 mg./kg. of isatin thiosemicarbazone, but as little as 10 mg./kg. of the 1-,or 7-, methyl homologs sufficed (Bauer et al., 1962 ) . The first clinical success followed the treatment of a 4-monthold baby who had contracted vaccinia from his parents while they were being vaccinated. The extent and severity of the infection made it improbable that the child would live. However, dramatic improvement was seen 3 days after oral medication with 1 gm. (daily) of N-methylisatin-b-thiosemicarbazone,and the child later recovered completely (Turner et al., 1962). However, an adult with malignant smallpox was not improved by 31 gm. of isatin thiosemicarbazone, orally given ( Marsden, 1962 ) . When both hydrogen atoms at the end of the side chain [i.e., those marked X in (XXVII)] are replaced by methyl groups, the activity against ectromelia increases greatly, but the action against vaccinia is completely lost (Bauer and Sadler, 1961). This fact carries the implication that antiviral drugs may be highly speciesspecific. Enteroviruses, which differ from pox viruses in many ways, e.g., in having RNA instead of DNA, seem to require still more lipophilic homologs of (XXVII) for inactivation. Thus type 2 poliovirus, in tissue culture, was inactivated by l-methyl-4’,4’-dibutylisatin thiosemicarbazone (XXVII, R = CH3, X = C4HB).Of a large number of benzimidazoles tested on type 2 poliovirus, only 2-( ahydroxybenzyl )benzimidazole ( XXVIII ) and its 5-chloro-derivative were found to have a selective action on the virus (I. Tamm
38
ADRIEN ALBERT
ct a]., 1961; Eggers and Tamm, 1961). It was later found that the isomeric 2- ( 2'-hydroxybenzyl ) benzimidazole ( XXIX) also has this selective action on the virus. It has been noted that intramolecular hydrogen bonding is a feature common to most of the antiviral agents in the benzimidazole and isatin series. This has led to some
s
II
{Q-$--,N\yNx2
H
\
c . , k
0
II
Isatin thiosemicarbazones
C,H,
(xxrx) speculation as to whether the biological activity is connected with chelation, particularly as the pox viruses often contain copper ( OSullivan and Sadler, 1961) .
V. Protozoa Protozoa are large monocellular organisms which, collectively, have even greater diversity of shape than fungi, and are usually motile as well. The life cycle is often complex, and some species pass, in alternation, through asexual and sexual forms, whereas others reproduce only by simple fission. A bewildering variety of organelles is often found, and it seems that, in a single cell, the protozoon is beginning to express many of the complex functions of a whole metazoon (higher animal) (Grimstone, 1961).
A. MICROSTRUCTURES OF PROTOZOA Often (as in Amoeba and T~ypanosoma)no celI wall is present, and osmotic pressure is low. Some other protozoa have walls of
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
39
cellulose about 230 A thick (e.g., Chlamydomonas, or of protein ( e.g., Eimeria), or of silica (e.g., the Foraminifera). A double cytoplasmic membrane, 70-100 A in thickness, is always present, also mitochondria. The latter may be few or numerous, and tend to be filled with tubules rather than cristae (cf. Section 111, A, 3). Their biochemistry has been little studied. The nuclear membrane of most protozoa resembles that of most metazoan cells: there is an inner and an outer membrane, each 70-80 A thick, and several hundred A apart. There are many apparent pores in this, 300800 A in diameter, and at the boundary of the pores the membranes are continuous. Distinct nucleoli are found, but the chromosomes are unusually poorly defined. Sac-shaped Golgi apparatus, secretory in function, are common in most species. Cilia and flagella grow out from kinetosomes (basal bodies), but little is known of their chemistry nor how their movement is produced. Myonemes and trichocysts (both consist of protein), and centrioles are three important kinds of organelles about which little is known. Vacuoles, including contractile vacuoles, are common; chloroplasts, pyrenoids, and eyespots occur in some species, also minute tentacles. In flagellates, a complex assembly of organelles is found at the base of the flagellum, namely a basal body (see above), a parabasal body, and a kinetoplast. The kinetoplast contains DNA, arises by division of earlier kinetoplasts, and seems to produce the cell’s mitochondria ( Steinert, 1960; Vickerman, 1962). Treatment with acriflavine abolishes the kinetoplast. The “volutin” granules, which occur profusely, seem to be ribosomes in this organism (Nath and Dutta, 1962). Trypanosomes go through a cycle in which they first multiply in a vertebrate host in the large “trypanosoma” form, and then multiply in an invertebrate host in the smaller “crithidia” form. The latter eventually changes to a “metacyclic” form which reinfects the first host. In culture, the “trypanosoma” forms become crithidial unless urea ( 10-3M) is present. Urea, which is the natural transforming factor (Steinert and Steinert, 1960), inhibits synthesis of DNA, and in its presence both cell and nucleus grow larger. No sexual reproduction has been unequivocally demonstrated in trypanosomes. Other protozoa, which assume a sexual form in insects, often owe this change to the presence of the insect molting hormone ecdysone (Cleveland, 1960).
40
ADRIEN ALBERT
B. BIOCIDES Many effective biocides, selective for protozoa, had been discovered before any selective antibacterial and fungicidal substances were known. Hence it is not surprising that the chemotherapy of protozoal diseases employs substances that are unfamiliar in other contexts. The principal forms of protozoal illness are caused by the following species: Entamoeba; Trypanosoma ( a flagellate, many species, some infecting man with sleeping sickness, and others, domesticated animals); Leishmania ( a flagellate); Plasmodium ( a sporozoite, many species, some infecting man with malaria, others infecting birds and wild animals); Toxoplasma ( a sporozoite, infecting man and other animals), Eimeria (and related sporozoites, causing coccidiosis in poultry); Babesia (causing red water fever in cattle) Theileria, Histomonas, Toxophsma, Trichomonas. In spite of quite good results with antibiotics and new synthetic drugs, amoebiasis in man seems to respond best to derivatives of 8-hydroxyquinoline such as chiniofon (see Section VI), plus (if the liver is affected) chloroquine or even the alkaloid emetine. Trypanosomiasis is usually treated with suramin, pentamidine, or (advanced cases) pentavalent arsenicals, in man, and quinapyramine or the phenanthridines, in cattle. The high selectivity of arsenicals in trypanosomiasis is related to the very high metabolic turnover of trypanosomes compared to that of their hosts (Albert, 1960). Arsenicals act by attacking thiol groups, apparently those involved in carbohydrate catabolism. Trypanosomiasis can usually be prevented by injections of either suramin, or one of the aromatic diamidines, at three monthly intervals. It is pertinent to ask how these two types of drug, one polyanionic and the other dicationic, can have the same action, and how the long duration is effected. It has been suggested that both types uncouple nucleoprotein, the anionic type seizing the histone to which DNA is normally attached, and the cationic type seizing the nucleic acid ( Ormerod, 1951, 1952). The prolonged action seems to arise from an immune reaction, somewhat as follows. Suramin, stilbamidine, and antrycide increase the volutin granules (RNA) of trypanosomes and, possibly as a consequence, the trypanosomes release much more than the usual meager amount of antigen: to this the host responds with an excess of antibody ( Ormerod, 1981 ) ,
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
41
This is an extension of earlier work with trypanosomes, in the rat as host, in which it was found that stilbamidine produced fluorescent granules (presumably of the stilbamidine-RNA complex salt) at once, but the infection continued for some time, and then was suddenly eliminated 4 days later (Fulton and Grant, 1955). Careful observation showed that the drug acted as such, i.e., not in the form of a metabolite, also that it was capable of killing only a few of the trypanosomes. However, the dead parasites liberate antigen, which causes the host to make antibody and this is responsible for the eventual sudden cure (Fulton and Grant, 1956). When the immune response of a rat is blocked by splenectomy, or by injecting copper, trypanosomiasis could no longer be cured by stilbamidine, suramin, or quinapyramine ( antrycide ) ( Ormerod, 1961) . Parallel to this work on trypanosomes, the similar but more easily cultured Strigomonas oncopelti has been investigated in vitru. The phenanthridine drugs, e.g., ethidium bromide ( 3,8-diamino-6-phenyl-5-ethylphenanthridinium bromide), inhibit growth gradually, and the number of organisms doubles before growth ceases (Newton, 1957). Only growing organisms are attacked. Antrycide only slows growth and does not abolish it. The phenanthridines were then found to inhibit formation of new DNA, but allow RNA and protein synthesis to continue for some time. Antrycide merely slows DNA formation, but it also inhibits incorporation of purine nucleotides into ribosomal RNA ( Newton, 1958, 1962). It was later found that the pyrimidine, and not the quinoline, ring in this drug was the part preventing purine incorporation. Tests with a variety of pyrimidines show that a quaternary nitrogen atom is essential for inhibition (Newton, 1960). Strigomonas, when made resistant to antrycide, is not cross-resistant to phenanthridines. At least some species of trypanosomes cannot synthesize the purine nucleus. Hence if the early action of antrycide on trypanosomes involves inhibition of purine incorporation, a good synergic effect should be found with purine analogs, e.g., (XIV). Leishmaniasis in man is treated mainly with the aromatic diarnidines and antimonial drugs which act on thiol groups. Histomoniasis, another flagellate disease (it causes blackhead in turkeys) is cured by nitroheterocycles, such as 1,2-dimethyl-5-nitroimidazole. Malaria prophylaxis is best achieved with minute doses of the
42
ADRIEN ALBERT
antifolic acid drug, pyrimethamine (see Section 11, €3, 3, c ) . Proguanil (“Paludrine”) is another, but less powerful drug of the antifolic type. If the disease is contracted, it is usually treated with chloroquin, a 4-aminoquinoline with a basic side chain. This drug is essentially a modified form of quinacrine (mepacine, “Atebrin”), the acridine derivative used so successfully on the battlefields of the second world war. Chloroquine and quinacrine attack only the schizonts, i.e., the nonsexual forms that parasitize the host’s red blood cells, Gametocytes (sexual forms), although causing no symptoms, are responsible for infecting new mosquitos and hence spreading the disease. Hence, in mosquito-infested country, the patient is also given an 8-aminoquinoline drug, such as primoquine, to eliminate the gametocytes. Eimeria (in domestic poultry) responds well to sulfonamides (see Section 11, B, 3, e ) . Some structure-action relationships among antiprotozoal drugs suggest worthwhile research topics. The tendency for effectivc cationic drugs to have two basic groups, as seen in the diamidines, antrycide, the phenanthridiniums, quinacrine, chloroquine, plasmoquine, and primaquine, requires explanation. This trend may be related to special nucleic acid conformations in the protozoa. These drugs act on protozoa, in general, at much lower concentrations than those at which they are antibacterial. On the other hand, quinacrine becomes much more strongly antibacterial without the side chain. The need for quaternization in such trypanocidal drugs as antrycide and the phenanthridiniums is yet to be explained. Acridines are also trypanocidal if quaternary, and if a second basic center is provided, but this center must be in the 3-position (Browning, 1951). Such quaternary acridines can penetrate to the nucleus, at least in mammalian cells, doubtless passing through the cytoplasmic membrane as the corresponding pseudobases with which they are in equilibrium (De Bruyn et al., 1950).
VI. Appendix on Chelation Many valuable antibacterial and antifungal snbstances which act by chelation (metal-binding) are known. Also a possible chelating action for some antiviral and amoebicidal drugs has been indicated above. Although the sites of action of chelating substances are unknown, it seems worthwhile to recount a number of well established facts concerned in their mode of action (Albert, 1960).
MICROBIOLOGICAL MORPHOLOGY AND BIOCIDES
43
The first chelating biocide to be recognized as such was 8liydroxyquinoline [oxine, chinosol, (XXX) 1. It was shown that, of the seven isomeric monohydroxyquinolines, only the 8-isomer could chelate, and only this isomer was bactericidal and fungicidal (Albert et al., 1947). Likewise, blocking either the oxygen or the nitrogen atom of oxine abolished both the chelating and the antibacterial actions. It remained to be settled whether the biocidal action depended on the removal of an essential heavy metal cation from the organism, or on carrying into the organism an excess of a metal accidentally present in the medium. The latter explanation was established by the experiments in distilled water shown in Table 111. Oxine is, of itself, harmless to bacteria, and a metal of variable valence (iron or copper) must also be present; the complex is the true toxic agent (Albert et al., 1953). In the experiments done in broth, iron was present as a contaminant. When this was removed, oxine became inactive.
OH
0
(xxx)
(xxxrr1
OMXI)
In any given series, where the affinity for metals remains stationary, the antibacterial action rises with the liposolubility up to a plateau value (Albert et al., 1954) (see Table IV). This suggests that the destructive action is taking place in the bacterial membrane which contains nearly all of the lipid present in the cell (Section 11, TABLE 111 METALIN THE BACTERICIDAL ACI'IONOF OXINE Stophylococcus atireus (20°C.; PLATEDOUT AFTER1 HOUR)=
THE NEED FOR h H E A V Y
Oxine ( 1/M)
Fez+ or Fe3+ (1/M)
Nil
Nil Nil
100,000 Nil
100,000 Symbols:
100,000 100,000
Growth
In distilled water
+ + + + + + ++ +
+ + + prolific growth; - no
In meat broth
growth.
++ + ++ +
44
ADRIEN ALBERT
A, 2 ) or at an alternate site inside the cell. It is understandable that highly hydrophilic chelating agents, like EDTA, are not biocidal. The biocidal action of oxine seems to take place by an oxidativc chain reaction, inhibited by cobalt ions, and death often occurs in 5 minutes. Fungi are not so rapidly affected as bacteria, but are equally susceptible. TABLE IV THE DEPENDENCE, IN A SERIESOF RELATEDCHELATINGAGENTS, ON SUFFICIENTLY HIGH LIPOPHILICPROPERTIES FOR BACTERIOSTATIC ACTION Lowest dilution inhibiting Streptococcus
pyogenes
Substance 8-Hydroxycinnoline 4-Methyl8-Hydroxyquinazoline 4-Methyl4-Propyl4-Allyl-
Partition coefficient ( oleyl alcohol/ water)
(20”;pH 7.3) 1/M (reciprocalof molarity )
Log of first stability constant (N?+ )
6 I6
13,000 25,000
7.8 8.1
5
13,000 50,000 100,Ooo 100,000
7.6 7.9 7.9 7.9
17 135 310
Many other chelating agents behave exactly as oxine. Thus 2mercaptopyridine-N-oxide ( XXXI ) and dimethyldithiocarbamic acid (XXXII) are inactive in the absence of metals. Whereas either copper or ion converts these substances into bactericides, only copper is effective for fungi (Anderson and Swaby, 1951; Nordbring-Hertz, 1955; Block, 1956). Oxine diffuses freely into the cytoplasm of bacteria and of fungi without causing harm if the requisite metal is absent (Beckett et al., 1958). Salts of (XXXII) are, perhaps, the most successful of all agricultural fungicides, and are manufactured in ton lots. The copper salt of oxine is much used in mildew-proofing of wood and fabrics. Many other biocides are known to chelate strongly. Among them are isoniazid (the most valuable of all drugs in treating tuberculosis) biallylamicol, hexachlorophane, “Ethambucil,” and the tetracyclines. Yet chelation seems to be only a part of their mode of action (Albert, 1960). For the iron-containing antibiotics (sideramines), see Section 11, B, 4.
MICROBIOLOGICAL MORPHOLOGY AND BIOCDES
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Generation of Electricity by Microbial Action J. B. DAVIS Socony Mobil Oil Company, Inc., Field Research Laboratory, Dallus, Texas
I. Introduction ........................................... 11. Redox Potential ........................... A. Source of Electric Power . . . . . . . . . . . . . . . B. Early Work ........................................ 111. Fuel Cells . . . . . . . . . ............................... A. Enzymes ........................................... B. Attempt to use Hydrocarbons .......................... C. Eschen'chiu coli ..................................... D. Salt Bridge versus Cellophane Membrane . . . . . . . . . . . . . . . E. Potassium Ferricyanide . . . . . . . F. Further Research ............. ................... IV. Corrosion Cell . . . . . . . . ............................. V. Summary .............................................. References .............................................
1.
51
54 55 55 58
57
S9 61 82, 83 64
Introduction
Open circuit voltage or electromotive force increases as microorganisms utilize organic compounds in a relatively anaerobic environment, particularly with reference to an oxygen electrode. By closing the circuit between the oxygen electrode and an electrode at the site of microbial action an electric current may be measured. The current is quite small, in the order of a few milliamperes with a single electrolytic cell of even one liter volume and large electrode surface; the current density is about 10W2 ma. per The small amount of electrical energy available in past work is principally why biological sources of electric current have not been exploited. The powerful bioelectric shock of the electric eel is quite different from the above. The shock is due to a discharge of several hundred volts derived from a membrane potential generated similarly to electric current in nerve tissue. The electric organ is composed essentially of striated muscle cells, each surrounded by a polarized membrane with a positive charge on the outside and a negative charge on the inside. The cells represent several thousand plates in series and there exists normally a concentration gradient 51
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J. B. DAVIS
of sodium and potassium ions, the source of electromotive force, on the two sides of the membrane. Loss of membrane resistance triggered by the chemical reaction of acetylcholine with membrane receptor protein (Nachmansohn, 1951) allows a rapid flow of sodium ions through the membrane, markedly increasing the concentration gradient, and the charge is reversed. The same sequence occurs in the cells in series, hence the strong bioelectric discharge. Obviously, the membrane potential in tissues is different from the oxidation-reduction potential developed in microbial cultures. Conversion of chemical energy to electrical energy as a practical process is being actively investigated again, after a lull of some fifty years, and continuous feed battery or fuel cell technology is progressing fairly well under this recent attack with modern methods and materials. Fuel cell research has actually focused attention on electron transport in biological, or more specifically microbiological, systems as an interesting and novel avenue. Potter (1911), of Great Britain, published a comprehensive paper on the electrical effects accompanying the decomposition of organic compounds. Thirty-one years ago Cohen (1931) reported at the national meeting of the Society of American Bacteriologists on the bacterial culture as an electrical half-cell. Harris (1960), much more recently reported the generation of small electrical currents in studies of the corrosion of steel in nutrient broth by common soil bacteria. This year, in a paper devoted to considerations of a microbial fuel cell, Davis and Yarbrough (1962) reported experiments employing bacteria, nocardia, or glucose oxidase as the respective biological agent in the decomposition of glucose yielding electrical currents up to 2 ma. usually with methylene blue serving as the electron transport mediator. Within the past year or two, newspapers have reported that experimental work on the generation of electricity by microbial systems is underway in various laboratories. These accounts characteristically point to the practical utilization of microbially produced electricity, ranging from the operation of sea buoy radio-beacons to the eventual production of enough electrical power to serve “entire communities.” These reports suffer from the usual newspaper flair for publicity and from their failure to cite earlier work and the practical problems involved. A general discussion of biochemical fuel cells including a typical
GENERATION OF ELECTRICITY BY MICROBIAL ACTION
53
diagram of the classic means for determining redox potential of bacterial cultures appeared in an article by Sisler (1961).
11.
Redox Potential
A. SOURCEOF ELECTRIC POWER Electrical energy derived from the metabolic activities of microbes has as its source the oxidation of organic compounds and the consequent transfer, “flow,” of electrons ultimately to oxygen, if complete oxidation occurs. Anaerobic bacteria, and facultative microbes in the absence of molecular oxygen, do not employ oxygen as the ultimate electron (or hydrogen, proton plus electron) acceptor. Electron transfer in microbial metabolism is not a straightforward “flow” since the electrons do not pass as through a wire but through a series of complicated intermediate transfer reactions involving specific enzymes dependent upon the substrate being metabolized; upon hydrogen carriers, diphospho- or triphosphopyridine dinucleotides; upon phosphate transfer agents, adenosine di- and triphosphate; and upon reduction-oxidation agents such as the flavins and cytochromes specific to the microbe and the conditions for metabolism involved. Despite this array of mediators, the process of electron transfer does occur stepwise through a chain of compounds, each pair of components in essence a battery connected in series. A drop in voltage depending upon the reduction-oxidation potential of each pair occurs when the electrons are transferred, and special storage batteries in the form of high energy phosphate bonds reserve energy. To utilize this flow of electrons in order to perform work outside the microbial cell is another matter. Electron transport within the microbial cell involves a potential change, an increase in voltage, and a consequent source of current; but much of the electrical energy is consumed in synthetic processes, for cellular movement, in cell division, and much is dissipated as heat. The external environment of the cell is actually the principal site and source of electrical energy for man’s use, not the interior of the cell. A reduction in electrode potential occurs in culture media during microbial utilization of the substrates. The substrate is oxidized, electrons are removed from the substrate, reduced products are formed, and the culture solution is reduced relative to its original state. If the culture electrode is connected to an electrode at higher
54
J. B. DAVIS
electrode potential, and there is ionic union between the two halfcells, a flow of current from the lower potential to the higher potential occurs as the system tries to reach equilibrium, hence electrical energy is available to do work.
B. EARLYWORK Potter, fifty-one years ago, was apparently the first to demonstrate the reduction in electrode potential ( increase in electromotive force, E.M.F.) due to microbial activity. He was successful with yeast, and tested with some success bacteria, particularly Escherichia coli var. communis, and also the hydrolytic enzymes extracted from yeasts, namely, invertase and diastase. He found further that a current could be detected when he short-circuited a plantilium electrode in a bacterial culture to a platinum electrode immersed in the sterile culture medium. Ionic union of these two half-cells was assured since they were separated by only a simple dialysis membrane. Potter employed gold, nickel, tin, zinc, aluminum, and carbon electrodes. All showed a difference of potential between a yeast-glucose solution and the glucose solution, and an electric current passed in the cell from the yeast-glucose half-cell to the glucose half-cell. The highest open circuit voltage obtained by Potter with yeast was about 0.4 volt. On closed circuit the voltage dropped to about 0.1 volt. He did not report the resistance of the circuit. But he did set up a battery of 6 cells with carbon electrodes connected in parallel and measured a current of 1.25 ma. Potter stated in conclusion: “The electrical effects are an expression of the activity of the microorganisms and are influenced by temperature, concentration of the nutrient medium, and the number of active organisms present. These effects are only found within the limits of temperature suitable to the microorganisms and under conditions which are favorable to protoplasmic activity.” By the early thirties, the study of redox changes caused by microbial activity in culture media became commonplace. Hewitt (1950) wrote an excellent monograph summarizing his own studies and those of numerous workers. These investigations had two principal objectives: ( 1 ) to observe changes in electrode potentials caused by microbial activity, and ( 2 ) to study the effect these changes had on the activity of the microbes. Hewitt studied in particular the bacterial production of hydrogen peroxide and its effect upon electrode potentials under various conditions of aer-
GENERATION OF ELECTRICITY BY MICROBIAL .%CTION
55
ation. Hewitt made reference to the work of Cohen in (what is now) an amusing manner: “but Cohen (1931) has carried the matter to an extreme point. He has built up a bacterial battery by connecting in series a number of cells each composed of 10 cc of culture coupled to a sterile control. The culture medium contains a poising agent such as potassium ferricyanide or benzoquinine. Each unit (now quoting Cohen) ‘yields about 0.2 ma. at a pressure of 0.5 volt with very small polarization for at least 5 minutes. By this means we have been able to build up a bacterial battery furnishing current of about 2 ma. at a pressure of 35 volts’.” Hewitt apparently took a rather dim view of this means of generating electrical energy, at least from a practical standpoint. Hewitt’s view must have been shared by Dr. Cohen, since he reported only briefly on his bacterial battery and then presumably turned his attention to other, more interesting, things.
111. Fuel Cells Obviously, recent attention toward microbial activity as a source of electricity was stimulated by current fuel cell research. The lag in interest between the observations of Potter and of Cohen concerning the generation of electricity in microbial cultures, and a subsequent interest lag after Cohen’s report until very recently, must be due to the reaction, “what are you going to do with such a small amount of electrical energy” or “so much for so little.” It would be better to generate electricity indirectly by burning the methane microbially produced in sewage disposal plants to operate steam-powered dynamos, and we know this is now done.
A. ENZYMES Electron transport in biological systems is a fascinating subject which has not escaped the attention of physical chemists also interested in chemical fuel cells. It is particularly intriguing that electrons are transported catalytically at ambient temperature and at neutral pH. An efficient transfer of these electrons ultimately to oxygen or other electron acceptor via a wire could indeed be an electrical circuit of some consequence. Limitations due to the above-mentioned inaccessible cellular reactions of microbes, let alone other biological species, may be circumvented at least theoretically by employing enzymes, particularly soluble ones
56
J. B. DAVIS
which, again theoretically, are comparable to any soluble chemical compound at an electrode. When one leaves the realm of theory and enters the realm of reality the paucity of enzymes presently available to us in anything like stable form becomes evident. The rate of reaction between enzyme and substrate at the biological electrode (anode) and the effect of changes at the anode on the activity of the enzyme become matters of concern. Davis and Yarbrough (1962) using glucose oxidase at the anode found that glucose in phosphate-saline solution was not oxidized at the anaerobic anode (nitrogen flushed). Electrons were not accepted by a platinum anodic surface and transferred to an aerated platinum cathodic area to react with molecular oxygen. Ionic union of the two “theoretical half-cells” was through a cellophane membrane impermeable to the enzyme. Lack of a reaction admittedly was not surprising. And it was interesting that upon addition of methylene blue to the biological half-cell a 0.05 ma. current was measured preceded, of course, by a drop in potential of about 0.1 volt. The methylene blue became decolorized and did not return to its oxidized (colored) state. However, it was obvious that electron transfer from methylene blue to the anode was slow and inefficient. Efforts are underway in this laboratory to increase the speed and efficiency of the electron transfer from mediators to the anode. DelDuca et al. (1962) although recognizing the possibilities of a direct enzymatic mechanism for releasing electrical energy have pursued the indirect approach. The enzymic ( urease ) formation of ammonia from urea at the anode in a 3 M potassium chlorideTris buffer electrolyte was employed in a urea-oxygen fuel cell to yield a current density of about 3 ma./cm.z at 0.4 volt. The enzyme reaction in this case served a function only indirectly, the electrochemical reactant being the product ammonia.
B. ATTEMPT TO USE HYDROCARBONS The use of hydrocarbons in a “microbial fuel cell” was the original objective of Davis and Yarbrough ( 1962). Their experiments with ethane gas and nocardia (see Fig. 1 ) were unsuccessful, but a brief summary might be worthwhile since the principles were successfully employed in experiments with glucose. Since electron transfer from glucose oxidase to the anode was unsuccessful without an electron mediator (methylene blue) it was
GENERATION OF ELECTRICITY BY MICROBIAL ACTION
57
not surprising that ethane was not oxidized at the anaerobic anode by an ordinarily highly oxidative cell suspension of ethane utilizing nocardia. But when methylene blue was added to the anode there was still no reaction. That is, the electrode potential did not decrease, nor was methylene blue decolorized due to ethane oxidation. This was interpreted to mean that methylene blue could not serve as an electron acceptor in the metabolism of ethane by nocardia. When glucose in the absence of methylene blue was substituted for ethane there was still no decrease in electrode AMMETER
1
CZH6
NOCAROIA,
20
CzH4 + 2 H
+
:/I
Oz+t$O -2OH-
2 H -+-2Ht+2a
2H++m 20H-+2H
0
FIG. 1. Hypothetical microbial fuel cell involving dehydrogenation, ethane as fuel.
potential, but after adding methylene blue, the electrode potential dropped rapidly, the E.M.F. increasing from about 0.1 volt to 0.3 volt. Current increased from 0 to 2 ma. Note that in Fig. 1 the dehydrogenation of ethane is hypothetically proposed. Recent evidence suggests ( Stewart et at., 1959) that the physical incorporation of molecular oxygen into the hydrocarbon molecule is the initial oxidation step. This requires the direct participation of molecular oxygen mediated by an oxygen transferase or oxygenase enzyme as discussed by Mason (1957). This eliminates the use of ethane as fuel in the manner proposed since even small amounts of oxygen at the anode prevent a decrease in electrode potential. C. Escherichia coli Some bacteria such as E . coli are versatile in that they oxidize compounds in the presence or absence of molecular oxygen or
58
J. B. DAVIS
methylene blue. They are facultatively anaerobic since they use “built in” electron acceptors under conditions of anaerobiosis. Employed in a biological half-cell these bacteria metabolize glucose and the redox potential changes very rapidly. Table I Er FECT
OF THE
TABLE I METABOLISM 01. GLUCOSE ’ BY Escherichia coliB
Biological half-cell
Oxygen half-cell
Electromotive force, millivolts
Glucose, N, Glucose, N, Added E . coli Added 2.5 mg. methylene blue Added 2.5 mg. methylene blue
Glucose, N, Glucose, 0,
148
0 625
At 1000 ohms, millivolts 0 33 511
No further change No further change
%Davis and Yarbrough (1962). The half-cells were separated by il semipermeable cellophane membrane, each had a volume of about 400 ml. Platinum foil was used for electrodes in both half-cells, connected to potentiometer.
summarizes an experiment in which nitrogen was bubbled into an oxygen half-cell at the beginning of the experiment, as well as into the biological half-cell. The system throughout contained an aqueous solution of 1% glucose and 1% NaCl in 0.05 molar phosphate buffer at pH 7. Upon addition of oxygen to the oxygen half-cell a potential difference occurred (Table I ) which was normal for the system. When nutrient agar grown, washed cells (75 mg., dry wt. ) of E. coZi were added to the biological half-cell, dramatic results occurred. The increase in E.M.F. was rapid and the approximate 500 millivolts measured at 1000 ohms resistance (approx. 0.5 ma.) were sustained for over an hour, at which time the experiment was terminated. It was of particular interest that the addition of methylene blue, which decolorized immediately, had no effect on the voltage. As mentioned above, E . coli does not require methylene blue to serve as an electron (hydrogen) acceptor substitute for oxygen. Therefore, when E. coli cells are in a very active state, even though methylene blue is readily reduced, it apparently has no measurable additive effect on the reducing conditions of the system. If the cells metabolize at a somewhat slower rate the addition of rnethylene blue causes a decrease in
GEXERATION OF ELECTRICITY BY MICROBIAL ACTION
59
the electrode potential and a consequent increase in available current.
D. SALTBRIDGE VERSUS CELLOPHANE MEMBRANE
A marked decrease in current was observed when a salt bridge was substituted for the cellophane membrane employed as described above (Table 11). The data of Table I1 show ( 1 ) that methylene blue at the electrode in the absence of bacterial cells has no effect; ( 2 ) methylene blue in the biological system produces a TABLE I1 COhfPAHISON OF CURRENT OBTAINED WITH M E M H R A N E SYSTEM VERSUS A
SATURATEDSALT BRIDGE^
Biological half -cell Membrane system: No cells, 190 glucose, N, Added 10 mg. methylene blrich Added E. colic Added additional E. coli Added 15 mg. methylene hlucc Added 25 mg. methylene bluec Added 25 mg. methylene bluec Transferred to saturated salt bridge . svstcm , n b c
Electromotive force, millivolts
9s 98 240 267 275 265 265
-
Milliamperes
0.05 0.05 0.6 0.5 1.4 2.0 2.0
0.4
Davis and Yarbrough ( 1960). Methylene blne did not decolorizc. Methylene blue decolorized.
constant current which may be increased stepwise by further additions of methylene blue; and ( 3 ) a maximum current is finally reached, following which the addition of methylene blue has no effect. Further experimentation with salt bridge and cellophane membrane systems showed that, alternately, a current of 0.2 ma. at a pressure of 0.3 volt (with salt bridge) increased to 2.5 ma. when the system with cellophane membrane was used.
E. POTASSIUM FERRICYANIDE The effect of a strong oxidizing agent on the system was tested while nocardia were metabolizing glucose. After the experiment was underway 1 mg. of K3Fe(Cn)6was added to the biological half-cell. The potential and current immediately dropped to zero
60
J. B. DAVIS
and the methylene blue which had been previously reduced became oxidized (blue). After 10 minutes the methylene blue became reduced again and the potential and current returned to their respective values. When K3Fe(Cn), was added to the oxygen half-cell strong current increase occurred. The data are summarized in Table 111. A test was then made of the effect of relatively large TABLE I11 EFFECT OF POTASSIUM
FERHICYANIDE ON THE
Electromotive force, Millimillivolts amperes
0, half-cell
Biological half-cell 1%glucose, N, Added nocardia Added 1.5 mg. methylene blue Added 1 mg. K,Fe( Cn), After 10 minutes Added 25 mg. methylene blue
CURRENT MEASUREMEN@
1% glucose, 0,
Added 1 mg. K,Fe( Cn) t) Added 1mg. K3Fe( Cn ) Added 1mg. K,Fe( Cn),
,
a
72 97 338 0 340 340 580 580 600
0 0 0.2 0 0.2 1.2 2.5 4.0 4.5
Davis and Yarbrough ( 1980).
amounts of potassium ferricyanide in the absence of microbial cells. The current increased 16-fold when E . coli cells were added (see Table IV). The data show that microbial cells are required to TABLE IV EFFECT OF POTASSIUM FERRICYANIDE IN THE PRESENCE AND ABSENCEOF E. coli CELLS~
0, half-cell
Biological half-cell 1%glucose, N,
1% glucose, 0, Added 50 mg. K3Fe(Cn ) tl
Added 50 mg. K,Fe( Cn ) Added 450 mg. K,Fe( C n ) ti Added 5 mg. methylene blue Added E. coli: After 5 minutes After 15 minutes After 30 minutes a
Davis and Yarbrough ( 1960).
Electromotive force, Millimillivolts amperes 92 260
0 0.2 0 0.1 0.I
0.5 1.2 1.6
GENERATION OF ELECTRICITY BY MICROBIAL ACTION
61
obtain a significant current measurement; fairly large amounts of potassium ferricyanide alone, or with methylene blue, do not result in much current in the absence of metabolizing cells. Actually a greater current was measured in the presence of microbial cells when potassium ferricyanide was present at both electrodes (see particularly Table 111). Iron may act as a better “absorber” of electrons at the electrode than oxygen, accounting for the increase in current when potassium ferricyanide is added to the oxygen electrode: plus electron FeS+
\
+ Fez+ gained from electrode /
regenerated by oxygen
The biological half-cell system must be reduced in order to produce a potential difference and furnish a source of current. However, methylene blue acting as a hydrogen (proton plus electron) acceptor in this system may not readily give up electrons to the electrode. If potassium ferricyanide is also present at the biological electrode the reverse of what happens at the oxygen electrode may occur: Fea+
\
minus electron lost to electrode
>
Fe3+
/
regenerated by methylene blue
Thus, iron may act as a catalyst at each electrode.
F. FURTHER RESEARCH Obviously there is a great deal of experimental work to be done when one considers the large number of metabolic systems that have not been tested. But it would appear that great advance in electrode design and materials are required, that extremely efficient electron mediators are another requirement, and that soluble enzymes afford the best physical state, theoretically, for efficient reactivity in the biological half-cell. Biological activity may have auxiliary uses in the operation of batteries or fuel cells. Hydrogenase conceivably might be employed as a hydrogen depolarizing agent at the cathode. As an example,
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J. B. DAVIS
methylene blue could be used at the cathode to accept hydrogen from hydrogenase. By cycling, the methylene blue could be reoxidized by oxygen and alternately reduced by polarization hydrogen mediated by hydrogenase. Sadana and Morey (1959) reported, interestingly, that the hydrogenase of DesuZfouibrio desidfuricans required added iron to react with methylene blue.
IV. Corrosion Cell Iron can be used as a fuel to generate electricity with bacteria serving indirectly as the anodic depolarizing agent and also, theoretically, as the cathodic depolarizing agent. When iron corrodes an electrolytic cell is established. In the presence of oxygen (no bacteria) the reactions are simply: (Anodic) (Cathodic)
+ +
Fe + Fez+ 2e 2e S O , H,O + 20H20H- -tFez+ + Fe( OH), corrosion product
+
Under strictly anaerobic conditions corrosion of iron can proceed with the aid of sulfate-reducing bacteria, for example, D. desulf uricans: (Anodic) (Cathodic)
F e + Fez+ + 2e 2e 2H+ + 2H (hydrogen of polarization)
+
Sulfate-reducing bacteria use hydrogen in the reduction of sulfate ion to sulfide ion. In this manner, the cathodic hydrogen of polarization may be used to reduce sulfates to hydrogen sulfide, which then reacts with ionic iron at the anode to produce iron sulfide, the corrosion product. Actually a driving force in this corrosion process is the concomitant oxidation of organic compounds by the sulfate-reducing bacteria. Autotrophic metabolism by D. desulfuricuns, although essentially demonstrable in cultivation ( Postgate, 1959) has not been demonstrated as being responsible for the principal corrosion process. To the contrary, the presence of organic compounds utilizable by sulfate-reducing bacteria greatly accelerate bacterial corrosion. This, in effect, emphasizes the importance of the anodic depolarization reaction. That is, the oxidation (dehydrogenation) of organic matter coupled with the reduction of sulfate ions generates hydrogen sulfide which acts as the depolarizing agent
GENERATION OF ELECTRICITY BY MICROBUL ACTION
63
at the anode, producing iron sulfide, thereby driving the corrosion reaction. Because of the very active hydrogenase present in the sulfatereducing bacteria (Sadana and Morey, 1959; Riklis and Rittenberg, 1961) these bacteria can serve, at least incidentally, as hydrogen depolarizing agents at the cathode. Thus, both organic compounds and iron serve as fuel in the corrosion process involving the activities of sulfate-reducing bacteria. The amount of current that can be derived from such a process is dependent on the electrode potential developed at the anode. Horvath (1960) published an excellent paper on anaerobic microbiological corrosion in which he presents data on electrode potentials observed in laboratory cultures of sulfate reducers. Mild steel electrodes showed an increase in E.M.F. of about 0.1 volt (calomel scale) due to bacterial activity. He did not measure closed-circuit voltage, but it undoubtedly would be quite small. At the resistance inherent in the circuit the current probably would be in the order of microamperes. However, the corrosion cell involving the activities of bacteria is obviously a source of current. Modifications in electrode design, the use of bacterial hydrogenase as depolarization agent at the cathode in conjunction with an effective sacrificial anode, or other means may be found to increase the generation of electricity from such a corrosion process. Goldner et al. (1962) reported recently the use of a sacrificial (magnesium) anode and sulfate-reducing bacteria at a porous iron cathode, ostensibly to act as depolarizing agents. The effectiveness of this depolarization in increasing current densities over those achieved by the magnesium-iron couple in the absence of bacteria was not specified in their report.
V.
Summary
Over fifty years ago the pioneering work of Potter (1911) indicated the effect microbes and extracellular microbial enzymes have on reduction-oxidation potential, and he pointed to a consequent small utilizable source of electrical energy. Recently due to a revival of interest in fuel cells or continuous feed batteries attention is turned again toward this biological source of electricity. But this source should be kept in perspective with scientific facts and data.
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J. B. DAVIS
There are at least three means by which biochemical reactions may be employed to either produce or promote the production of electricity. ( 1) Microbial (or specific enzymic) activity at the anode exemplified by biochemically catalyzed dehydrogenation results in activated hydrogen which dissociates at the electrode. Electrons are absorbed and hydrogen ions are formed which via electrolyte reach and react with oxidant ions. ( 2 ) Biochemical (enzymic) reactions may be employed for the purpose of producing the electrochemical reactant, i.e., the biochemical reaction is not intrinsic to the generation of current but to the generation of the fuel, and may be employed in or at the electrode. ( 3 ) Microbes or enzymes may be employed as depolarizing agents, e.g., in the utilization of cathodic hydrogen, thereby increasing the efficiency of an already established electrolytic cell.
REFERENCES Cohen, B. (1931). J. Bacterial. 21, 18-19. Davis, J. B., and Yarbrough, H. F. (1960). Unpublished data. Davis, J. B., and Yarbrough, H. F. (1982). Science 137, 815-818. DelDuca, M. G., Fuscoe, J. M., and Zurilla, R. W. (1982). Symposium: “Biochemical Fuel Cells,” 19th General Meeting of the Society for Industrial Microbiology, Corvallis, Oregon. Goldner, B. H., Otto, L. A., and Canfield, J. H. (1932). Symposium: “Biochemical Fuel Cells,” 19th General Meeting of the Society for Industrial Microbiology, Corvallis, Oregon. Harris, J. 0. (1980). Corrosion 16, 441t-448t. Hewitt, L. F. ( 1950). “Oxidation-Reduction Potentials in Bacteriology and Biochemistry,” Williams & U’ilkins, Baltimore, Maryland. Horvath, J. (1980). Actu Chirn. Acad. Sci. Hung. 25, 65-78. Mason, H. S. (1957). Aduun. EnzymoZ. 18, 79-233. Nachmansohn, D. ( 1951). In “Phosphorus Metabolism” ( W. D. McElroy and B. Glass, eds.), Vol. I, 568-585. Johns Hopkins Press, Baltimore, Maryland. Postgate, J. (1959). Ann. Reu. Microbiol. 13, 505-520. Potter, M. C. (1911). Proc. Roy. SOC. (London) B84, 280-278. Riklis, E., and Rittenberg, D. (1961). J. Biol. Chem. 236, 2528-2529. Sadana, J. C., and hlorey, A. V. ( 1939). Biochim. Biophys. Acta 32, 592-593. Sisler, F. D. (1981). New Scientist No. 258, 110-111. Stewart, J. E., Kallio, R. E., Stevenson, D. P., Jones, A. C., and Schissler, D. 0. (1959). J. Bncteriol. 78, 441-448.
Microorganisms and the Molecular Biology of Cancer G. F. GAUSE lnstitute
of
Antibiotics, Academy of Medical Sciences, Moscow, U.S.S.R.
I. Introduction ........................................... 11. The Molecular Biology of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . A. Some Problems of Methodology ....................... B. The Deletion Hypothesis ............................. 111. Microbial Models of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Respiratory Deficient Mutations in Yeast . . . . . . . . . . . . . . . B. Respiratory Deficient Mutants in Staphylococcus uureus ... C. Respiratory Deficient Mutants in Staphylococcus afermentuns D. Respiratory Deficient Mutants in Eschetichiu coli . . . . . . . . IV. Microbial Models of Cancer as Sources of Biological Inhibitors V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
65 68
66 68 69
69 71 82 87 a7 90 91
I. Introduction It is comparatively recent that microorganisms have emerged from their role as eccentric and exceptional manifestations of life, of interest mainly for their importance in medicine and agriculture, to become model systems for probing general biological phenomena at the level of molecular organization and behavior (Hayes, 1962). The present dramatic advances in the field of molecular biology stem mainly from the study of bacteria and of their viruses. At the same time it becomes increasingly clear that cancer belongs to the realm of molecular diseases, and should be attacked from the viewpoint of its molecular biology. In spite of some controversies, there is a growing body of evidence that some specific defects or deletions in biosynthetic systems occur in tumor cells, as well as in the systems responsible for energy-yielding metabolism. I t is therefore very attractive to attempt to elicit some similar defects at the molecular level in the cells of microorganisms, and to use the “equivalents” of cancer thus obtained for fundamental studies concerning the nature of malignancy. There is good reason to anticipate that microbial equivalents of cancer could very substantially contribute to better understanding of the molecular biology of malignant change. As Francis Bacon noted long ago, 65
66
G . F. GAUSE
“No one successfully investigates the nature of a thing in the thing itself; the inquiry must be enlarged, so as to become more general” (Novum. Organunt, 1 0 ) .
11. The Molecular Biology of Cancer A. SOMEPROBLEMS OF METHODOLOGY
There are two points which should be taken into account before any consideration of the molecular biology of cancer. The first is that human cancer represents a multitude of diseases with subtle similarities and striking divergencies, and there may be a multitude of molecular distortions at the bottom of these malignancies; nevertheless, there may also be some key alterations similar or even identical in various cases. The second point is concerned with “controls” for tumor cells. i.e., all biochemical measurements on tumors should be carefully compared with appropriate control values. This point was discussed by S. and A. Fiala (1959), and the following is a quotation from their paper (p. 149): “It has been argued that in certain tissues such as pancreas or lymph nodes the amount of mitochondria is also low and that it is incorrect, therefore, to ascribe any importance to the small quantity of mitochondria in tumors. This criticism is invalid because it is meaningless to compare pancreas with hepatoma; pancreas should be compared with pancreatic tumor and lymph nodes only with lymphoma. . , . In special instances it may be wrong to assume that the homologous normal tissue is the adequate control of a tumor. Thus by comparing the respiration of normal mouse epidermis with skin tumors it was concluded that tumor growth leads to an increase of succinoxidase activity and a greater respiration. Notwithstanding the fact that any comparison must be done on a per cell basis, one must point out that all the normal epidermal cells are not the natural counterpart of the skin tumor. Much of the epidermis consists of very differentiated cells the cytoplasm of which is almost completely filled out with tonofibrils with only few remaining preformed cytoplasmic elements. These cells are keratinized and, consequently, have a low respiration. There the loss of metabolic function is the result of normal differentiation in order to assume a specialized protective function. Skin tumors, on
MOLECULAR BIOLOGY OF CANCER
67
the other hand, stem from the cells which preceded normal differentiation. It is from the basal cells that both normal differentiation and malignant transformation stem and with which the only valid comparison can be made. It seems, thus, that all main objections against the thesis of mitochondria1 depletion in tumors can be answered satisfactorily.” There is no doubt that many controversies in the field of biochemistry of cancer may depend upon the inaccessibility of adequate control cells. Most, if not all, of the neoplastic cells previously studied by many investigators in fact had no adequate controls; therefore little confidence can be placed in many comparisons made up to now. A plea for extensive biological experimentation in this ficld has been made by Potter (1962), and the following suggestions made by this author are very significant (p. 3 7 2 ) : “What are the minimal deviations that must be effected in a normal cell to make it a malignant cell? We must be able to define malignancy and to test for it. . . . What is beginning to emerge are the ideas that in the over-all transition from normal cells to cancer cells there is an accumulation of changes, that evolution by selection occurs throughout the life of a neoplastic cell population. . . . It seems likely that successive changes occur in the conversion of normal cells to cancer cells and that many of the observed differences between cancer cells and normal cells may be completely irrelevant to the transformation. Thus there are two aspects to the biochemistry of cancer cells. One is the definition of the strategic changes that are able to result in carcinogenesis, and the other is the definition of the most commonly evolved end result5 of cancer evolution, i.e., the most frequently encountered chemotherapeutic problems.” The significance of biochemical observations made on tumor cells that surely represented advanced stages of cancer evolution is another subject for discussion. The continued selection in cell populations for enhanced malignancy goes on in a certain definite direction, and the end result of this selection may make the direction of the process more clearly visible than it is at its very beginning. It may therefore happen that essential molecular aspects of malignancy could be more easily recognized at the advanced stages of evolutionary orthoselection going on in the specific direction.
68
G.
F. GAUSE
B. THEDELETION HYPOTHESIS
In a carefully written short book, Chmistry of Enzymes in Cancer, Bergel (1961) summarized some important aspects of the molecular biology of malignant growth. Discussing the present state of knowledge with respect to certain important groups of enzyme systems, he concludes that those concerned with nucleic acid catabolism are deficient in tumors; those concerned with protein and amino acid catabolism are deficient at least in some tumors; and in the enzymes of carbohydrate breakdown, among many other differences, phosphorylase is deficient. All these observations support the “deletion” theory of cancer, developed mainly by Potter (1957, 1958), according to which the cancer cell has lost certain important constituents, probably concerned in the synthesis of enzymes and of tissue specific surface proteins, thereby permitting metastasis and invasion. Although the architecture of these deletions is still far from clear, it appears that the fundamental difference between normal and cancer cells must lie in the chemistry of their deoxyribonucleic acids (DNA), and that the changes in enzyme concentrations which are observed must necessarily result from this change in the DNA, through mechanisms the study of which is still in its infancy. Among many deficiencies and deletions, the distorted respiration of tumors is of particular significance (Warburg, 1930, 1956). It is significant not because “faulty” or “impaired” respiration is fundamental to the nature of cancer and represents the “true cause” of malignancy, but because malignancy is often accompanied by some specific distortions in the energy-yielding metabolism and can better be recognized in the wide biological perspective from the viewpoint of these specific distortions. It appears to be clearly established that the oxidative rate of tumors is fixed, and quite refractory to the stimulation seen when excess carbohydrate or fatty acid substrate is added to most normal tissues (Aisenberg, 1961a). It might be said, therefore, that the respiratory activity of neoplastic material is a maximal one, while normal cells have ample reserves. This specific impairment of tumor respiration is accompanied by the complete or nearly complete deletion of some components of the cytochrome system (Monier et al., 1959). The study of the way in which these secondary distortions in the energy-yielding metabolism are related to
MOLECULAR BIOLOGY OF CANCER
69
the primary alterations in the nucleic acids represents one of the most important problems of contemporary cancer research. The impaired respiration represents only a special case of deficiency in biochemical control mechanisms. The control mechanisms of enzyme synthesis of the tumor cell are not working as they do in the normal cell (Pitot et al., 19Sl). It is expected that the study of microbial “models” of cancer could be particularly useful for the further advancement of knowledge in this fundamental field.
111.
Microbial Models of Cancer
A. RESPIRATORY DEFICIENT MUTATIONS IN YEAST The discovery of respiratory deficient mutations in yeast stimulated the interest in possible microbial models for cancer cells and was also helpful in the search for similar mutations in other microorganisms. The occurrence of respiratory deficiency in yeast was for the first time described by Stier and Castor (1941). The mutant culture was produced by exposure to cyanide, and possessed the following metabolic characters: Qo2
Qco2air
31.8 (parent)
4.7 (mutant)
158.5 (parent)
238.5 (mutant)
Stier and Castor (1941) noted that metabolic organization of mutants was remarkably different from that of their parents, as far as mutants possessed impaired respiration and enhanced glycolysis. Similar mutants were induced in yeast by ethylene oxide (Whelton and Phaff, 1947), and it was noticed that cytochromes a and b are deleted in mutants. Ephrussi et al. (1949) induced these mutants by acriflavine and studied them in great detail. They observed that respiratory deficient mutants of yeast are less efficient in the utilization of sugars than the normal yeast and form small colonies. It is well known that Warburg since 1930 has claimed that cancer cells are biochemical mutants of normal cells which acquired enhanced glycolysis and impaired respiration. From the point of view of his observations the metabolic organization of yeast mutants with impaired respiration and enhanced glycolysis has much in common with metabolic organization of tumor cells.
70
G . F. GAUSE
This is the reason why yeast mutants were repeatedly discussed as a microbial model of cancer (Lindegren, 1959). Although Warburg’s theory of cancer stimulated the investigation of the first microbial model of cancer, both the theory and the model appear to be oversimplified at present. The yeast model represents a very special case from the point of view of its metabolism, as far as respiration in mutants is not only impaired but practically abolished. It is also very special genetically, as far as the frequency of respiratory deficient mutants spontaneously appearing in ordinary yeast cultures is in the neighborhood of 1%. Such a frequency of spontaneous mutations is unusually high as compared with that of other biochemical mutants. On the other hand, Warburgs theory in its original form did not take into account that the glycolysis in cancer cells is not always enhanced; it may in some cases be very low ( Aisenberg and Morris, 1961). At the same time respiration in tumor cells is not abolished, as in yeast mutants, but impaired in a more subtle way; it may be not so much quantitatively deficient as “fixed,” and quite refractory to the stimulation seen when carbohydrates are added to normal tissues (Bergel, 1961). The similarity of the respiratory deficient mutants of yeast to cancer cells is therefore a rather remote one, but it may have heuristic value in the search for better models in other microorganisms. Respiratory deficient mutants in yeast have been studied recently in many laboratories and in great detail, particularly from the point of view of their induction by various agents. The literature in this field has been reviewed by Nagai et aZ. (1961), and there is no need to repeat it here. Mutants with impaired respiration in yeast can be induced by ultraviolet radiation, as well as by three groups of chemical agents, namely, basic dyes, heavy metal salts, and respiratory enzyme inhibitors. The mechanism of action of these inducers is not known, but there are some speculations concerning the primary interaction of basic dyes and of ultraviolet radiation with nucleic acids (Raut and Simpson, 1955; Nagai et al., 1961). It is also interesting that mutants of bacteria with impaired respiration have not been produced so far by many good inducers of the respiration deficiency in yeast (Gause et al., 1958). On the other hand, carcinogenic agents such as benzpyrene and methylcholantrene failed to produce the respiration deficient mutants in yeast and bacteria (Gause et al., 1957a,b; Nagai et aZ., 1961). It
MOLECULAR BIOLOGY OF CANCER
71
appears therefore that indncers of respiration deficiency are rather specific for various groups of organisms (Gause, 1959). Some interesting observations have been published recently on the induction of heritable respiratory deficiency in yeast by pantothenate starvation (Sarachek and Fowler, 1961). The extensive mutation which pantothenate-starved yeast cells undergo at the inception of the stationary phase of aerobic growth can be attributed to their oxidative depletion of that limited supply of endogenous acetate which is essential for biosynthetic purposes. Thus acetate metabolism does appear to be of basic importance in the genetic maintenance of cellular respiratory competence. The respiratory activity of the normal mitochondrion depends uniquely on the structural orientation of its constituent enzymes. Lipids and ribonucleic acid along with the enzymic proteins themselves contribute to the preservation of this organization. While it may be assumed that the biosyntheses of mitochondrial enzymes, like those of other enzymes, are genetically directed, the fact that mitochondria do not arise de nouo shows that their fabrication is contingent on the structural organization of preexistent mitochondria. Thus treatments which upset normal mitochondrial organization might be expected to give rise to lines of mutant mitochondria exhibiting depressed respiratory activities. The heritable loss of respiratory competence induced in yeast cells by pantothenate starvation can most simply be ascribed, at present, to such a disorganization resulting from a cellular deficiency for particular fatty acids. The reduction in amount of mitochondria during carcinogenesis, which results in the damaged respiration of tumor cells, has been demonstrated for various animal tumors in different laboratories (Lettre and Sachsenmaier, 1957; Fiala and Fiala, 1959; Aisenberg, 1961b). It appears therefore that the study of induction of mitochondrial depletion in the respiratory deficient mutants of yeast might be helpful for better understanding of some aspects of molecular biology of cancer.
DEFICIENT MUTANTSIN Staphylococcus aureus B. RESPIRATORY Respiratory deficient mutants in S. aureus were first induced by ultraviolet radiation (Gause et at., 1957b), with the idea of obtaining a new microbial model of cancer different from the yeast model. To some extent this attempt has been successful, as far
72
G . F. GAUSE
as the system induced in this organism appeared to be truly remarkable among many other microbial models of cancer. It was later observed that mutants with impaired respiration in the culture of S. aureus 209 also appear spontaneously, with the frequency 1 x 108 cells, and these spontaneous mutants are practically identical with those induced by ultraviolet radiation ( Gause el: al., 1960). The best way to observe spontaneous mutants is to use the liquid nutrient medium containing 5-fluoro-2-deoxyuridine ( 100 pg./ml. ), selectively inhibiting the growth of parent staphylococci and in this way favorable for the multiplication of mutants with impaired respiration which are not inhibited (Gause et ul., 1961). If, after 5-7 days of incubation in this medium at 37°C. the contents of the tubes were plated on nutrient agar containing 5-fluoro-2-deoxyuridine (25 pg./ml.), in some cases pure cultures of mutants were observed instead of the parent culture. Numerous experiments have shown that the quantity of inoculum added to the selective medium is of great importance. If one adds too many parent cells, these acidify the medium and inhibit the growth of mutants, which require a neutral or slightly alkaline medium for their multiplication. If the parent cells are too few, the probability of occurrence of mutants among them is negligible. It was observed that the respiratory coefficient on glucose is decreased in various strains of mutants of S. aureus by 40-600/0 as compared to parent values, while respiration is less sensitive to cyanide (Gause et al., 1957b). It is known that the consumption of oxygen in many strains of cancer cells may often decrease to half the value characteristic for normal cells. In other words, the impairment of the respiratory system of cancer cells is quantitatively more like that of biochemical mutants of staphylococci than it is like the respiratory deficiency in the mutant strain of yeasts, where respiration is practically abolished under aerobic conditions. Further studies have shown (Gershanovich et al., 1962) that endogenous respiration in parent and mutant staphylococci is of the same order, but that respiration rates ( Q O , ) , measured in presence of such added substrates as glucose, lactate, acetaldehyde, ethanol, and formic acid, increase to a much higher degree in normal staphylococci than in their mutants (Table I ) . In other words mutants are quite refractory to the stimulation seen when glucose and some other substrates are added to normal staphy-
73
MOLECULAR BIOLOGY OF CANCER
lococci. This kind of respiratory deficiency has been observed in tumors ( Bergel, 1961) . It was recorded that respiratory-defective mutants of S . aureus contain much less catalase activity than the cells of the parent strain (Gause et al., 1961). It is remarkable that Warburg (1959) considers the decreased catalase content as one of the most important biochemical features of tumor cells. Because of it, tumors are preferentially sensitive to hydrogen peroxide, which shows up in tissues under the action of X-rays. TABLE I THE EFFECTOF ADDITIONOF VARIOUS SUBSTRATES UPON RESPIRATION IN PARENTSTAPHYLOCOCCI ( S . aureus 209) AND IN MUTANTSuv-3 WITH IMPAIRED RESPIRATION^
Qo2 Substrate None Glucose Lactate Acetaldehyde Ethanol Formic acid a
Parents * 30 169
77 95 91 180
Mutants 36 42 40 51 61 38
From Gershanovich et al. (1962).
Whereas normal staphylococci reveal three bands of cytochromes, a2 (630 mp), al (600 mp), and bl (557 mp), in mutants the band a2 is not visible, either under usual conditions of observation or when liquid nitrogen with the subsequent devitrification is employed. Instead of band bl, two new bands are observed in mutants, at 563 and 553 mp, which are easily oxidized and reduced in the presence of succinate (Gause et al., 1961). In mutants of S. auww with the impaired respiration not only various distortions in the energy-yielding metabolism have been recorded; what is of particular significance is the specific vulnerability of nucleic acids in these mutants, which was observed for the first time on this model using various inhibitors with the already known mechanisms of action (Gause and Kochetkova, 1961). Three groups of inhibitors were used in this work, namely inhibitors of protein synthesis, inhibitors of cell-wall synthesis, and inhibitors affecting the nucleic acids. I shall summarize here the results of these studies in some detail.
74
G . F. GAUSE
1. Efects of Inhibitors of Protein Synthesis Gale and Folkes (1953) observed that chlortetracycline and oxytetracycline stopped the growth of staphylococci in nutrient broth by selective inhibition of protein synthesis; nucleic acid synthesis, however, continued in the presence of bacteriostatic concentrations of these antibiotics. Gause and Kochetkova ( 1961) TABLE I1 BACTERIOSTATIC ACTIONOF VAHIOUSINHIBITORSUPON THE GROWTHOF PARENTAND MUTANTCULTURES OF S. aureusa Inhibitors of: ( 1)
Protein synthesis Tetracycline Chlortetracycline
Parentz
Mutants (uv-2, uv-.3 I
0.09-0.10pg./ml.~~ 0.15-0.18 pg./ml.
0.09-0.15~g. ml. 0.20 pg./ml
0.008 units/ml. 0.007 units/ml.
0.06-0.07 irnitr/ml. 0.06 units/ml.
0.0750 pg./ml. 0.250 bg./ml. 3 Ltg./ml. 2 mg./ml.
0.0015 pg./ml. 0.005 pg./ml. 0.01 FLg./ml. 0.2mg./ml.
6-8 pg./ml.
2-3 pg./ml.
( 2 ) Cdl-wall synthesis
Penicillin G Phenoxymethylpenicillin ( 3 ) Nucleic acid synthesis
Mitomycin C Actinomycin C Trypaflavine Nitrogen mustards (Degranol) ( 4 ) Other inhibitors
Chloramphenicol a
b
From Cause and Kochetkova (1961). Minimum inhibitory concentrations in broth.
showed that tetracycline and chlortetracycline inhibited the growth of the parent and mutant staphylococci to the same degree (Table 11). 2. Eflects of Inhibitors of Cell-Wall Synthesis
Bacteriostatic concentrations of penicillin which selectively inhibit biosynthesis of cell wall do not disturb the synthesis of proteins and nucleic acids in staphylococci (Strominger et al., 1959; Rogers and Perkins, 1959). It is clear from Table 11 that the mutant staphylococci, as compared with the parents, were about eight times more resistant to the inhibitory action of the peniciIlins in nutrient broth. It may therefore be suggested that mutants differ from the parent or-
75
1IOLECULAR BIOLOGY OF CANCER
ganism in the biosynthesis of the cell-wall material, which is selectively affected by penicillin. Cell-wall material in the mutants appeared to be more resistant to the disruptive action of penicillin. This is in accord with chemical studies which have shown that the amino acid composition of the cell-wall material in parent staphylococci is different from that of their mutants (Gause et al., 1961). In the tell-wall material of mutants only traces of lysine were recorded while diaminopimelic acid was observed. Since many bacteria possess the capacity to decarboxylate diaminopimelic acid into lysine (Rhuland, 1960), the substitution of lysine by diaminopimelic acid in the cell wall of respiratory-deficient mutants of staplivlococci might indicate the loss by mutants of this specific decarboxylase.
3. EfFects of Inhibitors of Nucleic Acid Formation a. Mitomycin C . Shiba et at. (1959) observed that mitomycin C in dilute solutions selectively inhibited the formation of deoxyribonucleic acid (DNA) in bacteria, while synthesis of protein and ribonucleic acid (RNA) remained unaffected. This was confirmed by Laiko (1962) for parent culture of S. aureus 209, and the corTABLE 111 EFFECTSOF MITOMYCINC AND OF ACTKNOMYCIN C UPON THE GROWTHAND SYNTHE5lS OF NIJCLEIC k I D S I N PARENT STAPHYLOCOCCI ( s. UUTeUS 209)"r" Time of incubation (hours)
DNA
RNA 11
0
Actinomycin C None
0.35 bg./ml.
2
0.247 0.485 0.715
0.247 0.486 0.725
0.238 0.420 0.712
0.238 0.339 0.451
0
0.150
0.150
1
0.201
0.149
I
3
0.247
0.147
0.121 0.151 0.208
0.121 0.128 0.158
0 1 2
0.495 0.720 1.130
0.495 0.740 1.140
0.526 0.899 0.871
0.536
0
Turbidity
- Mitomycin C None 0.1 kg.iml.
1
0.473 0.479
From Laiko ( 1962). Numbers in italics indicate selective inhibition.
responding data are presented in Table 111. Gause and Kochetkova (1961) showed that mutant staphylococci were 50 times more vulnerable than the parent culture to the action of mitomycin C (Table 11).
76
G . F. GAUSE
b. Actinomycin C . Reich et aZ. (1961a) reported the selective action of actinomycin C on RNA synthesis in bacteria. This was also recorded by Laiko (1962) for parent culture of S . aureus 209, and data are given in Table 111. Gause and Kochetkova (1961) observed that the mutants were 50 times more vulnerable than parents to the inhibitory action of actinomycin C (Table 11). c. Trypaflavine. Trypaflavine ( 3,6-diamino-lO-rnethylacridine chloride) is a mitotic poison which selectively inhibits DNA synthesis (Morthland et al., 1954). Gause and Kochetkova ( 1961) observed that with this compound the mutants were 300 times more vulnerable than the parents (Table 11). d. Nitrogen Mustards. The nitrogen mustards as a group arc potent inhibitors of DNA synthesis. By carefully grading the doses, a value can be chosen where synthesis of DNA is blocked completely, while synthesis of protein and RNA continue (Shepherd, 1958) . Degranol [ 1,6-bis-( P-chloro-ethylamino) -1,6 deoxy-d-mannitol] was used by Gause and Kochetkova (1961) as a represcntative of this group of compounds; it is a combination of nitrogen mustard with mannitol and is soluble in water. The mutants were 10 times more vulnerabIe than the parents to the action of Degranol (Table 11). The selective inhibition of growth of mutant staphylococci has been recorded for other nitrogen mustards ( Gause, 1960). Triethylene melamine, which specifically affects pyrimidines in the synthesis of bacterial DNA (Szybalski, 1960a), also selectively inhibits the growth of mutant staphylococci (Gause et al., 1961). e. Halogenated Pyrimidines. This group of compounds is of particular interest for the study of the vulnerability of nucleic acids in mutant staphylococci with impaired respiration. It is known that 5-fluorouracil acts as a specific inhibitor of RNA synthesis; it can be incorporated into bacterial RNA, where it may replace, in part, the normally present uracil. The synthesis of DNA, however, is not affected (Horowitz and Chargaff, 1959). Gause and Kochetkova ( 1961) observed that 5-fluorouracil at 125 pg./ml. inhibited the growth of the parent staphylococcus in broth. Addition of thymine (100 pg./ml.) to the nutrient medium produced no effect, but addition of uracil annulled the inhibitory action of 5-fluorouracil. The annulment of the inhibitorv action of 5-fluorouracil in the presence of uracil was competitive (see data presented in Table IV).
77
MOLECULAR BIOLOGY OF CANCER
TABLE IV CONCENTRATIONS O F 5-FLUOROURACIL
REQUIRED
GROWTHOF PARENTSTAPHYLOCOCCI AND PRESENCE OF \'ARIOUS
Microorganisms Parent staphylococci The same The same The same
u
OF
TO CAUSE INHIBITION OF THEIRMUTANTSIN THE
CONCENTRATIONS OF URACILu
Uracil (%./Id. ) 0 10 100 1000
5-Fluomuracil (yg./ml. 1 125 200 500 750
Mutants UV-3 0 The same 10 The same 100 The same 1000 From Gause and Kochetkova (1961).
2 6 25 75
For the mutant staphylococci (uv-2, uv-3) the inhibitory concentration of 5-fluorouracil was 2 pg./ml.; this inhibitory action was not affected by thymine (100 pg./ml.), but with uracil it was competitively annulled (Table IV). It may be concluded that in the parent and mutant staphylococci 5-fluorouracil specifically inhibited RNA synthesis, but did not affect DNA synthesis. It appears, therefore, that the synthesis of RNA in mutants was about 60 times more vulnerable to the action of 5-fluorouracil, as was the parents. It is of interest that 5-fluorodeoxyuridine (FUDR), in experiments with various bacteria, specifically inhibited synthesis of DNA (Cohen et al., 1958). Game and Kochetkova ( 1961) observed that FUDR at 25 pg./ml. inhibited the growth of the staphylococcus in broth. Addition of uracil (100 pg./ml.) produced no effect, but addition of thymine competitively annulled the inhibitory action of FUDR on the parent staphylococcus (Table V). TABLE V CONCENTRATIONS OF FUDR REQUIRED To CAUSEINHIBITION OF GROWTHOF OF VAF~IOUS PARENTSTAPHYLOCOCCI IN THE PRESENCE CONCENTRATIONS OF THYMINE^
a
Thymine
FUDR
(w/ml.)
(PLR./ml.)
0 10 100 1000
25 60 100 200
From Gause and Kochetkova ( 1981).
'is
G . F. CAUSE
By inhibiting the synthesis of thymine, FUDR apparently interfered with the formation of DNA in the parent staphylococci. It is remarkable, therefore, that FUDR did not inhibit the growth of the mutant staphylococci with impaired respiration (uv-2, uv-3, and all other available mutants of this type), up to maximal concentration tested (1000 pg./ml.). If FUDR is a specific thymine synthetase inhibitor, the absence of inhibitory action on the mutant staphylococci points to some deficiency in the enzymic mechanism of synthesis of thymine in these mutants. Gause and Kochetkova (1961) also observed that certain bromiiiated pyrimidines ( e.g., 5-bromouracil, 5-bromodeoxyuridine, BUDR) did not inhibit the growth of either parent or mutant staphylococci in nutrient broth, up to the maximal concentration tested (500 pg./ml. ). Lorkiewicz and Szybalski ( 1960) observed that in the presence of FUDR thymine synthetase was inhibited and a state of thymine deficiency was produced in the cells, and the incorporation of BUDR, the thymidine analog, into the bacterial DNA can be observed. The substitution of thymidine in the DNA by its brominated analog (BUDR) renders the cells highly sensitive to the killing action of ultraviolet radiation ( Szybalski, 1960b). Gause and Kochetkova were able to reproduce this phenomenon with the parent S . uureus 209. The cocci were grown for 18 hours in nutrient broth containing FUDR (10 pg./ml.), BUDR (10 pg./ ml.), or a mixture of FUDR BUDR. Separately, as well as in admixture, these substances did not inhibit the growth of the staphylococci in the concentrations tested. Then the suspensions of staphylococci were adjusted turbidimetrically to a population density of 108/ml. of organisms, 0.05 mi. of dilution poured on to the surface of nutrient agar plates, and the plates ultraviolet irradiated for different times. The results are shown in Table VI. It is dear that with the parent staphylococcus grown in the BUDR the sensitivity to ultraviolet radiation presence of FUDR was markedly increased. According to Szybalski ( 1960b this sensitization directly follows the incorporation of BUDR into the bacterial' DNA as a result of substitution of the halogenated analog in place of thymidine. It is remarkable, therefore, that this phenomenon was not observed in the mutant staphylococci. The data presented in Table VI show that the mutants were more resistant than the parent to the killing action of ultraviolet
+
+
79
MOLECULAR BIOLOGY OF CANCER
radiation. The interesting point, however, is that in mutants grown BUDR no sensitization was observed. in the presence of FUDR This suggests that in the mutants the incorporation of halogenated thymidine analogs into DNA did not take place. It can be sup-
+
TABLE VI ULTRAVIOLET LIGHT SENSITIVITY OF PARENT AND MUTANTSTAPHYLOCOCCI GROWNIN THE PRESENCEOF FUDR. BUDR. OR BOTH^ Mean number of colonies per plate
FUDR Irradiation time
+
FUDR
Control
Secnnds
BUDR
Parent ciilture
15
> 5000
30 45 60
4400 1700 220
4100 1500 340 20
> 5000 3000 1200 30
540 14 2 0
Mutants UV-3
Minutes
1 2 3 4 5
B'C7DR
> 5000
> 5000
> 5000
> 5000
660 80 35 17
650 55 35 12
600 80 25 20
550 75 37 16
From Gause and Kochetkova (1961).
posed that the mutant staphylococci lacked the enzymic mechanism necessary for effective incorporation of halogenated pyrimidines into the DNA precursor pool. 4 . Eflects of Other Znhibitors a. Chloramphenicol. Chloramphenicol is an inhibitor of protein synthesis. It is probable that the sensitive stage lies somewhere between the activation of amino acids and the polymerization of these components into macromolecular structures (Gale, 1959). Gause and Kochetkova (1961) observed that the mutants were about 2 to 3 times more sensitive than the parent to this compound (Table 11). These figures are similar to those for the tetracyclines, which inhibited the growth of the parent staphylococcus and of the mutants to the same degree. It might be supposed that the mechanism of the inhibitory action of chloramphenicol is similar to but not identical with that of the tetracyclines.
80
G . F. CAUSE
b. Effect o j Heating. Suspensions of staphylococci (lOB/ml. of organisms) were heated in a water bath at 55°C. for different periods of time, and then poured on to nutrient agar. Gause and Kochetkova (1961) showed that the mutant staphylococci were much more sensitive to heat than was the parent. It is difficult to ascribe a reason for this phenomenon, but it is interesting to note that defects in nucleic acids make bacteria more sensitive to the lethal effect of elevated temperatures (Lorkiewicz and Szybalski, 1960). c. Conclusions from the Action of Inhibitors. An analysis of the action of various inhibitors with the already known mechanisms of action clearly indicates that mutant staphylococci with impaired respiration reveal some disturbances in nucleic acids, which make them specifically vulnerable to the action of substances selectively affecting DNA and RNA in the bacterial cell. It may be suggested that respiratory defects, as well as other hereditary metabolic alterations, can be related to some disturbances in the synthesis of deoxyribonucleic acid in mutant staphylococci. As it was mentioned earlier in this review, the study of the way in which the secondary distortions in the energy-yielding metabolism may be related to the primary alterations in the nucleic acids represents one of the most important problems of the contemporary cancer research. It seems that mutant staphylococci could be exploited as a convenient model for more penetrating studies along these lines. 5. Autonomy of Protein Synthesis in Mutant Staphylococci with Zmpuired Respiration Recent experiments indicate that control mechanisms of protein synthesis in the ribosomes of mutants are not working as they do in the normal cells (Gause and Laiko, 1962). According to present concepts about the mechanism of protein synthesis, the ribosomes act as nonspecialized structures which synthesize the protein dictated by the transient attachment to the ribosome of a short-lived “messenger” RNA (Jacob and Monod, 1961). It has been observed recently that the synthesis of messenger RNA in the bacterial cell is selectively inhibited by the actinomycins (Reich et al., 1961a). This specific mechanism of action of actinomycins gives a unique opportunity for analysis of the effects of blocking the synthesis of messenger RNA in the cell upon the synthesis of proteins in the ribosomes. These effects were comparatively investigated in normal
81
MOLECULAR BIOLOGY OF CANCER
staphylococci, as well as in mutant staphylococci with impaired respiration. Table VII shows the data published by Gause and Laiko (1962). In the work with parent culture of staphylococci the concentration of actinomycin used was 0.3 yg./ml., and in the work with muTABLE VII OF PROTEIN AND NUCLEIC EFFECTOF ACTINOMYCINC ON THE SYNTHESIS ACIDSIN THE PARENTSTAPHYLOCOCCI AND IN THE MUTANTSWITH IMPAIREDRESPIRATION^
Culture
1. Parent 2. Thesame 3. Thesame
Time Aotinoincubated mycin (hours) (Fg./ml.)
0 3 3
RNA
DNA
0.272 0.781 0.291
0.081 0.152 0.122
56
60
96
42
0 0 0.006
0.117 0.439 0.417
0.052 0.183 0.190
0.288 0.498 0.270
0.087 0.113 0.110
7
0
99
6
Per cent of inhihiton a
0.058 0.209 0.138
0.168 0.548 0.336
Per cent of inhibition
4. M u t a t s ~ v - 2 0 5. Thesame 17 8. Thesame 17
Turbidity Protein
0 0 0.3
From Gause and Laiko ( 1962).
tants 0.006 pg./ml., as far as the latter were 50 times more sensitive to the action of actinomycin (see Table 11). It is clear that actinomycin C in the concentration of 0.3 pg./ml. completely blocks (96% of inhibition) the synthesis of RNA in the cells of parent staphylococci. Under these conditions the protein synthesis in the same cells is decreased by 60%. An entirely different picture was observed in mutant staphylococci with impaired respiration. Actinomycin C in the concentration of 0.006 pg./ml. completely (by 99%) inhibits the synthesis of RNA in the cells of mutants, but this block produces no effect upon the protein synthesis, which continues at the same rate as in the control culture to which no actinomycin has been added. The significance of these observations is self-evident. In the cells of parent staphylococci the protein synthesis is controlled by messenger RNA, and as soon as the synthesis of messenger is blocked by the actinomycin, the rate of protein synthesis decreases
82
G . F. GAUSE
immediately. In the cells of mutant staphylococci this control mechanism is evidently lost, and the protein synthesis for some time goes on autonomically, in spite of complete suppression of the synthesis of messenger RNA. In other words, the protein synthesis in the cells of mutant staphylococci is not controlled to the same degree as in the cells of parent staphylococci. The validity of this conclusion is supported by some additional evidence. In accordance with some recent observations on the mechanism of streptomycin action in bacteria, streptomycin impedes the attachment of messenger RNA’s to bacterial ribosomes, and in this way blocks the synthesis of proteins in the cell (Spotts and Stanier, 1961). As far as in the mutant staphylococci studied by Gause and Laiko (1962) the protein synthesis in the ribosomes is more autonomic and does not depend upon the messenger RNA to the same degree as the protein synthesis in the ribosomes of parent cells, one could theoretically expect that these mutants should be less vulnerable to the action of streptomycin than the parent cells. In fact, Gause and Kochetkova (1960) observed that mutant staphylococci with impaired respiration uv-2 and uv-3 are five times less vulnerable to the bacteriostatic action of streptomycin than the parent cells. At that time this observation was left without an explanation, but at present one can see that it well agrees with observation on the autonomy of protein synthesis in the cells of mutant staphylococi with impaired respiration.
C. RESPIRATORY DEFICIENT MUTANTSIN Staphylococcus afermentans The microbial model of cancer in the case of S . aureus is remarkable in many respects, and primarily by a very considerable difference between parents and mutants in some important characters. Some very strong differences were recorded also, for example, in comparative studies of the liver cell and its neoplastic counterpart. On the other hand there are cases where differences between normal cells and their neoplastic counterparts are minima!. Such cases are of particular significance for recognition of the more important manifestations of malignant change. Microbial models of cancer could also be helpful for reeosnition of possible minimal changes in a series of mutants with impaired respiration, and in this connection the system of mutants with “small colonies” and impaired respiration in another species of
83
MOLECULAR BIOLOGY OF CANCER
micrococci ( Staphylococcus afermentans) represents considerable interest (Gause et al., 1962a). In this species two types of respiratory deficient mutants were observed: minimal and colorless, with some distortions in DNA, and more fundamental and colored, with more distortions in DNA. Both types of mutants of S. afermentans are much more similar to their parents than are the mutants in S. uureus. Vulnerability of RNA to the action of specific inhibitors observed in the mutants of S. aureus was not recorded in the mutants of S. afermentans. The results obtained in the work with S. afermentans will be summarized here in some detail. 1. Induction of Mutants with lnipaired Respiration
Various mutagenic factors were tried, but positive results were observed only in experiments with 5-fluorouracil (Gause et al., 1962a The parent culture of S. afermentans 7503 was grown at 37" C. in test tubes in the nutrient broth containing 60-70 pg./ml. of 5-fluorouracil,and the samples from the tubes were daily streaked ) v
TABLE VIII MUTANTSOF S tuphylococcus afernientcmsn
RESPIHATlON IN PAHENT CULTUHE A N 0
Number of experiments
-
Parent culture Mutant 42 (orange colonies) Mutant 22 (orange colonies) Mutant 19 (colorless colonies) Mutant 44 (colorless colonies) u
12 7 5 5 5
Qo, 82.5 83.3 56.2 54.5 63.9
From Gause et al. (1962a).
in various dilutions on plates with nutrient agar. After 4-5 days of incubation of the culture in broth, the subcultures on plates showed the appearance of small colonies of mutants, dispersed among the colonies of normal bacteria. Some of the mutants were growing in the form of sectors upon the colonies of normal organisms. Numerous mutants with small colonies were isolated in the course of this work, and classified into two groups: with colorless colonies and with colonies of orange color of various shades. Of the former group, mutants 19 and 44 were taken, and of the second group mutants 42 and 22 were used. Table VIII shows that the consumption of oxygen in colorless mutants attained 66-77%, and in mutants
84
G . F. GAUSE
with orange colonies, 68-77% of normal values, i.e., the respiration in these mutants was impaired in the same degree.
2. Effects of Inhibitors Measurement of minimum inhibitory concentrations of various compounds in the nutrient broth showed that the inhibitors of protein synthesis in the bacterial cell affected the growth of the parent and mutant organisms to the same degree (chloramphenicol, 0.4-0.5 pg./ml.; tetracycline, 0.2,-0.3pg./ml.; chlortetracycline, 0.4-0.5 pg./ml. ). Mutant and parent cultures were affected also to the same degree by inhibitors of cell-wall synthesis (penicillin G, 0.03-0.05 units/ml.; phenoxymethylpenicillin, 0.02-0.03 units/ml.) TABLE IX EFFECT OF MITOMYCIN C AND ACTINOMYCIN C ON THE GROWTHAND NUCLEIC ACIDSYNTHESIS OF S. afer7nentama.b Incubation time (minutes)
Compound (W m l .1 None
Mitomycin C,
0 75 150
0.1
None
0 75 150 0
90 180 Actinomycin C,
0.1
0
90 180 a b
Turbidity
RNA
DNA
0.190 0.265 0.337 0.190 0.270 0.342
0.411 0.481 0.578 0.411 0.487 0.577
0.091 0.113 0.132 0.091 0.086 0.086
0.171 0.261 0.369
0.399 0.477 0.581
0.085 0,109 0.136
0.171 0.209 0.259
0.399 0.359
0.085 0.092
0.372
o m
From Gause et al. ( 1962a). Numbers in italics indicate selective inhibition.
as well as by 5-fluorouracil. Addition of thymine (100 pg./ml.) or uracil (100 pg./ml.) produced no effect upon the inhibitory action of fluorouracil. Gause et d.(1962a) were particularly interested in the analysis of inhibitory action of actinomycin C and mitomycin C , as far as these compounds selectively affect the formation of nucleic acids in the bacterial cell. Experiments showed that mitomycin C selectively inhibited formation of DNA, and actinomycin C selectively affected synthesis of RNA in the logarithmic phase of growth of
85
MOLECULAR BIOLOGY OF CANCER
S . afermentans (Table IX). It is of interest that actinomycin C inhibited the growth of parent and mutant cultures of S . ufermentans to the same degree, the minimal inhibitory concentration in broth attained 0.003-0.004 pg./ml. In distinction from this, the substances selectively affecting DNA formation, namely mitomycin C, Degranol [ 1,6-bis-(p-chloroethylamino) -1,6-deoxy-d-mannito1], and myleran mannitol ( 1,6-dimethanesulfonyI-d-mannitol) also seTABLE X MINIMAL INHIBITORY CONCENTRATIONS ( ~,G./ML.) OF V ~ I O U SCOM~OUNDS IN THE NUTRIENT BROTHFOR S. afermentans AND ITS MUTANT+ Mutant 42 Mutant 22 Mutant 19 Mutant 44 Compound Parent (orange) (orange) (colorless) (colorless)
0.0500
0.0015
0.0011
0.0140
0.0180
Degranol
210
22
28
25
29
Myleranmannitol
4000
620
520
620
410
Mitomycin C
a
From Gause et al. ( 1962a).
lectively inhibited the growth of mutants with the impaired respiration (Table X). The action of mitomycin C is of particular interest, as far as this compound selectively and irreversibly depolymerizes DNA in bacterial cells (Reich et al., 1961b). It is therefore of significance that colorless mutants were 2.7-3.5 times, and mutants with orange colonies 33-45 times more vulnerable to the action of this compound than the parent culture. It may be suggested that some alterations in DNA of mutants with impaired respiration made the cells more vulnerable to the action of mitomycin C, and that these alterations are more fundamental in mutants with orange colonies as compared to colorless mutants. 3. EfJect of Heating
It was observed that mutants with impaired respiration in S . ufementans, both orange and colorless, were much more sensitive to heat than was the parent (Gause et al., 1962a). In this respect the mutants in S . ufermentans and in S . aureus are similar (Gause and Kochetkova, 1961), and it should be remembered that yeast mutants with small colonies and impaired respiration are also very sensitive to heating (Sherman, 1956).
86
C. F. CAUSE
4. Diflerences in the DNA-Protein Relationship of Norm1 and
iMutant Organisms The extraction of DNA from bacterial cells by Schmidt and Thannhauser procedure (in the modification described by Spirin et al., 1957) showed that the capacity of DNA for extraction from a complex with proteins was decreased in colorless mutants by 2.8-2.9 times, and in mutants with orange colonies by 5.0-7.3 times (Table XI). This may point to the increased firmness of DNATABLE XI DECREASE OF EXTRACTION OF DNA AND INCREASE OF SENSITIVITY TO MITOMYCIN C IN MUTANTS OF S. afernentansa Parent
Mutant 42 (orange)
Mutant 22 (orange)
0.262
Mustant 19 Mutant 44 (colorless) ( colorless)
Extraction of DNA
0.036
0.052
0.094
0.090
Decrease of extraction of DNA (parent = 1 )
x 7.3
x 7.5
x 2.8
x 2.9
Increase of sensitivity to mitomycin (parent = 1 )
x 45.0
x 33.0
x 3.5
x "7
D595-Da5o
From Gause et al. ( 1962a).
protein bonds in the cells of mutants, which accompany their vulnerability to the action of mitomycin C. This suggestion was confirmed by the results of use of another method for extraction of DNA from the cells of parent and mutant cultures. Instead of mild separation of DNA from proteins by extraction at pH 8 by 10% NaCl, as prescribed by Schmidt and Thannhauser, Gause et al. (1962a) used 0.5 N HC104 at 70" C. for 20 minutes, as recommended by Ogur and Rosen (1950). Acid hydrolysis completely separated DNA from proteins in normal and mutant cells, and the concentration of DNA estimated by this method in normal and mutant cultures was practically identical. However, DNA was partially destroyed by this procedure, and the appearance of free nucleotides was observed. For the extraction of DNA from the cells of mutants the following reagents were used instead of 10% NaCl, which might be ex-
MOLECULAR BIOLOGY OF CANCER
87
pected to facilitate the separation of DNA from protein: 20% sodium benzoate; 0.25%, l % , and 2% sodium dodecyl sulfate; 10% urea; 10% formamide. The results in all cases were negative. It is interesting that Kirby (1961) found recently some differences in the DNA-protein complexes of normal and cancer cells.
D. RESPIRATORY DEFICIENT MUTANTS IN Escherichia coli Mutants with small colonies and impaired respiration were induced in E. coli strain B by copper sulfate added to a synthetic medium in a low concentration of 5 x M (Weed and Longfellow, 1954; Hirsch, 1!351). In other strains these mutants were induced by ultraviolet radiation and by urethane (Gause et al., 1958). The rate of oxygen consumption in mutants was reduced to about one-third of that of the normal strain. Some characteristic alterations in cytochromes of the mutant cultures of E. coli were also recorded (Gause et al., 1958). The parent strains possessed three distinct a bands, namely a2 at 625 mp, a at 605 mp, and the strong bl band at 560 mp. In all mutant strains the intensity of the a band was strongly increased, and in some of them it was shifted from 605 to 600 mp. Most important of all, however, was the fact that the bl band at 560 mp had disappeared in all mutant cultures, and was actually replaced by two new strong bands, at 550 and 565 mp. In respect of splitting of bl band of cytochromes, the mutants of E. coli with impaired respiration resemble the respiratory deficient mutants of staphylococci (Gause et al., 1961) . Mutants of E. coli are selectively inhibited by actinomycin C and by Degranol (Gause, 1959). These mutants deserve much more detailed investigation in the future.
IV. Microbial Models of Cancer as Sources of Biological Inhibitors
In this section a new possible approach to obtaining antimetabolites of tumor cells will be discussed, with the idea of attaining this goal not by chemical synthesis but by means of producing biochemical analogs of tumors cells in microorganisms and isolating from them antimetabolites that inhibit the growth of tumor cells (Gause, 1962). This new line of investigation is based upon two concepts: (1) the possibility of producing in microorganisms some mutants with biochemical alterations in the cells which are similar
88
G . F. GAUSE
to but not identical with corresponding biochemical alterations in the cells of malignant tumors of higher organisms; ( 2 ) the fact that progression from metabolite action to antimetabolite potency can be attained with small change in the structure of a molecule,
4.5 After
24 hours = 4.65
lnrtial = 3.15
5 !
5
'
3
3.0
.c
-! 2.5
\Mutants
20
40
uv
60
-2
lo(
80
5.5
5 5 .O
3.5 1
I
50
I
100 150 200 Dilutions of extrocts
L
250
I
30
FIG. 1. The destroying action of extracts from mutants of Staphylococcus aureus uv-2 upon multiplying tumor cells of the NK/Ly strain in test tubes. From Gause et al. (196213).
and the fact that metabolites of some biochemical systems show antimetabolite properties when tested in similar biochemical systems of other species of organisms. Progression from metabolite action to antimetabolite potency is passing from one biochemical system to another similar but not identical biochemical system has been described by Woolley (1952)
89
MOLECULAR BIOLOGY OF CANCER
in great detail. Woods (1953) also reviewed a number of cases of inhibition of growth of cells by analogs of growth factors which are themselves metabolites in some other systems. This is due to the fact that essential metabolites are very exacting in their structural requirements. Even closely related molecules interfere with the normal functioning of the metabolite and are thus antimetabolites. 140
After 2 4 hours = 134% Boci. porocoli
c; 100
E
; I , ,, 2 $ 1 E
u
140
20
,
,
, 60
40
,
,
,
80
- After 24 hours= 139%
[ 120 -
Socch. cerevisioe
- \
-
0
0
Initial = 100%
I00
” ‘Muionl
I
I
I
20
I
I
I
40
s - 23 I
60
80
Dilutions of exirocis
FIG. 2. The destroying action of extracts from mutants of Bacterium paracoli pc-43 and mutants of Saccharomyces cerevisiae s-23 upon multiplying tumor cells of the NK/Ly strain in test tubes. From Game et al. (1962b).
In view of these principles of antimetabolite inhibition, the components of biochemical systems of “analogs” of cancer cells in microorganisms may in some cases behave as antimetabolites in the similar but not identical biochemical systems of cancer cells. In this respect the microorganisms appear particularly promising, since the biochemical potentialities of microbes cover a wider spectrum than the cells of animal tissues. The theoretic potentialities discussed above were used recently for the development of new biological approaches in the search for antimetabolites of malignant growth (Gause et al., 1962b). The following microorganisms were used in this study: S. uureus
90
G. F. GAUSE
209 and its mutant with impaired respiration uv-2 (Gause et al., 1957b); Bacterium paracoli and its mutant with impaired respiration pc-43 (Gause et al., 1958); Saccharomyces cerevisiae and its respiratory deficient mutant s-23 (Gause et al., 1957a). These cultures were grown in liquid media on shaking machines for 48 hours. Afterward the cells were sedimented on centrifuge, washed by distilled water and ground with glass powder for 30 minutes. After centrifugation and filtration through glass filters the transparent watery extracts from bacterial cells were obtained. These extracts were tested on multiplying tumor cells in test tubes (ascitic lymphoma of mice, strain NK/Ly, described by Nemeth and Kellner, 1960). The details of this method of testing were published by Toropova (1962). Figure 1 shows that extracts from mutant cells of S. aureus contain an inhibitor which is absent in normal staphylococci and which not only prevents multiplication but even kills multiplying tumor cells, up to dilution of 1:160. Similar but less active inhibitory action was detected in the extracts from mutant yeast cells (in the dilution 1:80, Fig. 2a), and mutants of B. paracoli with impaired respiration (in the dilution 1:40, Fig. 2b). It is therefore possible to conclude that respiratory-deficient mutants of various microorganisms can be used as sources of obtaining new biological inhibitors, and that these potentialities should be studied in the future in more detail.
V. Conclusion It appears that the study of microbial models of cancer can at present serve a number of useful purposes. In connection with the deletion theory of cancer it could be useful for better understanding of the architecture of deletions in cellular biochemical mechanisms. In particular, it can contribute to better understanding of the way in which the secondary distortions in the energy-yielding metabolism may be related to the primary alterations in the nucleic acids. Microbial models of cancer can also be helpful for recognition of possible minimal changes in a series of mutants with impaired respiration which may be of interest for understanding of different manifestations of malignant change. The autonomy of protein synthesis in some mutants with impaired respiration, i.e., the fact that control mechanisms of protein synthesis in the ribosomes of mutants are not working as they do in the normal cells,
MOLECULAR BIOLOGY OF CANCER
91
represents another example of theoretical significance of the studies along these lines. The second field of potential interest of mutants with impaired respiration is their use for testing of various inhibitors. Mutants of S. uureus, for example, are specifically vulnerable to inhibitors selectively affecting the synthesis of both DNA and RNA in the microbial cell; to this group of substances belong the antitumor agents at present available. On the other hand, mutants of S. ufermentuns are specifically vulnerable only to inhibitors affecting the synthesis of cellular DNA. By using the variety of mutants it is possible to differentiate the mechanisms of action of different inhibitors at an early stage of the screening work. Finally, the microbial models of cancer can be exploited as sources of new biological inhibitors. Biochemical analogs of tumor cells in microorganisms are in fact producing antimetabolites that inhibit the growth of tumor cells, and their potentialities in this field should be studied in more detail. REFERENCES
Aisenberg, A. C. (1961a). “The Glycolysis and Respiration of Tumors.” Academic Press, New York. Aisenberg, A. C. ( 1 9 6 l b ) . Cancer Res. 21, 295. Aisenberg, A. C., and Morris, H. ( 1961). Nature 191, 1314. Bergel, F. ( 1961 ) . “Chemistry of Enzymes in Cancer.” Thomas, Springfield, Illinois. Cohen, S., Flaks, J., Bamer, H., Loeb, M., and Lichtenstein, J. (1958). Proc. Natl. Acad. Sci. U S . 44, 1004. Ephrussi, B., Hottinguer, H., and Chimenes, A. M. (1949). Ann. Inst. Pasteur 76, 351. Fiala, S., and Fiala, A. (1959). Brit. J . Cancer 13, 236. Gale, E. F. (1959). Ciba Found. Symp., Amino Acids Peptides Antimetab. Activity. Gale, E. F., and Folkes, J. P. (1953). Biochem. J . 53, 493. Gause, G.F. (1959). Biol. Rev. Cambridge Phil. SOC. 34, 378. Cause, G. F. (1960). “The Search for New Antibiotics.” Yale Univ. Press, New Haven, Connecticut. Gause, G. F. (1962). Vestnik Acad. Med. Sci. U.S.S.R. 3, 8 . Gause, G.F., and Kochetkova, G . V. (1960). Antibiotiki 5, 63. Gause, G. F., and Kochetkova, G. V. (1961). Antibiotiki 6, 643. Gause, G. F., and Laiko, A. V. (1962). Dokl. Akad. Nauk S.S.S.R. 149, 711. Gause, G. F., Kochetkova, G. V., and Vladimirova, G. B. (1957a). Dokl. Akad. Nauk S.S.S.R. 117, 138. Gause, G. F., Kochetkova, G. V., and Vladimirova, G. B. (19571~).Dokl. Akad. Nauk S.S.S.R. 117, 720.
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Gause, G. F., Ivanitskaia, L. P., and Vladimirova, G. B. (1958). Izo. Akad. Nauk S.S.S.R. Ser. Biol. 8, 719. Cause, C. F., Kochetkova, G. V., and Sarbaeva, N. A. (1980). Dokl. Akad. Nauk S.S.S.R. 130, 200. Cause, G. F., Kochetkova, G. V., and Vladimirova, G. B. (1981). Dokl. Akad. Nauk S.S.S.R. 139, 223. Cause, G. F., Kochetkova, C. V., Vladimirova, G. B., and Landau, N. S. (1962a). Microbiology 32, 280. Cause, G. F., Vladimirova, G. B., Zimenkova, L. P., and Landau, N. S. ( 1962b). Antibiotiki 7. Gershanovich, V. N., Palkina, N. A., and Kaz, G . I. (1962). Biokhimiya 27, 109. Hayes, W. (1962). Nature 193, 208. Hirsch, H. M. (1981). J. Bacteriol. 81, 448. Horowitz, J., and Chargaff, E. (1959). Nature 184, 1213. Jacob, F., and Monod, J. (1981). J. Mol. Biol. 3, 318. Kirby, K. S. (1961). Progress Erp. Tumor Research 2, 291. Laiko, A. V. ( 1962). Antibiotiki 7, 801. Lettre, H., and Sachsenmaier, W. ( 1957). Natunoissenschaften 44, 335. Lindegren, C. (1959). Nature 184, 397. Lorkiewicz, Z., and Szybalski, W. ( 1960). Biochem. Biophys. Res. Commun. 2, 413. Monier, R., Zajdela, F., Chaix, P., and Petit, J. (1959). Cancer Res. 19, 927. Morthland, F. W., DeBruyn, P. P., and Smith, N. H. (1954). Erptl. Cell Res. 7, 201. Nagai, S., Yanagishima, N., and Nagai, H. (1961). Bacteriol. Reo. 25, 404. Nemeth, L., and Kellner, B. ( 1960). Naturwissenschaften 47, 544. Ogur, M., and Rosen, C. (1950). Arch. Biochem. 25, 262. Pitot, H. C., Potter, V. R., and Ono, T. (1961). PTOC. Am. Assoc. Cancer Res. 3, 259. Potter, V. R. (1957). Univ. Mich. Med. Bull. 23, 401. Potter, V. R. (1958). Federation Proc. 17, 891. Potter, V. R. (1962). “The Molecular Basis of Neoplasia.” Univ. of Texas Press, Austin, Texas. Raut, C., and Simpson, W. (1955). Arch. Biochem. Biophys. 57, 218. Reich, E., Franklin, R., Shatkin, A., and Tatum, E. L. (1961a). Science 134, 558. Reich, E., Shatkin, A., and Tatum, E. L. (1961b). Biochim. Biophys. Acta 53, 132. Rhuland, L. (1980). Nature 185, 224. Rogers, H. J., and Perkins, H. R. (1959). Nature 184, 520. Sarachek, A., and Fowler, C. (1961). Nature 190, 792. Shepherd, C. J. (1958). 1. Gen. Microhiol. 18, IV. Sherman, F. (1958). Erptl. Cell Res. 11, 859. Shiba, S., Terawaki, A., Taguchi, T., and Kawamata, J. (1959). Nature 183, 1056. Spirin, A. S., Belozersky, A. N., Shugaeva, N. V., and Vanushin, B. F. (1957). Biokhimiya 22, 744.
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Spotts, C. R., and Stanier, R. Y. (1961). Nature 192, 633. Stier, T., and Castor, J. (1941). J . Gen. Physiol. 25, 229. Strominger, J. L., Park, J. T., and Thompson, R. E. (1959). J . Biol. Chern. 234, 3263. Szybalski, W. ( 1960a). “Developments in Industrial Microbiology.” Plenum Press, New York. Szybalski, W. ( 1960b). “Progress in Photobiology.” Copenhagen, Denmark. Toropova, E. G. (1962). Antibiotiki 7,598. Warburg, 0. ( 1930). “The Metabolism of Tumors.” Constable Press, London. Warburg, 0. (1956). Science 123, 309; 124, 269. Warburg, 0. ( 1959). Natunoissenschaften 46, 25. Weed, L. L., and Longfellow, D. (1954). J . Bacteriol. 67, 27. Whelton, R., and PhafF, H. (1947). Science 105, 44. Woods, D. D. (1953). “Symposium on Nutrition and Growth Factors.” 1st. Super. Sanita, Rome. Woolley, D. W. (1952). “A Study of Antimetabolites.” Wiley, New York.
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Rapid Microbiological Determinations with Radioisotopes GILBERT V. LEVIN Resources Research, Incorporated, Washington, D.C. I. Classic Microbiological Techniques ........................ A. General Techniques ................................. B. Time Factor ........................................ 11. Radioisotope Technique ................................. A. Basic Considerations ................................ B. Applications ........................................ 111. Conclusion ............................................ References ............................................
95 95 96 100 100 102 132 132
1. Classic Microbiological Techniques A. GENERAL TECHNIQUES The microscope had been known for nearly four centuries when Leeuwenhoek made his startling discovery of bacteria in 1676. Ever since this historic event, direct observation has been one of the principal methods by which microorganisms have been detected, identified, and studied. For nearly another hundred years, this was the sole method available. Then Pasteur conducted his brilliant experiments. In proving that fermentation was caused by bacteria, he simultaneously provided a powerful new tool for bacteriological determinations-the culturing of bacteria in nutrient media. Masses of bacteria and the effects produced by them on inoculated materials could be observed. The two techniques, microscopy and culture, thus provided means for the micro and macro study of organisms invisible to the naked eye. The introduction of staining by Weigert, Ehrlich, and Salomonsen helped bacteriological microscopy to reach its present state of attainment. When mechanical improvements refined the microscope to the limit imposed by optical resolution, the invention of the electron microscope greatly increased useful magnification. Correspondingly, the development of transparent, solid media by Koch in 1881 implemented the development of the colony technique. The introduction of selective media further enhanced the usefulness of culturing. More recently, serological and enzymatic reactions have offered important new methods for the study of microorganisms. 95
96
GILBERT V. LEVIN
B. TIME FACTOR The identification and enumeration of microorganisms by these now classic techniques are determined by: inspection of the cells, inspection of colonies, the development of turbidity, the formation of gas bubbles, color changes or other physical changes in the reaction mixture. Despite the wide diversity of these criteria, they have one aspect in common: all are of a direct visual nature. AS a consequence, the quantitative examination of an unknown sample by these methods consumes considerable time. Microscopic inspection permits rapid identification of morphological types, but different species with the same morphology cannot be distinguished. The enumeration of a representative sample is exceedingly tedious, especially where small numbers of bacteria are concerned, because statistical confidence requires the time-consuming observation of a great many fields. Dilution and plating techniques permit quantitative determinations by the colony method. However, since each cell must give rise to a visible colony, time must be allowed for the reproduction of many generations. The same incubation time requirement confronts quantitative application of the gas bubble and turbidity methods in liquid media. On the other hand, enzymatic reactions can produce color or other changes in the reaction mixture rapidly. Their use, however, has been limited to the examination of materials, principally milk, in which relatively large numbers of bacteria are normally found. The method is inherently very sensitive and its sensitivity can be greatly extended by fluorescent techniques and the use of photomultiplier apparatus ( Laurence, 1957). Although enzymes generally react with specific substrates, the various enzymes are so widespread in nature that the use of this technique for the identification of specific microorganisms seems generally precluded. Serological reactions are specific, but, as used, require large numbers of cells. As a consequence of the above considerations, all standard methods used for the quantitative identification of small numbers of cells of a particular species or group of organisms in unknown samples require from 24 hours to several days for completion. 1. Significance of Delay For most research studies, this time delay is inconvenient, but seldom constitutes a serious problem. In the fields of medicine and
RAPID DETERMINATIONS WITH RADIOISOTOPES
97
public health, however, the matter of time is frequently paramount. In monitoring a product or an environment for bacteria, even a one day period of ignorance may constitute a serious hazard. a. Water Supplies. The bacteriological control of public water supplies is an outstanding example in this category. The established (Public Health Serv., 1962) index for the bacteriological quality of drinking water is the coliform organism group. These organisms live in the intestinal tracts of warm-blooded animals and are, consequently, present in great numbers in sewage. They are discharged in such quantities that, even when sewage is diluted to the point where the receiving water is aesthetically acceptable, the presence of the coliform organisms can readily be established. Although most of the bacteria in the coliform group are not pathogenic, their demonstrated presence is grounds for rejection of the water for drinking purposes on the assumption that pathogenic organisms are also present. The most widely used standard method (Am. Public Health ASSOC., 1962) for the quantitative determination of coliform organisms is the most probable number technique based on serial dilution of the sample. The quantitative aspect of this test relies upon the isolation of a single cell in a diluted aliquot of the sample. Such aliquots are incubated in lactose broth where the production of gas constitutes positive evidence. To produce a 1 mm. diameter bubble, a population of 1.7 x lo9 cells must result from the single bacterium. Forty-eight hours must be permitted to elapse before the test can be presumed to be negative. Should the test become positive after either 24 or 48 hours, a transfer must be made into a more selective medium for confirmation. Confirmation requires another 48 hours before negative results can be accepted, although the tubes may produce gas for a positive result after 24 hours, Thus, a minimum of 48 hours must elapse for a positive determination, and a period of 72 or 96 hours must elapse for a negative sample which gave a positive presumptive test. As a result, in most municipalities, the water is consumed by the public before the bacteriological quality is ascertained. Although careful process control safeguards the water, this ignorance of the principal criterion of potability has resulted in disease outbreaks. Recently, a second standard method (Am. Public Health ASSOC., 1962) for the quantitative determination of coliform organisms in water was adopted. This method uses a submicron filter through
98
GILBERT V. LEVLN
which the sample is drawn. The “membrane” filter is then placed on a pad saturated with a coliform group selective medium which rises through the pores of the filter and permits the supposedly isolated cells on the filter to develop into visible colonies. Twenty t two hours of incubation are required for the completion of this test. Even this delay relegates bacteriological results to the realm of historical information in most municipalities. Rapid bacteriological determinations in water supply quality control would also be helpful in determining raw water quality. Such information would assist in intelligent process control, and, in event of gross contamination, the source could be rejected. Here again, the time required by the standard bacteriological methods prevents use of bacteriological data except in retrospect. b. Swimming Pools. At swimming pools and natural bathing areas, the time delay in obtaining bacteriological results also creates a public health problem. Water quality control based on bacteriological results is impossible, Nonetheless, the primary criterion for bathing waters is the bacteriological one. Here, the need is not only for a rapid method, but for a simple one which can be administered by the pool or beach operator. Otherwise, inspectors from the health department must transport samples back to the central laboratory for determinations. Such visits can at best be only infrequent with respect to the public exposure time. The ideal method for meeting the public health requirements for determinative bacteriology at water treatment plants and swimming pools would be a periodic sampler and analyzer which would obtain analytical results rapidly enough to operate feedback mechanisms controlling the water production process. Specifically, the coliform organism level might be used as a direct control of the level of chlorination. Until such a method is available, actual reliance on bacteriological quality control will continue to be careful process operation, particularly the maintenance of an adequate chlorine residual in the water. With modern treatment methods, this is normally satisfactory. However, if this were a completely reliable safeguard, the public health standards wouId be couched in terms of chlorine residuals, which can be determined immediately, rather than in terms of bacteria. c. Food. Another important area of public health bacteriology is that of food processing and serving. The packaging of sea food, dairy products, vegetables, and poultry would benefit from a
RAPID DETERMINATIONS WITH RADIOISOTOPES
99
bacteriological method rapid enough to permit early measurement of the quality of the raw foods and the quality maintained through the various process steps. The packaging of unpasteurized, frozen foods has greatly increased this need in recent years. Moreover, frozen foods should be bacteriologically analyzed in storage and on display. Freezer power failures or improper temperature control frequently place food which has been defrosted a number of times in the hands of the consumer. The problem in food serving establishments is much like that of the swimming pool, where periodic-or sporadic-sampling of food and utensils by health department inspectors supplies information suitable only for identifying habitual offenders. d . Selection of Antibiotics. A second major field requiring rapid bacteriological methods is medicine. In many instances, rapid identification of bacterial infection would make treatment more effective and could even save lives. If the infectious organism could be identified rapidly, this would permit selection of the preferred chemotherapeutic agent. However, a method which would not identify the organism, but would determine the treatment agent of choice would be equally effective. Either method would have benefits beyond those associated with treating the particular infection. Because such knowledge is not readily available in time for its effective use, broad spectrum chemotherapeutic agents are frequently used. Many times, knowledge of the infection would indicate against such use. Administration of these agents sometimes sensitizes the patient, resulting in considerable hazard being associated with his future use of the agent. Furthermore, widespread use of some antibiotics has rendered them less effective by promoting the selection of resistant strains of organisms. The elimination of nonessential use of antibiotics would alleviate this problem in many instances. e. Bacteriological Warfare. There is, regrettably, a third major demand for rapid bacteriology. This is the requirement for adequate defense against bacteriological warfare. Before appropriate measures can be taken to protect populations, the attack must be detected. Means for delivering BW agents through air or water have become sophisticated to the point where there may be no overt indications of an attack. Only through detecting the bacteria themselves can knowledge of such an attack be ascertained reliably. Only the briefest time, perhaps several minutes, will be
100
GILBERT V. LEVIN
available for protective measures. Thus, the attack must be detected almost immediately. This formidable problem has been approached along several avenues. Identification of the specific pathogen would require perfection of a rapid test for each of the species suitable for bacteriological warfare. With less difficulty, an alarm might be based upon the detection of a rapid rise in the background count of microscopic particles. Dust or other particles could produce false alarms with such a system. Chemical identification of the media in which the bacteria were grown or transported might be used as an index. However, such analyses would still not prove the presence of living organisms. While much of the information on BW defense is classified, published accounts indicate that the two principal approaches are the development of rapid particle size analyzers which will signal an alarm when the background levels significantly change, and the development of a device which stains and microscopically detects living particles. Although current state of the technique must remain obscure for security reasons, the Army Chemical Corps has frequently and publicly announced its urgent need of improved BW detection methods.
II. Radioisotope Technique A. BASIC CONSLDERATIONS To eliminate the growth period in a quantitative bacteriological determination requires a method with a resolving power at least as great as that permitted by visible light, and a means for the rapid examination of a statistically significant portion of the unknown sample. The two requirements tend to be mutually exclusive. A practical consideration adds to the problem: the method or instrument must be simple enough to serve as a routine laboratory tool. The great jump in analytical sensitivity provided by the introduction of radioisotope techniques and a fortuitous aspect of biochemistry combine to make the desired test possible. The increased sensitivity offered can be appreciated by the fact that radiation detection instruments can detect a beta particle ejected from an atomic nucleus. The beta particle is many trillions of times smaller than a bacterium. The physical elements of the technique are thus satisfied. The remaining requirement is the biochemical one-to achieve the desired selectivity in applying the method to determinative microbiology. The simplest approach is to rely on
RAPID DETERMINATIONS WITH RADIOISOTOPES
101
the selectivity of existing tests by using the same media and conditions, the only innovation being appropriate labels. This is possible where the procedure permits only the organisms of interest to grow. The problem is more complicated with organisms that are identified by color or sheen developed in media which also permit growth of other organisms. In such cases, new criteria must be applied based on other distinguishing characteristics of the species. Some of these characteristics might, themselves, be determined through the use of isotopes. It is conceivable that a new array of selective, radioactive media could be developed in much the same manner as was the present arsenal of the microbiologist. That bacteria could be induced to incorporate substrates containing radioactive atoms which could then be followed to elucidate metabolic pathways has been demonstrated by Cowie et al. (1950, 1951, 1952a, b ) and in the extensive work of Roberts et at. (1955). Massive quantities of bacteria were used in these studies and the principal method of determining the disposition of the radioactive atoms was by radioautography of chromatograms. A practical bacteriological test using isotopes requires that the radioisotope be easily introduced and easily recovered from the bacteria or metabolic products. If the bacteria or the metabolic products retained in the medium are to be sought as evidence, the problem becomes difficult. This is because a physical separation of the unused, labeled substrate from the bacteria or metabolic products would have to be accomplished before results could be obtained since the isotope detection equipment cannot distinguish any one of these fractions from the others. Moreover, the separation is complicated by the fact that, after a brief exposure to the bacteria, most of the label remains in the unused substrate. Therefore, unless separation is complete, traces of the substrate will mask the presence of the organisms and the products produced. Such separation from the medium would be extremely difficult to accomplish with the desired rapidity. The fortuitous circumstance making the isotope method readily applicable is the fact that a substantial portion of carbohydrate carbon taken in by cells utilizing the Krebs cycle is oxidatively metabolized to carbon dioxide. Thus, converted to a gas, metabolized radioactive carbon readily separates from the liquid culture medium for easy collecting and counting. The same advantages, of course, hold for other isotopes producing other gases. The tech-
102
GILBERT
V.
LEVIN
nique implied by these facts is sensitive enough to detect the respiration of small numbers of resting cells in a matter of several minutes or hours. B. APPLICATIONS 1. Coliform Test The first goal of the radioisotope technique was a rapid test for the coliform group of organisms. The standard method (Am. Public Health ASSOC.,1962) multiple tube fermentation test offered the possibility of direct adaptation. This is the test in which the fermentation of lactose with the production of gas constitutes a positive finding. The test is generally applied in two steps, one presumptive and the other confirmatory. The tube portions positive in the presumptive test are transferred to tubes of lactose broth containing dyes inhibitory to noncoliform organisms. Production of gas in the latter tubes confirms the test. Approximately one-third of the gas produced by coliform organisms is carbon dioxide. Appropriate labeling of the lactose with C14 results in the production of C1402. The C1402 can be captured readily with barium hydroxide or other “getters.” The radioactivity collected on the getter can then be measured and is an index of the metabolic activity in the sample. As the method was first reported (Levin et al., 1956), a portion of a water sample in question was inoculated directly into 10 ml. of lactose broth in which the 0.5% lactose content was supplied with 1act0se-l-C~~ synthesized by Frush and Isbell ( 1953). The apparatus consisted of a train through which filtered air was bubbled into the inoculated culture. The air entrained CI4O2 produced by the culture and carried it through a vapor trap, to reduce possible aerosol carry-over, and finally through a porous paper pad impregnated with several drops of a saturated solution of barium hydroxide. At suitable intervals, the pad was replaced and the exposed one dried and counted in an internal flow counter. This process paralleled the standard presumptive test for coliform organisms, merely substituting a more sensitive method for the detection of the gas evolved. The sensitivity achieved is demonstrated in Table I. As few as 125 cells were detected in 1 hour. The principal drawback of this approach was its high cost imposed by the large quantity of isotope used. In the course of development (Levin et al., 1957, 1961), this problem has been met by reducing the quantity of isotope required and by substituting the much less
103
RAPID DETERMINATIONS WITH RADIOISOTOPES
expensively prepared formate-C14for the lactose-l-C14. The possibility of using formate was indicated by the standard (Am. Public Health ASSOC., 1962) formate ricinoleate broth and by the determination by Roberts et al. (1955, p. 166) that 86% of the carbon utilized by Escherichia coli as formate was converted to Cog. While the incidental developmental details can be obtained from the references cited, it is felt that a description of the method in its current form may be worthwhile. TABLE Ia PRESUMPTTVE TESTOF SAMPLECONTAINING APPROXIMATELY 125 E. coli Radioactivity of testa (counts per minute)
Radioactivity of controlb (counts per minute)
Time (hour)
Increment
Cumulative
Increment
Cumulative
1 2 3 4 5
172 3 09 1,154 4,075 12,579
1720 481 1,635 5,710 18,289
67 38 36 36 27
67 105 141 177 204
a From Levin et al. Reproduced courtesy J. Am. Water Works Assoc. 48, 1, 77 (1956). b Radioactivity measured above a background of 21 counts per minute. c Point of presumptive determination.
The apparatus consists of a commercially available membrane filter assembly, membrane filters, and paper absorbent pads, all of one-inch diameter; a vacuum pump or aspirator; a shaker; aluminum planchets one inch in diameter by one-fourth inch deep with a flat lip one-eighth inch wide; 35 mm. by 50 mm. glass cover slips; calibrated pipettes; a hot plate or heat lamp; and a commercially available end window or gas flow radiation detector with associated scaler. Most of these items are shown in Fig. 1. The method is a one-step, confirmed test for fecal coliform organisms. Narrowing of the coliform group to those coliforms of fecal origin increases the sanitary significance of the test. British M F MacConkey broth ( Membrane Filtration) to which sodium formate-C14 is added is the medium used. The ingredients are: 3% lactose, 1% peptone, 1%bile salts, 0.5% NaCl, 0.0012% brom cresol purple; 0.002% sodium formate-C14 (8 mc./millimole) . Sterilization of the medium is accomplished by autoclaving for 15 minutes at 15 p.s.i. or by membrane filtration. The flask containing the medium is then
104
GILBERT V. LEVIN
stoppered with sterile cotton and shaken for several hours or overnight to reduce, by atmospheric exchange, small amounts of nonmetabolic C1402 generated in the sterile medium. The desired quantity of the water sample is drawn through a filter membrane. The membrane is aseptically placed into a sterile planchet. Then, 0.5 ml. of the medium is pipetted onto the membrane and a cover slip is immediately placed over the planchet.
FIG. 1. Apparatus for rapid coliform organism test. Left to right (foreground ) : membrane filter apparatus, C1102 collection planchet, cover slips, culture planchet with cover slip in place, assembled culture-collection unit; (background): scaler, internal flow counter.
The oxygen restriction thus enforced increases the specificity of the test. Together with a sterile control, the test portion or portions are incubated at 44°C. After 3% hours, the planchets are removed from the incubator. A tightly fitting paper pad is pressed into the bottom of each of an equal number of planchets. Five drops of a settled, saturated solution of barium hydroxide are then delivered onto each pad. The planchets containing the pads are quickly inverted on the cover slips of the culture planchets. The cover slips are slid out from between the planchets which then enter into direct communication with each other. Carbon dioxide evolved
105
RAPID DETERMINATIONS WITH RADIOISOTOPES
from the culture planchet will leave the broth and travel to the absorbent pad under the impetus of the concentration gradient created by the fixation of the gas on the pad in the form of barium carbonate. Immediately after being united, the paired planchets are returned to the incubator for 30 minutes, providing a total incubation period of 4 hours. The planchets are then removed from the incubator and the pairs separated. Those planchets containing the pads are placed on a hot plate or under a heat lamp for several minutes. Still in their original planchets, the dried pads are counted for radioactivity. Counting to a satisfactory degree of significance can generally be achieved within several minutes. TABLE 11"
RESULTSOF 4 - H o u ~RADIOISOTOPETESTON E . coli ATCC 8739 USINGNONFILTERED INOCULA AND M F MACCONKEY BROTH ____~
_____
Inoculum (no. cells)
12 28
77 83
85 975 1,170 2,460 9,820 41,600
____~
~
~
Average counts per minute
Counts per minute per initial cellb
57 263 625 807 391 7,120 5,540 16,600 70,600 2 11,000
4.75 9.40 8.12 9.73 4.60 7.30 4.73 6.75 7.19 5.08
a From Levin et al. Reproduced courtesy 1. Water Pollution Control Federation 33, 10, 1024 (1961). Average counts per minute per cell 1= 6.77.
Because the use of the membrane filter has been shown (Levin et al., 1961) to reduce markedly the sensitivity of the test, the ultimate sensitivity is best demonstrated by showing data obtained using this method with the exception that the inocula were applied by pipetting 0.1 ml. portions of test suspensions rather than by filtration. Table I1 lists values obtained with E . coli ATCC 8739. Each value is an average of 5 replicates with background and sterile control levels subtracted. The numbers of cells producing the responses were determined by nutrient agar pour plates. When several commercial types of filter membranes were used, the C1402produced in the 4-hour period was approximately onetenth of that evolved by equal inocula applied by the pipette
106
GILBERT V. LEVIN
method. Figure 2 illutrates this effect. One type of filter membrane, Gelman Type 27A, was found to produce only a twofold reduction in C1402production. Figure 3 compares equal inocula applied by filtration and pipetting. Quantitative data on fecal coliforms in water samples have been collected using this filter, but have not
10'
-
5-
-8 I
0
5-
-u 0
>
w
N
'P -0
lo3
-
5 -
-
Nontiltertd
102
-
-Filtered
lnacufum E a c h point Is o v e r a g e of 8 i K r ~ p l l c o t e t wlth bockpround o n d t t e r l l e c o n t r o l I. v C l 8 Subt r a c l e d
5 -
0
Inocutum
I
2
5
4
3 Incubation
Time
6
(hour)
FIG.2. Effect of membrane filter on E . coli ATCC 8739. C1402 evolved as a function of time for filtered and nonfiltered equal inocula. Almost perfectly straight line of nonfiltered inoculum curve demonstrates accuracy of gas production as index of growth. From Levin et QZ. Reproduced courtesy of J . Water Pollution Control Federation 33, 10, 1026 (1961).
RAPID DETERMINATIONS WITH RADIOISOTOPES
107
yet been published. By way of interest to the quantitative aspect, Table I11 shows the relationship between various ranges of cell populations and the 4-hour responses obtained by an earlier version (Levin et al., 1959) of the test. a. Factors AfJecting Accuracy of Standard and Rapid Tests. The radioisotope coliform test has some advantages beyond those cited
-
to6
5 -
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:lo3
0
:5 0
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W
N
0 *
-0
4
10
-
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-
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5
-
levels
subtroc l a d
FIG.3. Effect of Gelman Type 27A membrane filter on E . coli ATCC 8739. evolved as a function of time for filtered and nonfiltered equal inocula. From Levin et al. Reproduced conrtesy of 3. Water Pollution Control Federation 33, 10, 1035 (1961). C1402
of
r e p l i c a t e s w i t h background a n d s l e r l l c C O n l r Q l ¶IS
F 0 03
TABLE IIIa CORRELATION BETWEEN E .
COli POPULATION AND
EVOLVED RADIOACTIVITY
Evolved radioactivity counts per minute0 Run no.
2550 Cells
1-25 Cells
27
1 C
2 3 4 5 6 7 8 9 10 11 12 13
Average Standard deviation 0 c
7 11
16 15
*
37 37 24 24 28 15
50-100 Cells
100-200 Cells
200-400 Cells
400-800 Cells
800-1600 Cells
160K3200 Cells
3200-6400 Cells
64
94
181 106 224
400
792
1625
4290
449
908
1820
627 1
391 164 224 245 109 167 117 149 358
673 298 472 755 254 321 247 285 488 342
1373 768 858 1676 669 771 520 480 985 762
2283 1336 1621 4000
6060
0
6262
M *I +I
31 89 52 39 57 40 14 37
155 135 93 96 105 36 66
1871
c r
?
2 2400
5450
12
27
47
98
203
415
880
2119
5667
4
7
21
37
92
163
340
84 1
839
From Levin et al. Reproduced courtesy Journal Am. Water Works Assoc. 51, 1 (1959). Average of three replicates without sterile controls. Blanks indicate no data available.
E
RAPID DETERMINATIONS WITH RADIOISOTOPES
109
above. Although replicate portions have routinely been used in testing samples, these are for the purpose of increasing statistical reliability rather than, as in the case of the multiple tube dilution test, to satisfy the requirement for a quantitative determination. As its name implies, the multiple tube technique requires that replicate portions ( generally five) of several dilutions of the sample (generally three) be inoculated to permit the determination of the most probable number of coliform organisms in the original sample. The comparative simplicity of the rapid test makes it convenient to test several replicates of each sample. In effect, each replicate is equivalent to one complete set of dilution tubes in the most probable number technique, Fundamental to the statistical approach of the multiple tube method is the assumption that the gas bubbIes in each of the highest dilutions found to be positive originated from a single cell inoculum. As will be discussed, this assumption is not valid. Accuracy of the multiple tube dilution test also suffers from the fact that a substantial number of false positives frequently results, even through the confirmed step. A third principal source of error is introduced by statistical effects and bias in the quantitative determination as shown by McCarthy et al. (1958) and McCarthy (1961). Quantitative results with the membrane filter test are also subject to error (Levin et al., 1961;McCarthy, 1961). Some of the difficulty may arise from toxic manifestations with some types of filter membranes (Levin et uZ., 1961). The numerical aspect of this test likewise depends upon isolation of single organisms. Jones and Jannasch (1956) have shown that, in reality, a high percentage, probably the majority, of the cells exist and are deposited as clumps, hence giving rise to fewer colonies than the initial number of cells. Clumping does not operate against the radioisotope method since the quantitative aspect of the latter is not derived from direct visual evidence. The total quantity of gas produced by the organisms present constitutes the parameter measured and is probably not materially influenced by clumping. The lack of the dilution requirement in the rapid test likewise serves to its advantage in a comparison with the membrane filter method. For statistical reliability in the membrane filter technique, it is recommended that the number of colonies developed be within the range of 20 to 200, preferably 20 to 80. Unless the approximate quality of the water to be tested is known in advance, several
110
GILBERT V. LEVIN
different quantities or dilutions of the original sample must be filtered and incubated to achieve this narrow range. Heavily polluted waters are frequently difficult, or even impossible, to test by the membrane filter. This is because noncoliform organisms, which greatly exceed the coliform organisms in the sample, also grow on the membrane filter and, while not exhibiting the identifying sheen of the coliform organisms, physically crowd out the latter. Sometimes, when the total organisms are sufficiently diluted, the colifoms are extinguished. A further disadvantage of the dilution technique for either of the current standard methods is that dilution imposes nutrient, osmotic, temperature, and sometimes pH changes which result in the death of organisms. Finally, the radioactive method is sensitive enough to measure the respiration of "dead" organisms which do not achieve growth during incubation periods of the standard tests. Butkevich and Butkevich (1936) state that, at least in sea water, bacteria which do not respond to the usual media may constitute a significant fraction of the total organisms. Having listed the advantages in accuracy that the radioisotope test enjoys over the current standard methods, it must now be said that the quantification of the rapid test has been one of its most difficult developmental problems, Much of the problem is a chicken or egg paradox. Against what can the sensitivity and the quantitative accuracy of the radioisotope method be calibrated? The sensitivity of the rapid test is greater than that of either standard method, and the quantitative accuracy of both standard methods has been shown to be considerably clouded. Because of the lack of an absolute standard, accurate calibration of the radioisotope test poses a quandary. Replication by the rapid test is good when inocula of the same strain are compared within a single run, but not quite as good when different runs are compared. Good replication is also obtained with wild cultures within a single run, but considerable variation in counts per minute per cell is produced in different runs on wild cultures. Figure 2 demonstrates the excellent quantitative results obtained from E. coli ATCC 8739 within a single run as a function of time. The exquisitely straight line through more than four orders of magnitude plotted for the nonfiltered inoculum is in complete agreement with the theoretical exponential growth curve which would be expected under the test conditions. Returning to Table 11, the counts per minute per initial E . coli ATCC 8739 cell are seen to
RAPID DETERMINATIONS WITH RADIOISOTOPES
111
range somewhat less than +-50% of the average. Considering the broad range of inoculum, 12 to 41,600 cells, the results are excellent as measured by current bacteriological standards. When wild cultures obtained from surface waters were used, the range of counts per minute per cell for different runs extended to approximately one-half an order of magnitude on either side of the average. In these cases, calibration was made by the membrane filter method using British MF MacConkey broth. The question that so far has not been answered is how much of the variation is inherent in the radioisotope test and how much of it represents errors produced by the other methods. The error-inducing factors associated with the standard methods certainly implicate them. There are also potential sources of error characteristic of the radioisotope test. Among the various strains of coliform organisms tested in pure cultures, two have been found to differ in rate of CO:! production by as much as an order of magnitude. The ranges of per cent abundances of the extreme strains in natural waters are not known so that the significance of the difference cannot be fully assessed. Another source of error may be the immediate history of the wild cultures. Cells in lag phase have been found to produce considerably more CO, per capita than exponentially growing cells. This, however, may not be significant in that exponentially reproducing cells would be expected to occur in surface waters only under rare conditions and then in such quantities that their presence would be readily detected. Although MacConkey broth is believed to be highly specific for fecal coliform organisms (Taylor, 1959-1960), if present in sufficient numbers during the period while noncoliforms are being inhibited, the latter may produce detectable quantities of C1402.The problem of the toxicity of the membrane filter introduces a common error into the radioactive and nonradioactive methods. Finally, results will differ if media of different specific activities are used. Care should be exercised in ordering the labeled compound and in mixing the medium. The half-life of carbon is sufficiently long so that no correction for shelf storage need be applied. While additional research may correct some of the causes of variation in C1402 production on a unit cell basis, a realistic appraisal of the tenfold variation discussed must conclude that this range of quantitative accuracy is as good as any, and better than most, conferred by currently accepted bacteriological tech-
111,
GILBERT
V.
LEVIN
niques. Jannasch and Jones ( 1959) compared direct microscopic methods and culturing methods, including the membrane filter, on total bacteria in sea water. It was found that there were 13 to 9,700 times more bacteria by direct counts than by cultural methods. A mean of more than 125 times as many cells were found on membrane filters by microscopic counting than by subsequent counting of visible colonies. The interests of absolute accuracy might be served by calibrating the radioisotope test by means of micromanipulation of one or several cells. Such a technique might permit accurate knowledge of the size of the inoculum producing a detected quantity of C1402under controlled conditions. It is conceivable that the radioisotope method could then be used as a standard for the other methods. While not normally a source of error, another characteristic of the radioisotope coliform test somewhat reduces its sensitivity and creates a minor annoyance. This is true not only of the rapid coliform test, but of any technique using labeled organic compounds. Beta disintegrations impart sufficient energy to adjacent molecules or ions to break bonds. Fragments are thus produced, generally free radicals, which enter into one or a series of reactions, some of which terminate in the production of C1402. The nonmetabolic C1402 in the medium can be reduced by promoting exchange with the atmosphere through shaking or bubbling with carrier gas. Sterile controls are routinely run with tests for the purpose of determining the levels of nonmetabolic C1402. b. Isotope Hazards. A word is in order concerning the hazards associated with handling radioisotopes in the test. The levels of activity used are so small, several microcuries per culture planchet, that a laboratory can conduct experiments with the method without requiring sufficient CI4 to be kept on hand to warrant a permit from the Atomic Energy Commission although the latter is readily obtainable. Other than normal, sensibIe care, no special precautions are required with the method. The beta particles emitted by CI4 are of relatively low energy and are completely attenuated by the flask and planchets containing the radioactive medium. Even in open flasks or planchets, the C14 cannot project beta particles beyond several centimeters in air. The gas produced by the test has a C14 content in the order of micromicrocuries. Small amounts of gas which may escape collec-
RAPID DETERMINATIONS WITH RADIOISOTOPES
113
tion are vastly diluted with air. Probably the principal concern associated with the use of isotopes is the realization that “aseptic” techniques must be used out of consideration for isotopic contamination of the test as much as out of consideration for bacteriological contamination. Nonetheless, in keeping with the general philosophy of isotope handling, routine mop-up counting to check against accidental spills is recommended, as is the use of a hood to carry off the minute traces of C1402. Upon completion of the test, a drop of disinfectant is added to each planchet to prevent further generation of C1402. Although the planchet contents could readily be washed down the sink in accordance with AEC standards, the practice followed has been to store all spent radioactive materials and containers for shipment to a commercial isotope disposal center. c. Radioactive Test Cost. Another factor generally associated with the use of isotopes is high cost. This was true in the early days of the rapid coliform test. The isotope for a single test cost $300.00. The changes reported in the use of the labeled compounds and volumes required have reduced this cost to approximately 10 cents per test. Materials and labor for the test are now less expensive than those for the standard methods. 2. Total Bacteria Test In addition to the needs for the rapid determination of particular species or groups of bacteria, there is also a need for the rapid detection of total bacteria present in a given sample. Classic techniques for total bacteria tests are used in the food processing and serving industries, in testing water supplies and in other public health applications. Of the six principal elements comprising life (carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur ) carbon, hydrogen, phosphorus, and sulfur occur in unstable forms. The short half-lives of the phosphorus radioisotopes make their use difficuIt for routine tests. Furthermore, phosphorus is not evolved in a metabolic gas. S35 has a half-life of 87 days which, far short of the convenient 5,568year half-life of C14, nonetheless, permits its practical use. Obviously, corrections must be applied for the age of compounds containing S35, but the compounds are useful for several half-lives. S3j, like carbon, is a beta emitter. Although protein has an average carbon to sulfur atomic ratio of approximately 150, sulfur has 9,300 times the specific activity of carbon. On this basis, sulfur would
114
GILBERT V. LEVIN
possess a theoretical advantage of 62 to 1 over carbon for use as a label in living material. In actual practice, carrier-free compounds are never used so that the theoretical specific activity comparisons do not come into direct play although they indicate the relative specific activities that may actually be attained in carbon and sulfur compounds. Like carbon, sulfur, in appropriate compounds, can be offered to living organisms and subsequently detected in the organisms themselves or in metabolic products. When S35 is assimilated by organisms producing H$35, the envolved gas can be trapped and counted in the same manner as C1402using either barium hydroxide or lead acetate to fix the sulfide. The radioactive isotope of hydrogen, tritium, offers greater problems to radiomicrobiology, despite its satisfactory half-life of 12.5 years. On disintegration, H3 yields a very low average energy, 0.018 m.e.v., making counting difficult compared to carbon and sulfur which yield betas with average energies of 0.155 and 0.167 m.e.v. respectively. Tritium exchanges readily, resulting in a loss of the tag from the desired sites and a corresponding contamination of sites where the deposition of radioactive atoms may interfere with the test. However, it has become relatively simple to tritiate organic compounds by virtue of this otherwise undesirable tendency to exchange. Tritium counting has been made easier by commercially available instruments, but the complexity and expense of these instruments still substantially exceed those suitable for carbon and sulfur counting. Nonetheless, the specific activity of H3 is 2000 times that of CI4. It is the most abundant species of atoms present in living material and exceeds carbon 12:7. On this basis, it possesses a theoretical advantage over C14 of approximately 3400. This figure and the corresponding one previously cited for H3 pertain to the apparent advantages. In the case of radioactive gas detection, they would have to be further modified by the fraction of the labeled compound converted to gas. Each application must be considered on its own grounds. Thus, there will be circumstances which will warrant the use of tritium labeling in rapid microbiological determinations. Labeled tritium in appropriate compounds assimilated by cells might conveniently be recovered in gaseous form as HZ3,H23S,CH43,or NH33.Appropriate getters would be required. The use of one, two, or all three of these radioactive isotopes in a nonselective medium can produce a total bacteria test. However, unless multiple dilution techniques are
RAPID DETERMINATIONS WITH RADIOISOTOPES
115
used, it is difficult to conceive of a means for making such a test quantitative. The vastly differing metabolic rates and lag periods of diverse species otherwise make it impossible to relate the radioactivity detected to the numbers of cells in the inoculum. 3. Bacteriological Warfare Defense
Shortly after publication on the rapid coliform research (Levin et al., 1956, 1957) the U.S. Army Biological Laboratories became
FIG. 4. Apparatus developed for radioisotope test during aeration. The apparatus used by the US. Army Biological Laboratories in its radioisotopic total bacteria test was patterned after this. Left to right: filter membrane, culture planchet, base, chimney, porous pad, top. Unit assembles in same order.
interested in the possibilities of radioisotopic bacteriology. At this time, the presumptive coliform test was being developed in the apparatus shown in Fig. 4. After inoculation of the culture planchet, the apparatus was assembled. Air was introduced through the side arm, sweeping across the surface of the medium and entraining any gas produced. The air containing the radioactive gas was then exhausted through a barium hydroxide-impregnated, porous paper, collecting pad secured in the top of the device. Using apparatus patterned after this, Yee et al. (1958) at
116
GILBERT V. LEVIN
the Army Biological Laboratories tested the method by incorporating ~ y s t e i n e - Sinto ~ ~ a medium developed at their laboratory to induce H2S production by many species of organisms. The paper pad was impregnated with lead acetate. Using Serratia marcescens as a test organism, the results shown in Fig. 5 were
% MINUTES
i 1
FIG. 5. Response of S. marcescens to rapid test using cysteine-W. Plot was made from data obtained by Yee et al. (1958). Courtesy U.S. Army Biological Laboratories.
obtained. Although the labeled cysteine was more than 6 months old, and therefore considerably reduced in activity, as few as 10,000 cells were detected in less than 3 minutes. It is of interest to note that this early production abated after 2 or 3 minutes. A similar early “burst” of CO, production has been noted with coliform organisms, but since the test for the latter is designed to
RAPID DETERMINATIONS WITH RADIOISOTOPES
117
detect very small numbers of cells, the gas is collected for 4 hours to take advantage of growth. Also worthy of note are the low sterile controls achieved with c y ~ t e i n e - S ~ ~ .
4. Exobiology Turning from the grim prospects of biological warfare, there is a happier use for a total microbes test. With amazing rapidity, the grandest era of adventure in the history of mankind has dawned. The earliest accounts of man show his yearning for knowledge of the celestial bodies. Within the past few years, fantasy on this subject has been reduced to reality. At this writing, two instrumented vehicles are spanning interplanetary voids, one bound for Mars and the other for Venus. These vehicles, laden with scientific instruments, have been set on courses that will take them close enough to their destinations to obtain reliable scientific data on surface conditions and transmit them back to earth. These “fly-by” vehicles will soon be followed by other space craft which will land instruments on the surfaces of the planets. The question of paramount interest and importance is “Does life exist beyond our planet?” Tentatively designated by the ( U.S. ) National Aeronautics and Space Administration for the first Mars landing is “Gulliver” (Levin et al., 1962; Levin and Carriker, 1962), a life detection experiment based upon the rapid radioisotope test. a. Gulliver. An instrument designed to detect life must be based on certain assumed characteristics of that life. Although our imagination can conjure up various exotic forms and mechanisms which would fit our definition of “living,” we cannot ignore the rather amazing fact that all the diverse forms of life on earth share common metabolic processes at the cellular level. Despite the manifold possibilities afforded by the range of chemical, physical, climatological, and other environmental conditions on earth, only aqueous and carbonaceous life exists and, to our knowledge, ever existed. No element approaches carbon in its ability to form complex chains, offering almost an infinite variety of macromolecules from which evolution could choose those best serving it. Similarly, water has no peer as a universal vehicle for solutes and, thus, is best qualified to serve the life processes. It is only logical, then, that our first extraterrestrial life explorations be directed toward types of life resembling those we know. Logic also dictates that extraterrestrial biological forms should be
118
GILBERT V. LEVIN
sought at the micro level. These are more likely to be ubiquitous than would macro forms, and would therefore be easier to obtain by the limited sampling techniques permitted by remote or automatic operations. Any biosphere at or approaching equilibrium and containing macro forms would require a device, such as microorganisms, to perform the catabolic processes. Otherwise, the system wouId be unidirectional, going in the unlikely direction of decreased entropy. The odds against such a short-lived system being in operation at the time of a landing would be great. Having thus determined to seek microbiological forms possessing a biochemistry similar to or approximating our own, other factors influencing the experiment must be considered. The most likely candidate for extraterrestrial life is Mars. To get there with propulsion equipment now available or under development will require approximately 8 months. It is planned that the space vehicle will fly by or to Mars, and, enroute, dispatch an instrument capsule to land on the planet. Impact will be reduced by the opening of a parachute when the capsule enters the Martian atmosphere. The various instruments contained in the capsule will perform their experiments on battery power and transmit the data to earth by radio. Because of the great thrust required for the journey, most of the space craft will consist of fuel, limiting the capsule to approximately 100 to 300 pounds, including instruments. This imposes severe limitations on the size and weight of the instruments, the amount of power they may draw and the length of time power will be available from the batteries which also suffer from the weight limitation. Added to these stringent conditions imposed upon a life-seeking experiment are those of shock and vibration experienced upon launch and impact, the hard vacuum of space, and the wide temperature range that must be withstood during flight and on the target planet. Simplicity and reliability must be such that the results can be unambiguously interpreted. The type of signal produced must be simple enough to be accommodated by the capability of the telemetry which will be considerably restricted by the power limitation. To preclude contaminating the planet or spoiling the experiment, it is imperative that the entire capsule and contents be completely free of earth organisms. The instrument and reagents, therefore, must be capable of withstanding severe heat sterilization.
RAPID DETERMINATIONS WITH RADIOISOTOPES
119
While experiments by Hawrylewicz et al. (1962) have indicated that it is possible for earth organisms to grow under Martian conditions, it seems likely that the stringency of the Martian environment would result in fewer organisms per unit surface than on earth. Because of the cold climate, the Qlo law would anticipate a lower average rate of metabolism. The weight and power limitations and the rotation of Mars, which will interrupt radio communication when the instrument is on the far side of the planet, indicate that the duration of the experiment may be as little as 4 hours and probably no more than 24 hours. These considerations require that the life detection experiment be sensitive to small numbers of cells and that it have a rapid response time. The radioisotope technique is capable of meeting all of the above criteria. As opposed to the desired specificity of the rapid coliform test, an early extraterrestrial life detection experiment should support and detect the growth of all types of microorganisms. Thus, it should be even more general than the total bacteria test. Gases are common products of metabolism of microorganisms. Moreover, most species, and possibly all, produce carbon dioxide. This includes photosynthetic organisms. Other gases of metabolic origin which can be readily labeled are methane, ammonia, hydrogen sulfide, and molecular hydrogen. Research efforts have been directed toward developing a broadly nonselective microbiological medium. The inclusion of appropriately labeled compounds results in the production of labeled gas. Radioactive substrates tested in complex media have included sodium formate-C14,uniformly labeled glucose-C14, sodium acetate1-ci4, sodi~m-pyruvate-l-C’~,glycine-2-U4, c y ~ t e i n e - S ~a~ ,yeast extract randomly labeled with C14, and an E . coli extract randomly labeled with C14.A combination of formate-C14and uniformly labeled g1uc0se-C’~has produced the best results in the medium shown in Table IV. With this medium, rapid responses have been obtained from a wide range of representative microorganisms including bacteria and other fungi, streptomycetes, and algae. Species successfully detected include aerobes, anaerobes, facultative anaerobes, thermophiles, mesophiles, psychrophiles, heterotrophs, phototrophs, autotrophs, spore formers, and nonspore formers. Table V presents results obtained from a wide range of test organisms. Response times and activity levels are shown to illustrate the type of data obtained by the tests. No attempt was made to relate the response
120
GILBERT V. LEVIN
to the size of the inoculum. These responses have been obtained by the detection of C1402 only. This does not, however, preclude the possibility of including additional labeled compounds to produce other radioactive gases. Doing so would improve the probability of response from unknown organisms and also increase the sensitivity. Because of their metabolic importance, methane and hydrogen sulfide are the two most likely candidates in this regard. Figure 6 is a photograph of the instrument1 being developed for the TABLE IV RADIOACTIVETESTh4EDlUhl USED Component K,HPO, KNO, MgS0,.7H20 NaCl FeC13 Amino acid hydrolyzate Yeast extract Soil extract Proteose peptone No. 3 Malt extract Ascorbic acid L-Cystine Beef extract Glucose-C14 Na Formate-Cl4 Distilled H,O Total activity
IN
“GULLIVER” Amount 1.0 gm. 0.5 gm. 0.2 gm. 0.1 gm. 0.01 gm. 4.0 gm. 13.0 gm. 250.0 d. 20.0 gm. 3.0 gm. 0.2 gm. 0.7 gm. 3.0 gm. 0.05 gm. 0.02 gm. u p to 1 liter 10 pc/ml.
experiment, An exploded view showing the parts and assembly, together with a schematic diagram, is shown in Fig. 7. The instrument weighs approximately 1% pounds. Two units will be contained in the space capsule, one to serve as a test and the other as a control. The experiment will proceed as follows: When the capsule containing Gulliver comes to rest on the surface of Mars, a glass ampule containing sterile radioactive broth is broken. Carrier gas is bubbled through the broth to remove traces of nonmetabolic radioactive gas which may have formed due to some breakdown of the medium by internal and external sources of radiation during the long voyage. While the broth is purged, the 1 Instrumentation is being performed by American Machine and Foundry Company, Alexandria, Virginia.
RAPID DETERMINATIONS WITH RADIOISOTOPES
121
two projectiles are fired. Each extends a 25-foot-long string over the surface of the planet. The strings are coated with silicone grease so that particles contacted are retained. A tiny motor then TABLE Va
ORGANISMS EVOLVING C1402
WHEN
TESTED IN
THE
Organism
MEDIUMOF TABLEIV Activity above that of control (counts per minute)
Response within 3% hours
Arthrobacter simplex Azotobacter agilis Azotobacter indicus Bacillus subtilis spores Bacterium bibulum Chlorella sp. Clostridium pasteurianum Clostridium roseurn Clostridium sporogenes Escherichia coli Micrococcus cinnubareus Mycobacterium phlei Pseudomonas delphinii Pseudomonus fhotescens Pseudomonas maculicola Rhodopseudomonas capsulata Rhodospirillum rubrum Saccharomyces cerevisiae Staphylococcus epidermidis Streptmyces fradim Xanthomonas beticola Xanthomonus campestris
1,629 28,956 1,868 11,784 7,221 323 1,698 5,367 664 65,389 479 1,913 971 6,701 16,266 365 420 858 3,219 560 58,189 537
Response within 6 hours
Photobacterium phosphoreum Thiobacillus novellus Thiobacillus thiooxidans
2,423 141 102
Response between 6 and 24 hours
Rhizobium leguminosarium
1,123
From Levin et al. Reproduced courtesy of Science 138, 3537, 117 (1962). a
winds the strings into the incubation chamber together with their precious cargo of soil. This operation takes several minutes, during which the culture chamber is free to exchange its atmosphere with
122 FIG. 6. Gulliver. A view of the complete instrument and associated electronic systems. Projectile guns would be mounted adjacent to capsule wall. Base plate and projectile gun mounts are for display only. Total fight weight is approximately 1.5 pounds. Instrumentation by American Machine and Foundry Company, Alexandria, Virginia, Division.
INCUBATION CHAMBER
Shm‘PlE COLLmw
INJECTOR
FIG. 7. An “exploded’ diagram of the instrument now being used. 1, Nonexplosive motor; 2, ampule breakers; 3, pressure release valve; 4, normally closed valve; 5, normally open valve; 6, throat baffle; 7, radiation detector, 8, antimetabolite injector; 9, electric motor and gear box; 10, thermostat; 11, acid ampule; 12, broth ampule; 13, string port. From Levin et al. (1962), p. 118.
124
GILBERT V. LEVIN
that of Mars. During the test, the only condition which will be imposed on the ambient environment of Mars will be the maintenance of the broth above the freezing temperature. After the string has entered the chamber, the chamber is sealed and a background count of the radioactivity is made. The radioactive broth is then injected into the culture chamber, saturating the string. A radiation detector is mounted directly above the culture chamber. A baffle intervenes so that the detector cannot see the radioactivity in the broth. The face of the detector is thinly coated with barium hydroxide. If organisms which can utilize the medium are present on the soil particles, C1402should be evolved. The gas wilI migrate from the culture chamber through the baffle arrangement and deposit on the coating of barium hydroxide where it will be fixed as carbonate. The radiation detection instrument will make periodic counts of the amount of C1402 thus deposited. Should other gases be sought through the use of additional labels, appropriate getters will be applied to the face of the radiation detector. The second instrument will be identical to the first and will be operated in the same manner with the exception that an antimetabolite will be injected shortly after introduction of the Martian soil. The data from each will be transmitted to earth. The generation of the classic biological growth curve when radioactivity is plotted as a function of time will constitute evidence of life in the test instrument. If an inhibition in the growth curve is produced in the control, a very strong case will have been made for life on Mars. The instrument shown in Fig. 6, containing an ampule of the medium cited in Table IV, has been field tested. The results of such a test, performed on frozen soil, are shown in Fig. 8. After collection outdoors, the instrument was removed to the laboratory where the experiment continued at room temperature. The curve produced is interesting from several aspects. The rapid response is evident. Moreover, three exponential phases of the curve are evident on the semilog plot. The indication is that three different groups of organisms predominated in the sample, each having its distinct lag period, exponential growth phase, and stationary phase. In addition, the average generation period for each group of organisms is given by the slope of the respective portion of the curve. Furthermore, it is interesting to note that the generation
RAPlD DETERMINATIONS WITH RADIOISOTOPES
125
periods for the exponential phases increased from left to right on the time scale. This is as might be expected since it associates those organisms which took longer to come out of lag phase with slower rates of metabolism.
FIG. 8. Evolution of C14o2 by microorganisms in soil collected and cultured in a field test with the instrument shown in Fig. 6. The soil was collected from frozen ground. From Levin et al. (1962), p. 119.
Rapid responses have been obtained with the method when strings were drawn across various adverse environments such as a pile of sand, an asphalt street, and a plate glass window. As little as 10 mg. of soil supplied from a remote area of the Mojave Desert and aseptically stored for several months produced the response seen in Fig. 9. In this case, the labeled compounds in Table IV were increased fivefold in concentration. Despite the gratifying
126
GILBERT V. LEVIN
rapidity of the various results shown, Gulliver is still one to two orders of magnitude less sensitive than the planchet method. This is due to factors introduced by the geometry and components necessary to the function of the instrument. Attempts are underway 60,000~
I
n
0
10 Time
20
(Hwrr)
FIG. 9. Evolution of C1402 by organisms in various quantities of Mojnve Desert soil inoculated into medium shown in Table IV with formate-Cl4 and glucose-Cl4 concentration increased fivefold. Curves are drawn through points obtained from readings taken every 24 minutes. From Levin et al. (1962), p. 116.
to increase the sensitivity of the instrument until it closely approaches that offered by the basic technique. Similarly, considerable effort will yet be spent in improving the medium to increase sensitivity and the broadness of response to it. When the final medium has been developed, those labeled and unlabeled compounds which have optical activity will be racemized in the event
RAPID DETERMINATIONS WITH RADIOISOTOPES
127
that Martian organisms require isomers opposite to those utilized on earth. Should Gulliver find life on Mars, the door would be open to extensive microbiological determinations on Mars. Rather than an “organic smorgasbord,” specific labeled substrates would be offered to the organisms. Temperature and light responses would be measured. The organisms would be tested under aerobic and anaerobic conditions and under atmospheres of various compositions. Specificity for optical isomers would be determined, DNAlike compounds would be sought. These and other experiments might determine whether the Martian organisms shared their origin with life on the earth, or whether life evolved independently on the two planets. Such genetic relationship or independence would be of major importance in the search for the origin of life.
5. Selection of Antibiotics Another application of the radioisotope technique may provide clinical medicine with an important tool. Just as the introduction of a tag can quickly detect growth in microorganisms, the inhibition of growth can be detected with equal ease. A method has been developed by Heim et al. (1960) which demonstrates this. The apparatus is the same as that described for the rapid coliform test. However, filtering through a membrane filter is not required since the numbers of organisms available are large. Two-tenth ml. replicates of organisms isolated from the infected person are placed into a set of the one-inch planchets, each of which contains 0.8 ml. of trypticase-soy broth to which has been added 0.003% sodium f0rmate-U’ ( 2 mc./millimole). Two or more replicates are used for the inoculated control. Into a series of sets of replicates of the culture, various concentrations of the antibiotics to be tested are added. Replicate sterile controls are also run. All planchets are then incubated at 37°C. in petri dishes. At the end of 2 hours, planchets containing pads impregnated with a saturated solution of barium hydroxide are inverted over the incubating planchets. Collection of C1‘02 thus proceeds for 30 minutes after which the collection planchets are removed, dried, and counted for radioactivity. Incubation is continued and a similar collection of C1‘02 is made at the fourth hour, terminating the test. The effects of various concentrations of penicillin and tetracycline on an E. coli are shown in Table VI. The table includes the results
128
GILBERT V. LEVIN
obtained when replicate concentrations of the organisms and antibiotics were tested by the conventional 24-hour tube dilution technique. An inoculated control, to which no antibiotics had been administered serves as a reference. All values reported for the radioactive test are averages of duplicates from which sterile control and background levels have been subtracted. A decrease in fourth hour activity over that of the second hour indicates effectiveness of the antibiotic as applied. The difference between penicillin TABLE VIa EFFECTS OF ANTIBIOTICS ON C*402 RELEASEDBY E. coli Antibiotic None Penicillin
Tetracycline
Concentration0
1 unit/ml. 5 units/&. 50 units/&. 1 Wml. 5 pg/d. 50 pg/ml.
IN
Counts per minutec 2 hours 4 hours 1915 2654 1379 4665 273 348 17
15,810 25,406 23,105 9,854 1,588 16 12
2
AND
4 HOURS
Tube dilution, 24 hours
Growth Growth Growth Growth No growth No growth
a From Heim et al. Reproduced courtesy Antimicrobial Agents Ann. p. 124 ( 1960). b Concentration of cells, 10-2 dilution of an 18- to 20-hour culture. c Counts per minute have background and sterile controls subtracted and are averages of duplicates.
and tetracycline is thus readily apparent. The increase in activity of the portions containing the lesser quantities of penicillin over those containing no antibiotic has been frequently observed. This is an interesting revelation of the radioisotope technique and requires further study for interpretation. The results of the 24-hour tube dilution test were in complete agreement with the rapid test and verified that tetracycline became effective at a concentration of 5 pg. per ml. Table VII shows the results obtained by the test performed on Proteus. Both the E . coli and the Proteus were isolated from hospital patients. Other tests have been successfully performed directly on body fluids, including urine. Such direct application is the ultimate objective of the rapid method.
6. Prospecting for Petroleum and Gas A radioisotope technique for prospecting for petroleum has been developed by Davis (1957). Earth overlying oil or gas deposits
TABLE VlIa DETERMINATION O F ANTIMICROBIALACTIVITY: EFFECTSOF ANTIBIOTICS ON
Antibiotic None Penicillin
Tetracycline
1 Hour 230
2 Hours 1,500
1 unit/ml. 5 units/ml.
200 193 248
1,362 1,274 3,441
8,820 7,552 18,597
167 154 88
401 144 37
721 311 28
1 pg/ml.
5 pg/ml. 50 pg/ml. b
d.Reproduced
PrOteUS OVER 6 HOURS
5 Hours 10,839
6 Hours 6,243
19,766 30,625 28,605
18,020 14,690 8,545
3,967 6,403 1,556
2,613 418 14
5,286 1,781 11
17,118 9,543 10
courtesy Antimicrobial Agents Ann. p. 125 ( 1960). Concentration of cells, 10-2 dilution of 18 to 20-hour culture. Counts per minute have background and sterile controls subtracted and are averages of duplicates.
a From Heim et 0
Counts per minutec 3 Hours 4 Hours 9,805 27,982
Concentrationb
50 units/ml.
c1402 RELEASED BY
z U
Tube dilution, 24 hours
8z
Growth Growth Growth
0
Growth Growth No growth
5 2 5 z
v)
5 2 a 0
B
2!2
130
GILBERT V. LEVIN
is permeated with hydrocarbons emanating from the deposits. The earth in these areas contains hydrocarbon-consuming microorganisms which have been selected by the environment. A sample of earth overlying a suspected petroleum or gas deposit is exposed to radioactive hydrocarbons in gaseous or liquid phase. If such organisms are present, the label will be detected in the metabolic products. The detection of these products indicates the presence of the deposit.
7. Experimental Uses UntiI now the discussion of application of the radioisotope technique has been limited to its use in practical test methods. However, the sensitivity and simplicity of the method make it valuable in fundamental as well as applied research in microbiology. It can be useful in conjunction with conventional respirometric methods. It also offers some advantage over these. For example, it permits results to be obtained within minutes, or even seconds, of the onset of an experiment. This makes possible a study of the early kinetics of metabolic reactions otherwise difficult or impossible to observe. An example of this is the early “burst” of activity detected when coliform organisms or S . marcescens (as reviewed herein) are given a source of energy. It is possible to study minute details of respiration rate changes in the organisms when making the transition from lag to growth phase. Automatic instrumentation such as that shown in Fig. 10, developed especially for laboratory research on the Mars life probe, makes it possible to follow gas production as it occurs. This permits experiments to be conducted on organisms at specified points in the development of the culture. It is possible to time and measure the effects produced by the introduction of various metabolites or antimetabolites of interest. An interesting effect observed by the radioisotope technique was that C 0 2 is required by growing coliform organisms. When the planchet apparatus was first used, the barium hydroxide planchet was inverted over the culture planchet immediately after inoculation. This seemed logical to effect maximum Cl4OZ collection. However, cultures containing small numbers of cells failed to go into exponential growth under these conditions. This inhibition was removed when the culture was permitted to incubate before application of the C 0 2 collection planchet. Further evidence of this
RAPID DETERMINATIONS WITH RADIOISOTOPES
131
effect was produced by comparison of C1*02production by sterile controls and test cultures several minutes after inoculation. It was found that less C1402 was collected from the cultures than from the sterile controls. The nonmetabolic C1402 in the sterile controls had been incorporated by the cells. This is in keeping with recent
FIG. 10. Automatic radioisotope microbiological monitoring apparatus. Multichannel recorder is at left. Eight culture chambers, same size and design as in Gulliver, are shown in center. A geiger tube (black cylinder) is mounted directly over each chamber. Log count rate meter is at right. Gas produced by cultures in each of the chambers is sequentially counted and recorded. Each chamber is counted at 24 minute intervals.
findings cited by Roberts et al. (1955, p. 95) of the importance of COz in anabolism. Another type of experimental application is the study of toxicity resulting from the membrane filter. Since the membrane filter is an integral part of the rapid coliform test, it is important that the toxic effect be reduced to a minimum. At the same time, insight thus gained will help elucidate the results obtained with the standard method membrane filter test.
132
GILBERT V. LEVIN
Ill. Conclusion It is abundantly clear that there is great room and need for improvement in classic procedures for quantitative microbiological determinations. Radioisotope techniques which, through their extreme sensitivity, render extended incubation of organisms unnecessary, may meet the important requirement for speed. Accuracy, another major requirement, may also be significantly improved by using isotopes to investigate “biological vagaries” and errors introduced through imperfect methodology. Finally, continued exploitation of isotopes in microbiological research promises to elucidate many fundamental biochemical processes. REFERENCES Am. Public Health Assoc. (1962). “Standard Methods for the Examination of Water and Wastewater,” 11th ed. Am. Public Health Assoc., New York. Butkevich, N. V., and Butkevich, V. S . (1936). Mikrobiologiya 5, 322. Cowie, D. B., Bolton, E. T., and Sands, N. K. (1950). J. Bacteriol. 60, 233. Cowie, D. B., Bolton, E. T., and Sands, N. K. ( 1951). J. Bacteriol. 62, 63. Cowie, D. B., Bolton, E. T., and Sands, N. K. (1952a). J. Bacteriol. 63, 309. Cowie, D. B., Bolton, E. T., and Sands, M. K. (195213). Arch. Biochem. Biophys. 35, 140. Davis, J. B. (1957). US. Patent 2,777,799. Frush, H. L., and Isbell, H. S . (1953). J . Res. Nat2. BUT. Standards 50, 133. Hawrylewicz, E., Gowdy, B., and Ehrlich, R. (1962). Nature 193, 497. Heim, A. H., Curtin, J. A., and Levin, G. V. (1960). Antimicrobial Agents Ann. p. 123. Jannasch, H. W., and Jones, G. E. (1959). Limnol. and Oceanog. 4, 128. Jones, G. E., and Jannasch, H. W. (1956). Limnol. and Oceanog. 4, 269. Laurence, D. J. R. ( 1957). In “Methods in Enzymology” ( S . P. Colowick and N. 0. Kaplan, eds.), Vol. IV, p. 174. Academic Press, New York. Levin, G. V., and Carriker, A. W. (1962). Nucleonics 20, 71. Levin, G. V., Harrison, V. R., and Hess, W. C. (1956). J. Am. Water Works Assoc. 48, 75. Levin, G. V., Harrison, V. R., and Hess, W. C. (1957). J. Am. Water Works Assoc. 49, 1069. Levin, G. V., Harrison, V. R., Hess, W. C., Heim, A. H., and Stauss, V. L. (1959). J. Am. Water Works Assoc. 51, 101. Levin, G. V., Stauss, V. L., and Hess, W. C. (1961). J. Water Pollution Control Federation 33, 1021. Levin, G. V., Heim, A. H., Clendenning, J. R., and Thompson, M. F. (1962). Science 138, No. 3537, 114. McCarthy, J. A. (1961). Proc. Rudolf* Res. Conf. Public Health Hazards of Microbial Pollution of Water (and discussion), p. 123-180, Dept. Sanitation, Rutgers Univ., New Brunswick, New Jersey.
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McCarthy, J. A., Thomas, H. A,, Jr., and Delaney, J. E. (1958). Am. 1. Public Health 48, 1628. Membrane Filtration. Baoteriological and other Applications of Oxoid Membrane Filters, Oxoid Membrane Media, The Oxoid Div., 0x0. Ltd., London. Public Health Sew. (1962). “U.S. Public Health Service Drinking Water Standards,” U.S.P.H.S., Dept. of Health, Education, and Welfare, Washington, D.C. Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T. Bolton, and R. J. Britten ( 1955). “Studies of Biosynthesis in Escherichia coli.” Camegie Inst. Wash. Publ. 607, Washington, D.C. Taylor, E. W., “Thirty-Ninth Report on the Results of the Bacteriological, Chemical and Biological Examination of the London Waters for the Years 1959-1960,” p. 20. Metropolitan Water Board, London. Yee, G. S., Taylor, E. R., Jr., and Bolduan, 0. E. A. (1958). Private communication, U S . Army Biol. Labs., Fort Dietrick, Maryland.
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The Present Status of the 2,3-Butylene Glycol Fermentation STERLINGK. LONGAND ROGERPATRICK University of Florida Citrus Experiment Station, Lake Alfred, Florida I. Introduction ........................................... 11. Types of Substrates ..................................... 111. Organisms Producing 2,3-Butylene Glycol . . . . . . . . . . . . . . . . . . A. Comparison of Aerobacter aerogenes and Bacillus polymyxa IV. The Fermentation ...................................... A. Acclimatization of Cultures .......................... B. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Aeration .......................................... E. Concentration of Carbohydrate ........................ F. Nitrogen and Miscellaneous Supplements . . . . . . . . . . . . . . . . V. Recovery of 2,SButylene Glycol .......................... VI. Potential Uses of 2,3-Butylene Glycol ...................... VII. Probable Future of the 2,3-Butylene Glycol Fermentation .... References ............................................
135 136 138 139 142 142 144 144 146 147 148 149 150 152 153
1. Introduction The authors would like to direct attention to the fact that this article should not be considered as a comprehensive review of studies on fermentation production of 2,3-butylene glycol, but rather as an attempt to explore the more recent interesting development on this fermentation, especially since 1945. Naturally, some overlapping is necessary and desirable; however, this will be held to a minimum. By contrast with some of the older fermentations, 2,3-butylene glycol (2,3-butanediol) is a relative newcomer to the field and as such has yet to attain importance as a commercial product, although we may rely upon the ingenuity of man eventually to develop processes for utilization of this interesting component. The earliest report of production of 2,3-butylene glycol by bacterial fermentation was that of Harden and Walpole (1906) using cultures of Aerobucter aerogenes, while it remained for Donker ( 1926) to explore similar fermentation abilities of Bacillus polymyxa. During the next 16 years, occasional studies served to maintain a low level of interest in butylene glycol, but it was not until 135
136
STERLING I<. LONG AND ROGER PATRICK
1942 that anticipated wartime shortages of a strategic compound, 1,3-butadiene, again focused attention on the fermentation. From 1942 to 1945 a number of processes were developed as a result of accelerated investigations by Canadian and United States agencies in cooperation with private fermentation industries. Although several of these investigations led to pilot-plant operations, none reached commercial production. A more comprehensive discussion of the wartime development of the fermentation may be found in the excellent review by Underkofler and Hickey (1954). Although the development of the butylene glycol fermentation received its greatest impetus due to the urgencies of World War 11, basic and applied studies continue to the present at perhaps a more leisurely pace, but no less imaginatively. It is these postwar years with which we will be concerned in this paper.
II. Types of Substrates Although a number of bacterial species produce 2,3-butylene glycol, past studies have essentially eliminated all but Bacillus polymyxa and Aerobacter aerogenes as potentially of industrial importance. Therefore this discussion will be limited to the substrates which can be utilized by these organisms. The more versatile of the two organisms, in a variety of suitable substrates, is B. polymyxa. This species is actively diastatic and fermentations may be run using starchy raw materials such as corn or wheat as well as the common sugar-containing substrates. Aerobacter aerogenes, however, is not diastatic and can ferment only sugars. The economics of the glycol fermentation limit the carbohydrate sources to the various types of molasses, the cereal grains, or industrial waste liquors. Purified carbohydrates such as lactose, glucose, and sucrose, while readily fermentable, are much too costly for this use. It is obvious that major factors in selection of a raw material are the comparative cost of the material and the value of the final fermentation product. It seems worthwhile to consider these carbohydrate sources in somewhat greater detail. Naturally each of these raw materials has certain disadvantages which may require considerable modification to render them suitable for fermentation; however, for convenience, they will be considered equivalent in fermentability.
2,3-BUTYLENE GLYCOL FERMENTATION
137
The most desirable raw material, i.e., least expensive, for the glycol fermentation would probably be an industrial waste material of little or no other utility, e.g., sulfite waste liquor or canning plant press juice. The primary advantage here lies in the fact that, in addition to being of little economic value, such wastes may actually constitute a disposal problem to the industry. Sulfite waste liquor has been successfully used as a substrate for glycol production by several bacterial species (Murphy and Stranks, 1951) although only A. aerogenes gave good yields of glycol. While this substrate is plentifu1 and obviously cheap, it was suitable for fermentation only upon supplementation with molasses. Another disadvantage is the low average sugar content (3.8%) and the small yields of glycol obtained (O.Q-l.O% ). This waste could very likely become a very useful substrate upon further study. Citrus canning plant press juice as well as sugar-containing wastes from other fruit canneries seem to have considerable potential for production of butylene glycol. Citrus press juice is fairly high in sugar content ( 4 4 % ) (Hendrickson and Kesterson, 1951) and has been used successfully for the glycol fermentation (Long and Patrick, 1960). Extraction of the glycol from the fermentation beer would be somewhat more of a problem due to the low concentration of the product; however, the increased production costs here might be offset by the cheapness of the raw material. Approximately 7 gallons of press juice were required to produce 1 pound of glycol in A. aerogenes fermentations. The cheapest types of molasses are blackstrap and citrus molasses. Both are readily fermented to butylene glycol by A. aerogenes and B . polymyxa (Bahadur and Ranganayaki, 1960; Owen, 1950; Long and Patrick, 1960; Freeman and Morrison, 1947). Blackstrap molasses has a sugar content of approximately 52% whiIe citrus molasses contains about 45% total sugars. Although these molasses compete for the same market, the price per ton of blackstrap is considerably higher than citrus molasses; however, blackstrap enjoys a slight advantage due to higher sugar content. Using the most active glycol-producer, A. aerogenes, butylene glycol can be produced from citrus molasses at a substrate cost of around $.09 per pound of product, and from blackstrap at $0.13-$0.18 per pound of product. Since both types of molasses are utilized in other fermentations (e.g., ethyl alcohol) and for animal feed, an-
138
STERLING K. LONG AND ROGER PATRICK
nual variation in supply and demand will result in some fluctuation in substrate cost per pound of product. The fermentation of sugar beet molasses to butylene glycol has been rather extensively investigated with favorable results. Yields as high as 1 pound of glycol per 1.8 pound of sugar beet sucrose (McCall and Georgi, 1954) and 1 pound of glycol per 6-7 pounds of sugar beet molasses have been reported. Good yields of glycol have also been reported by Murphy et al. (1951b) and Anastassiadis and Wheat (1953). This type of molasses seems well suited to the fermentation, although cost of the substrate is a factor to be considered. The market price of beet molasses may exceed that of blackstrap (cane) by $3-$5 per ton and that of citrus molasses by $%$lo per ton. Corn molasses (“Hydrol”), while no doubt equivalent to the other types of molasses in fermentability, occupies about the same price position as beet molasses. A considerable amount of study has been devoted to the use of surplus cereal grains, primarily corn and wheat, for the butylene glycol fermentation. It should be noted, however, that it is doubtful that these raw materials would be economically feasible. As stated previously, only B . polymyra can produce glycol from unhydrolyzed grain and this organism characteristically produces low yields of glycol in all substrates. As much as 8.9 pounds of Z-2,3butylene glycol per bushel of wheat has been obtained (Blackwood et al., 1949) which, at 1960 market prices, would amount to a substrate cost of about $0.24 per pound of product. It is obvious that the substrate cost alone far exceeds the last reported market price (1960) for butylene glycol of $0.15 per pound. The use of corn (or cornstarch) creates a similar problem since substrate cost per pound of glycol would be approximately $0.08$0.10, assuming complete conversion (Kooi et al., 1948; Ward et al., 1944). Hydrolysis of corn or wheat starch to glucose (Ward et al., 1944) which might thus be fermented by the more active A. aerogenes offers little hope of industrial application due to the added costs of hydrolysis to already rather expensive substrates.
111. Organisms Producing 2,3-Butylene Glycol A number of species of microorganisms produce butylene glycol but only a few do so in what might be considered significant quantities. Species which are noted for this ability include those belong-
139
2,3 -BUTYLENE GLYCOL FERMENTATION
ing to the genera Aerobacter, Bacillus, Serratia, and Pseudomonas (Table I). It is expected that future studies will reveal other glycol-producers of potential industrial value but the most promising results have been obtained with only two of the above genera. The following discussion will be limited to these two organisms. TABLE I STEREOISOMERS PRODUCED BY VARIOUS SPECIES Organism
Bacillus polymyxa Bacillus subtilis Aerobacter aerogenes
Serratia marcescens, Serratia spp. Pseudomom hydrophila
Distillers yeast
Stereoisomer of butylene glycol D-(-)
(leuo)
Approx. 65% D-(-) remainder meso S l 4 % L-(f) remainder meso Primarily meso
Reference Neish (1945) Blackwood et al. (1947) Morel1 and Auemheimer (1944); Freeman (1947) Neish ( 1947); Neish et al. (1948) Murphy et al. (1951b)
50% racemic 48% meso 2% levo Neish (1950) 67% leoo remainder meso (possibly some dextro )
A. COMPARISON OF Aerobacter aerogenes
AND
Bacillus polymyxa
Only two organisms, A. aerogenes and B. polymyxa have been shown to be potentially useful in the industrial production of 2,3-butylene glycol, In view of this, it seems to be desirable to consider the advantages or disadvantages of each in somewhat greater detail. The major attribute of B. polymyxa is its ability to produce the pure 1-isomer of butylene glycol. The special properties of this isomer in the preparation of antifreeze (Clendenning, 1946) would provide the only justification for using this species in commercial operations. A number of serious disadvantages would probably lessen the importance of this isomer. Bacillus polymyxa, in addition to glycol, produces large quantities of ethanol. This cannot be considered particularly advantageous since ethanol can be produced much more efficiently by yeast ( Liebmann, 1945). The value of the capacity of this organism to ferment unhy-
140
STERLING K. LONG AND ROGER PATRICK
drolized grain mashes is minimized by the greater difficulty in recovering the glycol from the high solids content mashes. Where the problem of availability of raw materials is of no great importance, the use of some type of molasses would be more satisfactory in this respect. Even this type of substrate would present some difficulties due to characteristically high residues of sugar which interfere with recovery of the glycol. Loss of fermentation activity or loss of cultures must also be considered in using B. polymyxa for commercial production of glycol. The organism is subject to a loss of activity upon repeated transfers or storage especially on media containing sugars (Blackwood et al., 1949; Long and Patrick, 1962). Preparation and acclimatization of cultures for use in fermentations must be done carefully to prevent such occurrences. The organism is also subject to loss of fermentation activity upon storage on ordinary stock culture media (Ledingham and Stanier, 1944). The only satisfactory method of carrying stock cultures is lyophilization. Another disadvantage of considerable importance is the sensitivity of B. polymyxa to attack by bacteriophage (Katznelson, 1944a, b ) . The yield of butylene glycol by B. polymyxa, in all types of substrates, is uniformly much lower than with A. aerogenes. Aerobacter aerogenes will usually produce at least twice the amount of glycol obtainable from B . polymyxa. This would result in considerably higher production costs per pound of glycol than would be obtained with the former organism. Aerobacter aerogenes is more stable under a wider range of environmental conditions than B. polymyxa and produces significantly less ethanol. Difficulties in recovery of the glycol due to residual sugar are considerably less with Aerobacter due to the completeness of fermentation. This organism possesses no diastatic properties and therefore cannot be used for fermentation of whole grains; however, in view of the greater yields of glycol obtainable, it would probably be more practical to hydrolize such starches to sugars, which could then be fermented, than to use B . polymyxa. Aerobacter aerogenes retains its viability on a variety of stock culture media and may even be carried on media containing the fermentation substrate without loss of activity. A practical value of this would be the convenience of maintaining acclimatized stock cultures. Consideration of butylene glycol as a simple industrial chemical, without reference to the isomer produced, would neces-
ORGANISMS
TABLE I1 PRODUCING 2,3-BUTYLENE GLYCOLAND RAW MATERIALSUSED
Organisms
Raw material
(SINCE
1944)
References
Bacillus polymyxa, Aeromnas liquefaciens, Bacillus subtilis, Psmdmnonus hydrophila, Aerobacter aerogenes
Blackstrap, cane molasses
Freeman and Morrison ( 1946); Freeman and Morrison (1947); Bahadur and Ranganayaki (1960)
B. polymyxa, B. subtilis, Serratia marcescens, A. aerogenes, P. hydrophila
Beet molasses
Freeman and Morrison ( 1947); Simpson and Stranks (1951); Murphy et al. (1951a,b); Wheat (1953); Anastassiadis and Wheat (1953); McCall and Georgi (1954)
B. polymyxa, A. aerogenes B. polymyxa
Citrus molasses
Long and Patrick ( 1960, 1961 )
Whole wheat
Ledingham et al. (1945); Clendenning (1946); Wheat et al. (1948); Blackwood el al. (1949)
A. aerogenes B. polymyxa
Acid-hydrolized wheat Cornstarch
Olson and Johnson (1948) Kooi et al. (1948)
A. aerogenes B. polymyxa
Acid-hydrolyzed cornstarch Barley
Ward et al. (1945) Tomkins et al. (1948)
A. aerogenes B. polymyxa, B. subtilis, A. aerogenes, Serratia spp., P. hydrophila B. subtilis, Bacillus cereus, P. hydrophila, A. aerogenes, Serratia spp., Yeast
Wood hydrolyzate Sulfite waste liquor
Perlman (1944) Murphy and Stranks (1951)
Glucose
Stanier and Adams (1944); Blackwood d al. (1947); Rose (1947); Tyson (1948); Blackwood et al. (1949); Neish (1950); Neish and Blackwood (1951); Tamboline (1953)
A. aerogenes
Sucrose
Freeman ( 1947)
, N (r,
$ L-
M
3
P n
8
3
i5
z8
2,
w
142
STERLING K. LONG AND ROGER PATRICK
sarily lead to selection of A. aerogenes as the culture of choice for commercial production (Table 11).
IV. The Fermentation A discussion of the optimum conditions for any fermentation must take into account a variety of factors which, collectively, are responsible for determining a single optimum condition. In addition, it must be kept in mind that even a slight change in any given fermentation condition will alter the amounts of the individual products formed. In view of the complexity of the problem, it is possible only to make certain generalizations regarding what might be considered as “optimum.” Exceptions to the following are to be expected, especially for untested substrates; still, the data provided may be useful for approximating ranges of suitable growth and fermentation conditions. A. ACCLIMATIZATION OF CULTURES
A generally accepted practice in the fermentation industry is acclimatization of the culture to the substrate. Whether this may be required depends, to a large extent, upon the vigor of the culture and the suitability of the substrate. For example, Aerobacter aerogenes is a very active fermenter of most carbohydrates under a variety of conditions. Bacillus polymyxa, by contrast, is somewhat less vigorous in the fermentation of carbohydrates and is considerably more demanding in its other environmental requirements. Experimental evidence indicates that acclimatization of A. aerogenes may be unnecessary although it is frequently done, while it is generally recommended for €3. polymyxa. The suitability of the substrate employed in the glycol fermentation is the determining factor in whether acclimatization may be required. The presence of toxic factors or even excessive concentrations of certain nutrients or carbohydrates will often inhibit growth of unadapted cultures or favor the production of undesirable end products. Freeman and Morrison (1947) suggested that the retarded growth rate of A. aerogenes in blackstrap molasses was probably due to toxic substances in the substrate. This toxicity was minimized by acclimatization of the culture. Occasionally it has been reported that prior adaptation of the
2,3 -BUTYLENE GLYCOL FERMENTATION
143
culture has resulted in an increase in yield of glycol, although the usual effect is that of decreasing the total fermentation time. Perlman (1944) obtained greater yields of glycol using cultures of A. aerogenes which had been acclimatized to the wood hydrolyzate substrate. He also observed that it was necessary to supplement the substrate with yeast extract or malt sprout extract if high sugar concentrations and unadapted cultures were used. These additives were not required if the cultures had been acclimatized. Somewhat higher yields of glycol were obtained by Freeman (1947) using a relatively small inoculum of unacclimatized A. aerogenes. He claimed this was due to better acclimatization of the culture during the early stages of growth in the fermentation mash. Wheat (1953) grew A. aerogenes and Psewdomonas hydrophila cultures on molasses agar sIants prior to inoculation of the molasses mash. Similar pretreatment of cultures was done by Anastassiadis and Wheat (1953) and Simpson and Stranks (1951) with P. hydrophila and B. polymyxa, respectively, in beet molasses substrates. Blackwood et al. (1949) found it desirable to serially transfer cultures of B. polymyxa on a wheat-starch-yeast extract medium prior to inoculating the fermentation mash. Other workers (Olson and Johnson, 1948, using A. aerogenes, and Kooi et al., 1948, with B. polymyxa) preferred acclimatization of inocula although it had little effect on the efficiency of the fermentation. The only difference noted in fermentation of citrus molasses by acclimatized and unacclimatized cultures of A. aerogenes was a somewhat more rapidly completed fermentation (Long and Patrick, 1961). Degree of utilization of sugar and total yield of butylene glycol were unaffected. Acclimatization of B. polymyxa to this substrate was required for maximum glycol production. It has been found undesirable to use fermentation media for carrying stock cultures of B. polymyxa since this organism has been observed to lose activity upon storage or repeated transfers under these conditions (Blackwood et al., 1949; Long and Patrick, 1962). The effect of storage on such media apparently must be determined individually for each substrate since Kooi et aL (1948) observed no adverse effects after repeated subculturing of B . polymyxa on a cornstarch medium. Similarly, Murphy et al. (1951b) obtained satisfactory results with P. hydrophila even after repeated transfers on a weak beet molasses stock medium. Aerobacter aero-
144
STERLING K. LONG AND ROGER PATRICK
genes was unaffected by prolonged storage on fermentation media, even upon standing at room temperature for several days (Long and Patrick, 1962). In an acclimatization of a different type, the effect on B. p02ymyxa of an accumulation of glycol in the mash was considered by Blackwood et al. (1949). Attempts to increase yields by acclimatizing the culture to butylene glycol were unsuccessful. Heatshocking had an adverse effect upon the production of glycol. B. TEMPERATURE The temperature of fermentation affects butylene glycol production in a predictable manner. As the temperature is increased, within certain limits, the rate of fermentation is increased. It should be noted however that this increased rate is accompanied by a decrease in the total yield of glycol. Thus, McCall and Georgi (1954) found the fermentation of sugar beet molasses by A. aerogenes to be more complete at 37"C., but much less efficient than at lower temperatures. Similar findings have been noted by Long and Patrick (1962). There seems little doubt that the greatest yields of glycol are obtained within the optimum temperature range for growth of the species employed. Naturally the type of substrate is a major factor affecting this optimum. Reports in the literature indicate a variety of temperature ranges for the glycol fermentation, but with considerable agreement within the same species and substrate combinations. The optimum range, in general, falls within the limits of 30°-37"C. (McCall and Georgi, 1954; Freeman, 1947; Murphy et al., 1951a; Perlman, 1944). Since the glycol fermentation is a strongly exothermic reaction, maintenance of the proper temperature during the course of the fermentation, after growth has been initiated, is primarily a problem of removal of heat. In the absence of some means of cooling the mash, the temperature increases until complete inhibition of growth and glycol production occurs. Satisfactory control may be obtained using cooling coils within the fermentor or, if a smallscale fermentation, by immersion of the fermentor in a flowing water bath. C. PH The observed pH optima for the glycol fermentation are similar in variability to other factors effecting the fermentation. Here again, the interplay of a number of factors, including the nature
2,3 -BUTYLENE GLYCOL FERMENTATION
145
of the substrate, aeration, temperature, and other environmental conditions, as well as the species used, governs the optimum pH range. In general, most studies agree that as the pH is increased above a certain level ( 6.3-6.5) butylene glycol production diminishes while organic acids increase. For most substrates, including wood hydrolyzates, citrus molasses, sulfite waste liquor, and cereal grain mashes, the optimum range was 6.0-6.2 (Perlman, 1944; Murphy and Stranks, 1951; Tompkins et al., 1948; Long and Patrick, 1960). Somewhat lower values were obtained by Freeman and Morrison (1947) who reported that the optimal pH range for fermentation of blackstrap molasses by A. aerogenes was 5.6-6.0. Olson and Johnson (1948) obtained good production of glycol from hydrolized wheat mash without pH control although a pH of 5.25 was attained during the course of the fermentation. Similarly, a rather broad optimum p H range was reported by Blackwood et a2. (1949) who found that alteration of pH within the range 5.8-7.0 during B. polymyxa fermentation of malted wheat mashes had no effect upon glycol yields. Tompkins et al. (1948) also obtained good results by maintaining a B. polymyxa fermentation using barley as a substrate within pH 5.7-6.5. It is, of course, difficult to compare one substrate with another in terms of a single factor which markedly affects the fermentation; however, it is worthwhile to note that Long and Patrick (1962) obtained complete inhibition of glycol production in citrus molasses at pH 5.25.5. These results suggest that the control of pH is desirable, although occasionally not necessary for laboratory-scale studies, for all culture-substrate combinations. In large-scale operations it is highly recommended. Although periodic checks and manual adjustment of pH is widely used, the use of the buffer, calcium carbonate, has found wide acceptance in controlling the pH of the fermentation. The presence of this compound in a majority of substrates had no adverse effects upon glycol production; however, in certain substrates, e.g., citrus molasses, addition of calcium compounds incident to the manufacture of the substrate may result in inhibition of glycol production through ion toxicity if additional calcium carbonate is added during fermentation. Under such conditions, the use of automatic controlling devices (Lee, 1951; Callow and Pirt, 1956) for dosing with the required neutralizing agents seem to be the methods of choice.
146
STERLING K. LONG AND ROGER PATRICK
Some evidence has been presented which indicates that the period of active glycol production in certain fermentations is accompanied by a rapidly decreasing pH (Olson and Johnson, 1948; Long and Patrick, 1960). If the pH of such fermentations was maintained at the optimum level (6.0-6.2), completion of the active glycol-forming stage was indicated by a slowly increasing pH which usually reached 6.5 or more. This phenomenon was sufficiently dependable to permit its use as a criterion for a completed fermentation.
D. AERATION Aeration is required for maximum production of butylene glycol by nearly all of the species studied. The function of aeration in the fermentation has been described in a number of ways, all of which are undoubtedly correct. McCall and Georgi (1954) observed that, based upon theoretical considerations, the formation of glycol should be an anaerobic process. They suggested that the stimulatory effects of aeration was due to the removal of COBfrom the medium. Substitution of inert gases for air had little effect upon glycol yields. Retention of COz in the medium apparently promotes the formation of organic acids and ethanol to the detriment of glycol production ( Underkofler and Hickey, 1954). Adams and Leslie (1946) did not consider mechanical aeration necessary but observed that fermentation was increased by a high surface:area:volume ratio. This factor could function in a manner similar to aeration by permitting rapid evolution of the COBfrom the medium. Although the stirnulatory effects of aeration may not be due to provision of oxygen to the fermentation, such treatment regardless of mode of action is definitely necessary for obtaining high yields of glycol (Freeman, 1947; Simpson and Stranks, 1951; Anastassiadis and Wheat, 1953; Long and Patrick, 1960). The side effects of aeration are probably of considerable importance in the glycol fermentation, especially the agitation produced in the medium. This stirring action increases the efficiency of the fermentation by continuously exposing new substrate to the culture and disseminating the metabolic end products throughout the medium. Blackwood et a2. (1949) found that agitation alone increased fermentation efficiency while aeration had no effect on the dio1:ethanol ratio.
2,3 -BUTYLENE GLYCOL FERMENTATION
147
Olson and Johnson (1948) and Wheat (1953) indicated that strong aeration of the medium at the beginning of the fermentation and progressively decreasing the rate at later intervals had no adverse effect on the efficiency of the fermentation. This suggests that the aeration requirements of the fermentation may be based upon the unfermented sugars remaining in the medium. Frequent analyses should be employed to follow the course of the fermentation since continued aeration after the sugar content of the medium has fallen to less than 1% results in the rapid conversion of butylene glycol to acetoin (Mickelson and Werkman, 1939; Blackwood et al., 1947; Freeman, 1947; Olson and Johnson, 1948). Acetoin is not produced under anaerobic conditions (Freeman, 1947).
E. CONCENTRATION OF CARBOHYDRATE It is difficult to make a general statement regarding the optimum concentration of sugar for the butylene glycol fermentation due to the variety of raw materials employed. In most studies, however, it seems that the usual concentrations employed were in the range of 510%. It is probable that these rather low concentrations are due in part to the fact that as the sugar concentration in the raw materials is increased, accompanying toxic substances are also increased. The latter substances are particularly apparent in wood hydrolyzates and various types of molasses. That this may be a partial answer is indicated by the use of somewhat higher sugar concentrations in laboratory-formulated media employing sucrose. Freeman ( 1947) and Olson and Johnson (1948) used 10% sucrose solutions for A. aerogenes. McCall and Georgi (1954) obtained maximum glycol yields from sucrose concentrations of 6% with the same organism although higher concentrations were readily fermented. With few exceptions, naturally occurring sources of carbohydrate are usually diluted to somewhat lower sugar concentrations. Perlman (1944) using A. aerogenes in a wood hydrolyzate medium obtained the most efficient fermentation in terms of glycol:sugar ratio at a sugar concentration of 4%. The optimum sugar concentrations for B. polymyxa in beet molasses was 5% (Simpson and Stranks, 1951). Murphy and Stranks (1951) used suKte waste liquor at a sugar concentration of only 3.8% for production of glycol by several bacterial species.
148
STERLING K. LONG AND ROGER PATRICK
Aerobucter aerogenes is usually capable of fermenting considerably higher concentrations of sugar than B. polymyxa. Freeman and Morrison (1947) obtained good fermentation of blackstrap molasses diluted to contain 1070 sugar and Long and Patrick (1960) found that A. aerogenes readily fermented citrus molasses at sugar concentrations of 17-22%.
F. NITROGEN AND MISCELLANEOUS SUPPLEMENTS A survey of the types of raw materials employed in the butylene glycol fermentation and the supplementation required to make them suitable for the fermentation indicates that the most frequent deficiency is the nitrogen content. This may be due to a loss of nitrogen-containing components during refinement and concentration of the natural raw material or it may be due to deficiencies inherent to the raw material, e.g., wood hydrolyzates, citrus molasses, or whole wheat. Ideally, a nitrogen supplement should be cheap and at the same time adequate to satisfy the deficiency of the medium. Fulfillment of these requirements is not always possible but must be kept in view. An excellent source of nitrogen as well as other growth factors is yeast extract, although the excessive cost involved probably will prevent its use on an industrial scale. Ledingham et al. (1945) found that addition of yeast extract to B. polymyxa fermentation of whole wheat mash stimulated the production of glycol. It would be an error, however, to observe that yeast extract or any other nitrogenous substance is always the one of choice for a given raw material used as a substrate. Long and Patrick (1962) found that use of yeast extract in a citrus molasses medium retarded glycol formation and promoted formation of organic acids. A nitrogenous compound which is both low in cost and adequate for the glycol fermentation is urea. This compound is probably more commonly used than any other in this fermentation. It has been used in such diverse substrates as hydrolized wheat mashes (Olson and Johnson, 1948) and wood hydrolyzates (Perlman, 1944). Long and Patrick (1960, 1961) found urea to be a satisfactory nitrogen source for fermentation of citrus molasses by A. aerogenes and B. polymyxa. Cornstarch alone is a poor substrate for fermentation by B. polymyxa but when supplemented with malt sprouts or brewers’ yeast
2,3 -BUTYLENE GLYCOL FERMENTATION
149
the fermentation proceeded rapidly with good glycol production (Kooi et al., 1948). In addition to nitrogen, occasional deficiencies in other growth factors are found in certain raw materials employed in this fermentation although this is rather unusual in most crude materials. Murphy et al. (1951a) observed a deficiency of phosphate in beet molasses which was satisfied by addition of orthophosphate or phosphate-containing natural materials such as yeast extract, corn steep liquor, or bran. Similar results were obtained by Simpson and Stranks (1951) with the same substrate. A more unusual approach was used by Bahadur and Ranganayaki (1960) to satisfy a deficiency of an unknown growth factor in blackstrap molasses. They obtained more rapid production of butylene glycol when Bacillus subtilis and Pseudomonas hydrophila were grown in the fermentation mash in the presence of Serratia: marcescens. In general, Aerobacter aerogenes and related species are less demanding in their nutrient requirements than Bacillus polymyxa. The former organisms will frequently give satisfactory glycol fermentations in media which contain sugar and inorganic salts. Bacillus polymyxa, however, has limited synthetic properties and therefore must be supplied with a utilizable nitrogen source and required growth factors.
V. Recovery of 2,3-Butylene Glycol No recent research has been directed toward one of the greatest problems of the butylene glycol fermentation, that of economical recovery of the product from the fermentation mash. There is little doubt that perfection of a simple, inexpensive method of recovery would have a great effect upon the industrial future of this compound. Since no new information can be presented on methods of recovery, a number of the problems involved might be more profitably reviewed. An excellent discussion of existing methods may be found in Underkofler and Hickey (1954). The major difficulties in recovery of the glycol are due to its high boiling point, its great affiity for water, and the presence of dissolved and solid constituents of the fermentation mash. It is unfortunate that the high boiling point (range 180"184°C.) of glycol renders simple vacuum distillation practically
150
STERLING K. LONG AND ROGER PATRICK
useless. Before the temperature at which glycol distills is reached the dissolved constituents of the beer have been concentrated into a thick tarry mass which effectively binds and retards vaporization of the desired compound. A possible solution to this difficulty might be the incorporation of a suitable high-boiling liquid to maintain at least a semiliquid state. Some success by this approach has been obtained by Long and Patrick (1962) using glycerol as the supporting medium, although considerable improvement would be necessary to make the method practicable on a large scale. Solvent extraction has been used as a method of recovery with some success, primarily on a laboratory scale. A number of solvents are suitable for this purpose including ethyl acetate, diethyl ether, and n-butyl alcohol. The usual practice is to make repeated extractions with these solvents since single-step extractions remove only part of the glycol. Subsequent extractions encounter the problem of increasingly dilute concentrations of glycol and less intimate contact between solvent and solute. A further complication of this method is the rather extensive treatment required, such as filtration, precipitation of dissolved organic constituents which might be coagulable in the presence of the solvent, and concentration of the glycol in the beer by removal of water prior to exposure to the solvent. One of the more successful methods of recovery was described by Blom et al. (1945). They devised a counter-current steamstripping column which was actually developed through the pilotplant stage. The steps involved in this process included removal of ethanol during concentration of the fermentation beer and introduction of the resulting syrup into the top of a packed column through which it passed counter-current to steam at 55-pound pressure. The resulting glycol-laden vapors were passed through a scrubber and the glycol was recovered by rectification. No appreciable difficulties were encountered even after extended periods of operation.
VI. Potential Uses of 2,3-Butylene Glycol A complete list of all the compounds which have been derived from butylene glycol would be too extensive for the purpose of this paper; therefore, consideration will be given only to developments since World War II.
2,3 -BUTYLENE GLYCOL FERMENTATION
151
The stimulus for the extensive wartime studies on commercial production of 2,3-butylene glycol was a search for compounds which might be convertible to butadiene, the major constituent in the production of the strategic buna-S rubber. It was found that this conversion was possible (Underkofler and Hickey, 1954) although termination of the war prevented commercial application of the process. The observations of Boroff and Van Lanen (1947) on the economics of wartime conversion processes indicated that 13 to 14 pounds of butylene glycol, which could be produced from a bushel of corn, could be converted to 6.3 to 6.8 pounds of butadiene a t a cost approximately equivalent to that for butadiene from alcohol. It is unlikely that this application of butylene glycol will ever achieve industrial importance due to the availability of cheaper constituents. Neish (1944) felt that the potential usefulness of butylene glycol was sufficient to justify founding a chemical industry based upon this compound and other fermentation products. He noted that a variety of products were obtainable from butylene glycol in two ways. Catalytic dehydrogenation gave acetoin, diacetyl, and hydrogen in almost quantitative yields while catalytic dehydration gave methyl ethyl ketone. The low cost of this ketone indicated commercial feasibility. This compound is an excellent solvent for glyptal, vinyl resins, and lacquers. It was observed that methyl ethyl ketone could be condensed to 8-C ketone which, upon hydrogenolysis, would yield isomers of octane. This type of synthesis of hydrocarbons suggested the possibility of obtaining pure compounds which could be blended to yield high grade aviation fuel. It is very doubtful that this will ever seriously compete with petroleum products; however, the above proposal serves to indicate the broad range of useful products which might be derived from butylene glycol. An interesting possibility for utilization of butylene glycol in the preparation of antifreeze was proposed by Clendenning ( 1946) and Clendenning and Wright ( 1946). These workers found that 60-70% aqueous solutions of the Z-isomer of butylene glycol resisted freezing to 4 0 ° C . Somewhat greater depression of freezing point was obtained in a ternary system of butylene glycolmethyl alcohol-water which protected automobile radiators down to --5O"C. Liebmann (1945) described a number of reactions which could
152
STERLING X. LONG AND ROGER PATRICK
be used to produce an impressive number of derivatives from butylene glycol. This compound can be employed in the production of halogenated substitutes, esters of monobasic and dibasic acids, oxides, nitrogen, ether, and ketone derivatives. A partial list of the uses for these products included printing inks, lacquers, synthetic perfumes, organic solvents, synthetic rubber, drugs and pharmaceuticals, fumigants, emulsifying agents, dynamite modifiers, explosives, plasticizer in synthetic resin preparation, and softening and moistening agents. A more immediate and promising future for butylene glycol may be seen in the expanding plastics industry. Fedor (1961) notes that production of complex linear polyesters represents the fastest growing segment of the plasticizer industry. Typically these polyesters contain a dibasic acid or mixture of dibasic acids, a glycol, and a fatty acid. Polyesters range from viscous liquids to hard plastics. Butylene glycol has been used in the production of a variety of polyesters including a plastic obtained with the Z-isomer which had about the same hardness as Lucite (Watson and Grace, 1948) and a large number of new amorphous resins or balsams from both the Z- and m-isomers (Watson et al., 1950). Another interesting possibility might be the use of butylene glycol in nonaqueous foams for use in drugs, cosmetic products, lotions, ointments, and antiperspirants ( Sanders, 1960). Neish (1947), Robertson and Neish (1947)) and Underkofler and Hickey (1954) provide additional information on a variety of compounds which may be readily obtained from butylene glycol.
VII. Probable Future of the 2,3-Butylene Glycol Fermentation
The future industrial importance of 2,3-butylene glycol depends upon several factors, among which the most important is its ability to compete with similar synthetic products upon an economic basis. Fortunately there is no feasible method for synthesis of 2,3butylene glycol and therefore commercial production of this compound would be limited to fermentation. The greatest competition for fermentation glycol may be expected from established synthetic products such as ethylene glycol and 1,3-butylene glycol. These compounds now satisfy about the same industrial requirements as 2B-butylene glycol. The major potential of 2,3-butylene
2,3 -BUTYLENE GLYCOL FERMENTATION
153
glycol, therefore, appears to depend upon the development of new products based upon properties not common to the synthetic compounds. Such differences might include the increased synthetic capabilities conferred by the adjacent hydroxyl groups or perhaps the unusual stereoisomerism of the glycol produced by BuciEZus p~lymyxu. The most likely possibilities for using such different properties seem to be in the pharmaceutical or plastics industries. If these special properties are disregarded it is unlikely that existing methods of production will allow fermentation glycol to compete with the synthetic glycols on a cost basis. A factor which might alter this situation could include development of greater eaciency in the fermentation, possibly with as yet uninvestigated strains of microorganisms. A related approach could be the production and selection of mutants of existing cultures. The steadily increasing production costs of the various synthetic glycols may eventually be responsible for a more favorable outlook for the fermentation process, provided that the raw material costs for the latter do not increase appreciably. The most promising raw materials would include blackstrap or citrus molasses, or industrial wastes. Not to be overlooked is the future development of new fermentations yielding 2,3-butylene glycol as a by-product of secondary importance. In this manner a major portion of the production costs could be borne by the primary product, leaving the glycol in an excellent competitive position. A corollary may be found in the primary butyl alcohol obtained from the acetone-butanol fermentation of World War I.
REFERENCES Adams, G. A., and Leslie, J. D. (1946). Can. J. Res. 24F, 12-28. Anastassiadis, P. A., and Wheat, J. A. (1953). Can. J. Technol. 31, 1-8. Bahadur, K., and Ranganayaki, S. (1960). lndian J. Appl. Chem. 23, 3-8. Blackwood, A. C., Neish, A. C., Brown, W. E., and Ledingham, G. A. (1947). Can. J. Res. 25B, 56-64. Blackwood, A. C., Wheat, J. A., Leslie, J. D., Ledingham, G. A., and Simpson, F. J. (1949). Can. J. Res. 27F, 199-210. Blom, R. H., Reed, D. L., Efrom, A., and Mustakas, G. C. (1945). lnd. Eng. Chem. 37, 865-870. Boroff, C. D., and Van Lanen, J. M. (1947). Id.Eng. Chem. 39, 934-937. Callow, D. A., and Pirt, S. J. (1956). J. Gen. Microbiol. 14, 661-671. Clendenning, K. A. (1948). Can. J. Rcs. 24B, 269-279. Clendenning, K. A., and Wright, D. E. (1946). Can. J . Res. 24F, 287-299.
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Donker, H. J. L. (1926). Ph.D. Thesis. Delft, Netherlands. Fedor, W. S. (1961). Chem. Eng. News 39, No. 46, pp. 118-138. Freeman, G. G. (1947). Biochem. J. 41, 389-398. Freeman, G. G., and Morrison, R. I. (1946). Analyst 71, 511-520. Freeman, G. G., and Morrison, R. I. (1947). J. SOC. Chem. Ind. 66, 216-221. Harden, A., and Walpole, G. S. (1906). Proc. Roy. S O C . (London). B77, 399405. Hendrickson, R., and Kesterson, J. W. (1951). Florida, Unio. of, Agr. Expt. Sta. (Gainesoille), Bull. 487. Katznelson, H. ( 1944a). Can. J. Res. 22C, 235-240. Katznelson, H. (1944b). Can. J. Res. 22C, 241-250. Kooi, E. R., Fulmer, E. I., and Underkofler, L. A. (1948). Ind. Eng. Chem. 40, 1440-1445. Ledingham, G. A., and Stanier, R. Y. (1944). J. Bacteriol. 47, 443. Ledingham, G. A., Adams, G. A., and Stanier, R. Y. (1945). Can. I. Res. 23F, 48-71. Liebmann, J. (1945). Oil dt Soap 22, 31-34. Lee, S. B. (1951). Ind. Eng. Chem. 43, 1950-1951. Long, S. K., and Patrick, R. (1960). Proc. Florida State Hort. SOC. 73, 241246. Long, S. K., and Patrick, R. (1961). Appl. Microbiol. 9, 244-249. Long, S. K., and Patrick, R. (1962). Unpublished data. McCall, K. B., and Georgi, C . E. (1954). Appl. Microbiol. 2, 355-359. Mickelson, M. N., and Werkman, C. H. (1939). Iowa State College J. Sci. 13, 157-160. Morell, S. A., and Auemheimer, A. H. (1944). J. Am. Chem. SOC. 66, 792796. Murphy, D., and Stranks, D. W. (1951). Can. J. Technol. 29, 413-420. Murphy, D., Stranks, D. W., and Harmsen, G. W. (1951a). Can. J. Technol. 29, 131-143. Murphy, D., Watson, R. W., Muirhead, D. R., and Barnwell, J. L. (1951b). Can. J . Technol. 29, 375-381. Neish, A. C. (1944). Can. Chem. Process. Ind. 28, 862-866. Neish, A. C. (1945). Can. J. Res. 23F, 10-16. Neish, A. C. (1947). Can. J. Res. 25B, 423-429. Neish, A. C. (1950). Can. J. Res. 28B, 660-661. Neish, A. C., and Blackwood, A. C. (1951). Can. J. Res. 29B, 123-129. Neish, A. C., Blackwood, A. C., Robertson, F. M., and Ledingham, G. A. (1948). Can. J. Res. 26B, 335-342. Olson, B. H., and Johnson, M. J. (1948). J. Bacteriol. 55, 209-222. Owen, W. L. (1950). Intern. Sugar J. 52, 120-121. Perlman, D. (1944). lnd. Eng. Chem. 36, 803-804. Robertson, F. M., and Neish, A. C. (1947). Can. J. Res. 25B, 491-501. Rose, D. (1947). Can. J. Res. 25F, 273-279. Sanders, P. (1960). Chem. Eng. News 38, No. 45, p. 64. Simpson, F. J., and Stranks, D. W. (1951). Can. 1. Technol. 29, 131-143. Stanier, R. Y., and Adams, G. A. (1944). Biochem. I. 38, 168-171.
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Tamboline, F. R. (1953). Can. J. Technol. 31, 70-71. Tomkins, R. V., Scott, D. S., and Simpson, F. J. (1948). Can. J. Res. 26F, 497-502. Tyson, R. S. (1948). J. Am. Chem. SOC. 70, 3610-3613. Underkofler, L. A,, and Hickey, R. J. (1954). “Industrial Fermentations,” Vol. 11. Chem. Publ. Co., New York. Ward, G. E., Pettijohn, 0. G., Lockwoocl, L. B., and Coghill, R. D. (1944). J. Am. Chem. SOC. 66, 541-542.’ Ward, G. E., Pettijohn, 0. G., and Coghill, R. D. (1945). Ind. Eng. Chem. 37, 1189-1194. Watson, R. W., and Grace, N. H. (1948). Can. J. Res. 26B, 752-762. Watson, R. W., Grace, N. H., and Barnwell, J. L. (1950). Can. J. Res. 28B, 652-659. Wheat, J. A. (1953). Can. J . TechnoZ. 31, 42-56. Wheat, J. A., Leslie, J. D., Tomkins, R. V., Mitton, H. E., Scott, D. S., and Ledingham, G. A. (1948). Can. J. Res. 26F, 469-496.
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Aeration in the Laboratory W. R. LOCKHART AND R. W. SQUIRES Department of Bacteriology, Iowa State University, Ames, Iowa, and Antibiotics Manufacturing and Development Division, Eli Lilly and Company, Indianapolis, Indiana I. Introduction .......................... A. Nature of the Problem .............................. B. Purpose of This Review ....................... 11. The Necessity for Aeration .............................. A. Effects of Oxygen Availability ........................ B. Possibilities for Control of Aeration . . . . . . . ..... 111. Methods of Measurement ................................ A. General Considerations ............................... B. Chemical Methods ........... ... C. Physical Methods ................................... D. Electrometric Methods . . . . . . . . . . . ................... E. Applicability of Methods . . . . . ............ IV. Control of Aeration .............. A. Means of Control .................................... B. Equipment for Control .............................. V. Summary .............................................. References ......................... ...............
1.
157 157 159 159 159 161 162 162 163 165 166 167 169 169 171 185 185
Introduction
A. NATUREOF
THE
PROBLEM
The aeration of cultures often seems a difficult field which the microbiologist tries to avoid. Finding apparently conflicting advice in the literature, he may decide to ignore aeration altogether rather than become involved. It may be useful to consider the factors which have given rise to this state of affairs. The effects on biological systems of oxygen availability, and of oxidation-reduction potentials in general, are complex and not we11 understood. Problems encountered in delivering oxygen to cultures involve technical and sometimes obscure concepts in physical chemistry and engineering. The investigator must choose among a great variety of equipment and techniques for carrying out aeration. Finally, the measurement of aeration efficiency is not easy. Although we have a number of analytical techniques, each of them is subject to severe limitations. The investigator may not be certain that his method 157
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of measurement was appropriate, or even that he knows what he has measured. These difficulties command the attention of groups of investigators who have approached the field from widely varying points of view and who have directed their efforts toward different and sometimes incompatible ends. Several types of expertise may be distinguished, A number of laboratory theorists, finding it necessary to maintain controlled aeration in their experiments, have produced elaborate theoretical treatments of the topic and have designed a variety of specialized equipment. The value of such work may be restricted, for often it does not find general acceptance by satisfying the somewhat broader needs of other workers. The engineers are more likely to be concerned with large-scale applications, and may regard aeration as a unit process to be handled generally in mechanistic fashion; they sometimes neglect biological considerations. Such work, while essential to development of industrial applications, may be of little use to the microbiologist who wants to know how fast he should run his rotary shaker to obtain the best yields. Finally, there is a large group of fermentation technologists, who on varying scales and for many reasons must control aeration in order to hasten or improve some microbiological process. An interesting subgroup among technologists are the intuitionists who, like the brewmasters of old, manage fermentations by instinct-tasting or smelling the contents of the flask or vat, cracking a valve a trifle, fussing with this or that control. They often achieve, through this empirical (or inspirational) technique, consistently better results than anyone else, but their mystique is not communicable and furnishes scant comfort to those who lack the touch. These types are not mutually exclusive; fermentation technologists, for example, may have been trained either as biologists or as engineers. Approaches vary from the empirical to the abstract, and nearly everyone involved with aeration develops a touch of “artistry,” even if he usually manages after the fact to rationalize such contributions. Various students of aeration nevertheless have quite different objectives, and work which may be useful in one context is not necessarily valid in another. Persons not familiar with aeration theory and practice, confronted with all these conflicting viewpoints, may be forgiven if they find that much of the literature in the field is incomprehensible.
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B. PUHPOSE OF THISREVIEW To the microbiologist at large, aeration is in many cases a peripheral concern. His objective is to produce spores, or antibiotics, or reproducible results in any of a multitude of experiments involving microbial growth. He desires to provide adequate oxygen for his cultures and to control aeration just as he would control any other experimental variable. Most experts, on the other hand, are preoccupied with specialized problems such as “scale-up,” the practical and theoretical considerations involved in reproducing on a production or pilot-plant scale those conditions which have been found optimal in the laboratory. There are relatively simple procedures for manipulating, controlling, and measuring aeration in the laboratory, but they are scattered in the literature among more specialized information. Consequently, the investigator does not readily find answers to his questions, and may be left with the alternatives of accepting some techniques uncritically, becoming expert enough to evaluate them himself, or ignoring aeration altogether. We propose to address ourselves here to those microbiologists who are not fermentation specialists, but who nonetheless need to aerate cultures. We shall demonstrate the need for controlling and measuring aeration in the laboratory, summarize the general principles involved in sound aeration practice, and present practical guides for the application of these principles through commonly used equipment and techniques of measurement.
II. The Necessity for Aeration A. EFFECTSOF OXYGEN AVAILABILITY The first question of the microbiologist is whether or not it is necessary to provide aeration of his cultures at all, except when working with large populations of aerobic organisms. He soon finds that in many cases the oxygen demand of cultures is great enough, and the level of oxygen availability sufficiently critical, that pains must be taken to measure and control this variable with some precision. Aside from the observation that yields of microbial cells or products are improved by aeration of cultures, levels of oxygen availability may profoundly influence metabolic events (and, consequently, the products formed) during culture growth and may
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even-through genetic selection-determine eventually the nature of the cell population itself. In nonagitated cultures (i.e., tubes or flasks not specially aerated in some fashion) the rate of diffusion of air into the medium is rarely sufficient to meet the oxygen demand of increasing cell populations. The literature contains numerous reports of yields vastly increased, particularly in rich media, by even the most perfunctory efforts at aeration. There is no assurance in many cases that even this aeration was anywhere near sufficient. Since the solubility of oxygen is very low, there is no appreciable reservoir in solution; the problem is to provide oxygen at a continuous rate high enough to meet the increasing demands of the growing cell population. Finn (1954) has discussed this in detail, pointing out that the rate of supply into the shallow reservoir of dissolved oxygen must balance the demands made upon it by the cells at all times and in all regions of the culture vessel. If this is not done, one has a culture which-if not anaerobic-is at least severely limited by oxygen availability. Not only are yields restricted, but the nature of the population may be quite different than that found in cultures not so limited. The metabolic patterns observed in oxygen-limited cultures of Escherichia coli are distinctively different from those in cultures limited by availability of the carbon or nitrogen source; this pattern is found even under conditions where the oxygen limitation is not severe enough to be reflected in decreased growth rates (Ecker and Lockhart, 1961a). The fact that oxygen limitation affects the nature of metabolic products as well as yields has been well documented (e.g., Finn, 1954). In such cultures, occasional mutant cells may be found which have lesser oxygen requirements than the parent cell type; the selective pressure of reduced oxygen availability may then result in establishment of a mutant cell line as the predominating component of the culture. Reduced oxygen tension has been implicated, for example, in selection of rough mutants of Brucellu (Altenbern et al., 1957) and in loss of antibiotic resistance in staphylococci (Fusillo and Weiss, 1958). These difficulties cannot always be overcome simply by aerating cultures so vigorously that there could be no question of oxygen deficiency. Aside from the technical difficulty of providing sufficient aeration to make the investigator feel secure in the absence of any measurements of actual oxygen demand or aeration efficiency, there
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is evidence that an overabundant supply of oxygen may exert deleterious effects. Though some reported inhibitions of antibiotic and other fermentations might equally well be attributed to causes other than oxygen supply, overaeration itself probably is at fault in many cases (Finn, 1954). High oxygen tensions are reported to be inhibitory for numerous cell types, including thermophilic actinomycetes ( Webley, 1954), Mycobacterium tuberculosis (Knox et al., 1957) and animal cells grown in submerged culture (Cooper et al., 1958). The effects of some toxic agents are intensified at certain levels of oxygen availability (Lockhart and Weaver, 1960); oxygen tension may mediate the effects on cells of chemical or environmental stress (Lockhart, 1959). In many of the above examples it was hypothesized that, although sufficient oxygen must be provided to permit normal metabolism, aeration above this level may induce an oxidation-reduction potential unsuitable for formation and/or function of essential sulfhydryl groups. Thus there is an optimal level of oxygen availability which may be distinctive for each cell line. Animal cells in culture have oxygen requirements much lower than those of most aerobic microorganisms ( McLimans et al., 1957), but aeration providing oxygen tensions either above or below the critical level results in decreased growth response (Cooper et al., 1958). The effects of variations in oxygen tension are sometimes more subtle than such relatively simple qualitative or quantitative alterations in yields of products or cells. Baracchini and Sherris (1959) have reported an interesting example of an apparently chemotactic response of motile bacteria to an oxygen concentration gradient. When cell suspensions were placed in tubes of soft agar, the motile cells migrated to a narrow band at a fixed distance from the air-medium interface. The relative position of the cell band could be altered by manipulating the partial pressure of oxygen in the overlying gas mixture. A number of similar phenomena might be found if control of oxygen availability were more commonly practiced during microbiological experimentation.
B. POSSIBILITIES FOR CONTROL OF AERATION The first requisite for controlled aeration is that a reasonably reliable method must be at hand for measuring oxygen availability. Actually there are a number of such methods, and although each of them has certain limitations, appropriate and relatively simple measurements can be found for almost any experimental situation.
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The investigator thus is able to standardize aeration practices in his own laboratory, and to report these techniques in terms which will permit others to reproduce his experiments. Equipment and techniques exist for providing controlled aeration in laboratory culture volumes as small as 5 or 10 ml. or as large as several liters. Though the possible culture vessels vary in cost and complexity from ordinary flasks to the relatively large laboratory fermentors now available commercially, the same fundamental principles govern aeration control in each, and the same variables are manipulated in similar ways in all cases. The investigator may, when necessary, change to another type of equipment or another scale of operations without undue difficulty. The problems of “scaling” which plague those who are involved with industrial production are generally less severe within the range of culture volumes and for most of the microbial systems encountered in the laboratory. Problems which admittedly are quite complex may be simplified for most purposes in this narrower context. The laboratory worker needs only to know what he must avoid, and this we now hope to point out.
111. Methods of Measurement A. GENERAL CONSIDERATIONS Having demonstrated the desirability for controlling the level of aeration in microbial cell propagation, the next problem is that of accurately measuring aeration in terms of quantity or rate of delivery of oxygen to the culture system. Several factors should be considered before selecting a method. The investigator must first decide whether he needs to follow the actual concentration of dissolved oxygen in the medium during the course of his experiment, or to calibrate his equipment in terms of its capacity to deliver oxygen at a particular rate. The dissolved oxygen concentration (oxygen tension) can be expressed as per cent of saturation or as oxygen partial pressure. Oxygen delivery rates usually are determined as the maximum amount of oxygen which the aeration device can place in solution in a given volume of medium per unit time. Most microbiologists express this as oxygen absorption rate (OAR), in millimoles of oxygen absorbed per liter per minute (Corman et aE., 1957) or per hour (Ecker and Lockhart, 1959; Pirt, 1957). Many chemists and engineers prefer to use the absorption rate constant &,a (liters per hour), which
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may be converted to OAR by multiplying by the equilibrium concentration of dissolved oxygen (about 0.20 millimoles per liter for most culture media). Finn (1954) has discussed the use of these various terms, and listed many of them. Although most writers define aeration efficiency in terms of OAR or &a, with higher values representing better aeration conditions, “aeration efficiency” may have a more specialized meaning for anyone who is studying oxygen tension in relation to a particular biological system. Equivalent aeration rates obtained for different methods of agitation (e.g., reciprocal vs. rotary shakers) may not yield the same results in actual practice. This is due to the interaction of other factors such as amount of growth, viscosity, evaporation and the physical effects of agitation itself. The measurements employed will depend on the nature and purpose of the experiment and the type of aeration equipment to be used. We shall indicate as we proceed which methods of measurement are most appropriate for particular kinds of experimentation. At some point the investigator should determine the critical oxygen level of the biological process he is studying. Steel (1958), in discussing “critical oxygen levels,” points out that the rate of oxygen uptake by a microbial culture is independent of the concentration of dissolved oxygen in the medium (a zero order reaction) until the oxygen tension is reduced to a certain point. Below this “critical level” the respiration rate of the culture then becomes dependent on dissolved oxygen concentration, and respiratory activity decreases in a hyperbolic manner as the concentration of dissolved oxygen decreases ( a first order reaction). When dissolved oxygen concentration is to be studied during the course of an experiment, rapidity of measurement becomes most important, particularly when the demand rate approaches the delivery rate of the system employed. Oxygen is sparingly soluble in water; saturation represents only 7-12 parts of oxygen per million depending on temperature and the concentration of salts in the medium. With these facts in mind, let us turn to an examination of various methods employed for measuring aeration rates and dissolved oxygen levels.
B. CHEMICAL METHODS The procedure which has been used most frequently for aeration rate studies is the sulfite oxidation method of Cooper et al. (1944).
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Basically, this method depends on the oxidation of sulfite to sulfate by oxygen in the presence of a copper catalyst. The rate at which the reaction takes place is limited only by the maximum rate at which oxygen can be put into solution during aeration. A sulfite solution of known concentration is substituted for culture medium in the vessel to be calibrated, and aeration carried out for a measured time. A known excess of iodine is then added to react with the unoxidized sulfite remaining, and the unreacted iodine is titrated with thiosulfate. By calculation, the amount of sulfite converted to sulfate (hence the amount of oxygen going into solution during the measured time interval) may then be determined. Useful modifications of this procedure were proposed by Corman et al. (19571, who employed dry ice as a source of CO2 to flush air from shake flasks and to halt further oxidation of the sulfite solution, and by Ecker and Lockhart (1959), who substituted a colorimetric determination of iodine for the back-titration with thiosulfate. Both groups of investigators demonstrated that determinations could be simplified by judicious selection of the volumes and concentrations of the reagents used. The sulfite method determines only the maximum rate at which oxygen could be made available under calibration conditions, but provides no information about rates of oxygen utilization during actual cultivation of microorganisms. Further, there is no assurance that rates of oxygen solution in sulfite reagent will be the same as those in cultures, especially for viscous media or in the presence of mycelium-there is in progress an involved technical argument as to the nature of the chemical and physical factors controlling sulfite oxidation (see Finn, 1954; Phillips and Johnson, 1959). Despite these disadvantages this is the easiest method available for calibrating aeration equipment in reproducible, standardized terms, and it remains the method of choice in cases where its use is possible. Many modifications of the “Winkler” method (Public Health Assoc., 1960) have been employed for dissolved oxygen measurements. Generally these determinations require fairly large volumes of liquid for measurement as well as special glassware and long periods of time for analysis. Loomis (1954, 1956) developed a rapid microcolorimeter method for dissolved oxygen in fermentation broths, adaptable to most culture fluids which can be rapidly filtered. His technique requires only small volumes of sample and can be carried out in a relatively short time. The principle of the
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method lies in measuring the partial oxidation of a solution of indigo carmen dye. The color change ranges from yellow to blue depending on the degree of oxidation.
c.
PHYSICAL METHODS
Several physical methods can be applied to determine aeration rates in certain kinds of culture vessels. Bungay (1959) pointed out that since both humidification and aeration are gas-liquid contacting operations, evaporation rate should be a fairly reliable measurement of aeration. The principle is to determine the capacity of the circulating air to evaporate water and to equate the loss of water from a system to air capacity. For example, saturated air at 32°C. contains 0.0335 gm.of water per liter of air. If a room at 32°C. had a relative humidity of So%, it would contain only 0.0201 gm. of water per liter of air. Therefore, each liter of room air could evaporate 0.0134 gm. of water. liters air required
=
observed loss in grams
grams per liter evaporation capacity
x
time
In this example, an observed loss of 2.91 gm. of water in a 24-hour period would require air through the flask at a rate of at least 151 ml./minute. Another method employed by Bungay in the study of aeration rates in shaken flasks was the smoke clearance test. Although this test is fairly crude, it shows good agreement with the evaporation method. The flask is filled with cigarette smoke and covered with an aluminum cap. The cap is then removed, and the time required for the flask to clear completely is observed. For example, if it takes 15 seconds to clear a 500 ml. erlenmeyer flask, the aeration rate would then be 2,000 ml./minute. Since these tests yield only preliminary information, they should be correlated with sulfite oxidation data if results are to be expressed in terms of actual rates of oxygen absorption. Correlations with sulfite oxidation studies and with fermentation results have been found rather poor, and these methods are suitable primarily for only rough, preliminary calibrations. Phillips and Johnson (1961) have developed a method for determination of dissolved oxygen which appears to give accurate and reproducible results. A long piece of thin wall teflon tubing is placed below the surface of the medium, and a stream of nitrogen
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gas is passed through this tubing at a slow enough rate that oxygen in solution can pass through the wall of the teflon tubing, diffuse into the stream of nitrogen and come to a steady-state condition before being carried from the vessel to an oxygen analyzer. A Beckman oxygen analyzer is employed for analysis of the nitrogenoxygen mixture coming through the teflon tubing exhaust. These readings were shown to be in good agreement with dissolved oxygen levels determined by other means.
D. ELECTROMETRIC METHODS Although primarily useful for dissolved oxygen measurements, the electrometric techniques are also employed along with sulfite oxidation procedures for rate determinations. The methods to be described use a sampling technique where broth is either removed from the culture vessel and a nearly simultaneous measurement is made on the sample, or the medium is first “gassed out” by flushing with nitrogen and then the oxygen reabsorption rate is measured. For determining critical oxygen demands, it is customary to interrupt the flow of air to the culture and then determine the rate at which the microbial population removes or lowers the oxygen activity of the broth. Wise (1951) describes the various methods for measuring dissolved oxygen activity, and the reader is referred to his paper for further details. Polarographic methods employ some type of recording device which plots the current-voltage curve of the electrolysis of the system being employed. There is a linear relationship between oxygen concentration (partial pressure) and the height of the polarographic oxygen wave. The reader should refer to Umbreit et al. (1957) and to Hewitt (1950) for discussion of the principles of polarography. Several types of electrodes may be used for studying dissolved oxygen in fermentation broths; one such is the dropping mercury electrode employed by Hixson and Gaden (1950), Bartholomew et al. ( 1950) and Karow et al. (1953). In each case, live cultures were studied by removing samples from the fermentor or the vessel under study. A problem frequently encountered was that the sample might completely lack oxygen by the time the measurements could be made. Chain and Gualandi ( 1954), Wise (1951), Strohm et al. (1959), and Steel and Brierley (1959) installed “rotating or “vibrating platinum electrodes in the fermentation vessel so that it was not necessary to remove samples, and
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dissolved oxygen tensions could be followed throughout the fermentation cycle. More recently, a polyethylene-covered platinum electrode has been used to study aeration in yeast cultures (Strohm and Dale, 1961). Oxygen diffuses from the medium through the membrane, forming an “oxygen gradient” between the membrane and the platinum cathode. This type of electrode measures oxygen “activity” rather than concentration (Finn, 1959; Strohm and Dale, 1961). In practice, it is necessary to calibrate the electrode in the airsaturated solution in which dissolved oxygen measurements are to be made. A version of this electrode was employed also by Phillips and Johnson (1961) with the design modified so that it could be steam sterilized in the fermentation vessel. Another method is that of measuring the oxidation-reduction potential of media during aeration (Squires and Hosler, 1958; Tengerdy, 1961a). In these studies, it was shown that a linear relationship exists between the redox potential and the logarithm of the dissolved oxygen concentration in uninoculated broth. This method has been employed to follow the dissolved oxygen level during the production of 2-keto-l-gluconic acid in shake flasks (Tengerdy, 1961b).
E. APPLICABILITYOF METHODS The sulfite method has been compared with most of the other available techniques with good agreement being observed by some investigators (Bartholomew et al., 1950; Chain and Gualandi, 1954). However, Wise (1951) found a large disparity between results with the sulfite and polarographic methods when a rotating platinum electrode was employed. Steel and Brierley (1959) showed that absorption coefficients obtained by the two methods were essentially similar only over a very narrow range of operating conditions in stirred equipment. Solomons and Perkin (1958) reported that sulfite oxidation behaved as a “gas-film” controlled process. The applicable principles of physical chemistry are useful and important, but they give a realistic picture only when related to the total effect of oxygen on a biological system. The microbiologist must determine the effect of aeration on a particular culture and observe the influences of secondary factors on microbial re-
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sponse in relation to the amount of oxygen that can be absorbed by the growth system under specifically defined conditions. When selecting a method or methods for evaluating aeration, he should keep in mind several factors: 1. Many bacteria studied in the laboratory are propagated in media which have nearly the same physical properties as water during the entire growth cycle. In such cases, the investigator can employ sulfite oxidation methods in the calibration of his equipment. It is advisable to standardize the aeration rates between the various kinds of equipment being employed and thus establish a known “control condition” for the various sizes and shapes of culture vessels commonly employed in the laboratory. If the investigator needs to study dissolved oxygen levels, however, he must employ one of the electrometric methods. 2. The morphological character of the culture being studied and the medium in which it is propagated affects the choice of methods. Filamentous microbial forms and changes in liquid surface tension (i.e., increased viscosity) may soon alter the absorption rates established for a water system or for uninoculated broth. Many media, during the course of growth and product synthesis, undergo transitory states which have a marked influence on the capacity to deliver oxygen at a predetermined rate or concentration. Product synthesis may need to be correlated with oxygen absorption rates which have been established by altering several different physical factors (i.e., different types of shakers, baffles, sparger openings, etc. ), and several methods of measurement of both rate and dissolved oxygen concentrations may need to be compared. Many studies have been made of the effect of undissolved solids on aeration and/or oxygen absorption. Most of these experiments have been done with killed mycelium from either a streptomycete or a penicillin fermentation, or with other substances which either increase viscosity or interfere with absorption because of their density. One recent study (Steel and Brierley, 1959) showed that oxygen absorption was reduced by 90% by the addition of 2% dry mycelium. The problem of measuring oxygen transfer rates becomes much more difficult in mold fermentations or when high viscosity becomes a factor. In such systems, sulfite oxidation is generally considered to be inferior to the electrometric methods, which probably have greater application in the mold fermentations since they can be employed to monitor dissolved
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oxygen concentration or oxygen activity (Phillips and Johnson, 1961; Strohm and Dale, 1961). 3. The amount of “final product” required will help to determine the size and shape of the vessel in which the cellular growth or by-product is to be synthesized, and thus the method by which turbulence and/or mixing of medium, microorganism, and air is induced. These factors may influence the choice of calibration methods. 4. In animal or plant tissue culture studies, the chemical methods should be sufficient for calibration. However, the possibility of overaeration exists when dealing with these cultures. The mixing devices employed in most tissue culture apparatus are designed to keep cells in suspension, but contribute little to gassing of the medium. Ziegler et a2. (1958) studied the growth of mammalian cells in submerged culture and employed very low agitation rates with no gassing of the medium. Gas transfer was by diffusion at the liquid surface. In summary, the investigator should choose the method of measurement most readily available to him, subject to the special requirements imposed by the purpose of his experiments or by the microbial system he is studying. It is important to be aware that the suitability of any method of measurement also depends on the type of aeration device and growth vessel being used.
IV. Control of Aeration A. MEANSOF CONTROL Having at hand suitable analytical techniques for measuring, one must then select a method for effecting a controlled and predictable increase in the availability of oxygen to his culture system. Before discussing specific equipment for this purpose, the variables encountered during aeration and the ways in which they may be controlled will be examined. The rate at which oxygen is supplied to a growing culture may be increased either by increasing the total absolute quantity of oxygen dissolved in the culture fluid (i.e., the size of the reservoir) or by increasing the effective gas-liquid interfacial area available for diffusion of oxygen into solution. The first of these alternatives is little practiced. Temperature influences gas solubility (which decreases as temperature rises), but incubation temperatures usu-
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ally cannot be manipulated without affecting microbial growth. In most cases the investigator can only note that this relationship exists, remembering that equipment must be recalibrated if a change is made in incubation temperature. Though the solubility of oxygen may be increased either by increasing the total gas pressure in the system or by increasing the partial pressure of oxygen in the gas mixture, the former is not practicable in most laboratoryscale culture equipment. Regardless of the pressure under which air enters the culture vessel from a pump, tank, or compressed air line, the pressure at once equilibrates with the atmosphere unless the apparatus is sealed in some fashion so that back-pressure builds up within the culture vessel. While such pressurization is used extensively in pilot or production scale fermentation vessels, it is seldom employed in laboratory equipment. Apparatus designed to permit control of the composition of gas mixtures is useful for some research purposes, but ordinarily is not suitable for routine use. In most laboratory applications aeration is accomplished with air at atmospheric pressure, with a fixed and predetermined incubation temperature. Increasing the effective gas-liquid interfacial area is more easily managed, and aeration rate is often manipulated in this way. The volume of air which comes in contact with the culture is increased by splashing the liquid about in the culture vessel (as in shaken flasks) or by bubbling air through the culture. This process is inefficient at best since only a relatively insignificant portion of the oxygen content of air goes into solution before the bubbles escape from the liquid. Efforts are therefore made to increase the surface area of gas in contact with the culture fluid. Air may be introduced into the culture through a relatively small orifice or orifices (spargers). A given volume of gas has greater surface area as many small bubbles than as a few large ones. The effectiveness of aeration may be increased by prolonging the period of contact between gas bubbles and liquid. Air bubbles may be retained in the culture fluid for as long as possible by releasing the gas near the bottom of the vessel, by adjusting the ratios between vessel size, dimensions and liquid volume, by placing obstructions (baffles) in the culture vessel to impede the rise of bubbles through the liquid, and by agitating the liquid through the physical action of the bubbles or by stirring the liquid with propellerlike impeZ2ers. An almost infinite variety of combinations and interactions of
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the above factors would be possible, and one begins to appreciate the role of the artists who can manipulate all these. The fermentation technologist suffers from an embarrassment of riches-he seems to have at his disposal many means of improving aeration. But there exist actually only a very few general ways to increase oxygen availability. These are: ( 1 ) increasing the solubility of oxygen in the medium by manipulating temperature or pressure; ( 2 ) increasing the volume of air which makes interfacial contact with the culture fluid; and ( 3 ) prolonging the contact between air and liquid. These general principles may be applied to the various types of laboratory culture vessels. Results in most cases are more predictable than might be imagined, and the laboratory worker, choosing the type and size of vessels best suited to his purpose, may control, measure, and reproduce his aeration procedures with quite satisfactory results.
B. EQUIPMENT FOR CONTROL 1. Shaken Flasks Probably the most common method of aeration in laboratories is the shaken flask. The culture vessel (usually an Erlenmeyer flask) is clamped to a mechanical device which imparts motion to the flask, agitating the medium and thereby increasing the available gas-liquid interfacial area. Shaker machines may be reciprocating, rotary, or “wrist-action” (lateral motion through an arc). Most machines provide for variable speeds and some provide for several amplitudes of shaking. The constant motion of the culture vessels makes it awkward to equip them with sampling devices, and the methods appropriate for measuring aeration in shaken flasks are somewhat limited. Many investigators calibrate them in terms of oxygen absorption rates, using the sulfite oxidation method. “Smoke clearance” and evaporation data might be used to estimate the volume of air passing through the flask, but may have little relation to product synthesis. Factors which have a directly measurable influence on shaken flask performance either limit the air flow (size of the ‘flask aperture and type of closure) or affect the interfacial area (size and shape of the flask, baffling, type of shaking, rate of shaking and ratio of liquid volume to total flask volume). Several investigators have reported that the type of flask closure
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has a significant effect on oxygen absorption rates. Chain and Gualandi (1954), employing both sulfite oxidation and polarographic methods, were able to measure the diffusion rate of air through stoppers. Corman et al. (1957) reported that milk filter disc pads as a flask closure are far superior to cotton plugs where increased aeration efficiency is helpful. Since cotton plugs vary in their “fit” from flask to flask, many investigators consider it better practice to substitute two or three filter discs as a means of reducing variation between flasks as well as providing better flask aeration. The ratio of volume of medium to total flask volume influences the oxygen absorption rate. This factor is related to the method of shaking or agitation as well as to the size and shape of the flask. Auro et al. (1957) reported that only the volumes of the flask and liquid were required to predict the absorption rate under the experimental conditions they described. Generally, greater oxygen absorption rates are obtained at higher ratios of flask to liquid volume, and at greater shaker speeds. For much microbiological work, the optimum combination of volume and shaking speed is that which gives the highest absorption rate or greatest amount of the desired reaction or product, without wetting the flask closure. The effectiveness of the means by which the flask may be shaken or agitated is somewhat controversial. Several publications report the effects of shaker speed and length of stroke (or “throw” for rotary shakers) on flask aeration. Mass transfer rates within the liquid, and between the liquid and suspended particles, have been examined by Rhodes and Gaden (1957). They reported that very little mixing takes place in an erlenmeyer flask when placed on a rotary shaker, while bulk mixing is almost instantaneous on the reciprocating shaker. Gaden ( 1962) further showed that oxygen absorption by uninoculated media could be increased by employing flasks with baffles and an improved flask closure. Chain and Gualandi (1954) have probably made the most comprehensive study on shaken flask aeration by including nearly all possible combinations of flask sizes and shapes, liquid volumes, baffles, presence of mycelium and shaking methods. Most of these workers have concluded that the reciprocating shaker is equal or superior to the rotary shaker in relation to oxygen absorption. In actual practice, however, different pieces of equipment for which similar oxygen absorption rates have been
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established may yield entirely different results. Yields produced from antibiotic fermentor flasks agitated concurrently on both types of shakers will generally show lower potencies in flasks shaken on the reciprocating machine than in those agitated on the rotary shaker. Such discrepancies have led many microbiologists to question the sulfite oxidation method as a tool for calibrating oxygen absorption rates in shaken flasks. Such results do not necessarily discredit the method but serve to emphasize that in studying a biological system the total interaction between the microorganism and its environment must be considered. In his initial studies, the microbiologist will want to examine product synthesis in relation to oxygen absorption rates under various conditions to be assured that the several physical factors which influence aeration have not predetermined his results. Since most calibration studies are carried out on sulfite solution or uninoculated medium, secondary factors resulting from growth may complicate an established aeration rate and produce a different oxygen absorption rate when the culture is introduced. Factors such as cell morphology and medium metabolism usually interact to change the viscosity, surface tension, chemical composition, and residual medium solids. All of these may have an effect on the total interfacial area available at any time during the growth cycle of the microorganisms. Although “wrist-action’’ shakers are used in many laboratories ( especially for work requiring relatively few replicates), we have been unable to find any data in the literature about their oxygen absorption rates or mixing efficiency. The “wrist-action” motion would appear to be more analogous to that of reciprocating than rotary shakers, but the total effect on microbial growth might be different. The limited range of culture volumes obtainable in shaken flasks (usually between 50 and 100 ml.) is a disadvantage, particularly if one wishes to take several sequential samples during the course of an experiment. The range of control of aeration rates is somewhat limited. Many investigators question the validity of shaken flask data when employed for developmental studies which are to be “scaled into large-volume, stirred equipment. But the advantage of being able to study convenient culture volumes under fairly uniform conditions is most attractive. Flasks are easily handled in large numbers, which permits adequate replication with minimum effort
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and loss of time. Shaken flasks are especially useful for various types of “screening” or developmental studies which require rapid examination of large numbers of samples or variables. 2. Sparged Vessels Second in popularity to shaken flasks are culture vessels (tubes, flasks, or bottles) into which a stream of air is delivered through a tube, a fritted glass sparger, or some similar gas dispersion device. Culture vessels of various sizes and forms can be employed. They may be quite simple or may incorporate such refinements in sampling ports and aeration controls as are required by the exigencies of one’s experiments. This is the kind of aeration ordinarily used in continuous culture devices. While only a few investigators (e.g., Moss, 1952; Pirt, 1957) have studied specifically the effects of oxygen availability in such systems, most workers in continuous culture have taken pains to measure and control oxygen supply and to insure that aeration was at least adequate (e.g., Herbert et al., 1956). The discussion here concerns only the practice of aeration in batch cultures, but the same principles apply practically without modification to continuous cultures. Although air is the usual gas bubbled through the culture fluid, it is possible to increase the proportion of oxygen in the gas mixture (Ecker and Lockhart, 1961b). Gas flow rates from tanks or a compressed air line may be controlled with great precision by means of needle valves, and measured with flowmeters calibrated in liters per hour or milliliters per minute. If an air pump is used, delivering a constant flow when set for a particular rate, it may be possible to dispense with valves and meters. However, the actual volume of air delivered per unit time at any setting of valve or pump may fluctuate with changes in line pressure, size of orifices in the sparger or gas delivery tube, depth of delivery tube below the liquid surface in the culture vessel, and the amount of resistance encountered by the air stream in passing through any filtration or humidification devices used. Rather than try to maintain all these factors constant, it is advisable to meter the gas flow and adjust valve or pump to maintain the desired rate. Apparatus has been described for controlling and metering aeration both in culture tubes (Lockhart and Ecker, 1958; Heden and Malmborg, 1958) and in larger vessels (Ecker and Lockhart, 1961b). The effective gas-liquid interfacial area in such vessels may be
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increased in a variety of ways. Even the bubbles rising from a single, open tube will produce significant agitation at modest flow rates in 5 or 10 ml. liquid volumes in culture tubes. For larger volumes the air flow may be increased, and delivered through one or more tubes or spargers whose outlets are strategically placed near the bottom of the culture vessel. The number, size, position and depth of gas delivery tubes or spargers may be manipulated along with air flow rates to produce a wide range of oxygen delivery rates. The relationships between liquid volume and the shape and size of the culture vessel also influence aeration. The surface area of the liquid is a factor, as is the distance that air bubbles rise through the medium. Impeding the rise of bubbles by means of horizontal baffles placed in the culture vessel does not seem to have been practiced, but could prove useful. The rate of aeration in tubes is most conveniently determined by the sulfite method. In flasks or bottles almost any method of measurement could be used, although evaporation and smoke clearance determinations probably would not be very accurate. This system of aeration permits considerably greater flexibility in the design of experiments. Constant aeration may be achieved in culture volumes ranging from 5 or 10 ml. up to at least a liter. Use of a series of flowmeters (or the less expensive, small-volume purge meters) permits a number of replicates, particularly when tubes are used (Lockhart and Ecker, 1958).The progress of growth can be followed turbidimetrically if optically calibrated culture tubes are used. Periodic removal of samples for chemical or other determinations is possible from flasks or bottles without interrupting aeration or seriously reducing the culture volume during experiments. Great flexibility and precision of aeration control may be achieved in sparged vessels. Variable and metered air flows, combined with manipulation of such variables as liquid volume and sparger depth, permit continuous adjustment over a wide range of oxygen absorption rates. Since the factors affecting aeration in sparged vessels are at least relatively simple, the investigator may often “scale” his experiments into larger or smaller culture volumes or into a different sort of growth vessel, achieving the same level of oxygen availability (or a predictable alteration therein) with a minimum of empiricism. Ecker and Lockhart ( 1961b) developed theoretical expressions relating oxygen absorption rates in sparged vessels to a number of
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variables, and provided experimental confirmation. At low gas flow rates there was little agitation of the medium, and oxygen absorption rate was related inversely to the culture volume and directly to the proportion of oxygen in the gas mixture, the depth of the sparger orifices below the surface of the medium, the surface area of the liquid, and the rate of gas flow. At higher aeration rates, which produced appreciable turbulence in the liquid and prolonged the contact between gas bubbles and medium, oxygen absorption rate was proportional to the fourth power of the gas flow rate. Their equations contained a pair of constants which could be used to characterize the performance of individual spargers. The foregoing relationships hold strictly true only for straightwalled, unbaffled vessels of intermediate size, aerated through a single sparger having orifices of uniform diameter. Although these conditions apply to much laboratory culture apparatus, commercially available spargers often have a rather wide distribution of pore sizes. Only a few of the larger pores are used when air is passed at a low rate through such fritted glass filters or “gas dispersion tubes.” At greater flow rates, the gas begins to be forced through the smaller orifices, producing bubbles of smaller average diameter and increasing oxygen absorption more than had been predicted. If higher flow rates are to be used, it may be best to determine the oxygen absorption rates obtained under identical conditions with a series of spargers, and to derive for each a “sparger factor” (Ecker and Lockhart, 1959) which will predict its relative performance within reasonable limits. Sparged vessels are not useful for culture volumes much larger than a liter, or when very high aeration rates are needed. Aeration in volumes near a liter could be improved through use of several spargers, or by incorporating baffles in the culture vessels, but the quantitative effects on oxygen absorption rates then become unpredictable (though still measurable). Investigators who require high aeration rates in relatively large culture volumes should consider the stirred equipment discussed in the next section of this paper. A difficulty with the more versatile equipment is that it also is more elaborate. The design, assembly and calibration of culture apparatus for proper aeration takes some time, although its routine me thereafter presents no difficulty. Some special problems arise
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in the use of sparged vessels. Air must be sterilized before it enters the culture vessel, by passing it through a membrane filter of suitable pore size (Lockhart and Ecker, 1958), a length of tubing packed with sterile glass wool or some other filtration device. Passage of large volumes of air through the medium results in appreciable evaporation loss during the course of an experiment, and may necessitate humidifying the air before it enters the culture vessel. Undesirable foaming may occur. The risk of contamination is greater than with shake flasks, and sterilization of the apparatus is more difficult. It is sometimes necessary to autoclave the growth vessel (with medium) separately from the sparger assembly, and to assemble the apparatus aseptically before use. Flowmeters and gas flow controls need not be sterilized if air is filtered just before it enters the sparger, but the air from compressed air lines also should be filtered upstream from these controls in order to remove oil and particulate debris (Lockhart and Ecker, 1958). Passage of a stream of bubbles through culture media produces aerosols, and precautions must be taken when working with pathogens (Heden and Malmborg, 1958). It also has been reported (Ginsberg and Jagger, 1962) that cells may be trapped in fritted glass spargers, particularly at low aeration rates, thus introducing errors in determinations of population levels, as well as changes in air flow rates at constant pressure. Despite some disadvantages, however, sparged vessels are the most convenient method available to investigators who wish to work with culture volumes of between 100 and 1000 ml., or with very small volumes. They are particularly appropriate for cultivation of fairly small populations of nonmycelial organisms in defined, nonviscous media, and for experimentation in which it may be necessary to vary the culture volumes, conditions of cultivation or levels of oxygen availability. 3. Stirred Vessels
Stirred equipment of the type employed in industrial fermentations has now become common in university laboratories. While this paper is not concerned with the mechanics of operating such equipment, those aeration concepts which may be of aid to the microbiologist in the utilization of these devices are included. Most of the literature regarding aeration refer to stirred vessels, and this is where it is very easy to become overwheImed with the many
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conflicting viewpoints about aeration. It would be naive to suggest that the task of initiating experimentation in agitated equipment is a simple one, but the problem is not as formidable as it may appear superficially. Transfer of experiments from shake flasks into stirred jars is the point at which unsatisfactory results are most likely to occur. This will be particularly true in those cases where aeration factors have been neglected. Procedures for utilizing shake flasks simultaneously with stirred vessels for standardization of operations have been described by Squires and Kavanagh (1961). In this way, various operations such as inoculum development and sterilization procedures can be evaluated in different equipment. It is possible to arrive at a set of conditions where experimentation can be transferred from one piece of equipment to another with satisfactory results. Agitated equipment can be quite versatile in its application, providing that an optimum balance between air flow, mixing and power imput is provided for each group of microorganisms (bacteria, actinomycetes or filamentous fungi). It has the advantage of employing larger volumes of medium (up to 5 liters), which allows more flexibility in sampling during the experimental cycle. This affords a better characterization of the biological system through multiple analyses (carbohydrate utilization, growth, pH, nitrogen, product formation, etc. ) . Another advantage is that it is relatively easy to adjust oxygen absorption rates during the growth cycle to compensate for changes in the oxygen demand of the culture. This may be accomplished through regulation of the stirring speed and/or air flow rates. Product uniformity, as related to product isolating procedures, is usually better in stirred jars than in pooled media from shake flasks. The principal disadvantage of stirred equipment is the limitation in the number of replicates, with a possibility of large run-to-run variation. This factor can be diminished by proper experimental design and the maintenance of good standard conditions. Another disadvantage is the increased possibility for contamination. An aseptic operation is sometimes very difficult to attain and therefore some type of continuous monitoring for maintenance of good aseptic practices is mandatory (microscopic examination of stained slides, wet mounts for direct observation, plating techniques, etc. ). Contamination may arise from inadequate sterilization of equip-
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ment or of large volumes of medium, or it may enter during inoculation and sampling, or around the agitator shaft. Antifoam systems can also be a source of contamination, although ., this can be reduced through proper equipment design (Bungay et al., 1960). One of the most comprehensive reviews regarding aerationagitation in submerged fermentation was that by Finn ( 1954). Steel (1958) brought the field up to date and presented approaches to problems encountered in the “scale-up” from pilot plant to large industrial equipment. A recent review by Richards (1961) also covers aeration and agitation in stirred equipment. These authors discuss the engineering aspects of submerged fermentation in greater detail than we will present here. Control of the aeration process in stirred equipment will be considered only on the broadest basis. Those physical factors which contributed to oxygen absorption rates in previous sections apply equally well here. Factors which may affect the partial pressure of oxygen in the gas stream as well as solubility in the medium have an effect on observed dissolved oxygen concentration. Partial pressures of oxygen can be regulated in the gas stream through the use of mixing valves and manifolds. Most agitated equipment is operated with some back-pressure ( 5 to 10 p.s.i.) in order to prevent contaminants from entering around the agitator shaft. There is some question as to how much these pressures may contribute toward increased oxygen absorption rates during the actual fermentation process. Equipment for regulating gas flow to stirred jars is similar to that employed for the aeration of tubes and bottles. Flowmeters which measure air flow are placed on the nonsterile side of the air filter, Compressed air should be dry and of a constant supply and pressure. Maintenance of the interfacial area through agitation ultimately determines the oxygen absorption rate. Many factors interact to establish the interfacial area: the medium, gas flow, the sparger system, the type of impeller or agitator, agitator speed, power requirements, and tank baffling. The measure of the volume of air which should be sparged to a stirred vessel is usually estimated in one of two ways. The first method commonly employed is that of “superficial gas velocity.” This refers to the volume of air flow in cubic feet per second per cross sectional area (square feet) of the vessel. Air flows calculated
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on this basis will generally range from 0.002 to 0.01 ft./sec. The other method for estimating the amount of air to be sparged is a measurement in terms of volumes of air per volume of liquid per minute. Most industrial investigators employ a flow rate of one volume per volume per minute ( W M ) . Finn (1954) and Wise (1951) both object to the use of VVM for air flows since they feel that it is unrelated to gas “holdup” or to OAR. However, Solomons and Perkin (1958) point out that if one employs superficial gas velocity and keeps the flow constant, the volume of air per unit volume of liquid would be reduced by 50% upon doubling the diameter of the vessel. Since agitation contributes more toward the OAR than does gas flow rate, the use of W M as a measure of air flow in stirred equipment is probably as good as any other method. The number and size of sparger orifices is not as important in agitated equipment as in bottle or tube aeration. Sparging devices in stirred equipment usually consist of either an open tube which points into the flow of medium at the outer edge of the agitator or of a “ring” sparger which is placed beneath the bottom agitator. Their relative merits will not be discussed here, since adequate air-liquid mixing is assured from either type of sparger. Agitation is a difficult function to describe on a basis which microbiologists will find satisfactory, since a great deal of “artistry” has developed around this subject. Agitators are usually evaluated by calculating their turbulence in dimensionless units, power relationships, and oxygen absorption rates. These are usually further influenced by the effects of baffles, the ratio of impeller diameter to tank diameter, multiple impellers, and the relation of liquid level to impeller depth. Oldshue (1953) and Rushton et al. (1950) have made detailed studies of these factors and their interaction. These papers should be consulted if more detailed information is desired. Turbulence may be defined as that force which mixes air, medium solids, and microorganisms throughout the vessel. Stirred jars and tanks must be baffled to prevent the swirling of medium, allow better mixing in the container and load the impeller from a power standpoint. The term most often employed by engineers as a measure of turbulence is the Reynolds Number: D2N p / u , where D = diameter of the agitator in feet, N = impeller speed in revolutions per sec, p = density in pound mass per cubic feet (62.4 at 90°C. for water) and u = viscosity, pound mass per foot second
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(0.000672 at 20°C. for water). A Reynolds Number of at least lo5 is necessary for adequate turbulence in submerged fermentation equipment. The smaller the agitator, the higher the speed at which it must revolve for adequate turbulence. The power employed for agitation in most industrial fermentors ranges from 0.003 to 0.01 horsepower per gallon of medium. When air is introduced under the bottom agitator, power is Iost since the impeller is now stirring a mixture of air and medium. Michel and Miller (1962) recently made an effort to define, quantitatively, a relationship long known to those experienced with agitator dispersers; namely that the smaller the impeller selected to deliver a particular amount of power in a nongassed liquid, the greater is the reduction in power when gas is supplied to the impeller. These authors formulated an empirical equation for describing the general relationship; however, they point out that it does not necessarily hold for all sizes of equipment. Oldshue (1953) also noted a reduction of power due to gassing and has termed this a “ K factor. Gassing of the medium will normally cause an increase in the liquid height in the vessel. This is termed “holdup” and can be a useful criterion for gassing studies on uninoculated medium or water. Oxygen absorption rates are influenced by all the above factors. Solomons and Perkin ( 1958), employing sulfite oxidation measurements, derived a series of equations which enables one to calculate the required power, Reynolds Number, and stirrer speed for a chosen oxygen transfer rate. They concluded that the over-all controlling process of oxygen transfer is a function of the degree of turbulence and power per unit volume. The interrelationship between turbulence and interfacial area is one that is difficult to comprehend (and where the art of operating stirred equipment plays an important role). To understand this relationship it would be necessary to relate interfacial area to changes in turbulence. The effect of impeller speed and diameter on Reynolds Numbers can easily be calculated on water where the ratio of p / u remains constant. However, in a growing culture, this ratio undergoes continual change as the culture grows and media become metabolized. For this reason, it is very difficult to obtain any reliable measurement of turbulence once fermentation is initiated. The increase in liquid height (holdup) which results from gassing a vessel can be employed as a rough criterion for the amount of interfacial area formed. As growth and/or product
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increase viscosity ( u ) , turbulence becomes reduced and holdup falls in the vessel, indicating a reduction in interfacial area. Such conditions usually indicate a corresponding reduction in OAR and the approach of critical oxygen levels in the broth. During a later stage of the growth cycle, autolysis of the culture may occur with subsequent reduction of the viscosity ( u ) of the system. Turbulence then is increased and holdup begins to rebuild, indicating an expanded interfacial area and conditions which favor a correspondingly better OAR. Unfortunately, the oxygen demand of the culture has fallen by this time and foamy conditions persist. The effect of density ( p ) upon the ratio of p / u is even more difficult to measure. As the medium is gassed, density becomes reduced with a loss in turbulence but an increase in interfacial area. Density also continues to change during the fermentation cycle for the same reasons that viscosities change. When antifoam is added to an aerated culture, entrapped air is released and interfacial area reduced. As the foam system collapses, the density increases and produces higher turbulence in the system. As the effect of the antifoam becomes dissipated, interfacial area again rebuilds and causes a reduction in turbulence. Understanding and quantitating agitation is difficult because submerged fermentation is a dynamic system in which culture and medium interact to produce a continual change in viscosity, surface tension and medium solids. Engineers use the term non-Newtonian for such fluid systems since the consistency (viscosity) of the liquid (medium) is not constant but is a function of shear stress. Deindoerfer and Gaden (1955) studied this phenomenon employing penicillin broths and showed the rheological characteristics of such systems. However, the microbiologist at least has the advantage that he can determine the degree of physical damage to the culture introduced by agitation, Dion et al. (1954) studied the effects of agitator shear on mycelium formation. The morphology of Penicillium chrysogenum Thom was observed in submerged culture with several intensities of mechanical agitation in both baffled and nonbaffled systems. Mild agitation produced hyphae which were long and attenuated, with little branching. With vigorous agitation, the hyphae were short, thicker and highly branched. They also observed autolysis with mild agitation ( attributed to insufficient aeration) as well as with vigorous agitation (which they thought resulted from mechanical damage). Squires (1958) studied the
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effects of impeller design on Streptomyces orientalis and found that excessive fragmentation of mycelia was induced by the standard flat bladed impeller. This particular streptomycete has an extremely delicate mycelium which was damaged by the shearing action of the impeller. When curved blades were installed in place of the flat type, less mycelial damage was observed during the later hours of fermentation and yields were increased. Since there is no way to measure turbulence or interfacial area during submerged fermentation, the investigator must resort to trial and error in order to balance his system. Better scientific criteria are needed in this realm. Until that time, however, experienced operators will continue to develop the art of obtaining maximum interfacial areas with adequate turbulence for mixing and high OAR’s. The general practice is to employ water or uninoculated media in calibrating fermentors for oxygen absorption rates. Whether sulfite oxidation or one of the electrometric methods are employed, the investigator should realize that this is only a starting place and that additional refinements in conditions will depend on further experimentation. The various methods listed earlier in this paper can be utilized for measuring OAR’s in stirred equipment. Some controversy will always remain regarding the use of sulfite oxidation. If the microbiologist feels that the effects from growth and/or viscosity will not influence the turbulence of the system, then it seems fairly safe to employ sulfite oxidation for initial calibration studies. Sulfite concentration itself may produce false oxygen absorption rates. Yoshida et al. (1960) studied the effect of sulfite concentration on bubble size at high agitator speeds. They showed that when oxygen was bubbled through a sodium sulfite solution under mechanical agitation the absorption rate per unit interfacial area depended on diffusion. However, the rate per unit volume became accelerated because of the greater interfacial area induced by the presence of ions. Sulfite oxidation studies employed for the initial calibration of stirred jars should give some indication of the agitator speed and gas flow necessary for a desired absorption rate. Generally, such information can be employed with a great deal of confidence for studies involving bacteria. Most bacterial cultures remain nonviscous and the cell population seldom reaches a density where air bubbles become entrapped in the medium.
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The electrometric methods can be employed in stirred equipment not only for calibration but for monitoring dissolved oxygen levels. It must be admitted that the problem of maintaining asepsis in a vessel becomes more difficult when these probes are introduced. Perhaps the polyethylene-covered platinum electrode is the least expensive and most reliable of the current methods. Strohm and Dale (1961) sterilized their electrode chemically since it could not be steam sterilized. They employed this electrode to monitor dissolved oxygen content in production-sized baker’s yeast and food yeast (Candida utilis) fermentors. The junior author has employed this type of electrode to study aeration requirements in many antibiotic fermentations. These were “short-term” experiments with broths being transferred from production fermentors to smaller vessels. The electrode was mounted in a 10-gallon fermentor, which was sterilized with ethylene oxide and then filled with inoculated broth. The electrode life of such a system appears to be approximately one day; i.e., the “span” of the electrode drifted so that initial calibrations were no longer valid. Bare platinum electrode potentials were studied by Squires and Hosler (1958) and found to be a function of the log of the dissolved oxygen concentration in uninoculated medium. Tengerdy (1961b) also reported on this method and found it to be quite satisfactory for monitoring dissolved oxygen in the 2-ketol-gluconic acid fermentation. The platinum electrode potential method needs to be tested concurrently against a good method for monitoring dissolved oxygen during the entire fermentation cycle (such as the teflon tube method) before reliance can be placed on its indicated dissolved oxygen levels. However, the platinum electrode along with a steam sterilizable calomel electrode does offer a system that can be steam sterilized repeatedly with little worry from contamination. When studying those microorganisms which produce changes in viscosity or become very thick, initial calibration serves only as the point from which further empirical studies must be made in order to provide sufficient aeration to the culture. Those statements regarding product response discussed under shake flask aeration apply equally well to stirred equipment.
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V. Summary There is ample evidence that it always is desirable (and often it is necessary ) to control aeration during microbiological experimentation, to measure aeration as accurately as possible, and to report aeration procedures in quantitative terms which permit their reproduction by other workers. To do so is quite feasible, for relatively uncomplicated procedures, equipment and analytical techniques are available for this purpose. To be sure, we have oversimplified the interaction of factors involved in use of certain equipment, and we have glossed over some very real technical difficulties in the interpretation of measurements. Investigators who encounter complications will find it necessary to explore these matters in greater depth among the references we have cited. But, if aeration is not as simple and straightforward as we may have implied, neither is it quite so abominably difficult as many microbiologists suppose. Perfectly respectable aeration practices are well within the reach of any laboratory worker, be he specialist or not.
REFERENCES Altenbem, R. A., Williams, D. R., Kelsh, J. M.. and h'fanzy, W. L. (1957). 1. Bacteriol. 73, 697-702. Auro, M. A., Hodge, H. M., and Roth, N. G. (1957).Ind. Eng. Chem. 49, 1237-1238. Baracchini, O., and Sherris, J. C. (1959).J. Puthol. Bacteriol. 77, 565-574. Bartholomew, W. H., Karow, E. O., Sfat, M. R., and Wilhelm, K. H. (1950). I d . Eng. Chem. 42, 1801-1809. Bungay, H. R. (1959).Unpublished data. Eli Lilly and Company Development Reports. Bungay, H. R., Simons, C. F., and Hosler, P. (1960).J . Biochem. Microhiol. Technol. Eng. 2, 143-155. Chain, E. B., and Gualandi, G. ( 1954).Rend. Ist. Super. Sunitu (English E d . )
17, 5-60.
Cooper, C . M., Femstrom, G. A., and Miller, S. A. (1944).Ind. Eng. Chem. 36, 504-509. Cooper, P. D., Burt, A. M., and Wilson, J. N. (1958).Nuture 182, 1508-1509. Corman, J., Tsuchiya, H. M., Koepsell, H. J., Benedict, R. G., Kelly, S. E., Feger, V. H., Dworschack, R. G., and Jackson, R. W. (1957). Ay~pl. Microbiol. 5, 313-318. Deindoerfer, F. H., and Gaden, E. L. (1955).Appl. Microhiol. 3, 253-257. Dion, W. M.,Carilli, A., Sermonti, G., and Chain, E. B. (1954).Rend. Ist. Super. Sunita (English Ed.) 17, 187-205. Ecker, R. E., and Lockhart, W. R. (1959).Appl. Microbiol. 7, 102-105. Ecker, R. E., and Lockhart, W. R. (1961a).J. Bucteriol. 85, 511-516.
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Ecker, H. E., and Lockhart, W. R. (1961b). Appl. Microbiol. 9, 25-31. Finn, R. K. (1954). Bacteriol. Reu. 18, 254-274. Finn, R. K. (1959). Proc. 136th Meeting, Am. Chem. Soc., Atlantic City, New Jersey. Fusillo, M. H., and Weiss, D . J. (1958). Antlbiot. Chemotherapy 8, 21-26. Gaden, E. I,. (1962). J. Biotechnol. Bioeng. 4, 99-103. Ginsberg, D. M., and Jagger, J. (1962). J. Bacteriol. 83, 1361-1362. Heden, C. C . , and Malmborg, A. S. (1958). Acta Pathol. Microbiol. Scand. 44, 405-412. Herbert, D., Elsworth, H., and Telling, R. C. (1956). J. Gen. Microbiol. 14, 60 1-622. Hewitt, L. F. ( 1950). “Oxidation-Reduction Potentials in Bacteriology and Biochemistry.” Livingstone, Edinburgh and London. Hixson, A., and Gaden, E. L. (1950). Ind. Eng. Chem. 42, 1793-1801. Karow, E. O., Bartholomew, W. H., and Sfat, M. R. (1953). J. Agr. Food Chem. 1, 302-306. Knox, R., Lister, A. J., and Thomas, C. G . A. (1957). J. Gert. Jlicrobiol. 17, ix-x. Lockhart, W. R. (1959). Bacteriol. Reu. 23, 8-17. Lockhart, W. R., and Ecker, R. E. (1958). Appl. Microbiol. 6, 93-96. Lockhart, W. R., and Weaver, R. N. (1960). J. Bacteriol. 80, 331-335. Loomis, W. F. (1954). A w l . Chem. 24, 402-404. Loomis, W. F. (1956). Anal. Chem. 28, 1347-1349. McLimans, W. F., Giardinello, F. E., Davis, E. V., Kucera, C. J., m d Rake, G . W. (1957). J . Bacteriol. 74, 768-774. Michel, B. J., and Miller, B. J. (1962). Am. Inst. Chem. Engrs. J. 8, 262-266. Moss, F. (1952). Australian 1. Exptl. B i d . M e d . Sci. 30, 531-540. Oldshue, J. Y. ( 1953). “Application of Mixers to Bio-Engineering Processes.” Rose Polytechnic Inst., Terre Haute, Indiana. Phillips, D. H., and Johnson, M. J. (1959). Ind. Eng. Chew. 51, 83-88. Phillips, D. H., and Johnson, M. J. (1961). J. Biochem. Microbiol. Tcchnol. Eng. 3, 277-309. Pirt, S. J. (1957). J. Gen. Microbiol. 16, 59-75. Public Health Assoc. ( 1960). “Standard Methods for Examination of Water and Sewage.” Am. Public Health Assoc., New York. Rhodes, R. P., and Gaden, E. L. (1957). Ind. Eng. Ckerrt. 49, 1233-1236. Richards, J. W. ( 1961 ), “Progress in Industrial Microbiology,” pp. 143-172. Wiley ( Interscience), New York. Rushton, J. H., Costich, E. W., and Everett, H. J. (1950). Cheni. Eng. Progr. 46, 395-404, 487-476. Solomons, G . L., and Perkin, M. P. (1958). J. Appl. Chem. ( Loridon) 8, 251259. Squires, R. W. (1958). Unpublished data. Eli Lilly and Company Development Reports. Squires, R. W. and Hosler, P. (1958). Ind. Eng. Cheni. 50, 1263-1267. Squires, H. W. and Kavanagh, F. W. (1961). Deuelop. Ind. Microbiol. 3, 376-383. Steel, R. ( 1958). “Biochemical Engineering.” Macmillan, New York.
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Steel, R., and Brirrlcy, M. R. (1959). Appl. Microbiol. 7, 57-61. Strohm, J., and Dale, R. F. (1961). lnd. Eng. Chem. 53, 760-764. Strohm, J.. Dale, R. F., and Pepplcr, H. J. (1959). Appl. Microhiol. 7, 235-238. Tengerdy, R. P. (1961a). 1. Biochcm. Microhiol. Tcchnol. Eng. 3, 241-253. Tengerdy, R. P. (1961b). 1. Biochem. Microhiol. Technol. Eng. 3, 255-260. Llmbreit, U’. W., Burris, R. H., and Stauffer, J. F. (1957). “Manometric Techniques and Tissue Metabolism.” Burgess, Minneapolis, Minnesota. Webley, D. M. (1954). J . Gen. Microbiol. 11, 114-122. Wise, W. S. (1951). 1. Gen. A4icrobioZ. 5, 167-177. Yoshida, F., Ikeda, A., Imakawa, S., and Miura, Y. (1960). Ind. Eng. Chem. 52, 435-438. Ziegler, D. W., Davis, E. V., Thomas, W. J., and McLimans, W. S. (1958). Appl. Microhiol. 6, 305-310.
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Stability and Degeneration of Microbial Cultures on Repeated Transfer FRITZREUSSEX Resecirch Laborutories, The Upjohn Company, Kulumuzoo, Michigan
I. Introduction ........................................... 11. Mechanisms Mediating Genetic Recombination in Microorganisms ................................................. A. Deoxyribonucleic Acid .............................. B. Ribonucleic Acid .................................... C. Episomic Elements in Bacteria ........................ D. Transduction ....................................... E. Parasexual Cycle .................................... F. Sexual Cycle ........................................ G. Heterokaryosis ...................................... H. Mutation .......................................... 111. Examples of Culture Stability and Degeneration . . . . . . . . . . . . A. Streptomyces Fermentations .......................... B. Fungal Fermentations ................................ C. Yeast and Bacterial Fermentations . . . . . . . . . . . . . . . . . . . . . D. Mammalian Cells in Tissue Culture .................... IV. Prevention or Circumvention of Culture Degeneration . A. General ..................................... B. Freezing of Living Cells .............................. C. Soil Cultures . . . . . . . . . . . . . . . . D. Lyophilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Mineral Oil Slants ..................... F. Circumvention of Culture Degenei ation in Continuous Processes .............................................. V. Discussion and Conclusions . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
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introduction
The terms culture stability and degeneration refer to the ability of a given microbial population to retain desirable morphological or biosynthetic characteristics qualitatively and quantitatively from generation to generation. It was originally assumed that recombinational processes among microorganisms were limited to higher fungi which often possess sexual stages comparable to those observed in higher plants and animals. Bacteria and fungi imperfecti were considered to propagate purely vegetatively. However, during the last decade a multi189
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tude of genetic exchange and transfer mechanisms have become evident among microorganisms. These differ from the meiotic process and allow the organisms to bypass it. Except for a few cases, very little is known yet concerning frequency, distribution, significance, and chemical nature of these nonconventional mechanisms. However, the well-known variability and instability of microorganisms can be accounted for by these new mechanisms. This essay discusses the nature and function of various recombination mechanisms encountered in microorganisms. Structural aspects of deoxyribonucleic acid ( D N A ) and ribonucleic acid ( R N A ) are discussed in moderate detail since both represent the chemical basis of the genetic material. The biochemistry of the genetic processes is not fully understood at present but major advances in this field are anticipated for the immediate future. Specific examples of culture stability and degeneration as observed in fermentation processes of industrial significance are cited to exemplify the problems facing the investigator who attempts to synthesize and maintain strains with a desired biochemical capability.
II. Mechanisms Mediating Genetic Recombination in Microorganisms
A. DEOXYRIBONUCLEIC ACID Since the discovery by Avery et al. (1944) of the D N A nature of the transforming agent in bacteria, it has become more and more evident that DNA is the material responsible for passing the genetic information from generation to generation in living systems. In organisms devoid of D N A , such as tobacco mosaic virus, RNA is assumed to carry the genetic information. For recent reviews on D N A structure see Drysdale and Peacock ( 1961a), Burton ( 1962). D N A consists structurally of two intertwined helices, Each chain of the double helix is a repeating unit of 2-deoxyribose and phosphate with a nitrogenous base attached to each deoxyribose molecule. While the sugar-phosphates provide the backbone of the helix, the nearly planar nitrogenous bases are stacked together closely and lie approximately at right angles to the helix axis. Hydrogen bonds formed between the bases of each helix provide stability to the double helix. The four principal bases found among D N A are: the two purine bases adenine ( A ) and guanine ( G ) , and
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the pyrimidine bases thymine ( T ) and cytosine ( C ) . Structural exigencies necessitate specific pairing of adenine of one chain with thymine of the other chain and guanine with cytosine. Structural arrangements of these hydrogen bonds are shown in Fig. 1. This structure implies also that complementary bases ( A and T, G and C ) are always present in equal amounts. Therefore, regardless of the source of the DNA sample and despite wide variations among noncomplementary bases, the ratio of complementary bases
guanine
FIG. 1. Hydrogen bonding between complementary bases in DNA. Double bonds not shown.
on a molar basis will always be near 1. Figure 1 also indicates that three hydrogen bonds can be formed between G and C and only two between A and T. The stability of a given DNA helix should, therefore, increase with increasing relative content of the base pairs G C (Marmur and Doty, 1959; Sueoka et al., 1959). Unlike DNA derived from higher species (Sueoka, 1961), the ratio A T/G C in different microbial DNA preparations has been found to vary considerably, whereas preparations of DNA from a particular microorganism have proved remarkably homogeneous with respect to base quotients. This variation would indicate that different bacterial species will have no common identical DNA molecules and that the probability of overlapping is remote. Upon
+
+ +
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heat treatment, the double helix of DNA has been found to dissociate into its single strands (Doty et al., 1960; Marmur and Lane, 1960). Following appropriate cooling, the two strands will recombine partially. This phenomenon is paralleled by a partial restoration of the biological activity. As the carrier of genetic information, DNA controls the synthesis and base sequence of RNA. In cells of higher organisms DNA is mainly localized within the chromosomes in the nucleus. Bacteria and streptomycetes do not have a clearly defined nucleus but have localized DNA aggregates among their cytoplasm. Such aggregates are usually referred to as bacterial nuclei, nuclear bodies or plasma genoms. As a working hypothesis we will assume that the bacterial and streptomycete DNA is localized within chromosomelike structures not enclosed in a nuclear membrane. These postulated chromosomes would therefore be present free in the cytoplasm. The small dimensions of bacterial cells allow one to theorize that only a restricted number of chromosomes (one or two) should be present per cell. Because of their filamentous growth form, streptomycetes, even though of rather minute dimensions, should contain several chromosomes; however, the maximal number one would expect should still be quite limited. Little experimental evidence is available to sustain this hypothesis. However, the conjugation process in certain Escherichiu coli strains coupled with transfer of a single genetic linkage unit from donor to acceptor cell obviously sustains such a hypothesis. B. RIBONUCLEICACXD RNA occurs chiefly in the cytoplasm and the nucleolus of cells. There are some structural similarities between RNA and DNA. For recent reviews of RNA structure see Drysdale and Peacocke ( 1961b), Brown (1962). Usually only the purine bases adenine and guanine, and the pyrimidine bases cytosine and uracil occur in RNA. Instead of deoxyribose which occurs in DNA, ribose is found in RNA. The main chain is made up by repeating units of ribose and phosphate with a nitrogen base attached to each ribose unit. Again, as in the case of DNA, the sugar-phosphates form the backbone of the chain. The structure of the RNA strand is not as well defined as in the case of DNA. RNA is usually present as a single strand. Recent evidence suggests that short regions among the single chain may fold back on themselves and form limited local
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double helices stabilized by hydrogen bonding between matching base pairs such as adenine-uracil and guanosine-cytosine. Nonmatching nucleotides can be accommodated outside the helices as loops. Functionally, RNA directs the synthesis of proteins. Recent developments in this regard have definitely established that RNA is directly involved in the sequence specification of the amino acids in proteins. Three fractions of RNA have to be considered in this process: soluble RNA, “messenger” RNA, and ribosomal RNA. Soluble RNA activates amino acids specifically by forming soluble aminoacyl-RNA-complexes. These complexes transfer the activated amino acids to ribosomes. Specific enzymes then mediate their transfer to ribosomal RNA. Until very recently it was assumed that the base composition in the ribosomal RNA was implicit for specific amino acid sequences of the synthesized proteins. However, Nirenberg and Mathaei (1961) have discovered that synthetic polynucleotides can act as templates for protein synthesis in a cell free system. Thus, “messenger” RNA acts as an immediate template for amino acid sequence. Subsequent exploitation of this discovery by different authors with synthetic polynucleotides of known base composition has led to some decoding of the genetic information localized in RNA templates (Lengyel et al.,1961, 1962; Speyer et al., 1962a, b).
C. EPISOMIC ELEMENTS IN BACTERIA Episomes are self-reproducing genetic elements capable of existing either autonomously in the cytoplasmic state, where replication is not controlled by the other genetic material of the cell, or integrated with a bacterial chromosome. The integration process involves attachment of the episome at either end of the chromosome. Replication of the integrated episome occurs concurrently with the chromosome. Episomes do not carry any essential genetic information necessary for normal cell growth and function and may be completely absent. They are able to leave a particular cell and enter another during cell to cell contact. The best characterized episomes are the lysogenic bacteriophages, the F-factor of E. coli, and the colicinogenic factors. Except for bacteriophages, relatively little is known about the chemical nature of episomes, but one may assume that these carriers of genetic information consist of RNA or DNA. Recent reviews of bacterial episomes are given by Jacob e f al. (1960),Jacob and Wollman (196l),and Sneath ( 1962).
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1. Lysogenic Bacteriophages Lysogenic bacteriophages can enter a host cell and in the prophage stage integrate with the bacterial chromosome. Under these conditions, phage replication takes place whenever the chromosome is replicated. By irradiation or treatment with various chemicals the integrated phage-chromosome state can be dissociated, probably by rupturing the link between phage and bacterial chromosome. The free lysogenic material will then replicate autonomously and may eventually cause lysis of its host cell.
2. Conjugation in Escherichia coli Conjugation in E . coli involves mating of two cells and transfer of genetic material from donor to recipient. An episomic factor designated F-factor or sex factor controls the ability of a cell to act as donor or acceptor. Bacteria carrying the F-factor in its cytoplasmic form are called F f , those containing nv F-factor, F-. In its cytoplasmic form the F-factor multiplies at high frequency and can rapidly convert F- cells to F+ forms. Conjugal pairs between Ff and F- cells are formed readily. However, genetic recombination occurs very rarely under these conditions. The F-factor can also exist in its integrated form and such cells are called Hfr. Upon mating with F- cells, conjugation will occur and genetic material from the Hfr strain is transferred to the receptor cell. Genetic analysis of such recombinants has implied that E . coli contains only one chromosome and is therefore haploid. Within a cell the chromosome has ring structure on which the genes are arranged linearly. Upon integration of the F-factor with the chromosome, the latter is split at the particular point of F-factor attachment and the chromosome assumes a linear structure carrying the F-factor at one end. The location of the ring split and F-factor attachment has proved to be always at the same location for a given strain, but the location may vary between strains. During mating, the chromosome from the donor cell (Hfr) enters the recipient cell in such a way that the end opposite the F-factor attachment will enter first. It has proved possible to interrupt the process of chromosome transfer at will by dissociating the conjugates through vigorous shaking or by addition of a phage which specifically lyses only the male mate (Wollman and Jacob, 1955; Hayes, 1957). Thus, only genes located within the male chromosome portion and already transferred to the
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recipient cell can participate in recombination and express themselves in recombinants. Genetic maps of E . coli have thus been constructed by interrupting the mating process at different time intervals. Decreasing frequencies of inheritance of different genes, due to exclusion by chromosome breaking, therefore indicate directly the linear order of the genes on the chromosome.
3. Colicinogenic Factors Colicinogenic factors control the formation of the proteinaceous antibiotic substances, colicins, by some strains of Enterobacteriaceae. Such colicins are capable of killing other organisms of the same group. Colicinogenic factors exist either in their autonomous cytoplasmic form or integrated with the bacterial chromosome. Upon transfer to recipient cells, the latter can produce colicin. Acquisition of the colicinogenic character is a fairly stable phenomenon and is usually not lost. Colicinogenic episomes can also mediate mating coupled with transfer of chromosomes and recombination of genetic material between different cells similar to the case discussed for the F-factor. A recent review on colicinogeny and genetic recombination was published by Smith and Stocker ( 1962). 4 . Drug Resistance Transfer Factors
Of great practical importance is the discovery by Watanabe and Fukasawa (1960, 1961a, b, c, 1962), and Mitsuhashi and Harada (1962), of the episomic nature of antibiotic resistance transfer in Enterobacteriaceae. Evidence accumulating from their work suggests that multiple drug resistance, including streptomycin, chloramphenicol, tetracycline, and sulfonamide resistance in Enterobacteriaceae is mediated by an episomic element, which is transferred by cell to cell contact. D. TRANSDUCTION Transduction is always associated with a lysogenic system. During this process, phages liberated from a donor cell are transferred to an appropriate recipient cell of different genetic composition. The recipient cell must favor the prophage rather than the vegetative state of the particle. Rupture of the integrated state in the donor cell takes place by a break slightly proximal to the original
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point of phage attachment to the bacterial chromosome. Under such conditions, a phage particle may transfer a small segment of the chromosome originating from the donor strain to the recipient strain upon integration with the chromosome of the recipient.
E. PARASEXUAL CYCLE This phenomenon allows genetic studies with somatic cells devoid of a meiotic apparatus. It was discovered by Pontecorvo and Roper
( 1952) in Aspergillus nidulans and has since been studied primarily in the two fungi imperfecti dspergillus niger and Penicilliurn chrysogenurn ( Pontecorvo et ul., 1953; Pontecorvo and Sermonti, 1954). The cycle consists of the following successive stages (Pontecorvo, 1962): (1) Formation of a diploid nucleus by fusion of two haploid nuclei (karyogamy ) in a multinucleate mycelium. These events take place at very low frequency and are thought to occur purely by accident. The diploid fusion product will be heterozygous and will carry the complete genetic material of both haploid nuclei if the two nuclei taking part in the fusion differ genetically. ( 2 ) Multiplication of the diploid nucleus side by side with the remaining haploid ones. Occasionally purely diploid colonies may emerge. ( 3 ) The diploid nuclei can undergo somatic segregat'1011. Somatic segregation will either leave the diploid condition of the nucleus intact or cause haploidization. Karyogamy as well as subsequent haploidization is enhanced by the application of certain chemicals. The following independent somatic segregation processes have been observed in Aspergillus: somatic (mitotic) crossing-over, somatic nondisjunction, and haploidization. The first two phenomena do not affect the diploid state of the nucleus. 1. Somatic Crossing-oc;er
Somatic crossing-over between chromosomes heterozygous at several genes will yield progeny honiozygous in the chromosome arm distal from the point of crossing-over. The other heterozygous genes located on the same chromosome arm proximal to the centromere from the point of crossing-over, as well as heterozygous genes located on the arm of the other side of the centromere, will remain heterozygous. Thus, somatic crossing-over allows sequence and segment analysis for markers located on the same chromosome
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arm extending from the centromere to the distal end of the chromosome. 2. Mitotic Nondisjunction During this process diploid daughter nuclei are obtained which contain a linked group of homozygous gene loci which originally were heterozygous in the parent and located in the same chromosome pair. In this case homozygosis can include any linked gene region among the chromosome, irrespective of arm and centromere. Mitotic nondisjunction allows for identification of linked genes located within the same chromosome pair, but does not allow elucidation of their linear sequence along the homozygous region.
3. Haploidization The diploid nucleus is reduced to two haploid nuclei during haploidization. The chromosomes are segregated at random and no exchange of intrachromosomal genetic material takes place. Haploidization allows recognition of the genes contained within a particular chromosome, but does not yield any information concerning the linear arrangement of the genes contained within the chromosome. F. SEXUALCYCLE Most industrially important microorganisms do not have a sexual cycle, and the techniques to improve yields of their products by use of the sexual cycle, so successfully applied in higher organisms, are not applicable. Among the microorganisms, only the perfect fungi possess a sexual cycle which is basically identical with the meiotic process in higher organisms. Extensive use of this identity was made by Beadle and Tatum (1945) and Beadle (1947) in their elucidation of genetics in Neurospora. In this organism, hyphae of opposite sex may fuse or protoperitheciae will be fertilized by conidia of the opposite sex. Following nuclear fusion of the two parent nuclei, recombination takes place and the diploid nucleus undergoes reduction division yielding two haploid nuclei. In Neurospora, each of the two daughter nuclei will undergo two further mitotic divisions to yield a total of eight haploid nuclei. Each of these nuclei is closed off as a spore and the entire spore set is enclosed in linear arrangement within an ascus. The asci themselves are assembled within a secondary structure called the perithecium. The individual ascospores contained within a par-
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ticular ascus can be isolated and their linear arrangement within the ascus provides an excellent model for genetic segregation studies mediated by the sexual cycle in Neurospora. Many aberrations from the meiotic process described for Neurospora are encountered among other fungi but will not be discussed here.
G. HETEROKARYOSIS The heterokaryotic state is characterized by the occurrence of two or more genetically different nuclei within a common cyto0
.
0
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1
FIG.2. Scheme of heterokaryon formation. a, Genetically different hyphae; b, Anastomosis and exchange of nuclei; c, Completed heterokaryon; d, Dissociation of the heterokaryotic state during spore formation.
plasm. Such a condition is commonly observed in fungi, The mechanism of heterokaryon formation is illustrated in Fig. 2. The process is initiated by the fusion of two compatible hyphae containing genetically different nuclei. These nuclei will intermix within the common hyphae and the progeny will carry nuclei of both types within the same cytoplasm. Dissociation of the heterokaryotic state can occur during spore formation. If such spores are uninucleate, this progeny will carry nuclei of only one of the parents.
MICROBIAL CULTURES ON REPEATED TRANSFER
H. MUTATION Mutation phenomena include any permanent or semipermanent changes of the genetic material (DNA) in a given cell with the exception of rearrangements due to genetic segregation. Distinction is usually made between natural or spontaneous mutations and artificial mutations. Natural mutations are those known to occur continuously at very low frequency. Economically more important are artificial mutations induced by chemicals or radiation. Most mutagenic chemicals have been found to interact with DNA. For example, acridines were found to slide between double helices, thus causing some unwinding of the latter ( Lerman, 1961). Some chemicals exhibit some specificity in their reaction with the different bases. Nitrogen mustards, ethylene oxides, dialkyl sulfates and alkylmethane-sulfonates were found to react readily with guanine at the N-7 position and with adenine at the N-1 position (Burton, 1962). Reagents such as bromine or osmium react with C-5 and C-6 of the pyrimidines. Hydroxyperoxides are formed from pyrimidines under the influence of ionizing radiation. The principal effects of ionizing radiation on the purines adenine and guanine are 8-hydroxylation, breakdown of the imidazol ring system, and some deamination (Ponnamperuma et al., 1962). Results of Klouwen et al. (1962) indicate that separation and recombination of the DNA double helix is somewhat impaired after ultraviolet ( UV ) irradiation and is partially reversible. Irradiation of DNA with X-rays at a dose of 10,000r. suggested that cross-linking occurred at such high radiation levels (Guda et al., 1962).
111. Examples of Culture Stability and Degeneration A. Streptomyces FERMENTATIONS
Variation in regard to morphology and physiology are outstanding features of the members of the genus Streptomyces. Despite their importance in the production of antibiotics, relatively little experimental work has been published to date concerning the stability of highly productive streptomycete strains. Several investigators have established that genetic interaction occurs in streptomycetes, but the mechanisms involved are not yet fully understood (Bradley, 1959; Bradley et al., 1959; Braendle and
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Szybalski, 1959; Sermonti and Spada-Sermonti, 1959; Saito and Ykeda, 1959; Hopwood, 1959; Alikhanian et al., 1959). The genus Streptomyces represents the evolutionary link between bacteria and filamentous fungi. Both streptomycetes and bacteria share the common features of small dimensions and absence of clearly defined nuclei within the cytoplasm. We therefore assume that the Chromosomes are free in the cytoplasm. The filamentous growth of the streptomycetes is comparable to the growth form of fungi. The mycelium is practically coenocytic since cross walls are rare. Phenotypic expression in streptomycetes is therefore the result of interactions of different chromosomes in a common cytoplasm. The chromosomes are randomly distributed in the cytoplasm. The possibility should therefore be considered that the relative spatial distribution of heterozygous chromosomes in a common cytoplasm might also have some influence upon the manifestation of the phenotype. In other words the same genetic material present within a cell might result in different phenotypic expressions depending on the relative spatial distribution of the chromosomes among the cytoplasm. In commercial streptomycete antibiotic fermentations, maintenance of culture stability in high-yielding variants is imperative and usually poses a serious problem. Many such cultures gradually degenerate in their productive ability upon successive transfer. However, one has to consider that most high-yielding strains are artificial mutants selected on an empirical basis. “Wild strains found to produce a particular antibiotic are treated with mutagenic chemicals or radiation, and superior producers are selected from among the survivors. Since fermentation tests are commonly done in complex media, nutritional deficiencies of such mutants usually remain undetected. Few data comparing effects of different mutagenic treatments (single or combined) upon a single culture are available. The same is also true when the same mutagenic treatment is applied to different antibiotic-producing cultures. In both cases certain treatments might result in the emergence of similar types and frequencies of new mutants. Some experimental treatment of this problem is given by Alikhanian et al. (1959), and Alikhanian (1962). Treating oxytetracycline-producing Streptomyces rimosus and S. (iureofaciens strains with UV-rays, X-rays, and ethyleneimine singly and in combination, they found it very difficult to assign preference
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to any one of these treatments or combinations, In the case of S. rimosus, the majority of morphological mutants induced by mutagenic treatment proved to have lower antibiotic productivity, whereas the majority of variants with higher productivity did not differ markedly in morphology from the parent strain. The same authors also investigated genetic recombination in oxytetracyclineproducing strains of S. rimosus. Several mutants with nutritional markers were crossed. The recovered prototrophic colonies from the same cross were found to vary markedly in antibiotic yield and morphology. Yields obtained from such crosses were usually considerably higher than those obtained from the parent auxotrophs, but inferior to the productivity of the “wild strains from which the auxotrophs were derived. Stability studies with such prototrophs were done over 4 to 5 generations. Some of these crosses proved remarkably stable and did not segregate auxotrophs. Unstable prototrophs yielded segregants of only one of their respective parent types. In one instance, initial prototrophs obtained from the same cross, designated types No. 1 and No. 2, behaved quite differently. Among seven prototrophs of type No. 1 tested, 2 proved unstable, while from 51 tested of type No. 2, 42 showed segregation. None of the recombinants of S. rimosus reverted into both parent forms, and only one was obtained in segregants. Recombination frequencies (usually rather low in S. rimosus) increased considerably when one of the mutants was present in excess during crossing irrespective of predominance of either one. Perlman et al. (1954) investigated degeneration of a streptomycin-producing culture of S. griseus. They observed that antibiotic production decreased markedly upon successive transfers. However, no significant differences could be detected between highand low-producing cultures when other metabolic parameters, such as glucose utilization, growth rate, ammonia liberation, and vitamin BI2 production, were analyzed. However, titer reduction was correlated with increasing emergence of asporogenous colonial types when plated on soybean infusion agar. Williams and McCoy (1953) observed changes in morphology and decreased streptomycin production by serially subculturing several strains of S. griseus on yeast-glucose agar. Marked loss of antibiotic production by Streptomyces strains upon repeated transfer was also experienced during similar studies in our laboratory (Reusser et al., 1961). These strains were trans-
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ferred serially at short intervals (2-3 days). Mycelial growth was abundant but antibiotic titers were low, thus minimizing possible deleterious effects of the antibiotic upon the culture itself. Parallel flasks of each transfer were also prepared and incubated until peak activity of the antibiotic was reached. Several single spore strains of the following organisms were derived from high-yielding parent
I
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20
30
FIG.33. Subculturing of Streptomyces niveus producing novobiocin.
cultures: S . niveus producing novobiocin, S . fradiae producing neomycin, and S . achromogenes var. streptoxoticus producing streptozotocin. During initial transfers, all homolog strains were remarkably similar to each other and to their respective parents in morphology and extent of antibiotic production. However, following approximately 30 successive transfers, antibiotic titers had dropped sharply to barely detectable levels (see Fig. 3a, b, c ) .
I80C
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I20C i
E \
7 I ooc
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= e
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a
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FIG. 3b. Subculturing of Streptomyces fradiue producing neomycin.
TRANSFERS
Fie. 3c. Subculturing of Streptomyces achronwgenes var. streptozoticus producing streptozotociu. 20.3
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Parallel to the serial transfers of vegetative mycelium, sporc transfers of high-producing strains were made from agar slant to agar slant at time intervals which were long enough to permit abundant sporulation. A spore suspension of each transfer was tested for antibiotic production under submerged growth conditions. Again culture degeneration occurred at a rapid rate during serial subculture. A gradual loss of the ability to produce spores was observed parallel to the gradual reduction of titer when vegetative mycelia derived from different transfers were reinoculated onto agar slants, and eventually fully asporogenous colonies emerged. Mixed inocula with low- and high-yielding cultures of the organisms resulted in sharply depressed antibiotic formation. However, clear culture medium derived from low-yielding fermentations or culture medium containing heat killed low-yielding mycelium added to high-yielding fermentations at different time intervals during the fermentation course had no adverse effect upon yield of the highproducing culture. These experiments suggested that the emerging low-yielding strains derived from the same parent strain by serial subculturing were faster growing segregants or mutants of the parent culture. In the case of the novobiocin fermentation the growth rates of both a high-yielding and the corresponding low-yielding strain were determined. The results obtained indicated that the growth rates of the two strains were virtually identical. However, it was found that the low-yielding strain utilized carbohydrate much more efficiently than the high-yielder and could therefore overgrow the high-yielder by producing greater amounts of mycelium. This finding indicates that some unknown medium ingredient had probably become limiting in the case of the high-producing strain. No morphological differences between high and low producing cultures were observed in the cases of S . niueus, S. fradiae, and S . achromogenes var. streptozoticus. Sikyta et al. (1959) did not find any evidence of culture degeneration during continuous propagation of their streptomycin producing S. griseus strain. However, the duration of their continuous culture experiments were rather short and may have bern terminated before culture degeneration could occur. The regularity with which culture degeneration occurs among antibiotic producing streptomycete strains indicates that such high-
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yielding commercial strains are genetically quite heterogeneous and lose their particular biosynthetic capabilities rapidly by genetic segregation. We postulated that the genetic material in streptomycetes is localized in chromosomal structures which are present free in the cytoplasm. During growth, replication of different heterozygous chromosomes, present in the same cytoplasm, can conceivably take place at different rates. This would result in a constant shift of the genotype and consequently the phenotype of the progeny of a culture. We can also assume that formation of a particular metabolic product, not directly associated with the growth process of a streptomycete, is determined by a unique heterozygous genetic configuration. Such a genotype will consequently be subjected to a gradual and progressive change during the replication process and may cause rapid degeneration of a culture. This postulate would lend itself to a logical explanation for the rapid degeneration phenomena observed among streptomycetes.
B. FUNGAL FERMENTATIONS Fungi with a known sexual cycle have a meiotic apparatus and well defined nuclei. However, great variability in physiology and morphology are also widely observed. Unfortunately, most industrially important fungi do not have a sexual cycle and the geneticist must resort to such mechanisms as heterokaryosis, parasexual cycle or artificial mutation in order to effect genetic changes directed toward increasing yields. As in the case of streptomycetes, artificial mutation has been applied extensively, notably in the production of penicillin by strains of the Penicillium notatum-chrysogenum group. Several high-yielding Penicillium clargsogenum strains selected after ultraviolet irradiation were tested in our laboratories for their stability to produce penicillin during serial transfer under submerged culture conditions. Transfers were made at %day intervals when satisfactory mycelium growth was present but penicillin titer was still low. This measure was taken to minimize inhibiting effects of penicillin upon the cultures. Mycelium of each transfer was also inoculated into parallel flasks and allowed to ferment over the full fermentation cycle and the culture medium assayed for penicillin. Typical results obtained are shown in Fig. 4. Penicillin titers increased during initial transfers to reach a peak (1500 units/ml.)
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at about the fifth transfer. Thereafter culture degeneration occurred, and the yields dropped to approximately 400 units/ml. after 8 transfers. However, unlike in streptomycete fermentations, yields leveled off at a plateau between 200 and 400 units/ml. over 20 further transfers. Thus, degeneration in this case did not result in a complete or near complete loss of activity but simply to a levelingoff at a lower production plateau.
TRANSFERS
FIG. 4. Subculturing of Penicillium chrysogenum producing penicillin.
Results obtained by serial transfer of spores of the same strains essentially corroborated the results obtained by serial transfer of vegetative mycelium. Whiffen and Savage (1947) analyzed culture degeneration of Peiiicillium notatum with respect to penicillin production. Vegetative submerged mycelium retained full productivity over more than 50 transfers. However, if spores of the same strain were transferred serially, antibiotic yields were reduced drastically. It appears likely
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that the strain was heterokaryotic since decrease of bioactivity occurred as a result of the sporulation process. During spore formation the particular heterokaryotic condition favorable for penicillin production may have been dissociated resulting in decreased antibiotic production. The sexual cycle of P . chrysogenum is unknown. Nevertheless, genetic recombination by this mold was obtained by use of the parasexual cycle ( Sermonti, 1959). A low-producing mutant crossed with several completely nonproducing mutants selected from a penicillin producing P . chrysogenum parent strain yielded heterozygous diploid strains with penicillin productivities comparable to the parent strain. Thus, two different mutations proved to complement each other to yield a strain fully as productive as the parent strain. The high penicillin yield characteristic proved to be recessive as evidenced by the synthesis of heterozygous diploids with high- and low-yielding mutants. The yields of such diploids corresponded to the lower-yielding ancestor. Yields of homozygous diploids proved not to differ significantly from those of the corresponding haploids when high-yielding mutants selected from three different strains were used. Heterozygous diploids synthesized from these strains gave yields inferior or equal to that of the less productive parent. A heterozygous diploid derived from a completely nonproducing mutant and a high-producing mutant gave a yield similar to that of their common ancestor from which the two parent mutants were selected. The data obtained by Sermonti ( 1959) would therefore suggest that penicillin production by high-yielding mutants is recessive. Furthermore, homozygous diploids are not superior to haploids which indicates that the gene effects related to penicillin production are not additive. However, the possibility exists that diploid strains may be more stable producers upon successive transfer. Fantini and Olive (1960) and Fantini (1962) analyzed the effect of heterokaryosis, heterozygous diploidy followed by somatic segregation and meiosis upon antibiotic formation in Emericellopsis species. Members of this genus all produce the antibiotic synnematin B. Emericellopsis has a sexual cycle as well as an imperfect stage. The process of heterokaryosis proved to be of low incidence among the species tested. Prototroph heterokaryons synthesized from auxotrophic mutants with no antibiotic production yielded titers comparable to the productivity of the original “wild” strain from
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which the auxotrophic mutants were selected. Upon retest yields obtained were significantly lower, which indicates that the complimentary heterokaryons probably underwent some dissociation into their parental auxotrophs and appear to be unstable. Synnematin yields obtained from a heterozygous diploid were higher than the yields of its haploid auxotrophic parent but below the capacity of the original wild strain. Meiotic recombinants showed various degrees of antibiotic yields between zero and those comparable to the wild type culture. The activity increased by a factor of two as compared to the wild strain in one particular cross. Meiotic recombinants appeared to be rather stable upon transfer. Ciegler and Raper ( 1957) studied heterokaryosis in AspergiZZus fmsecaeus which produces citric and gluconic acids. Yields ohtained from heterokaryons were normally intermediate to the yields of the component mutants and lower than those obtained from the “wild type” strain, A high ratio of revertants to the parental types was observed in these studies, indicating a high degree of instability of the heterokaryotic state in this organism.
C. YEAST AND BACTERIAL FERMENTATIONS Yeast strains used in industrial processes are normally more stable in the particular biological property of interest ( ethanol production, cell growth, etc. ) than the antibiotic-producing streptomycetes or fungi. Loss of sexuality and inability to discriminate between opposite mating types of haploid yeasts following successive transfer were described by Lindegren (1949). Yeasts in the haploid phase are extremely variable on subculture, while diploid forms appear more stable. Among bacteria, one of the most troublesome problems concerning strain stability is the continuous emergence of drug resistant strains in pathogenic organisms. This is attributed to selection, mutation, and cell to cell transfer of resistance factors by episomic elements. The discovery of episomic elements and their effects upon variation in bacteria and autonomous cytoplasmic granules in yeasts (Lindegren, 1960) have provided a material basis to explain acquisition or loss of morphoIogical or functional characteristics upon transfer. Very little is known concerning the general occurrence of such autonomous genetic elements among bacteria and yeasts or of their nuclear-cytoplasmic interrelations at the cellular level. Therefore, culture stability discussions of fermentation
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processes involving yeasts or bacteria would be premature and are not discussed in detail. D. MAMMALIAN CELLSIN TISSUECULTURE
In rare cases, outgrowth from native tissue or cells subjected to only one or two transfers past initial isolation may retain some morphological and/or functionaI characteristics inherent to the original tissue. However, in general, chromosome composition, morphology, and specialized functions such as secretion of endocrine substances by cells of glandular origin, infectivity of malignant cells, etc. (Hsu and Moorhead, 1957; Reusser et al., 1962) are either absent or lost after the first transfer. Among the few exceptions are the functional in vitm cell lines of Buonassisi et al. (1962) derived from adrenal and pituitary rat tumors. The adrenal line apparently produces a A4-3-keto steroid, the pituitary line produces A4CTH. Permanently established tissue culture cell lines, derived from different tissues and subcultured over a number of years, appeared morphologically and to some extent biochemically similar (Foley et al., 1962). The chromosomal composition in cells of such permanently established lines usually differs drastically from the normal somatic constitution of the corresponding native tissue and also, to a lesser but significant extent, within individual cells of a given cell line. Such aberrations at the chromosomal level include both chromosome number and morphology of particular chromosomes per cell (Hsu and Moorhead, 1957). The high variability and lack of functioning within such tissue culture cell lines, where gross inhomogeneities are observable at the chromosomal level is self-explanatory if one realizes the effects small genetic changes within a chromosome may have. Potential factors causing such gross genetic variability are discussed by Chu (1962) and Westfall (1962). Some stabilization was obtained by repeated selection of diploid clones among undefined, mixed karyotypes. At the present, this process seems to be the method of choice for prolonged maintenance of actively growing and functioning cell lines of nearly uniform diploid karyotype.
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IV. Prevention or Circumvention of Culture Degeneration A. GENERAL Progeny of any cell is subject to eventual variation. This variation process is a consequence of genetic changes taking place during growth and reproduction and is a phenomenon imposed by nature. Cell strains with a particular genetic configuration can be maintained by suppression or prevention of genetic mechanisms or certain phases thereof during reproduction, or by continuous selection. However, the most efficient methods known to prevent culture degeneration are based on the principle of storing a particular culture in a viable but nonreproducing state. Since the reproduction process is suppressed or greatly minimized, little or no genetic recombination will take place.
B. FREEZING OF LIVING CELLS Storage of living specimens at extremely low temperaturcs is rapidly becoming the method of choice. Upon fast freezing, icc crystals form inside the cell, thus causing cell rupture. Upon slow freezing, however, the intracellular water is removed from the cells and crystallizes intercellularly. Extracellular ice formation will eventually lead to complete removal of all crystallizable water from within the cell. Under such conditions, cell distortion is extremc. However, such highly plasmolyzed cells seem to withstand thesc procedures, and upon thawing will rehydrate and function noimall y (Meryman, 1962). Freezing of mammalian cells as compared to microorganisms was not satisfactory until it was found that acldition of glycerol, and more recently dimethylsulfoxide, would prcvent freezing injuries of such cells. The mechanism of protection by glycerol is not known. However, at a temperature near 37"C., glycerol may replace water in the bonding of hydrophilic amide surfaces of peptide layers. Upon freezing, the hydroxyl groups of glycerol, as opposed to the oxygen position in water, do not vary appreciably in their distance of separation and thus will stabilize the peptide structure at low temperatures (Warner, 1962). The histologica1 distortion of the cellular structure might therefore be less severe and thus minimize the deleterious effects of freezing. Empirical data suggest that a temperature drop of 1°C. per minute is most satisfactory for mammalian cells. Most bacteria, streptomycetes and viruses can be frozen by direct immersion in
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liquid nitrogen. For storage, dry ice at -79°C. seems to perform satisfactorily for short storage periods. Lower temperatures are preferred for longer periods. To this end, specimens are placed into sealed ampules and submerged in liquid nitrogen at temperatures near -200°C. The main advantages of this method are the availability of uniform inoculum for experiments extending over periods of several years, high recovery of viable cells, and virtual absence of degeneration processes during long storage periods.
C . SOIL CULTURES This technique is widely used for the storage of fungi and streptomycetes. Air-dried garden soil is sieved and dispensed into small test tubes. These soil tubes are then autoclaved three times on successive days. A few drops of a mycelial or spore suspension are added per tube and mixed. The tubes are kept at room temperature for a few days to allow limited growth and sporulation and are then stored under refrigeration. This method has the advantage of being inexpensive, requires a minimum of work, and provides a uniform inoculum source for periods from several months to one to two years. Viability upon prolonged storage is usually not as good as by storage at extremely low temperatures.
D. LYOPHILIZATION This method of culture preservation is widely used for the storage of bacteria and yeasts. Spores, cells or mycelial fragments are suspended in a colloidal material such as skim milk, gelatin, or blood serum, placed into sterile vials, and freeze-dried under vacuum. The tubes are sealed under vacuum and stored at room temperature or in the refrigerator. E. MINERALOIL SLANTS This method is mainly used for the preservation of fungi. Fungi are inoculated onto agar slants and subsequently incubated to allow growth to occur, Sterile mineral oil of reasonably high viscosity is then added to the slants to such a level that the entire agar layer within the tube is completely covered. Whenever new inoculum is required, a loopful of spores or mycelium is removed from the agar surface and transferred to appropriate growth medium.
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F. CIRCUMVENTION OF CULTUREDEGENERATION IN CONTINUOUS PROCESSES
In continuous fermentation processes none of the aforementioned methods of culture preservation are useful. The total time such continuous operations can be maintained at a steady output rate of product will be less than the time required for culture degeneration to occur. Such productive periods can be increased by selection of more stable strains. In some processes such as antibiotic fermentations (Reusser, 1961a,b ) elaboration of the product is not directly associated with cell growth but lags behind cell growth. During continuous operation of such processes, culture degeneration was successfully circumvented by separating growth and antibiotic phases spatially in a multistage system. By proper adjustment of the holdup time, cell propagation was confined mainly to the first stage of the multistage system, while antibiotic production took place in subsequent stages. The continuously propagated culture in the growth stage was then replaced before culture degeneration occurred.
V. Discussion and Conclusions Investigations pertaining to the variability and marked instability of microorganisms have shown that microorganisms do have mechanisms for passing genetic information from parent to daughter cell. It is now also realized that beside the conventional sexual mechanism, a number of other mechanisms are instrumental in the transfer and rearrangement of genetic material in microorganisms. Because the generation times of microorganisms are short as compared to higher living forms, the effects of genetic processes among the progeny of a culture manifest themselves quickly, and microorganisms are therefore considered to be markedly unstable. The discovery of nonconventional genetic mechanisms among microorganisms, bypassing the sexual stage, has provided a very useful but as yet little applied tool for the synthesis of strains of economical significance. In a few cases where such mechanisms were applied, as in the synthesis of heterozygous diploids of antibiotic-producing organisms, the results were not very encouraging because of the instability of the diploid phase or low productivity. The main reason for this failure is probably that most commercially important organisms had been improved in their particular strain
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characteristic by empirical means such as treatment with mutagenic agents, or by continuous selection. In both cases variants were selected in which the desired metabolic processes were greatly enhanced. This is usually achieved by diverting a higher proportion of the total energy within the cell to this process rather than to reproduction. As a result, most high-yielding strains of industrial significance have lost growth-vigor as compared to the “wild strain, and have a longer generation time. Directed breeding experiments by conventional as well as unconventional genetic mechanisms necessitate the isolation of single cells or short mycelial fragments. Because of their low growth-vigor, such single isolates frequently fail to develop when plated out for colony development. On the other hand, maintenance of a particular genetic configuration in a strain is also very difficult and poses severe problems. The progeny of any living cell is subjected to gradual changes due to loss, exchange, and new acquisition of genetic material. These processes, imposed by nature, are irreversible and cannot be prevented completely. Exact knowledge of the dominant genetic process causing degeneration within a culture will, in some cases, allow the minimization of such effects, e.g., by suppression of the sexual cycle. However, in general, it is very difficult to assign any single process as causative for culture degeneration since several processes may be active at the same time. Also, the relative magnitude of different processes causing degeneration may vary in different organisms. Since all genetic processes are taking place during reproduction, suppression of the replicating processes is the method of choice to prevent degeneration of a particular culture. To this end, cultures must be stored under conditions where no growth and reproduction can take place but where all the structural and functional characteristics are retained. In practice, this goal is achieved by maintaining the cells in the dehydrated state or frozen at extremely low temperatures.
REFERENCES Alikhanian, S. I. (1962). Adcan. Appl. Microbiol. 4, 1-50. Alikhanian, S. I., Mindlin, S. Z., Goldat, S . U., and Vladimizov, A. V. (1959). Ann. N . Y. Acad. Sci. 81, 914-949. Avery, 0.T., MacLeod, C. M., and McCarty, M. (1944). J . ErptE. Med. 79, 137-158.
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Beadle, G. W. (1947). Zti “Science in Progress,” 5th Series (G. A. Baitsell, ed.), pp. 166-196. Yale Univ. Press, New Haven, Connecticut. Beadle, G. W., and Tatum, E. L. (1945). Am. J. Botany 32, 678-686. Bradley, S. G. (1959). Ann. N . Y. Acud. Sci. 81, 899-905. Bradley, S. G., Anderson, D. L., and Jones, L. A. (1959). Ann. N . Y. Acad. Sci. 81, 811-823. Braendle, D. H., and Szybalski, W. (1959). Ann. N . Y. Acud. Sci. 81, 824-8S3. Brown, G. L. (1962). Brit. Med. Bull. 18, 10-13. Buonassisi, V., Sato, G., and Cohen, A. I. ( 1962). Proc. Natl. Acud. Sci. U.S. 48, 1184-1190. Burton, K. (1962). Brit. Med. Bull. 18, 3-9. Chu, E. H. Y. (1962). Natl. Cancer Inst. Monograph 7, 55-62. Ciegler, A,, and Raper, K. B. (1957). Appl. Microbial. 5, 106110. Doty, P., Marmur, J., Eigner, J., and Schildkraut, C. (1960). Proc. Natl. A d . Sci. U . S. 46, 461-476. Drysdale, R. B., and Peacocke, A. R. ( 1961a). Bid. Reo. Cambridge Phil. SOC. 36, 542-554. Drysdale, R. B., and Peacocke, A. R. (1961b). B i d . Reu. Cumbridge Phil. SOC. 36, 554-560. Fantini, A. A. (1962). Genetics 47, 161-177. Fantini, A. A., and Olive, L. S. (1960). Science 132, 1670. Foley, G. E., Handler, A. H., Adams, R. A. (1962). Natl. Cancer Inst. Monograph 7, 173-203. Guda, H. E., Frajola, W. J., and Lessler, M. A. (1962). Science 137, 607-609. Hayes, W. (1957). J . Gen. Mkrobiol. 16, 97-116. Hopwood, D. A. (1959). Ann. N . Y. Acud. Sci. 81, 887-898. Hsu, T. C., and Moorehead, P. S. (1957). I. Natl. C m c . Inst. 18, 463-471. Jacob, F., and Wollman, E. L. ( 1961). “Sexuality and the Genetics of Bacteria.” Academic Press, New York. Jacob, F., Schaeffer, P., and Wollman, E. L. ( 1960). In “Microbial Genetics,” (W. Hayes and R. C. Clowes, eds.), pp. 67-91. Cambridge Univ. Press, London and New York. Klouwen, H. M., Appelman, A. W. M., and Berendsen, G. W. (1962). Nature 194, 554-555. Lengyel, P., Speyer, J. F., and Ochoa, S. ( 1961). Proc. Natl. Acad. Sci. U . S . 47, 1936-1942. Lengyel, P., Speyer, J. F., Basilio, C., and Ochoa, S. ( 1962). Proc. Natl. Acad. Sci. U.S . 48, 282-284. Lerman, L. S. (1961). J . Mol. Biol. 3, 18-30. Lindegren, C. C. (1949). “The Yeast Cell, Its Genetics and Cytology,” pp. 14-1-14-14. Educational Publishers, Inc., St. Louis, Missouri. Lindegren, C. C. (1960). Develop. Znd. Microbiol. I, 221. Marmur, J., and Doty, P. (1959). Nature 183, 1427-1429. Marmur, J., and Lane, D. (1960). Proc. N d l . Acad. Sci. U.S. 46, 453-461. Meryman, H. T. (1962). Nutl. Cuncer Inst. Monograph 7 , 7-13. blitsuhashi, S., and Harada, K. (1962). Nuture 195, 517-518.
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Nirenberg, M. W., and Mathaei, J. H. ( 1961). Proc. Natl. Acud. Sci. u. s. 47, 1588-1602. Perlman, D., Greenfield, R. B., and O'Brien, E. ( 1 9 5 4 ) . Appl. lA4icrobiol. 2, 199-202. Ponnamperumd, C. A., Lemmon, R. M., and Calvin, M. (1962). Science 137, 605-607. Pontecorvo, G. (1962). Brit. Med. Bull. 18, 81-84. Pontecorvo, G., and Roper, J. A. (1952). 3. Gen. Microbiol. 6, vii. Pontecorvo, G., and Sermonti, G. (1954). 3. Gen. Microbiol. 11, 94. Pontecorvo, G., Roper, J. A., and Forbes, E. (1953). 3. Gen. Microbbl. 8, 198-210. Reusser, F. ( 1961a). Appl. Microbiol. 9, 361-366. Reusser, F. ( 1961b). Appl. Microbiol. 9, 366-370. Reusser, F., Koepsell, H. J., and Savage, G. M. (1961). Appl. iMicrobiol. 9, 342-345. Reusser, F., Smith, C. G., and Smith, C. L. (1962). Proc. SOC. Exptl. Biol. Med. 109, 375-378. Saito, H., and Ikeda, Y. (1959). Ann. N . Y. Acad. Sci. 81, 862-878. Sermonti, G. (1959). Ann. N . Y. Acud. Sci. 81, 950-966. Sermonti, G., and Spada-Sermonti, I. (1959). Ann. N . Y. Acud. Sci. 81, 854861. Sikyta, B., Doskocil, J., and Kasparova, J. (1959). 3. Biochem. Microbiol. Technol. Eng. 1, 379-392. Smith, S. M., and Stocker, B. A. D. (1962). Brit. Med. Bull. 18, 46-51. Sneath, P. H. A. (1962).Brit. Med. Bull. 18, 41-45. Speyer, J. F., Lengyel, P., Basilio, C., and Ochoa, S . (1962a). Proc. Nutl. Acad. Sci. U . S . 48, 282-284. Speyer, J. F., Lengyel, P., Basilio, C., and Ochoa, S. (1962b). Proc. Nutl. Acad. Sci. U . S . 48, 441-448. Sueoka, N. (1961). 3. Mot. Biol. 3, 31-40. Sueoka, N., Marmur, J., and Doty, P. (1959). Nature 183, 1429-1431. Warner, D. T. (1982). Nature 196, 1055-1058. Watanabe, T., and Fnkasawa, T. (1960). Biochem. Biophys. Res. Commun. 3, 660-665. Watanabe, T., and Fukasawa, T. (1961a). 3. Bacteriol. 81, 669-678. Watanabe, T., and Fukasawa, T. (1961b). 3. Bacteriol. 81, 679-683. Watanabe, T., and Fukasawa, T. ( 1 9 6 1 ~ ) .J . Bacteriol. 82, 202-209. Watanabe, T., and Fukasawa, T. (1962). 3. Bacteriol. 83, 727-735. Westfall, B. B. (1962). N d l . Cancer Inst. Monograph 7, 147-157. Whiffen, A. J., and Savage, G . M. (1947). 3. BucterioE. 53, 231-240. Williams, A. M., and McCoy, E. (1953). Appl. Microbiol. 1, 307-313 Wollman, E. L., and Jacob, F. (1955). Compt. Rend. 240, 2449-2451.
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Microbiology of Paint Films RICHARDT. Ross Ruckman Laboratories, Inc., Memphis. Tennessee
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Paint Environment . . . . . . . . . . . . . . . ............... Microflora of Paint Films .......................... Microbiological Degradatio int Binders . . . . . . . . . . The Deteriorative Role of Microorganisms on the Durab
11. 111. 1V. V.
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A. Growth of Bacteria in Preserved and Unpreserved Paint
Films . . . . . . . . . . . . . ............................. B. Growth of Bacteria at t Wood-Paint Interface . . . . VI. Factors Contributing to Paint Film Deterioration and Thei tionship to Bacterial Degradation ......................... A. Type of Wood and the Nature of Its Grain Characteristics B. Chemical and Physical Characteristics of Paint Films . . . . . . C . Impermeability and Stress Paint . . . . . . . . . . . . . . . . . VII. Methods of Microorganism Con A. Selection of Paint Raw Materials . . . . . . . . . . . . . . . B. Formulating Techniques , . . .................. C . Chemical Inhibitors . . . . . . VIII. Effect of Preservation on Paint Durability References . . . . . . . . . . . . . . . . .
225 225 227 227 228 228 229 229 230 23 1 23 1 234
1. Introduction The sciencc of paint technology is dedicated to the manufacture of more decorative and durable paint films. The disfiguring and deteriorative action of microorganisms is conceded to be a major
obstacle in the achievement of that goal. This concession is attested by the yearly investment of many millions of dollars in products used partially or entirely for the control of fungi and bacteria in paints and paint films. It is the purpose of this presentation to discuss the nature of the microbiological problem and the steps which may be taken to achieve adequate microorganism control. The principal cause of the disfigurement or soiling of exterior painted surfaces and some interior paint films is the mycelial and spore cluster growth structures of fungi. A typical exterior house paint formulated with no consideration for the control of microorganisms is heavily attacked by fungi within several months’ exposure. The degree of fungal growth is, of course, influenced by 217
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geographical location, but represents a significant problem in practically all areas. The useful life of a paint film is determined not only by its cleanliness and color retention, but also by its resistance to cracking, blistering, and ultimate peeling from the substrate. While the degree to which bacteria and fungi contribute to film deterioration is debatable, investigations which will be discussed subsequently indicate that certain bacteria may be a major cause of film failure.
II. The Paint Environment To properly classify the microbiological problems of paints and paint films, it seems worthwhile to briefly discuss the types of paints involved and the different growth environments which they provide. Those paints which are most frequently attacked by microorganisms are generally described as consumer or trade sales paints, and include those products used for painting interior and exterior surfaces of houses and other commercial buildings. They include two basic types: (1) water-thinned paints, and ( 2 ) solvent-thinned paints. Water-thinned paints most frequently used are actually emulsions of film-forming resins or oil in water. Solvent-thinned paints employ drying oils or other oleoresinous materials as the film former, and are thinned with mineral spirits, turpentine, or other hydrocarbon solvents. While painting practices are undergoing evolutionary changes, it is safe to say that solvent-thinned paints currently make up the bulk of paint products used on exterior surfaces and that waterthinned paint is the type most frequently used on interior surfaces. Water-thinned paints are subject to attack by microorganisms in the container. Such paints include methylated or ethylated cellulose products, proteins, and numerous synthetic additives which make them ideal environments for the growth of a number of microorganisms. The pH and storage temperatures of water-thinned paints are in a range favorable for microbial growth, The types of spoilage which are encountered in water-thinned emulsion paints depend upon the types of emulsion stabilizers employed, and include putrefaction, fermentation, gassing and viscosity changes. While a number of organisms have been isolated from “spoiled’
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water-thinned paints, the organisms most frequently isolated are the pseudomonads, particularly Pseudomonas aeruginosa. Pseudomoms aeruginosa is easily controlled by a number of paint preservatives however, and the attack of water-thinned paints in the container is an industrial problem only when inadequate levels of these preservatives are incorporated in the paint. The nonaqueous nature of solvent-thinned paints prevents their being altered in the container by microorganisms. Fungi have occasionally been found in solvent-thinned paints but generally in a static condition. The conversion of liquid paints to paint films provides a radical change in both water-thinned paints and solventthinned paints as a substrate for the growth of fungi and bacteria. In addition to the chemical changes of the paints themselves, microbial growth is sharply influenced by the paint substrate and the general environment of the painted surface. The growth of fungi occurs more frequently and is more luxuriant on paint films applied over wooden surfaces than on paint films applied over masonry or metal substrates. Paint films on exterior surfaces represent the greatest number of microbiological problems, yet the severest disfigurement of painted surfaces by fungi occurs on the interior walls and ceilings of breweries, dairies, food processing plants, etc., where conditions of temperature and humidity are ideal for maximum growth.
111. Microflora of Paint Films The most complete surveys of the types of microorganisms associated with paint films were made by Rothwell (1958), who examined exterior solvent-thinned paint films, and by Drescher ( 1958), who examined exterior water-thinned paint films. A list of microorganisms isolated from paint films is presented in Table I and Table 11. While a large number of microorganisms were observed or isolated, most of them were isolated infrequently and were undoubtedly chance inhabitants. At least four fungi and one bacterium were isolated in sufficient numbers however to be considered major contributors to the disfigurement or deterioration of paint films. These include Pulluluria pullulans, Phoma glomerata, Cladosporiuni sp., Altcrnaria sp., and Flavobncterium marinum. \'arious geographical locations tend to support more extensive growth of certain of the fungi, Esamination of exterior paint films
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exposed at west coastal areas indicated that P . glomerata accounts for a major part of the fungal disfigurement in that area. Conversely, this particular fungus was rarely found in other areas of the United TABLE I MICROORGANISMS ISOLATED FROM OIL PAINTFILMSEXPOSED ON TESTFENCES AT SIX DIFFERENT LOCATIONS IN THE UNITED STAT= Fungi
Alternuria dianthicola Aspergillus flavus Botryodiplodia malorum Botrytis cinerea Cladosporium sphaerospermum Cladosporium sp. Cephalosporium carpogenum Fusarium flocciferum Helminthosporium spiciferuni Paecilomyces varioti Penicillium oxalicum Phoma glomerata ( Peyronelluea ) Pullularia pullulans Stemphylium consortiale
Bacteria
Alcaligenes recti Alcaligenes sp. Bacillus cereus Bacillus mycoides Bacillus sphericus Bacillus sp. Flavobacterium inoisibile Fhvobacterium marinum Micrococcus albus Micrococcus candidus Micrococcus ureae Sarcina flava
TABLE I1 hf ICHOOHCANISMS ISOLATED FROM EMULSION PAINT FILMSEXPOSED ON TEST FENCESAT SIX DIFFERENT LOCATIONS I N THE UNITED STATES Fungi
Alternaria dianthicola Aspergillus flavus Cladosporium sphaerospermum Cladosporium sp. Cephalosporium carpogenum Helminthosporium spiciferum Penicillium oxalimm Phoma glomerata ( Peyronellaea ) Pullularia pllulans Stemphylium consortiale Towla nigra Unknown yeasts
Bacteria
Bacillus mycoides Flauobacterium marinum Sarcina flaua
States. CkdospoTium sp. was isolated froin disfigured paint films exposed along the eastern seaboard, with a far greater frequency than from paint films exposed in other geographical areas, Pulltilaria pulluluns was isolated in every geographical location and
MICROBIOLOGY OF PAINT FILMS
221
represents the greatest single offender. It has been repeatedly isolated and reported as the fungus most frequently associated with exterior paint films (Go11 and Coffey, 1948; Klens and Lang, 1956). The microflora of interior paint films in breweries, dairies, canneries, etc., was reported by Krumperman (1958) to include extensive growth of many fungi rarely found on exterior surfaces. Prominent among these are Aspergitbs species and PenicilZium species. His investigation again indicated the frequent occurrence of F . marinum. In general, regardless of the type of exposures or geographical locations, the following observations concerning the growth of fungi were consistent: 1. Fungal growth was most profuse when associated with fissures in the paint films. 2. The presence of chemical preservatives in paint films had a decided effect on the gross appearance of fungi. Whereas unpreserved paint films generally supported mycelial growth forms, the fungal growth on preserved paint films was more often in the form of minute spore clusters, indicative of fungistatic or at least unfavorable growth conditions. 3. The presence of fungi on paint films increases the retention of dirt, particularly when present in the vegetative state. Dirt particles become firmly entrained by the mycelium. The association of fungi and dirt particles is an extremely important one since nutrients, in addition to that provided by the paint itself, are available to the fungi.
IV. Microbiological Degradation of Paint Binders Despite the obvious suitability of paint films as a substrate for microbial growth, little if any consideration was given to the potential deteriorative effects of bacteria and fungi on paint films prior to 1956. Normal painting practice of previously unpainted wooden surfaces call for application of two or three coats of paint. Rarely did those paints applied directly to the wood contain any inhibitor to retard the growth of microorganisms. It was the genera1 belief of the paint chemist that microorganisms observed on painted surfaces were associated with the outermost paint film only. The potential deterioration of paint films was first pointed out by Buckman and Stitt ( 1957). They reported that superior dura-
222
RICHARD T. ROSS
bility of paint films could be obtained when all coats of paint applied to a wooden substrate contained microbial inhibitors. The ability of bacteria and fungi to use solvent-thinned paint films as a part of their nutrition seems almost inevitable when the formulation of such paints are examined. Table I11 shows the composition of a typical solvent-thinned paint. TABLE I11 COMPOSITION OF A TYPICAL EXTERIOR HOUSEPAINTUSEDFROM 1930 TO 1960 Ingredient Titanium dioxide Magnesium silicate Zinc oxide Linseed oil Lead naphthenate Cobalt naphthenate Mineral spirits
Pounds per gallon 2.0 3.5 2.75 4.0 0.1 0.03 1.4
The film-forming constituent in the paint formulation is linseed oil, a naturally occurring mixed triglyceride extracted from flax seed. Other so-called drying oils commonly used in paints include soya oil, safflower oil, and dehydrated castor oil. The existence of the ester linkage in all oil paint films suggests that those organisms capable of utilizing paint films as a part of their nutrition possess lipases. A large number of fungi were found to possess these enzymes in investigations reported by Reese et al. ( 1955). Since oxidative polymerization of drying oils used in paints does not completely saturate the fatty acid moiety, oxidation of the remaining unsaturated carbon linkages represent a second point of attack for microorganisms associated with paint films. Investigations of the author (Ross, 1958) were initiated to determine whether or not those microorganisms which had been isolated most frequently from paint films could utilize liquid and polymerized drying oils as a carbon source, and whether or not such utilization actually contributed to the deterioration of exterior solvent-thinned paint films. These investigations included the substitution of linseed oil as the only carbon source in a completeIy defined growth medium and the measurement of hydrolytic and oxidative activities of the test organisms on liquid oil and polymerized oil films.
223
JLICROBIOLOGY OF PAINT FILMS
Pulluluria pullulans, Aspergillus n i p , Alternariu sp,, Cludosporiurn sp., and F . marinurn all grew abundantly on the substituted media and also on polymerized films when inoculated in a soil extract vehicle. Hydrolytic changes were recorded by measuring the titratable acidity of inoculated oil samples. Results were calculated as per cent COOH by the following formula: Per cent COOH =
ml. of 0.01 N NaOH (0.4502) weight of sample
(100)
Oxidative changes were measured by the Kreis and Schiff tests. The Kreis reaction is a colorimetric test that measures the presence of epihydrinaldehyde or its acetal. Epihydrinaldehyde is believed to be formed as a result of the decomposition of a peroxide of CHEMICAL .4ND PHYSICAL
TABLE IV ALTERATIONS IN LINSEEDO I L
FILMS
INOCULATED WITH VARIOUS MICROORCANIShfS ISOLATED FROM PAINT FILMS
Test organism
Uninoculated Alternaria sp. Aspergillus niger Cladosporium sp. Pullularia pulluluns Flavobacterium marinurn
Carboxyl per cent increased
0.360 0.640 0.250 0.425 0.810
Schiff test
Kreis test
Iodine value
Per cent of water soluble constituents
Trace
Trace
113.73 55.55 62.80 58.24 60.24 53.69
0.12 0.27 0.26 0.26 0.28 0.30
++++ +++ +++ +++ ++ ++ ++ +++ +++ +++
linoleic or other polyethenoid glycerides such as linseed oil. The Schiff reaction measures colorimetrically the presence of aldehydes formed primarily by the oxidation of glycerol. Inoculated linseed oil recovered from both the defined media and from polymerized films showed marked increases in titratable acidity and reacted positively in the Kreis and Schiff tests. Further indications of microbial utilization of linseed oil films were found in the increase in iodine value, volatility, and water solubility and the decrease in both conjugated and nonconjugated constituents of inoculated oils. These results are summarized in Table IV. The preceding results established the ester linkage of linseed oil
224
RICHARD T. ROSS
as the initial point of attack by the test organisms, but did not establish degradation of linseed oil beyond the oxidation of glycerol or partial saturation of the fatty acids released by hydrolysis. The Schiff reaction does not differentiate between glyceraldehyde and phosphoglyceraldehyde. The presence of either phosphoglyceraldehyde or glycerol phosphate would be indicative of further metabolism of linseed oil via the glycolytic series of reactions. Manometric and spectrographic techniques were employed to demonstrate a stoichiometric addition of inorganic phosphate to glycerol formed by the hydrolysis of linseed oil. This addition was established in cultures of P . pullulans and F . marinum in growth media containing an inorganic phosphate source and either glycerol or linseed oil as the sole carbon source. On the basis of these experimental results, it is hypothesized that microbial utilization of linseed oil enters the glycolytic cycle according to the accompanying abbreviated scheme: C;liicose I .L (21: ceraldehyde phosphate
Glycerol phosphate
T
I .1
H,PO,
Glyderol
Pyruvic acid - <
Fatty acid - <
t
Linseed oil
The ultimate utilization of linseed oil by P . pullulans and F . marinum was determined by paper strip chromatographic techniques. Following the entrance of glycerol and fatty acids into the glycolytic cycle, complete oxidation by F . marinum was found to occur with an eventual accumulation of oxalacetic acid when the carbon source was exhausted. The ultimate utilization of linseed oil by P . pullulans was found to be the formation of oxalic acid and acetic and acetoacetic acids.
V. The Deteriorative Role of Microorganisms on the Durability of Paint Films
The most consistent form of film failure of exterior house paints is that generally encompassed by the term “peeling.” Such failure has alternately been referred to as a “wood problem,” “moisture problem,” “film thickness problem,” and occasionally a “paint problem.” Whatever its reference, peeling is a result of adhesion loss,
225
MICROBIOLOGY OF PAINT FILMS
either between paint films or, as more frequently encountered, at the paint-substrate interface, Most paint technologists do not attribute loss of adhesion and subsequent peeling to any single factor but believe it to be influenced by: ( 1 ) type of wood and the nature of its grain characteristics; ( 2 ) chemical and physical characteristics of each paint film applied; ( 3 ) moisture accumulation behind and within paint films; ( 4 ) impermeability and stress created by additional coats of paint. The consistent isolation of bacteria from within paint films and their ability to utilize linseed oil binders as at least a portion of their nutrition suggested that degradation of paint binders by microorganisms at the paint-wood interface provides a more logical explanation for adhesion loss and subsequent peeling than that provided by deterioration from moisture alone. A. GROWTHOF BACTERIA IN PRESERVED AND UNPRESERVED PAINT FILMS Isolation of F . niurinuni from unpainted wood indicated that wood siding is probably the source of that organism in paint films, although the low static population of F . marinum in wood indicates that it is not an ideal substrate. However, when wood having a population of approximately ten organisms per gram was coated with an unpreserved linseed oil film, the population of this bacterium increased a hundredfold after a single year's exterior exposure. Similar studies, including wood panels coated with preserved and unpreserved paints, yielded the anticipated results. The bacterial population at the paint-wood interface of the unpreserved paint was between 100 and 1000 times as great as that found at the paint-wood interface of adequately preserved paints. Results of these studies are summarized in Fig. 1. B.
GRQWTH OF
BACTERIA AT
THE \vQOD-PAINT
INTERFACE
During the course of the investigations by the author and his associates, approximately 50 houses on which paint was failing by peeling to the wood were inspected. Examination of the peeling paint or paint chips removed from the surface adjacent to bare areas revealed that little or none of the original paint primer was adhering to the wood. An inspection of the backs of these chips
226
RICHARD T. ROSS
revealed only powdery remnants of the original prime coat. In some instances where the moisture content of the wood was at that time relatively high, the powder was damp to the touch. In other instances there was no apparent moisture present in the powder.
0
2
4
6 Months
8
10
I
12
FIG. 1. Growth curve of bacteria in preserved and unpreserved puints applied to wood panels; a, number of bacteria in unpreserved paitit film, b, number of bacteria in uncoated wood, c, number of bacteria in preserved paint film.
The remnants of the prime coat were removed and the bacterial population determined. A summary of information and results for 10 of the houses inspected is presented in Table V. The numbers of bacteria isolated from the deteriorated prime coats of paint ranged from approximately 500,000 to over 1,000,000 per gram. The exact numbers are relatively unimportant since the lowest number isolated is sufficiently high to establish the environment as suitable for microbial growth and reproduction at the expense of the paint film. There was no apparent correlation of the percentage of moisture in the wood at the time the peeled paint samples were collected and the numbers of bacteria isolated. This lack of correlation should be viewed with caution, however, since the percentage of moisture in the wood of each of the houses
227
hlICRORIOLOGY OF PAINT FILMS
TABLE V DATAOBTAINED FROMINSPECTING HOUSESON \.I’HICH PAINTFILMSHAD FAILEDBY PEELING TO THE SUBSTRATE
SUMMARY OF
Age of house in years 10 12 6 7 5 9 13
7 2 5
Number of coats of paint
Moisture reading in per cent
Number of bacteria per gram
5
35 14 29 16 18 22 25 13 10 19
1,060,000 650,000 700,000 500,000 620,000 830,000 1,020,000 610,000 660,000 780,000
7 4
5 3 5
7 5 3 5
could have been considerably higher or lower during prior periods of time.
VI. Factors Contributing to Paint Film Deterioration and Their Relationship to Bacterial Degradation
The factors attributed as being the most influential in bringing about loss of adhesion of paint films at the wood substrate were listed previously. As stated there, the common denominator of these factors is moisture and its ultimate effect on paint films. Since moisture is decidedly a determinant for the growth of microorganisms, it is perhaps obvious that the postulation of loss of adhesion due to microorganisms is not in conflict with these factors. However, it seems worthwhile to review them with reference to their correlation with microbiological deterioration at the woodpaint interface.
A. TYPEOF WOODAND THE NATURE OF ITS GRAINCHARACTERISTICS It is well known that paint peeling problems are more frequently associated with houses constructed of southern pine, Douglas fir, and western hemlock than with houses constructed of redwood and western red cedar. The superior paintability of the latter is attributed primarily to the fact that they show less tangential and radial swelling and shrinkage than do southern pine, Douglas fir, and western hemlock. There is no question but that the stress created On paints applied over wood siding is significantly influenced by
258
RlCHARD T. ROSS
swelling and shrinkage of the substrate. However, it does not seem to be simply coincidence that those woods which provide the best surface for paint also provide the poorest environment for the growth of microorganisms. Redwood and western red cedar both contain water-soluble constituents that are toxic to bacteria and fungi. These materials inhibit microbial growth at the paint-wood interface, just as they inhibit the growth of mold within and on the surface of paints applied over them.
B. CHEMICAL AND PHYSICAL CHARACTERISTICS OF PAINTFILMS Laboratory and exposure tests of different formulated primers have indicated that those paints which absorb the least amount of water, and thus undergo the least dimensional change, have the longest durability. The popular usage of primers made to meet the requirements of Federal Specification Paint TT-P-25a, a standard formulation used throughout the paint industry, is based on the inclusion of lead carbonate as a portion of the pigment and the general belief that primers containing lead carbonate have superior adhesion. The most accepted explanation for this superior adhesion is that provided by Browne (1955), who found that linseed oil paints containing lead carbonate absorbed less water and showed the least tendency for linear changes of the film due to swelling. The role of bacteria in bringing about loss of adhesion fits nicely with the theory and in fact offers a more logical explanation of adhesion loss than that provided by swelling alone. Since growth of bacteria within a paint film is dependent to the extent that water is absorbed and retained in the film, any material which would decrease water absorption and swelling of paint films would in effect reduce bacterial activity. It has been demonstrated in exposures of emulsion paints over primers made according to Federal Specification Paint TT-P-25~1 that increased water resistance in itself is not sufficient to prevent the growth of microorganisms. Adequate amounts of effective preservatives are needed in the primer to prevent the progressive deterioration of that primer as the result of microbial growth.
C. IMPERMEABILITY AND STRESSCREATED BY MULTIPLE COATSOF PAINT The contribution to peeling provided by additional coats of paint i, an obvious one. The thicker the paint film, the less chance there
MICROBIOLOGY OF PAINT FILMS
229
is for moisture vapor to escape, with a subsequent increase in pressure exerted against the multiple layers of paint. The weight and stress resulting from additional paint films complement this pressure to bring about a rupture in the film and subsequent peeling. Relating microbiological deterioration of the prime coat to additional coats of paint is not too difficult. The impermeability created by additional coats of paint provides a more suitable environment for their growth. In addition to the entrapped water at the interface, the thicker paint film buffers the temperature at the interface so that growth and reproduction of microorganisms can proceed at a more constant rate than would be possible in an environment of variable temperature and moisture content. The result of stress created by thicker paint films seems even more reasonable if at the same time a slow, progressive degradation of the primer is occurring.
VII. Methods of Microorganism Control in the Paint Industry
Having discussed the disfiguring and deteriorative roles of microorganisms on paints and paint films, it seems worthwhile to examine the methods employed to provide satisfactory microorganism control in the formulation and manufacture of paints. The total resistance of paint films to the growth of microorganisms depends not only on the inclusion of chemical antimicrobial agents, but on the types of raw materials employed and their volumetric ratio to one another as well. A. SELECTION OF PAINTRAW MATERIALS It was previously pointed out that paint binders are the filmforming ingredients of paints and consist of both naturally occurring drying oils and resins and synthetic polymers. Oil and alkyd binders are gellike structures which when wetted absorb varying amounts of moisture. Since the amount of moisture which is retained within a paint film is a critical determinant of the degree to which it is attacked by microorganisms, the greater the water resistance of paint biinders, the less favorable is the environment for microbial growth. The higher viscosity oils and alkyd resins generally provide more mold and bacterial-resistant paint films because of their increased water resistance. Synthetic vinyl-type
230
RICHARD T. ROSS
paint binders, such as polyvinyl acetate copolymers and acrylic resins, provide increased resistance to microorganisms not only because of their increased water resistance, but because of their chemical structure as well. However, in the preparation of emulsion paints using this type of binder, the inclusion of emulsifiers, surfactants, and other additives counteracts the improved resistance of the vinyl resins. The total amount of binder used in relation to the pigment of a paint film also influences the paint film’s over-all resistance to microorganisms. Any binder not directly in contact with the pigment is more easily hydrated and more readily accessible to microbial attack. The choice of nonfungicidal pigments and the pigment volume concentration affect the mold and bacterial resistance of a paint film. Lead pigments tend to decrease the water absorptivity of a paint film. Diatomaceous silica and other flatting pigments tend to increase water contact with the binder.
B. FORMULATING TECHNIQUES In paint terminology “pigment volume concentration” refers to the ratio of the volume of pigment to the sum of the volume of nonvoIatile binder and the voIume of pigment in a given paint. The “critical pigment volume concentration” refers to that pigment volume concentration at which there is just enough binder in the dry film to fill the voids between the pigment particles. Increasing the volume of pigment in a given paint closer to the critical pigment volume concentration reduces the amount of “free binder” and thus reduces the amount of moisture that can be trapped behind or held within a paint film. Other than the use of chemical preservatives, the most frequently used technique of paint chemists to formulate paints which will not become disfigured by fungi is to choose hiding pigments which will chalk or slough off surface pigment layers after a short period of exterior exposure. “Chalking” or sloughing-off of unbound pigment at the surface of paint films obviously have little effect on the bacterial population concentrated at the paint-substrate interface. It does, however, remove much of the fungal growth on the paint film surface by physical removal of spores and dirt particles. It is extremely important that “chalking” not be excessive such that chalk wash or rapid errosion results.
MICROBIOLOGY OF PAINT FILMS
231
C. CHEMICAL INHIBITORS
The most effective and frequently employed method of controlling microorganisms in paint films is by the use of chemical inhibitors. With the exception of two pigments, barium metaborate and zinc oxide, the preservatives most frequently used have been phenylmercury compounds, copper compounds, and chlorinated phenolic compounds. For adequate control, barium metaborate and zinc oxide must be used at levels exceeding 1 pound per gallon of paint. The amount used will depend upon the microbial-resistant properties otherwise formulated into the paint by selection of raw materials. The same holds true for phenylmercury compounds and copper compounds which are used at concentrations of 0.05 to 2%, based on the total weight of the paint. The choice of paint preservatives is dictated by a number of considerations. They must not only be effective microbial inhibitors, but must be limited in solubility so that protection is afforded for long exposure periods. Regardless of the effectiveness of a given preservative, it must be compatible with other components of the paint, produce no color changes, or interfere with the drying or other performance properties of the paint. Preservatives must have a low order of toxicity for mammals at their use concentrations. Lastly, but of significant importance to the paint manufacture, a preservative must be of a cost that will not increase excessively the over-all cost of the finished product.
VIII. Effect of Preservation on Paint Durability As a part of an extensive exposure study of paint films to measure their resistance to the growth of mold, exposures made of a single linseed oil topcoat applied over a preserved and unpreserved linseed oil primer coat were made. This study was patterned after that reported by Buckman and Stitt (1957) referred to earlier in this presentation. The paints were applied on 6 inches by 36 inches southern pine panels and exposed both to the north and to the south on paint test fences in Memphis, Tennessee. The microbial inhibitor used in the preserved primer coat was barium metaborate pigment. No essential differences in the film integrity of the two paint systems was observed during the first two years of exposure. However, during the third year cracks in the paint system with the unpreserved primer were observable. These cracks penetrated
232 RICHARD T. ROSS
M
w
LO
Fic. 2.
(For legend see p. -333.)
MICROBIOLOGY OF PAINT F I L M S
233
through both topcoat and primer coat. Failure by additional cracking and peeling was progressive during the third and fourth years of exposure. The appearance of the two paint systems at the end of 49 months’ exposure is shown in Fig. 2. While bacteria were isolated from the paint-wood interface of both paint systems, the population was significantly higher in the unpreserved primer indicating a correlation between bacterial population and paint durability.
REFERENCES Browne, F. L. (1955). Forest Prod. J. 5 ( 3 ) , 192-200. Buckmsn, S. J., and Stitt, W. D. (1957). Am. Paint 1. 41(39), 80-118. Drescher, R. F. (1958). A m . Paint J. 42(27), 80-102. Coll, M., and Coffey, C. (1948). Paint Oil Chem. Rev. 111(l6), 14, 16, 17. 30. Klens, P. F., and Lang, J. F. (1958). J. Oil Colour Chemists’ Assoc. 39( 12), 887-899. Krumperman, P. H. (1958). Am. Paint J. 42( 38), 72-84. Reese, E. T., Cravetz, H., and Mnndels, G . R. (1955). Faslowia 4, 409-421. Ross, R. T. (1958). Ofic. Dig. Federation Paint Varnish Prod. Clubs 30 (399), 368-376. Rothwell, F. M. (1958). Ofic. Dig. Federation Paint Varnish Prod. Clubs 30( 399), 377-391.
.____
FIG.2. Superior film durability of a preserved oil paint applied over a preserved primer a s compared with the same paint applied over an unpreserved primer. The right half of each panel was painted with two coats of an oil paint preserved with the preservative pigment barium metaborate. The same paint was used as the topcoat on the left half of each panel and was applied over a primer similarly formulated, but with magnesium silicate replacing barium metaborate. Thus, both primer and topcoat on the right half were preserved; whereas, only the topcoat on the left half was preserved. The panels are southern pine. The paints had been exposed for 49 months at the time they were photographed.
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The Actinomycetes and Their Antibiotics SELMANA. WAKSMAN Institute of Microbiology, Rutgers, The State Unifiersity, New Brunswick, New Jersey
.
I. Introduction . . . . ...................................... 11. The Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Occurrence of Actinomycetes in Nature . . . .... B. Pathogenic Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Structure and Composition of Cells of Actinomvcetes . . . . . . D. Genetics of Actinomycetes . . . .. .... ............ E. Characterization and Classification of Ac etes . . . . . . . F. Streptomyces Groups and Species .. . . . . . . . . . . . . .. . . . . . G . Thermophilic Actinomycetes . . . . . ..... H. New Species of Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . I. Production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .................. 1. Production of Pigments . . . . . . K. Transformation of Steroids . . . . . . . . L. Production of Actinophages . . . . . . . . . . , . ...... M. Problems of Growth and Nutrition of Act N. Effect of Nutrition on Biogenesis of Antibiotics . . . . . . . . . . . . 0. Physiology of Autotrophic Actinomycetes 111. The Antibiotics . .. .. .. . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . .. . . A. New Antibiotics . . . . . . . . . . . .. .. . . . . . . . . . . . . . .. . . .... . B. New Information on Old Antibiotics . C. Antitumor Substances , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Antiviral Agents ........... IV. Mode of Action and A. Mode of Action B. Utilization of Antibiotics in Clinical Medicine C. Effect of Antibiotics upon Animal Diseases . , . . . . . . . . . . . D. Antibiotics and Animal Growth . . . . . . . , . . . . . . . . . . . . . . . . . E. Effect of Antibiotics upon Plant Growth and Plant Diseases . . F. Antibiotics in Food Preservation . . . . . . . . . . . . . . . . . . . . . . . References . . . ......
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235 236 236 239 240 241 242 245 247 249 252 255 255 255 256 257 258 259 259 264 27 1 280 280 280 286 292 292 293 293 293
1. Introduction The literature on the actinomycetes and their antibiotics has been accumulating at such a rapid pace and is of such important theoretical and practical significance, that the need for frequent reviews of this subject has become of paramount importance. The literature prior to 1960 has been largely covered in three volumes published 235
236
SELSIAN A. WAKShlAN
by Waksman (1959, 1961a) and by Waksman and Lechevalier (1962) under the title of “The Actinomycetes and Their Antibiotics,’’ as well as in a number of other publications in various countries and in different languages. It is sufficient to mention the following: (1) A treatise by Korzybski and Kurylowicz (1961), largely devoted to the antibiotics of actinomycetes. ( I t is a pity that the authors found it desirable to include, among the antibiotics, also antimicrobial substances produced by higher plants and animals. ) ( 2 ) A comprehensive treatise (third edition) by Shemiakin et al. (1961) on the chemistry of antibiotics. (Unfortunately, in this work all products of living systems, including higher plants and animals, possessing antimicrobial properties were placed among the antibiotics. ) ( 3 ) A two-volume treatise in Japanese by Sumiki ( 1961) on the subject of antibiotics. (4)A volume by M. W. Miller (1961),dealing largely with the chemistry of microbial metabolites, of which the antibiotics form a constituent part. (5) A treatise by Brunner and Machek (1962) on antibiotics. ( 6 ) A report by Krassilnikov (1961) on the application of antibiotics to plant growth. ( 7 ) A review by Humphrey and Deindoerfer (1!362) covering the antibiotic literature for 1960. The following review will be limited largely to the publications that have appeared during the latter part of 1960 to the latter part of 1962. For the sake of completeness, however, a few papers that appeared prior to these dates but were overlooked in this author’s previous three volumes will also be cited. II. The Actinomycetes A. THE OCCURRENCE OF ACTINOMYCETES IN NATURE The extensive screening programs that have been initiated as a result of the discovery that actinomycetes form one of the most important groups of antibiotic-producing organisms continue at an unabated pace. Relatively little attention is being paid in these studies to the abundance of actinomycetes in various soils, in their
ACTINOXIYCETES AND THEIR ANTIBIOTICS
237
relation to type and treatment, or to plant growth. The same may be said of the distribution of actinomycetes in water basins, composts, and other natural substrates. Marton (1962) demonstrated the existence of thirty-two types of streptomycetes in a profile of chernozemlike riverside soil. In the subhorizons of the A- and A/C-horizons the existence of four different communities of actinomycetes were found. A fifth community was isolated from the topmost layer of the C-horizon. There was increasing sensitivity of the organisms isolated from the deeper subhorizons towards higher temperature values. This suggested a close ecophysiological correlation between the microorganisms and the space required. The survival of Streptomyces albus in mixed artificial and natural populations and the general ecology of streptomycetes were studied by Rehm (1958, 1959, 1961). The occurrence of actinomycetes in the rhizosphere of various plants was examined for legumes by M. E. Brown (1961), for wheat by Andriiuk (1960), for alder by Uemura (1961), for corn by Horst and Herr (1962), and for other plants by Jagnow (1961) and Hirte (1961). Thirty-two cultures of actinomycetes were isolated by Uemura from nodules of alders and certain other leguminous plants and were divided into three groups: ( 1) those forming a yellowish-brown to brownish-black pigment in organic media; these belonged largely to the genus Streptomyces, although some could possibly be considered as Nocurdia species; ( 2 ) those forming a purplish soluble pigment ( phenazine derivative resembling iodinin) in nitrate sucrose solution and not liquefying gelatin; ( 3 ) those forming orange-colored growth on various media (none on nitrate sucrose agar) with white powdery aerial mycelium. By the use of buried-slide technique inoculated with spores of plant pathogenic Helminthosporium, Rangaswami and Ethiraj (1962) were able to demonstrate the production of an antibiotic substance by a Streptomyces in unamended soil. Characteristic malformations of germ tubes and hyphae were identical with those produced in sterilized soil in which the streptomycete was grown and in the cell-free culture filtrate of the antagonist grown in dilute soil extract. According to Lindenfelser and Pridham ( 1961), Streptomyces zdridochromogenes is able to maintain itself in a complex competi-
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tive environment due to prolific sporulation, a broad carbon-utilization pattern, and the ability to form antibiotics, thus tending to explain the wide distribution of this organism and the relative ease with which it can be isolated. The idea that the ability to form antibiotics plays an important role in the survival of different organisms in nature was questioned, however, by Waksman ( 1961b). Stout (1962) emphasized that sensitivity to antibiotics is not a major factor in the discontinuous distribution of free-living bacteria. The principal groups of bacteria commonly absent from soil are no more sensitive to antibiotics than the closely related common soil forms. The most sensitive group are the aerobic sporeforming bacilli which are among the most widespread and numerous soil organisms. The wide seasonal fluctuations, in kinds and in numbers, of yeasts on leaves were found (diMenna, 1962,) to be due rather to nutritional and physical factors than to antibiotic ones. Safferman and Morris (1962) developed methods for the screening of antialgal substances. The extracellular products of 545 actinomycete isolates showed a wide distribution of agents having algicidal activities, 20.4% possessing selective antagonistic effects against particular groups. A comprehensive screening program for actinomycetes capable of producing antibiotics having nematocidal properties against the root knot nematodes was carried out by Mori (1961). Other screening methods have been examined by Vhlyi-Nagy et al. (1961a), Hernhdi et al. (1961), Shibata (1961), Kalyuzhnaya et al. (1962), Grossbard (1962), I. Szabo and Marton (1962b), and Griffin (1962). The occurrence of A. levoris in soil was studied by Marton and Szabo ( 1962). Actinomycetes in sea water were investigated by Stoyanovski et at. (196l), in marine sediments by Demny et al. (1961), and in highmoor bogs by Klosowska and Pawlowska (1960). Lingappa and Lockwood (1961) proposed a chitin medium for the growth and characterization of actinomycetes. The effect of various carbon sources in medium for the isolation of Streptomyces from soil was studied by Porter and Wilhelm (1961). The problem of continuous culture of actinomycetes, especially from the point of view of antibiotic production has been receiving much attention (Bekhtereva and Kolesnikova, 1959; Sikyta et al., 1961; Reusser, 1961).
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B. PATHOGENIC ACTINOMYCETES Only a few investigations dealing with pathogens need be mentioned here, one dealing with the formation of actinomycosis in carnivorous animals (Prevot et al., 1961), and one dealing with the pathogenicity of Actinoplanes philippinensis ( Torre and Baroni, 1961 ) . Christie and Porteous ( 1960) developed a stirred-culture technique for obtaining uniformly small colonies of Actinomyces ismelii. This organism usually grows in the form of discrete colonies in static liquid media. The microcolonies obtained by the new technique sedimented rapidly and could be kept in even suspension by bubbling nitrogen through the suspension. These gave uniform inocula from a graduated Pasteur pipette, and showed the characteristic growth of A. ZsraeZii in static liquid media. Pine et al. (1960) reported that Actinomyces bovis fails to form a true mycelium in vitro; it has a lesser tendency than A . israelii and A . naeslundii to produce mycelial growth in lesions in inoculated animals. Actinoniyces bovis produces two types of colonies when first isolated from animals, one a smooth, glistening, transparent microcolony and another raised, opaque, and rough; it is a catalasenegative anaerobe, does not hydrolyze gelatin or casein; in contrast to the others, it hydrolyzes starch rapidly and completely; it forms acid but no gas from starch; it does not form acid or gas from xylose, raffinose, and mannitol, nor does it reduce nitrate to nitrite. A further study of the cultural and physiological propertics of anaerobic actinomycetes was made by Kalakoutskii ( 1961). Grasser ( 1962) recognized among the microaerophilic actinomycetes isolated from udder actinomycosis of the pig two clearly separated basic groups, one almost identical with A. imaelii from human actinomycoses, with their long-threaded, branched mycelium and rough shapes of the colonies, and another called Actinomyces suis. Buchanan and Pine (1962) isolated an anaerobic actinomycete from the lachrymal duct of a case of human lachrymal canaliculitis. The organism was found to be similar to Actinomyces in its production of true branching mycelial elements, catalase negativity, pathogenicity for experimental animals, and amino acid cell-wall composition. On the other hand, it was similar to the propionic acid bacteria in its fermentation of glucose to propionate, acetate,
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and C02, formation of a dull orange color, and thc presence of diaminopimelic acid in its cell wall. The organism was named A. propionicus. The causation of endocarditis by A. bovis was discussed by \%'alters et al. (1962). The production of diseases in cattle by Nocardia asteroides was studied by Pier ct al. (1961). Nocardin cultures were examined in detail by Arai and Kuroda (1959), Edwards et aZ. ( 1961, 1962), Adams and McClung ( 1962a), and Gordon and Mihm (1962); hr. astcroides and N. brasiliensis were identified by Georg et al. (1961); developmental cycles of Nocardia were studied by Adams and McClung (1962b). Nolof and Hirsch (1962) suggested the name N . lzydrocarbonoxydans for a culture capable of utilizing gaseous aliphatic hydrocarbons ( C6-CI4) for growth; it differed from other oligocarbophilic actinomycetes in its colony form, its ability to oxidize hexane, heptane, and octane, and its inability to grow on glycerol slants. The oxidation of alkanes by N. petroleophilu was studied by Seeler (1962). Lessel (1962) proposed to change thc name N . caviae to N. otitidis-cauiarum. The oral nocardias (A7. rtentocariosus) were studied by Roth and Thurn (1962). Oliver et al. (1962) isolated an antibiotic, chelocardin, from a culture of Nocnrdia sulfuria. See also Rodrignez-Villanueva ( 19601. The oxidation of steroids by N . italica were reported by Spalla ct al. (1961). The importance of differentiation between Nocardin and Actinomyces was brought out by Intile and Richert ( 1962), who demonstrated that a patient with cervicofacial actinomycosis caused by an Actinomyces, complicated by purulent meningitis, was successfully treated with penicillin. Nocardiosis, on the other hand, is best treated with sulfonamides. AND COMPOSITION OF CELLS OF ACTINOMYCETES C. STRUCTURE Cummins (1962a,b) made a detailed study of the chemical composition of the cell walls of actinomycetes and related organisms. They were found to contain glucosamine and muramic acid, and occasionaIIy galactosamine. The aerobic forms allow the differentiation of two types, called tentatively the Streptomyces and the Nocardia. The cell walls of the Nocardia type contain arabinose, galactose, alanine, glutamic acid, and DL-diaminopimelic acid; those of the Streptomyces type often do not contain any sugar and yield, upon hydrolysis, alanine, glutamic acid, glycine, and LL-
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diaminopimelic acid, the m-form of the latter being occasionally present. The microaerophilic or anaerobic actinomycetes of human origin contain galactose, alanine, glutamic acid, and lysine; those of the bovine type contain rhamnose, fucose, 6-desoxyl-~-talose, alanine, glutamic acid, aspartic acid, and lysine. The chemical nature of the cell wall skeleton does not change with age of cultures and composition of media. By the use of phase-contact microscopy of living material, the primary mycelium of 3 Streptomyces was shown capable of forming apparent anastomoses (G.H.G. Davis, 1960). However, no enlarged structures that would be interpreted as “initial cells” were detected. The large “sporelike bodies” described by various investigators were believed to represent protoplasts. The surface structure of the Streptomyces spore was studied by Niida and Hamamoto (1961). The presence of volutin in cells of actinomycetes was demonstrated by F’rokofieva-Belgovskaya and Kaz ( 1960). The use of dyes for staining antibiotic-forming cultures was studied by Sahay ( 1960) and Sugano et al. (1960); the mitotic activity of actinomycetes under the influence of streptomycin and colchicine by Rosini (1958); the internal structure of Streptomyces by Edwards et al. ( 1962). The surface structure of Streptomyces coelicolor was studied by the use of the electron microscope by Glauert and Hopwood (1960) and Hopwood and Glauert (1960, 1961); the cytology of actinomycetes by Arai et al. (1960); the structure of the spiny spores of Streptomyces by Arai and Kuroda (1962). Shamina (1962) studied the colony and hyphal structure of Streptomyces violaceus. D. GENETICS OF ACTINOMYCETES Bradley and Anderson ( 1960) demonstrated recombination between the derivatives of strains of different origin and between derivatives of a single wild-type strain of Streptomyces uiolaceoruber. Vegetative hyphae produced about twice as many prototrophs as aerial hyphae per plating unit of mixed growth, thus demonstrating that sporogenesis was not a prerequisite for recombination (see also Horvith, 1962). Alikhanian et al. (1960) analyzed the phenomenon of transduction among actinomycetes. Alikhanian and Borisova ( 1961) obtained biochemical mutants of S. aureofaciens on treatment with ultraviolet irradiation or with ethyleneimine; biochemical mutants with
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either the same or with different amino acid requirements resulted in prototroph formation. A mutant of S. griseus, unable to synthesize streptomycin, was obtained by treating the spores of a highly active strain with ultraviolet light ( Alikhanian and Teterjatnik, 1962) . The growth of the mycelium of the mutant in a 24-hour culture fluid of the active strain, not containing streptomycin, resulted in the appearance of the antibiotic in four days; it was suggested that the active strain produced a precursor for streptomycin synthesis, named substance “S,” which was used by the mutant for building the streptomycin molecule. The hereditary structure of the mutant controlling the first stage of streptomycin synthesis was believed to be inactivated. The phenomena of induced mutagenesis was reviewed in detail by Alikhanian (1962,)The genetic recombination of Streptoniyces was studied further by H. Saito (1960) and by Hopwood (1961).The use of CoBDfor the irradiation of antibiotic-producing organisms was discussed by Schonwalder ( 1961) . A comparative study of streptomycin-producing and nonproducing strains of S. griseus was made by G. Szabo et al. (1960, 1961); of active and inactive strains of oxytetracyclineproducers by Nyiri (1962).
E. CHARACTEREATION AND CLASSIFICATION OF ACTINOMYCETES Hesseltine (1960) proposed a new evolutionary scheme for actinomycetes and related organisms, by analyzing their morphological and physiological relationships, beginning with a sporangial ancestor with motile spores, evolving to reduced spore production, and finally to nonmotile spores. The actinomycetes were placed in a separate phylogenetic line, believed to be more closely related to the fungi than to the bacteria. These conclusions were based on the following properties: ( a ) spores and spore germination of actinomycetes resemble those of fungi; ( b ) vegetative growth in surface and submerged culture is funguslike; ( c ) mycological characteristics used in identification of the organisms are considered as most fundamental; ( d ) methods required for maintaining vigorous and stable cultures are similar to those used for fungi; and ( e ) colonies of actinomycetes and their mode of growth are more funguslike than bacterial in nature. Lechevalier et al. (196lb) pointed out that the difference between the families Actinomycetaceae and Streptomycetaceae cannot possibly be based on fragmentation, since variations in this
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respect can be observed between isolates of the same strains of organisms now designated as Nocardia and Streptomyces. A new genus, Micropolyspora, which fragments like the Actinomycetaceae and sporulates like the Streptomycetaceae, by forming chains of conidia on aerial hyphae, was described. The genus also forms chains of conidia on the substrate mycelium, located in and on agar media. It was suggested that the family Streptomycetaceae be dropped and that the family Actinomycetaceae be enlarged to include the genera Actinomyces, Micromonospara, Thermoactinomyces, Waksmania, Micropolyspora, Nocardia, and Streptomyces. Micropolyspora brevicatenu was said to represent a novel morphological type easily distinguishable from the previously described forms. The thermophilic organism described by Henssen as Pseudonocardia thermophila was considered to be a facultative thermophilic Nocardia. Kalakutski and Krassilnikov ( 1960) examined the phenomenon of sclerotia formation by species of Streptomyces, and could not distinguish any important differences between these cultures and the typical Streptomyces. The formation of sclerotia was not considered as a sufficient property for the creation of new genera. In spite of this they proceeded to create a new species which they designated as Chainia zjiolaceus. Another species of Chainia, C. ocfiraceu, was described by Kusnezov ( 1962). Jones and Bradley (1961b) examined the relationship and justification of the genera Streptozjerticillium and Jensenia to the other actinomycetes. Krassilnikov ( 1962) created a new genus, Actinopycnidium, belonging to the Actinomyceteae. It is characterized by the formation of fruiting bodies of the pycnide type; inside of these bodies there are formed pycnidospores of spheroid, or oval and oblong shapes. The organisms produce typical spores on the branches of the aerial mycelium. Three species belonging to this genus were described: Actinopycnidium globosa, A. coeruleum, and A. elongatum. A comparative summary of criteria used in characterization of actinomycetes by an international subcommittee (Kuster, 1961) brought out the fact that the morphology of the sporophores and the shape of the spores are significant and constant characteristics, so that they can be used as taxonomically useful criteria; the melanin reaction is an unequivocal characteristic and could be used for classification purposes; the colors of the aerial as well
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as that of the vegetative mycelium are more complicated. Gottlieb (1961) also analyzed the criteria used in the characterization of actinomycetes. Various methods were proposed by digerent investigators, ranging from the quantitative procedures of Gilardi et nl. (1960), Hill and Silvestri (1962), and Moller (1962), to the use of paper chromatography by Koreniako et al. (1960a). Dietz and Mathews (1962) studied taxonomy by carbon replication. Arai et al. (1962b) suggested the ultilization of antibody technique for serological identification; Solovieva and Delova (1961) used a precipitation in agar reaction for this purpose. The suggestion that the property of antibiotic formation be used in the classification of actinomycetes, with particular attention to the genus Streptomyces, proposed by Krassilnikov, could not be accepted by Baldacci (1961). The difficulty lies in the fact that a study of antibiotic formation is within the domain of the chemist and not the taxonomist, who is primarily a morphologist. Since patent protection requires the applicant to give species identification, the tendency is to create continuously “new species.” Unfortunately, this does not take into account the Bacteriological Code of Nomenclature. Baldacci suggested that patent protection be given at levels below that of the species. The effect of metabolites upon pigmentation of actinomycetes was studied by Krassilnikov and Egorova (1960). Colorless and pigmented species of Streptomyces in relation to antibiotic production were compared by Sahay (1960). Krassilnikov et al. (1961) analyzed the external features which are important in the taxonomy of actinomycetes. Nishimura et al. (1960) developed further the Mayama system of classification of actinomycetes. Hiitter (1961), Pridham and Lyons (1961), Lyons and Pridham (1962b) considered Streptomyces albus as the type species of the genus Streptomgces and suggested its proper characterization. Comparative studies of the genera Actinobacterium, Actinomyces, and Nocardia, and their relation to the true bacteria were made by Bedrynska-Dobek (1960). The family Actinoplanaceae was studied in detail by Taig et al. (1962). Bradley et at?. (1961) analyzed the phylogenetic relations of actinomycetes based upon actinophage susceptibility. Twentyfive strains representing six genera of actinomycetales and thirtyseven actinophages were used. Five of eight actinophages isolated originally on species of Nocardiu were able to attack strepto-
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mycetes; seven of eleven phages isolated against strains of Streptomyces lysed species of Nocardia. The actinomycetes were arranged on the basis of phage susceptibility. Strains of Streptomyces griseus formed a continuous series, whereas organisms designated as Nocardia were apparently not connected with one another. The conclusion was reached that although actinophages could not be used in separating species, they should prove useful in the identification of genera, group-species, and strains. Electron microscopic studies of the morphology of actinophage-producing strains were made by Rautenstein and Mach (1961). Pridham ( 1962) applied the species-subspecies concept to streptomycetes and streptoverticillia.
F. Streptomyces GROUPS AND SPECIES Various species of Streptomyces, notably the streptomycin- and tetracycline-producing organisms, continue to receive considerable attention from the point of view of their morphology, cytology, genetics, physiology, and production of antibiotics (G. Szabo et al., 1960; Dubinin and Shavel'zon, 1960; Nikitina et al., 1960b). Mindlin et al. (1961) isolated a new strain of Streptomyces rimosus which was characterized by a lower level of foaming and higher antibiotic activity as compared to the other active strains of this organism; it combined properties of different strains of S. rimosus. Alikhanian et al. (1961) produced inactive mutants of S. rimosus from the highly active form of this organism; when two such mutants were grown together in a corn extract medium with the usual mineral salts, an active antibiotic, apparently hydroxytetracycline, was obtained; this did not occur in cultures of individually grown mutant colonies. The variability of S. rimosus was examined further by Nyiri (1961). Rudaya et al. (1961) divided 120 strains of verticillate actinomycetes into five species: Streptomyces netropsis (most common), S . hachijoensis, S. circulatus, S . biverticillatus, and S. rubrireticuli (rare). A new variety S. circulatus var. roseus has been described. Verticillate branching of the aerial hyphae was considered as a stable character, under definite conditions of cultivation. However, primary or secondary disposition of the verticils are not characteristic of the cultures, since both modes of disposition )nay occur in the same culture (see also Konev, 1962). Hiitter
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( 1962b) classified the whorl-forming streptomycetes into w r ticillate, pseudoverticillate, and umbellate. The speciation of various groups of Streptomyces received much consideration. This is true of the S. griseus group (Nikitina et al., 1960b; Szabo et al., 1960; Lyons and Pridham, 1962a); the blue-spored group (Trejo, 1961; Hiitter, 1962a); the S. fluorescens group (Koreniako et al., 196Oc; Menshikov and Denisova, 1962); the brown to olive-green group (Kutchayeva et al., 1960); and the nonverticillate species ( 1. Szabo and Marton, 1962b). Krassilnikov and Ayre (1960) examined 11 cultures of actinomycetes belonging to a white-blue group; two subgroups were recognized: Streptomyces alhoc!yaneus and S. cyanoalbus. Artamonova and Krassilnikov (1960) divided the Actinomyces tiiolaceus group into seven species and two subspecies: A. uiolaceus, A. violarus, A. janthinus, A. violans, A. violatus, A. flavoviolaceus, A. violaceochromogenes, A. violaceus vicinus, A. violaceus confinus. The Actinomyces leuoris of Krassilnikov appears to be identical with Streptomyces coelicolor (Koreniako et al., 1960b). The Actinom yces globisporus group was subdivided by Krassilnikov et al. (1960a) into two subgroups, largely on the basis of antibiotic production, namely, A. globisporus and A. globisporus roseus; the first produces a lemon-yellow fluorescent pigment, forms an actinomycin type of antibiotic, and comprises Streptomyces fluorescens, S . citreofluorescens, and S. chrysomallus ( Koreniako et al., 196Oa). Kutchayeva et al. ( 1962) divided the Streptomyces lavendulac group into two subgroups: spiral spore bearers, with Actinomyccs pscudolavendulm, A. cyringae, S . venezuelae, and A. pseudoveneztrelae; nonspiral spore bearers, with seven types characteristic of the original S. lavendulae. New species were added: A. lavendulae avireus, A. latiendulae grasserius, A. virocidus, A. lavendocolor, and A. lavendofoliae. The red group of actinomycetes, possessing a mutating structure of sporiferous hyphae, ~ 7 a sexamined by Semenova (1962). Krassilnikov and Vinogradova ( 1960) divided fifteen ciiltures belonging to the group usually considered as S. chromogenes, 011 the basis of their ability to produce antibiotics, into A. robeus, A. robefuscus, A. robustrus, and A. pheochromogenes levoris. The suggestion o f Tresner and Danga that H2S production be
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used as a criterion for classification of actinomycetes was considered by Silvestri (1960) as of doubtful value. Only the chromogenic strains gave a positive reaction; the bluish-black pigment formed in presence of iron salts was believed to be due not to the formation of metallic sulphide, but to a reaction between Fe3+ ions and substances of phenolic and/or enolic nature of the quinoid compounds of the melanoid pigments. Tresner et al. (1961) made a detailed study of the spore surface of streptomycetes. Out of 600 cultures examined, comprising some 120 species or varieties, about one-third of the gray to brownish spored species were found to have either spiny, warty, or hairy spores; the remaining members of this spore color group were smooth-spored. All of the blue to blue-green spored forms had spiny spores. The white, yellow to cream, or buff cultures had smooth-walled spores. All of the pinkish-cinnamon to pinkishtan spored group had smooth spores, with the exception of S. erythraeus, S. purpurmcens, and two undetermined species which had spiny spores. The conclusion was reached that the size and shape of spores in most species tend to be variable, and appear to be of limited usefulness of taxonomic differentiation; the surface configuration of the spores is a remarkably constant species characteristic, and may provide a reliable and useful taxonomic aid. Lindenfelser and Pridham (1961) demonstrated that S. uiridochromogenes strains are subject to little variation, as shown by the constancy of morphology of sporophores, the color of the aerial mycelium, the ability to form hydrogen suIfide and brown diffusable pigments, the ability to use a broad range of carbon compounds for growth, and the qualitative nature of the antibiotics produced. A taxonomic study of the genus Streptomyces was also made by DeMorais and DAlia Maia (1961).
G. THERMOPHILIC ACTINOMYCETES The antagonistic properties of thermophilic actinomycetes were studied by Tendler and Burkholder (1961) and Agre and Orleansky (1962). In 1951, Schone isolated from soil kept at 60°C. a strain of S. thermophilus which produced an antibiotic, designated as thermomycin, active against Corynebacterium diphtheriae. This organism dissociated into the S and R types, the S-type strain being
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antibiotically active. Schuurmans et al. (1956) isolated a substance designated as thermoviridin. Kosmatchev ( 1956) reported that thermophilic actinomycetes are widely distributed in various soils, curative muds, manures, and composts. As many as 40 to 50% of the cultures possessed antagonistic properties. The formation of antibiotics by actinomycetes, growing at 50-60°C.,takes places two to four times as fast as that of mesophilic forms. Kosmatchev later (1960a) examined 2000 cultures of thermophilic actinomycetes belonging to several genera; 40% of all cultures depressed the growth of StuphyZococcus uureus and certain other microbes. By subjecting S. uureus to sublethal ) , slow growing strains were obtained temperatures ( 50°-53"C. resembling bacterial and yeast mutants with lowered respiration. The ability to bring about the lysis of dead cells of Escherichia coli and the bacteriostatic activity upon weakly growing Staphylococcus aureus were utilized for screening cultures of actinomycetes as antitumor agents. A preparation was isolated from a culture of Micromonospora vulgaris, designated as antibiotic 12, and found to be active against tumors in vitro, as measured by repression of dehydrase activity of Ehrlich's ascites. Kosmatchev ( 1959) summarized the literature on thermophilic actinomycetes. He argued that the thermophilic properties are not sufficient to establish independent genera, and recommended that they should be classified in the same genera as the mesophilic forms. When preserved in dry synthetic agar at room temperature, thermophilic actinomycetes survived for a period of 8 years with their morphological, physiological, and cultural properties, as well as their ability to produce antibiotics unchanged ( Kosmatchev, 1960b, 1962). In a more recent classification of thermophilic bacteria, Jacob ( 1961) included the thermophilic actinomycetes under the following two genera: Streptomyes: 1. Thermomesophilic 2. Medium thermophilic a. Brown aerial mycelium on synthetic media b. White aerial mycelium on synthetic media 3. Highly thermophilic
S . thcrmocasei
S. thermofuscus S . thermophilus S. thermodiastaticus
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Micromonospora: 1. Thermomesophilic a. White aerial mycelium b. Gray-green aerial mycelium 2. Medium thermophilic
M . thermothalpo~h~la M . thermomonospora M . thermovulgaris
H. NEW SPECIES OF ACTINOMYCETES The creation of new species of actinomycetes, predominantly belonging to the genus Streptomyces, continues at an unabated pace. It appears that, in search for new antibiotics throughout the world, the first discovery is bound to be associated with the naming of freshly isolated cultures, not always even accompanied by a proper description for identification purposes, as a “new” species. Frequently such new names are listed in patents. It would require a corps of experts to determine how “new” such species are. Even if such a corps existed, it is doubtful whether some of the creators of these “new” species would be willing to release the cultures before the lapse of several years, either after a patent has been obtained, or after the conclusion has been reached that the “new” antibiotic is not new at all and that even the “new” species may not be new. Baldacci and Locci (1961) reexamined ten cultures classified as Micromonospora melunosporea, and isolated from different substrates. They created a new subspecies, M . melanosporea subsp. corymbica nobis. Micromonospora sp. Strain No. 1 Waksman 1942 and Actinomonospora lusitanica Castellani, DeBrito, and Pinto 1958 were considered as synonyms of this new subspecies. Sebald and Prevot (1962) created a new species Micromonospora acetoformici, isolated from the posterior intestine of Reticulitermes. Streptomyces akiyoshiensis, resembling S. crythrochromogenes, was isolated by Tatsuoka et al. (1961) and found capable of producing a new antibiotic HON. Thirumalachar and Bhatt (1960), after listing eight species of Streptomyces (S. alboflavus, S . armillutus, S. gilvus, S. griseoflavus, S . griseolus, S. utilis, S. vendargensis, and S . vorosoviensis ) capable of producing oxytetracycline, created a new species under the name of S . albofaciens. S. atlas, producing rufomycin, was isolated by Nakazawa et al. ( 1961).
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S. atratus by Shibata et al. (1962a) as a producer of rufomycins A and B. Lindner et al. (1962) described S. barnbergiensis, an organism that produces an antibiotic moenomycin. S. bluensis was described by Mason et al. (1961) as a producer of a new antibiotic. S. conganensis by Lindner et al. (1960b) as a producer of antibiotics F 1370 A and B. S. clistallicus by Arcamone et al. (1958) as producer of antibiotics distacin and distamycin A, B, and S. efluuius was described by Lindner et al. (1957) as capable of producing a new fungicidal antibiotic. S . griseosporeus, said to be related to S. antibioticus and S. tanashiensis, capable of producing the antibiotic taitomycin, was isolated by Niida and Ogasawara (1960). Shinobu and Shimada ( 1962) described S. griseouerticillatus, an organism producing an antibiotic takacidin. S. haranomachiensis was described by Masumoto (1961 ). It formed a straight aerial mycelium and a pinkish soluble pigment on some organic media; it was chromogenic and produced vancomycin or a related antibiotic. S. humidus, described by Shibata (196l), capable of producing dihydrostreptomycin. S. indigoferus, producing a blue to green soluble pigment on certain synthetic media and forming a straight aerial mycelium, pinkish-gray in color, was described by Shinobu and Kawato (1960). It was said to be closely related to S . herbalicolor. S . kreotomyceticus, producing an antibacterial substance FE-1100, was described by Societa Farmac. (1960). S. lincolnensis, producing lincomycin, was described by Mason et al. (1962). S. lusitanus, producing a tetracycline, was described by Villax (1962). S. mariensis was described by Soeda (1959). It is an organism producing marinamycin, an antitumor substance. S. mauvecolor, a chromogenic organism resembling S. cinnamonensis, was described by Murase et al. (1961). The white to lilac aerial mycelium produced spirals, with spiny surface spores. S. misakiensis produces the antibiotics tubercidin A and B (G. Nakamura, 1961a).
c.
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S . mcditerranei, an organism that produced an antibiotic rifomycin, was described by Margalith and Beretta (1960). S. nashvillensis was described by McVeigh and Reyes (1961). S. neyagawaensis, producing folimycin, was described by Yamamot0 (1960). S. ogaensis, producing an antitumor agent cervicarcin, was described by Ohkuma et al. (1962b). S. ostreogriseus, producing factor B, was described by Ball ( 1961). S. parvisporogenes, producing a polyene antibiotic, by Pfizer & Co. (1960). S. paucisporogenes, producing antifungin 4915, was reported by Hagemann et al. (1959, 1961). S. peruviensis, producer of antibacterial 6798RP, was listed by Rhone-Poulenc ( 1960). S . pimpina, producing an antifungal antibiotic hamycin of the polyene (heptaene) type, was described by Bhate et al. ( 1960). S. polychromogenes, an organism producing sideromycin, an iron-containing type of antibiotic related to grisein, was emended by Zahner et al. (1960). Shibata et al. (1961) described S. pulveraceus, resembling S. $xvogriseus, and producing a group of basic substances, zygomycins A and B, belonging to the neomycin type. S. rameus, producing streptomycin, was described by Shibata (1961). S. rufochromogenes, producing rufochromomycin, was described by Rhone-Poulenc ( 1961). S. saraceticus was described by Berger et al. (1960). S . senoensis was described by Kanda et al. (1961). S. sioyaensis was described by Nishimura et al. (1961); it produced a crystalline polypeptide antibiotic, similar to thiostrepton in its elementary analysis, infrared and ultraviolet spectra, but from which it can be differentiated by its specific rotation, amino acid composition, and RI value. S. sparsogenes, producing an antitumor antibiotic sparsomycin, was described by Owen et al. (1962). S . tubercidicus, producing tubercidin, was described by G. Nakamura ( 1961b). S. untbrosus, producing phyllomycin, was described by SchmidtKastner et al. (1959).
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S . viridogriseus, producing an antifungal antibiotic dermostatin, was described by Thirumalachar and Menon ( 1962). A. rantholiticus, a member of a group of yellow actinomycetes, was described by Konev and Zyganov (1962). Various other species of Streptomyces have been described. One may mention: S. flavopersicus producing M-141, an antibiotic identical with actinospectacin (Oliver et al., 1961a); Szabo and Preobrazhenskaya ( 1962) described Actinomyces primicini producing primicine, A. flavofungini producing flavofungin, and A. enythreus var. speleomycini, producing speleomycin. Nogatsu et al. ( 1962) described Streptomyces albus var. pathocidicus, producing an antifungal antibiotic pathocidin. S. Kondo et al. (1961, 1962) described S. goshikiensis, producing bandamycin. Sakamoto et al. ( 1962) described S. griseofuscus, producing the antibiotics bundlin A and B. S. capreolus producing capreomycin was described by Stark et al. (1962). Monnier and Bourse (1962) described S. pristinae spiralis producing pristinamycin. Krassilnikov et al. ( 1960b) described an organism belonging to the Actinomyces globisporus group as A. pncumonicus. Koreniako et al. ( 1 9 6 0 ~ )described A. citrofluorescens, belonging to the fluorescent group. Krassilnikov and Koveshnikov ( 1962) described A. tumemacerans, producing a substance degrading tumors in plants. Various other organisms are listed in patent literature, or in compilations, such as S. varsoviensis (Korzybski and Kurylowicz, 1961) and S. alma-ataensis (Shemiakin et al., 1961). S. Kondo and Miyakawa (1961) studied several water-soluble basic antibiotics, in a screening of Streptomyces antibiotics, where 4000 cultures were isolated from soil samples and tested. S. lavendulae var. hypotoxicus produced a crystalline hydrochloride designated as A-249, differing from streptothricin antibiotics in some chemical properties.
I. PRODUCTION OF ENZYMES Streptomyces griseus produces a variety of enzymes (Otero Abalo and Regueiro Varela, 1960), especially proteases ( Capriotti, 1962; Ouchi, 1962), phosphotases (Kotelev, 1960)) and hydrogenases (Nesemann et al., 1960). According to Satake et al. (1961), the proteinase of S. griseus liberated two peptide fragments from
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native ovalbumin. Namiki et al. ( 1961) investigated the inactivation of crystalline Streptomyces protease by X-ray irradiation in an aqueous system. The inactivation of the enzyme was attributable mainly to the indirect action of radiation. Halogen ions, especially iodine, and nitrite were found to be most protective among various inorganic anions examined. Sulfur-containing and unsaturated compounds were generally effective for protection of enzyme activity against radiation damages. Chloroform and chloral acted as synergists for irradiation inactivation. Production of ribonuclease and phosphodiastarase by S. albogriseolus was studied by Yoneda et al. (1962). Preparations obtained from cultures of Streptomyces by differential centrifugation of sonic extracts contained the following enzymes : diphosphopyridine nucleotide ( DPNH ) oxidase, DPNH diaphorase, and succinic oxidase; the presence of cytochromes a, b, and c, and probably a flavoprotein were also demonstrated (Niederpruem and Hackett, 1961). The effect of ultrasonics upon the growth of Streptomyces was further studied by Fadeeva et al. (1961).Cell-free extract of S. olivaceus was found (Maitra and Roy, 1959) to contain glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, gluco-, ribo-, and gluconokinases, and phosphohexoisomerase. Indirect evidence has been obtained for phosphoriboisomerase, phosphoketopentose 3-epimerase, transketolase, and transaldolase. Streptomyces olivaceus grown in a synthetic medium yielded per 100 ml. of culture 0.4-0.7gm. dry mycelium and 131432 pg. of vitamin B12 (Konova and Borisova, 1961). Bezborodov (1961a) examined the role of aspartase in aspartic acid synthesis. According to Gregory and Shyu (1961), the production of tyrosinase by S. scabies is due to an autoreproducing constituent in the cytoplasm. On the basis of these findings, cytoplasmic inheritance of several characteristics in diverse organisms was suggested. K. Tanaka (1961) purified the RNase of S. erythreus by a combination of acrinol precipitation and by cellulose column chromotography. The enzyme preparation was thermostable. After exhaustive digestion of yeast RNA with this enzyme, resistant polynucleotides still remained, the terminal nucleotide being guanylic acid.
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Ghuysen et al. (1962) demonstrated in cultures of S . albus G. the presence of amidase and an enzyme active upon the cell walls of bacteria. Decomposition of pectic substances by enzymes of actinomycetes was studied by Bilimoria and Bhat ( 1961) , Batt and Woods (1961) found that Nocardia corallina attacks the pyrimidines, uracil, thymine, and cytosine by induced enzymes. Maurer and Batt (1962) studied the oxidation of polyols by N . coralha. Other strains oxidized thymine, but varied in their abilities to oxidize uracil and cytosine. Organisms adapted to pyrimidines converted uracil to barbituric acid and thymine to 5-metliylbarbituric acid. Oxidation of uracil by thymine-grown organisms was almost entirely by a pathway in which barbituric acid was an intermediate. Oxidation of thymine by uracil-grown organisms was similarly almost entirely via 5-methylbarbituric acid. Barbiturase preparations converted barbituric acid anaerobically to malonic acid, C02, and NH3, but barbituric acid was not degraded by whole organisms under anaerobic conditions. The culture grown on uracil degraded urea but did not oxidize malonic acid; acetic and propionic but not malonic or barbituric acids were activated by cell-free extracts as judged by hydroxamate formation. Batt also (1961) demonstrated that a uracil-thymine oxidase has been induced in N . corulfinu by uracil, thymine, barbituric acid, 5-methylbarbituric acid, 5-hydroxymethyluracil, and &methyluracil. Induction of the oxidase was inhibited by glucose chloramphenicol, and sodium azide. The oxidation of hydrocarbons by a Nocardia was further studied by Raymond and Davis (1!360), J. B. Davis and Raymond ( 1961), and Raymond (1961). The utilization of liquid n-alkanes, such as n-octadecane, gave a 95% conversion of the alkane into nocardial cells and a slime polymer. The cells cultivated on n-hexadecane or n-octadecane contained a large amount of lipid consisting of about 60% glycerides and 40% waxes. No waxes were formed by cells grown on n-tridecane, n-hexane, or glucose. Nitrogen sources, oxygen concentration, and hydrocarbon concentration influenced growth and lipid formation. The synthesis of thiamine and N . rhothii was studied by Harington (1960). Photoprotection against X-ray inactivation in A'. corullinu by Clark and Frady (1961) .
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J. PRODUCTION OF PIGMENTS Actinomycetes are characterized by a variety of highly interesting pigments, some of which are antibiotic in nature. Prodigiosin formation has been studied by J. J. Perry (1961b), actinorhodin by Bradley ( 1962), and a new flavin by Ciferri and Machado (1958). Musilek (1962) described a red pigment of the coproporphyrin type found in intramycelial cultures of Streptomgces griseus and S. fracliae. Variants giving a low yield of streptomycin, and in the absence of Fe++ in the medium, gave more intensive pigments. The production of carotenoids, designated as vitamycin, by actinomycetes, was studied by Koreniako and Gavrilova ( 1962).
K. TRANSFORMATION OF STEROIDS An extensive literature has accumulated on the oxidation of steroids by actinomycetes. No attempt will be made to review this literature. Attention may be called to the recent paper by Sih (1962). See also Casas-Campillo (1960), Feldman et al. (1962), E. Kondo et al. (1962), Shirasaka et nZ. (1961), and Mallett and Fukuda ( 1962).
L. PRODUCTION OF ACTINOPHAGES The formation of, and susceptibility to, actinophages by various species of Sfreptomyces received much attention. According to Rautenstein et a2. (1961), growth of the mycelium in lysogenic Streptomyces cultures is accompanied by reproduction of the prophage. The latter is uniformly distributed throughout the length of the mycelium together with nucleoids. Prophage is present in minute fragments of the mycelium able to grow7, thus securing the stability of the lysogenic status of the culture. Agre (1961) isolated four phages from lysogenic cultures of a thermophilic Micromonospora, which proved identical to one another and specific for the culture of this species. The optimum temperature for lysis was 55°C. The phages were sho\vn to have a spermlike shape. Further studies on actinophage of Streptomyces species were carried out by Jones and Bradley (1961a), Kutzner (1961), Nakata et al. ( 196l), and Mach ( 1962). Weindling et al. (1961) came to the conclusion that S. aureofaciens actinophage is species-specific. On this basis, they investigated three controversial organisms, S. viridifaciens, S. psam-
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moticus ( S . feofaciens), and S. sayumaensis. These were found to have similar morphological, cultural, and physiological properties which raised doubts as to their being distinct from S. aureofaciens. All three proved to be susceptible to the virulent, monovalent S. aureofaciens phage. It was suggested, therefore, that these three species should be reduced to synonomy with S. aureofaciens. The utilization of actinophages for the identification of actinomycetes was discussed by Rautenstein ( 1960). M. PROBLEMS OF GROWTH AND NUTRITION OF ACTINOMYCETES Maitra and Roy (1959, 1961) studied the pathways of glucose dissimilation by growing and resting cells of Streptomyces oZivaceus. Utilization of glucose and phosphorus was favored by aerobic conditions, the process being considerably suppressed by glycolytic inhibitors, resulting in accumulation of fructose-1,6-phosphate and triose phosphate in the presence of iodoacetate, and of phosphoglyceric acid in the presence of arsenate or fluoride. Resting cells oxidized glucose rapidly in a high phosphate medium; in presence of iodoacetate the rate of oxidation was about half. Glucose exerted a sparing action on nitrogen utilization under aerobic conditions, but not under anaerobic conditions; the latter did not favor synthesis of vitamin B12by S. oliwaceus (Maitra and Roy, 1959, 1960). The mechanism of glucose utilization by actinomycetes was studied further by Surikova (1960), Elbein et al. (196l), Niederpruem and Hackett ( 1961). Fixation of labeled COB was studied by Kanai et al. (1960) and Pine (1960). Respiration of Nocardia rubra was studied by McClung et al. (1960). Utilization of fats by actinomycetes was studied by J. J. Perry (1961a). The sources of nitrogen for the growth of Streptomyces species were given much consideration, notably nitrates and nitrites (Kawato and Shinobu, 1960, 1961; Hirsch et al., 1961). Utilization of organic compounds was studied by Ostrowska-Krysiak et ul. (1961) and Bezborodov (1961b). The transformation of specific amino acids, notably glutamic, was studied by Bekhtereva (1960); oxidative cleamination of L-arginine by Thoai et al. (1961). Glutamic acid was produced in high concentration ( 3 7 4 0 % ) in media containing glucose or beet molasses (Kobayashi et al., 1960). The role of amides in streptomycin biosynthesis was studied by Severina et al. (1961); formation of inositol by Charalampous
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(1959); formation of a new amino acid by S. aureofaciens by McCormick et al. (1961). The effect of chlorine on the production of antibiotics by S. aureofaciens by Kollar and Jarai (1960); of iron by Bachmann and Zahner (1961). The biosynthesis of cyanocobalamin by Nocardia rugosa was investigated by DiMarco et al. (1961); formation of H2S by Streptornyces species by Prauser and Meyer (1961). The effect of low temperatures upon their growth was studied by Vizir and Zhevchenko (1960). A study was made by Orlova et al. (1961) of the physiology of inactive mutants of S. rimosus.
N. EFFECTOF NUTRITIONON BIOGENESIS OF ANTIBIOTICS The role of nutrients in the medium upon the formation of specific antibiotics is highly significant. Bu’Lock ( 1961) reviewed in detail the effect of intermediary metabolism upon antibiotic synthesis and considered antibiotics as secondary metabolites. The structure and biogenesis of certain antibiotics were reviewed by Sexton ( 1960). The importance of inositol in the synthesis of streptomycin was examined by Majumdar and Kutzner (1962); the role of thymidine phosphates by Blumsom and Baddiley (1961).Musilek and Nomi (1962) demonstrated that the elimination or depression of cytochromes by changing the Fe++ content in the medium, or by varying aeration intensity, resulted in an inhibition of streptomycin formation without much effect on mycelial growth. The factors or conditions favorable for the formation and function of cytochromes, as the presence of iron and an adequate aeration intensity, increased the rate of streptomycin production. The elimination of the cytochromes also caused an accumulation of an aromatic substance which was considered to be a derivative of an intermediate in the streptomycin biosynthesis. Musilkova ( 1959) explained inhibition of biosynthesis of streptomycin, induced by the addition of atebrin to the fermentation medium of S. griseus, by a probable relationship of this synthetic reaction either to some enzymatic process mediated by flavins, or to the metabolic process linked with an enzymatic reaction catalyzed by flavins. The addition of diethylbarbituric acid largely abolished the inhibitory effect of atebrin and 2,4-dinitrophenol on biosynthesis of streptomycin.
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Birch et al. (1960) studied biological transmethylation by S. nizjeus. In the presence of L-methionine-Me-C", novobiocin was produced in which the radioactivity was distributed between the C-Me of the coumarin system (35%), the Q-Me (35%), and the C-Me2 groups (31%) of the noviose moiety; in the biosynthesis of actinomycin it provided the 2 nuclear C-Me groups (28% of the total antibiotic radioactivity), in addition to a portion of the peptide chains (presumably in the N-Me groups); the Me is transferred from methionine to a C in an aromatic ring system derived by the shikimic acid route. The role of methionine methyl group in the formation of cladinose was studied by Corcoran (1961). The biosynthesis of tetracyclines was studied by Sekizawa (1960) and of erythromycin by Kaneda et al. (1962). 0. PHYSIOLOGY OF AUTOTROPHIC ACTINOMYCETES
Kanai et aZ. (1960) found that autotrophically grown cells of Streptom yces autotrophicus are capable of fixing carbon dioxide; the organism utilized the energy derived from the oxyhydrogen reaction, the endogenous fixation of carbon dioxide in the absence of hydrogen gas being small. Growth of the organism and the process of COZ assimilation were slow, although the efficiency of the process, as expressed by the carbon dioxide/hydrogen ratio, was considerable. Hirsch ( 1961 ) tested 133 strains of actinomycetes comprising eleven genera for their ability to grow chemolithotrophically. Nine of the strains were stimulated by hydrogen in an organic medium under reduced partial pressure of 02.These strains grew well on mineral medium with H2 as the sole energy source and C 0 2 as the only carbon source. They included Mycobacterium phlei, Nocardin saturnea, N . petroleophila, N . ( Streptomyces) autotrophica, Streptomyces sp., and Streptosporangium sp. Three strains of Nocardin were regarded as facultative chemoautotrophs, because of their ability to activate molecular hydrogen (methylene blue reduction), of catalyzing an oxyhydrogen reaction, of utilizing energy gained from this reaction to incorporate COa in a highly reduced state, and because of their ability to grow on mineral medium with H--Q2-CQ2 as the sole source of carbon and energy. The hydrogenase activity of the three strains was approximately equal.
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111. The Antibiotics A. NEW ANTIBIOTICS The great majority of new antibiotics isolated in recent years have come from cultures of microbes that are usually classified with the genus Streptomyces. They are being announced at an unending pace. Some have later proven to be merely new names for old compounds, or chemical modifications of such compounds. Doskocilova and Vondracek ( 1961) described micromethods for the identification and characterization of freshly isolated antibiotics. Methods for making antibiotic sensitivity tests were reviewed by an Advisory Committee (1960). Various new and old compounds announced during the last two years are listed here. This list is far from complete. Some of these preparations have been announced previously, but were only recently described in greater detail. Actinobolin, C1:3H20.22N20G, was described by Haskell et al. (1962). It is produced by Streptomyces griseooividus var. atrofaciens. It contains one basic and one nonbasic N atoms, at least one OH and one CO groups; it forms a deep-red complex with Fe3+ and inhibits the growth of certain gram-positive bacteria. Actinospectacin, a basic antibiotic produced by S. spectabilis, was announced by Mason et al. ( 1961) and Bergy et al. (1961). The empirical formula for its sulfate was given as CI4HZeN2. 07-H2S042H20.It is active against a variety of grani-negative and gram-positive bacteria. Its activity in blood was described by Sokolski et al. (1961b). Acumycin was isolated by Bickel et al. (1962). Amidinomycin was isolated by S. Nakamura et al. (1961a), from a culture related to S. fravochromogenes. Its structure corresponded to N- ( 2’amidinoethyl)-3-aminocyclopentanecarboxamide. It showed partial inhibition of spore-forming bacteria. Its identity with myxoviromycin was later established (S. Nakamura et al., 1961b). Antibiotic No. 1415 was isolated by Sgarzi et al. (1960, 1961) from Streptomyces 1415. It showed a narrow spectrum of activity against gram-positive bacteria at 0.25 pg./ml, or less; it was inactive against gram-negative bacteria and nonhemolytic streptococci. It was said to be nontoxic. Antibiotic 2703 was isolated by Belikova et al. (1961) from strains of a Streptomyces described as Actinonzyces flziorescens.
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Antifungal antibiotic F-17-C, a heptaene, was isolated by Craveri et al. (1960) from the mycelium of S. cinnamomeus f. azacoluta. Antifungal antibiotic X-5079C, active against systemic mycosis, was studied by Emmons (1961) and Grunberg et al. (1961). Ascomycin, an antifungal antibiotic, was isolated by Arai et ul. ( 1962a). Avilamycin was studied by Gaumann et aZ. ( 1961a). Antifungin 4915 was isolated from a culture of S. paucisporogenes by Hagemann et al. (1959, 1961). Bandamycin, a nitrogen-free antibiotic active primarily against gram-positive bacteria, was isolated by S. Kondo et al. ( 1961, 1962). Caerulomycin, isolated by Divekar et a2. (1 9 6 l), gave with ferrous ions a color reaction typical of 2,2’-bipyridyl. Growth inhibition was approximately proportional to its chelating ability and was reversed by an excess of heavy metal salts. Capreomycin, a water soluble antibiotic, active against tuberculosis in mice, produced by S. capreolus (Stark et al., 1962); a peptide antibiotic, with five ninhydrin components corresponding to alanine, serine, a$-diaminopropionic acid, (3-lysine, and an analog of arginine. 0-Carbamyl-D-serinc produced by a culture of Streptomyces (Okami et al., 1962). Chalcomycin (Frohardt et al., 1960). Chelocardin was isolated by Oliver et al. (1962) from a culture of Nocnrdia sulfureu. It was active against both gram-positive and gram-negative bacteria. Cryptocidin was described by Arishima and Sakamoto ( 1961) . Demethylchlortetracycline was studied by Knothe ( 1961) . It was found to be superior to other tetracyclines, by possessing greater stability, by being more active in vitro against the majority of sensitive pathogenic organisms, and b y possessing a longer lasting activity in serum and a longer renal excretion time. Dermostatin was described by Thirumalachar and Menon (1962) as a new antifungal antibiotic, produced by S. viridogriseus. Desferrioxamine B, a typical sideramine Fe3+ trihydroxamate complex of the ferrioxamines, occurs in the desferri-form. It is produced by S. pilosus (Gaumann et d., 1962). Its extracellular secretion occurs mainly in the stationary growth phase. It is composed two molecules of three molecules l-amino-5-hydroxylaminopentane, succinic acid and one molecule of acetic acid, combined in a
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straight-chain arrangement. By adding one of these compounds to the culture medium, it acts as a precursor of desferrioxamine (Nuesch et al., 1962). The molecules react instantaneously with Fe3+ ions. Emimycin was listed as a new antibiotic by Sumiki and Umezawa (1961). Ferrimycin A, a new iron-containing antibiotic, was described by Sackmann et al. (1962). Its activity is enhanced by human serum (Maeda et al., 1962). Folimycin, an antifungal antibiotic, produced by S. neyagawaensis (Yamamoto, 1960). Gabbromycin (Genoese, 1961) . Glebomycin, an antibiotic belonging to the streptomycin group, was described by Okanishi et nl. (1962), Miyaka et al. ( 1962,), and Ohmori et al. (1962). Glumamycin was described as a new antibiotic by Shibata et al. ( 1962b). Glutarimide ring antibiotics of Streptomyces were reviewed by R. Brown ( 1962b). Gougerotin was described as a new antibacterial antibiotic by Kanzaki et al. (1962). Griseococcin (Takeuchi et al., 1962). Hamycin, a new antifungal heptaene, produced by S. pimprina, was described by Thirumalachar et al. (1961). Hortensin is produced by S. cersipellis (Oliver et al., 1961b). Lankamycin and Lankacidin, nonbasic, nitrogen-free macrolides, produced by S. violaceoniger were described by GBumann ef 01. ( 1960). Leucocidin was described by Fujiwara et al. (1961). Lincomycin, produced by S. lincolnensis, was described by Mason et al. ( 1962). Lucensomycin, an antifungal agent, by Graessle et al. (1962). Marcomycin, produced by S. hygroscopiczrs, was isolated by McGuire and Mann ( 1961) . Minomycin, produced by S. minoensis, was reported by Nishimura (1960). Moenomycin by Lindner et 07. (1962). Moldicidin and Onomycin I were listed as new antibiotics by K. Tanaka (1960). Myconomycin by Gaumann et 01. (1961b).
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Naramycin B was found (Okuda et al., 1958) to accompany naramycin A or cycloheximide in a culture of a Streptomyces. Niddamycin, a macrolide antibiotic, produced by S. djakartensis, was described by Lindner et al. (1960a). Olivomycin was described as a new antibiotic by Game et at. (1962) and Brazhnikova et al. (1962). Pathocidin was described by Anzai and Suzuki (1961) as a new antibiotic active against fungi. It was isolated from the culture broth of a blasticidin S-producing Streptomyces. This antibiotic did not decompose above 300°C. and had the empirical formula CIH4NB0,an acid compound having a weakly basic group or groups. Peptimycin, produced by S. mauvecolor, was isolated by h h a s e et al. (1961). Phenazine compounds, produced by S. griseoluteus, were studied by S . Nakamura et al. ( 1 9 6 1 ~ ) . Phleomycin, a copper form, was described by Umezawa (1961b). Phyllomycin, produced by S. umbrosus, was reported by SchmidtKastner et al., (1959). Phytobacteriomycin, effective against plant pathogens, was reported by Semenova et al. (1960). Polyaminohygrostreptin, produced by S. hygroscopicz4s, was reported by Ziffer et al. (1962). Pristinamycin ( Monnier and Bourse, 1962) belongs to the macrolides. Quinoxaline antibiotics, similar to the actinomycins, were isolated by Kuroya et al. ( 1961). 9-P-n-ribofuranosyl purine was isolated by G. Nakamura ( 1961b) from S. yokosukanensis. It possessed antituberculous activity. Rufochromomycin, isolated from S. rufochromogenzts by HhonePoulenc ( 1961) . Rufomycins A and €3, produced by S. atratus and active against acid fast bacteria, were isolated by Higashide et al. (1969), and Shibata et ul. (1962a). Rokugomycin, a product of an organism described as S. (Actinomyces) rokugoensis, was listed by Shemiakin et al. (1961). Sabriomycin was listed by Taguchi ( 1960). Secasine was reported by Ozeretskovsky (1961) to be effective in the treatment of pneumococcal and streptococcal sepsis, as well as of localized staphylococcal infection in white mice. When ad-
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ministered by mouth it exceeded 3- to 5-fold its activity by intramuscular administration. Semmimycin was said to inhibit growth of Candida albicans (Ito and Tamatoshi, 1960). Spinathricin reported by CIBA ( 1961). Synnematin B was isolated by I. M. Miller et al. (1962). Stendomycin (Thompson and Hughes, 1962) active upon fungi. Takacidin (Shinobu and Shimada, 1962) inhibits the growth of tubercle bacteria. Teichoic acid, isolated from a strain of S . griseus, was reported by Naumova et al. (1962). Teleocidin A and B, toxic substances active against aquatic organisms, isolated from a species of Streptom!/ces by Takashima et al. (1962). Triostin, isolated by Shoji and Katagiri (1961), is comparable to other quinoxaline antibiotics, such as echinomycin, actinoleukin, levomycin, and quinomycins. Tuberin was studied by Ohkuma et al. (1962a). Tylosin, listed previously ( Waksman and Lechevalier, 1962), has been described in further detail by McGnire et al. (1961). It is active against gram-positive and certain gram-negative bacteria, and mycobacteria. It gives partial cross resistance with erythromycin, but not with penicillin or the tetracyclines. Hamill et al. (1961) further reported that tylosin is a single compound having the empirical formula C45H77N017. It is a weak base, forms water-soluble salts and can be acylated to form esters. It appears to belong to the macrolide class of antibiotics. Uredolysin, isolated by Gattani (1961) from a strain of S. griseus. Venturicidin was isolated from a culture of a Streptomyces, related to S. griseolus and S . halstedii. It was active against various plant pathogenic fungi, Nocardia rubra, Bhtomyces dermatitidis, and Aspergillus fumigatus ( Rhodes et al., 1961). Nontoxic by oral and of a low toxicity by intravenous administration of animals. It was soluble in organic solvents and had a composition of C.sH7iOiJ’J. Vernamycin A and B, isolated from a strain of S. loidensis by Donovick et al. ( 1961). Active against gram-positive bacteria. Soluble in organic solvents, precipitated by addition of hexane. Zygomycin A was studied by Hitomi et al. (1961), Shibata et al.
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( 1961), Higashide et al. ( 1961), and Tatsuoka et al. ( 1962). It belongs to the neomycin group of antibiotics, more particularly to paromomycin and hydroxymycin. It is produced by an organism described as S. pulveraceus.
B. NEW INFORMATION ON OLD ANTIBIOTICS Numerous reports are appearing throughout the world dealing with previously described antibiotics produced by actinomycetes. To review them all, or even to list them is quite impossible. Only a few pertinent references may be given here. A survey of the distribution of antibiotic-producing streptomycetes isolated from the soil was made by Routien (1961). Thirty cultures, isolated from various samples of soil, collected about 30 feet apart, were tested for their ability to produce specific antibiotics. Some samples were devoid entirely, at least by the methods used, of antibiotically active cultures, whereas others contained many active cultures. Some antibiotics were produced by cultures having wide distribution, whereas others were formed by single strains found only once. Some organisms were present in several parts of the United States, whereas others were extremely limited in their geographical distribution. Methods for demonstrating antibiotics have been discussed by Davidek and Janizek (1961). Antibiotics with indicator properties have been reviewed by Blinov et al. (1961); a litmocidin-type was isolated by Balitskaya et al. (1962). Actinoidin separation into active variants was discussed by Lomakina et al. (1961). Actinomycetin, usually listed among the antibiotics, exerts a lysozyme effect not only upon various bacteria but also upon certain Streptomyces cultures ( Welsch, 1960). Actinomycin-producing species of Streptomyces continue to be isolated all over the world. Ahmad et a2. (1958) reported on a new species said to be related to S . antibioticus. The irradiation of actinomycin-producing cultures has been investigated by Schonwalder ( 1961). The biosynthesis of the actinomycin chromophore received particular attention (Katz and Pugh, 1961; Weissbach and Katz, 1961; Katz et d.,1962). The mode of action of actinomycin was discussed by Kirk (1960) and Hauen et d.(1960). Further
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information on recent developments concerning the nature and use of this antibiotic is found in Section 111, C. Warner (1961) proposed molecular models for the different actinomycins. They contain a substituted phenoxazine chromophore attached to two individual cyclic pentapeptide lactone rings through the amino group of threonine, the hydroxy group of threonine forming the lactone juncture. The synthesis of actinomycin CS by Brockmann and Lackner (1961) is said not to be unequivocal proof for the two individual pentapeptide lactone rings. According to Reich et al. (1961, 1962), actinomycin D inhibits the synthesis of ribonucleic acid in L cells and the yield of vaccinia virus containing deoxyribonucleic acid; it does not inhibit, however, cellular deoxyribonucleic acid synthesis or the multiplication of Mengo virus containing ribonucleic acid. The replication of virus ribonucleic acid could thus be distinguished from ribonucleic acid synthesis which is controlled by viral or cellular deoxyribonucleic acid (see also Goldberg and Rabinowitz, 1962; Levinthal et al., 1962; Kingsburg, 1962). Katz and Weissbach ( 1962) isolated radioactive actinomycin from the medium 3 minutes after the addition of ~-valine-l-C'~; this synthesis continued linearly for 30 minutes. D-Valine-l-C14was not incorporated into the actinomycin, although it produced an 80-90% inhibition of the incorporation of the L-isomer. Chloramphenicol markedly inhibited L-valine incorporation into protein but produced a =-fold stimulation of incorporation into actinomycin. Actinorhodin, an antibiotic pigment produced by S. violaceoruber, was isolated by Bradley ( 1962). It is produced in a chemically welldefined medium. Peptone-yeast water gave no pigment even though growth was vigorous. Aurantine production by S. aurantiacus has been studied by Nefelova (196l), Bitteeva (1961), and Belova (1961); its chemical structure by Kuznetsova et al. ( 1962). Aureothricin, thiolutin, and isobutyropyrrothine were isolated from a culture of Streptomyces by Bhate et al. (1960). Their total synthesis was reported by Schmidt and Geiger (1962). Blasticidin S was studied further by Sakagami (1961). Chloramphenicol: Haldar et al. ( 1960) compared this antibiotic with others in their effect upon the growth of bacteria, using turbidimetric curves. At a concentration of 1p.g./ml., it markedly inhibited growth and protein synthesis but appreciably stimulated
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ribonucleic acid synthesis; the deoxyribonucleic acid content remained unchanged. Gibson and McDougall ( 1961) demonstrated that in the biosynthesis of tryptophan by Escherichia coli, the formation of indole, anthranilic acid, and 5-dehydro-shikimic acid is inhibited by chloramphenicol and oxytetracycline. The decomposition of this antibiotic by certain bacteria was discussed by Miyazawa ( 1960). The incorporation of Cl4-1abeled precursors into chloramphenicol was reported by Gottlieb et al. (1962). Cycloheximide synthesis, as well as that of isocycloheximide, was investigated by Okuda et al. (1961). Its effect on the metabolism of Saccharoni yces cerevisinc and Rhodotorula gracilis was studied by Scardovi ( 1960), on Fomcs annosus by Gundersen (1962). Resistance of Saccharomyces to cycloheximide by Peynaud et al. (1962). Cycloserine stability at different temperatures and pH values received attention (Kolesinska, 1961); its activity was studied by Meier (1962). Endomycin activity was measured in presence of neomycin (Sokolski et al., 1961a). Problems involved in the production of erythromycin were reviewed by Stark and Smith (1961). Grisein was studied by Lugli et aZ. (1960). Halometabolites: The antibiotics containing halogens were reviewed in detail by M. A. Petty (1961). HON forms colorless needles and has a molecular formula of C5H8O4N.It inhibits the growth of human-type tubercle bacteria, including sensitive, isoniazid-resistant, and streptomycin-resistant strains (Tatsuoka et al., 1961). Kanamycin: Tejerina and Portolks (1960) reported that in the sera of rabbits treated with this antibiotic, there exists a substance which specifically inactivates it, acting proportionally to the “time of contact.” The substance cannot be included in the group of precipitins. Complement tests have shown the presence of certain specific antikanamycin antibodies at 1/5 sera dilutions against concentrations of 10 mg./ml. The separation of kanamycin from other antibiotics was studied by Cotta-Ramusino et al. ( 1!361). Lagosin (Bessell et al., 1959) was isolated from S. roseoluteus; believed to be identical with pentamycin. Macrolides: Hiitter et a!. (1961) made a comprehensive study of the Streptom yces species capable of producing macrolide anti-
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bioties. These include compounds that contain, in addition to one or more sugars, a many membered lactone ring. This group was divided into 2 subgroups: ( a ) polyene antifungal antibiotics, ( b ) antibacterial macrolide antibiotics. The second subgroup includes: picromycin, erythromycin, carbomycin, griseomycin, leucomycin, methymycin, spiramycin, narbomycin, angolamycin, tertiomycin, miamycin, oleandomycin, P.A. 108, P.A. 133A, 133B, P.A. 148, tylosin, lankamycin, and niddamycin. The proactinomycins and teruchiomycin probably also belong here. The pyrromycins, cinerubin, rutilantin, and aklavin are closely related. These antibiotics are active against gram-positive bacteria and certain gram-negative bacteria and protozoa. The majority of gramnegative bacteria and fungi are resistant. They vary in their in uivo activity upon staphylococci and in the development of resistance, The sensitivity of picromycin-resistant Staphylococcus aureus to various macrolides is shown in Table I. TABLE I MACROLIDE-SENSITIVITY OF PICROMYCIN-RESISTANT Stavhulococcus aureus
Full cross resistance
Reduced sensitivity
Picromycin Erythromycin Oleandomycin M ethymy cin Criseomycin Narbomycin Lankamycin
Spiramycin Tertiomycin Angolamycin h4iamycin
-~
Only slight reduction in sensitivity Carbomycin Leucom ycin Tylosin Niddamycin
The bactericidal action of macrolide antibiotics was discussed by Cluzel et al. (1960). Mikamycin: The various forms of mikamycin were found (N. Tanaka et nl., l!36la, 1962) to exhibit a marked protective effect against streptococcal and staphylococcal infections by oral administration. Mitomycin C is active on various strains of Eschericlzia coli (Reich et al., 1960). Myxoviromycin was examined further by Shoji et al. ( 1961). (See also Section 111, D.) Neomycin formation and isolation were studied in various laboratories ( Sokolski and Lummis, 1961). A chromatographic method for its determination in broth was developed by Emilianowicz-
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Czerska and Herman (1961); a quantitative method was described by Korchagin et al. (1962). The chemical structure of the neomycins was summarized by Rinehart et al. ( 1962a,b,c). The penetration of metabolites into Escherichia coli cells was found to be inhibited by neomycin (Sazykin and Borisova, 1961). This is measured by the reduction of methylene blue by intact cells. Using a neamine-dependent strain of Staphylococcus aureuS to determine if the requirement for neamine could be fulfilled by other antibiotics, growth was obtained with streptomycin, paromomycin, zygomycin, and neomycin C (all of which contain 2-aminohexoses ), but not with neomycin B, kanamycin, erythromycin, spectinomycin, or vancomycin. Streptomycin-dependent strains of Staphylococcus aureus and Salmonella paratyphi grew well on neamine, but strains of E. coli and Mycobacterium gave negative results (Sokolski et al., 1962). Novobiocin is composed of a sugar, noviose, a substituted coumarin, 3-amino-4,7-dihydroxy-8-methylchromone, and a substituted benzoic acid, 4-hydroxy-3(3-methyl-Z-butenyl ) benzoic acid. Analog formation was obtained by the addition of 4-hydroxy-3-isoamyl benzoic acid as a precursor; however, instead of novobiocin, dihydronovobiocin was formed. The benzoic acid moiety is joined with the remainder of the molecule through an amide linkage resembling the penicillin side chain (Walton et al., 1961). The production of novobiocin was reviewed by Hoeksema and Smith (1961). Nybomycin structure was reviewed by Rinehart and Renfroe (1961). The antibiotics of the tetracycline series were discussed in detail by Chernukh and Kivman ( 1962,). Oxytetracycline formation by Streptomyces rimosus was studied by Zygmunt ( 1%1). Aspartic acid, proline, threonine, valine, and p-alanine were utilized for both growth and antibiotic production. Glucose and glycerol supported the highest antibiotic yields; short chain organic acids were not utilized readily for growth in the absence of a readily fermentable carbon source. Polyenes continue to be isolated all over the world. This subject was discussed in detail by Vining ( 1960). Lechevalier et al. ( 1961a) summarized heptaene macrolide forms. The use of various synthetic and complex organic media was studied by Kohler (1962). Aburatani et al. (1959) tested 1000 strains of Streptomyces for their antifungal activities by the agar disc method, using Candidu albicuns as test organism; 129 cultures produced anticandidal sub-
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stances. Of the antifungal agents obtained from some of the active strains, 68% had polyenic characteristics according to their ultraviolet absorption spectra, positive sulfuric acid test and paper chromatography. These were classified as follows: 59.6% heptaenes, 34.0% tetraenes, and 6.4% of pentaenes. No hexaenes were found. Among the nonpolyenic substances, 5 belonged to the streptothricins, 3 to eumycetin, 1to actinomycin, and 13to others. The strains producing polyenes were found to belong to S. aureus, S. fungicidicus, S. albireticuli, S. uiridoflauus, and S. abikoensis. Nefelova and Pozmogova (1960) and Golyakov (1961) also reported on the isolation of antifungal antibiotics. The physiology of S . noursei was examined by Popova and Stepanova (1962). Gerke and Madigan (1961) found that the activities of polyenes are influenced by inoculum size, medium composition, pH, incubation time, and temperature; they were in inverse ratio to inoculum size and to incubation time. Amphotericin B and filipin are more active at 37°C. than at 25°C. The reverse is true for nystatin. The pH range for maximum activity for amphotericin A was found to be 4.0 to 6.0; for nystatin, 4.5 to 6.5; for amphotericin B, 5.5 to 7.0; magnesium and calcium ions decreased its activity, but sodium citrate increased it. The effect of nystatin on the growth of algae was studied by Lampen and Arnow ( 1961). Various Chlorophyta, Euglenophyta, Chrysopliytu, and Cyanophyta were inhibited, but one of the Bacillariophyceae was not. Nystatin was lethal at concentrations which completely prevented growth. The effect of monovalent cations on inhibition of yeast metabolism by nystatin was studied by Marini et al. (1961). Porfiromycin was believed to be identical with methyl mitomycin ( Wakaki et al., 1962). Primocarcin structure was studied by Isono ( 1961). The taxonomy of Nocurdiu fukayae producing primocarcin was examined by Nogatsu et al. (1962). Streptomycin and dihydrostreptomycin: These two forms of streptomycin continue to receive considerable attention. The mechanism of formation of the latter from the former was investigated by Tsuji (1961 ). Colorimetric determination of dihydrodesoxystreptomycin was studied by Katayama et al. (1960). The eflect of various substances on the production of streptomycin
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was examined by Galanina and Agatov (1960). The intrastrain transduction of a streptomycin resistance marker of phages in Proteus mirabilis was investigated by Coetzee and Sacks (1960). The addition of inositol to a synthetic medium increased streptomycin production by 40-4576, without affecting the growth of the organism. The addition of methionine and vitamin B12 also greatly stimulated streptomycin production ( Majumdar and Kutzner, 1962). The transformation of streptomycin resistance by deoxyribonucleic acid, isolated from resistant cultures of Mycobacterium auium, was studied by Tsukamura ( 1961a); conversion of streptomycin resistance of Staphylococcus aureus in stationary populations by Dobrzanski ( 1961). Streptomycin resistance was studied further by Grosset and Canetti (1961); streptomycin dependence by Engelberg and Artman ( 1961). Streptothricin and streptolin structures were elucidated by Carter et al. (1961). Streptozotocin chemistry and stability were studied by Garrett (1960). Tetracyclines: Streptomycetes producing oxytetracycline were studied by Cross (1962). Goodman and Matrishin ( 1961) demonstrated that 7-chloro-6demethyl-tetracycline is formed by Streptornyces aureofaciens in media containing certain sulfonamides. Birch ef al. (1962) found that all of the methyl groups in the oxytetracycline molecule can be derived directly from methionine. Perlman et al. (1961) found that small amounts of 6-demethyltetracycline can be produced similarly in fermentations yielding predominantly 7-chloro-6-demethyltetracycline (see also Szumski, 1960). Sekizawa ( 1960) investigated the biochemical chlorination in the biogenesis of 7-chlortetracycline. Bromide and organic thiol compounds exhibited an inhibitory action on the biochemical chlorination reaction allowing the accumulation of the tetracycline nucleus itself. Katagiri et al. ( 1961) reported that tetracyclines inhibit the metabolism of various carbon compounds participating in terminal respiration of E . coli; tlie formation of n-ketoglutarate diminished. It was suggested that tetracyclines inhibition may be related to the oxidative phosphorylation process. Snell and Cheng ( 1961) demonstrated the interference of tetracyclines with the metabolism
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of endogenously synthesized D-ghtamiC acid. The accumulation of radioactive D-glutamic acid from C-l'-acetate precursor takes place in cultures of inhibited oxytetracycline-sensitive E . coli cells, but not in resistant cultures. Parnas et al. (1960) isolated fifteen strains of Brucellu brucei that showed resistance against chlortetracycline and twelve strains that were resistant to streptomycin. The chlortetracycline-resistant strains showed instability while the streptomycin-resistant strains showed stability. There were no special bacteriostatic and serological changes. Highly virulent strains of Bmcella also possessed a greater urease activity when compared with the original strains. Thiostrepton was investigated by Kutscher and Seguin ( 1961). Toyocamycin was studied by Ohkuma ( 1961) , Tubercidin formation was studied by G. Nakamura (1961a). Tuberin was studied by Anzai et al. (1962). Vancomycin, produced by Streptomyces harunomachiensis, said to be different from S. orientalis, was studied by Masumoto (1961). It is an amphoteric substance, easily soluble in water and insoluble in usual organic solvents. The ultraviolet absorption spectrum indicated the maximum absorption at 2880mp. It showed a strong activity against gram-positive and acid-fast bacteria. The intravenous injection in mice of 300mg./kg. showed no toxic effects.
C. ANTITUMOR SUBSTANCES The search for antitumor and antiviral agents continues to occupy a leading place among the various screening programs carried out in this country and abroad. Certain metabolic products of different groups of microorganisms were found to possess remarkable antitumor properties, Only the metabolic products of actinomycetes are considered here (Gause, 1962; Sekizawa et ul., 1962). Various methods have been developed recently for testing the antitumor effects of microbial products. These comprise both the in vitro and the in vivo tests. The former were analyzed in detail by Vdyi-Nagy et al. (1961b). They are usually based on the discoloration of different redox dyes; these are useful in detecting cytotoxic or cytostatic agents possessing properties inhibitory to dehydrogenase. One particular test is frequently referred to as that of Miyamura. It can be carried out by using normal white blood cells of human and animal origin, as well as human white blood cells from pathogenic specimens.
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The dehydrogenase activity of various antitumor agents and crude microbial preparations has received considerable attention. Navashin et a2. (1960) performed comparative tests with strains of cells of various tumor cells, using a number of different antibiotics with known antitumor properties; some were found to possess definite activity. Various culture fluids and crude preparations were also active. Kato et al. (1961) described a method for screening antitumor substances found in filtrates of 200 cultures of Streptomyces. HeLa cells were used as the primary screening system; active preparations were then tested against Ehrlich ascites tumor cells, as well as against various bacteria. No correlation could be found between antibacterial and antitumor activity. The reproducibility of the results on the production of anti-HeLa cell factors was higher than that of the anti-Ehrlich factors, although both procedures were considered to be fairly good. The effect of antibiotics on HeLa cells infected with meningopneumonitis was studied by Galasso and Manire (19SO). Foley (1961) developed a method for directly assaying crude preparations, using chromatographic strips of disks with monolayer mammalian cell cultures under an agar overlay. By the use of this method potential antitumor activity of an antibiotic can readily be detected. The inhibitory activity in such mammalian cell assays, which is frequently independent of antibacterial activity, was found to correlate well with activity in experimental animal tumor systems. Foley emphasized that there are now more than two dozen antibiotics known to inhibit experimental tumor systems, some half-dozen of these exhibiting a certain inhibitory activity in human neoplasia. Umezawa (1961a, 1962) examined in detail the various screening procedures and came to the conclusion that the principal method is that of using animal tumors. Tumor cells in tissue culture, cylinder plate methods with tumor cells, methods using microorganisms or their mutants, and enzymatic methods were said to be also helpful. The known antitumor substances were classified into three groups, depending on their activity on ( a ) animal tumors, ( b ) tumor cells in tissue culture, and ( c ) microorganisms. Sarcomycin was used as a standard and the propenoyl group was said to be a group exhibiting antitumor activity. Hilamycin was found to be a simple compound exhibiting inhibition against mouse leukemia SN36, Ehrlich
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carcinoma, and HeLa cells. Peptimycin, a peptide, exerted no direct destruction on tumor cells, but exhibited inhibition against animal tumors. In vivo studies of antineoplastic agents were also made by Modarski ( 1960). Barnard ( 1961) investigated the antineoplastic properties of actinomycetes not producing any antibiotics. Laiko (1962) made a comparative study of the action of various antitumor antibiotics and synthetic compounds on the synthesis of nucleic acids in staphylococcal cultures. The content of DNA and RNA in the extracts was determined by means of diphenylamine and orcin reactions, respectively. Mitomycin C affected selectively the synthesis of DNA; the other antibiotics had the same effect on RNA. It was suggested that the method of studying the inhibiting action of antibiotics on the synthesis of nucleic acids in staphylococcal cells be used for the study of mechanisms of action of various new antibiotics. Further studies of screening procedures of antitumor agents were made by Schabel and Pittillo ( 1961), Arcamone et al. ( 1961) , Chorin and Lyashenko (1962), and Neelameghan (1961a). Ivanitskaya (1961) used Curyophanon Zatum as a test organism, since it is an intermediate form between bacteria and water plants. Out of 357 cultures of actinomycetes tested, 19 antibiotics were obtained, none of which had any effect on usual test organisms; 6 of these demonstrated an antitumor activity in animal experiments. Detailed reviews of the new developments in cancer chemotherapy have been made by Jones et al. (1960), and Sokoloff ( 1960). Among the individual antitumor preparations, some have received particular attention. These, comprising new preparations and well known substances, may be discussed further here: Actinobolin was found (Pittillo et aZ., 1%1) to be active against a variety of bacteria and a certain rodent neoplasm. Its inhibition of leukemia in mice does not seem to be related to the chelating properties of actinobolin, although certain metal salts affect actinobolin inhibition of bacteria; this effect may be due to metabolic interference, although it is not clearly associated with its chelating properties. Its formation and chemistry were discussed previously. Actinogan is a stable, water-soluble, amorphous polysaccharide (Bradner et al., 1961; Schmitz et al., 1962). It is active against bacteria and tissue cultures of tumors. Mouse antitumor assays were used to follow the production and isolation of the active
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principle. Growth of sarcoma 180 and adenocarcinoma 755 was inhibited in uiuo with a nontoxic therapeutic ratio of 2; Ehrlich ascites tumor was inhibited by a toxic dose. Activity was obtained by intraperitoneal or by intramuscular administration, but not orally. The most potent solid preparation was active at 0.5 mg./kg./ day. Actinogan enhanced the survival of mice with sarcoma 180, permitting an incidence of complete regressions above the controls. Actinomycin: Various new forms of actinomycin continue to be isolated and studied as potential antitumor agents. It is sufficient to mention actinomycin P2 (Young, 1961) and actinomycin K (Balitskaya, 1961). Burdette ( 1961) emphasized the alteration of mutation frequency following treatment with actinomycin D. According to Rauen et al. (1960), the cytostatic action of actinomycin C on the growth of Neurospora crassa and Streptococcus faeculis was abolished by the addition to the growth medium of nucleic acids. A concentration of 50yg./ml. of the antibiotic was inhibited by 50pg./ml. DNA with Nocardia crassa and by 1.2 pg./ml. DNA with Streptococcus fuecalis. Actinomycin C reacted with DNA and RNA to form complexes similar to the compounds produced by nucleic acids with acridine orange and rosaniline. Such complexes could be demonstrated in the ultracentrifuge. The abolition of the actinomycin inhibition was considered to be due to a direct reaction between the antibiotic and nucleic acids. A study of the sensitivity of mouse fibroblasts to the cytostatic action of actinomycin and other antibiotics brought out the fact that the cells were less sensitive when grown in media supplemented with calf serum than when grown on protease-peptone media (Giuffre et al., 1961).The transplant immunity has an effect on the action of actinomycin D on choriocarcinoma in hamsters (Li and Mann, 1961) , Lung perfusion techniques revealed the widespread damage caused by 75 and 50pg./kg. of actinomycin, but not by 30 pg./kg. (Pierpont, 1960). According to Kawamata et al. (1960), actinomycinic acid, a preparation obtained on treatment of actinomycin with methanolic sodium hydroxide, thus opening the lactone groups, and considered as not having any antibacterial effects, has a definite antitumor effect; the amount of the acid required for such action was much greater than of actinomycin. Actinomycinic acid was administered intraperitoneally daily for 5 days after the lapse of 3 days of intra-
ACTINOMYCETES An’D THEIR ANTIBIOTICS
275
peritoneal inoculation of Ehrlichs ascites tumor cells to mice. The treatment prolonged the life span of the experimental animals when 12.5100 mg./kg./day of the acid were given. Long-term administration also brought prolongation of the life span which could not be expected of actinomycin itself. Actinomycin was found to form a complex with deoxyribonucleic acid, a phenomenon that seems to pIay an important part in the mechanisms of action of actinomycin (Kawamata and Imanishi, 1960). Actinomycinic acid did not show such a complex formation. It was suggested that the mechanism of action of actinomycinic acid differs from that of actinomycin. The favorable effect of actinomycin C on Hodgkin’s disease was reported by Domoto and Kamijo (1960). The use of actinomycin D in the treatment of malignant diseases in children was examined by Colebatch ( 1960a). According to Leider ( 1961 ), perfusion with actinomycin D plus irradiation of the affected extremity of a patient with melanoma caused the disappearance of all nodules in 2 weeks (see also Sampey, 1962). Bailey et al. (1961) made a survey of the treatment of rhabdomyosarcoma with actinomycin D. It was found to be the most effective chemotherapeutic agent for this type of tumor. It was used (75-100 pg./kg. over a 5-day period) in six patients after surgery, when excision was incomplete or when recurrence or metastases had occurred. Response to the drug was good in three cases, as evidenced by shrinking or disappearance of the tumor, as well as a case where embryonal tumor of the peritoneum had responded moderately well to irradiation and to the drug. Some of the children survived for varying lengths of time, after a course of actinomycin D was given; metastases to the spine and lungs responded dramatically to actinomycin D therapy only. The clinical effect of actinomycin D on Wilms’ tumor was examined by Momose and Aito (1961). Further information on the response of certain forms of cancer to actinomycin D chemotherapy is found in the work of Tan et al. (1959), Straffon (1961), and others. The effect of actinomycin C on normal and neoplastic cells has been studied by Awa ( 1961). Actinoxanthine effect upon human cancer cells was studied by Navashin et al. (1961). Angustmycin A was found (Miyairi et nl., 1961) to inhibit incorporation into nucleic acid fraction of Bacilltrs subtilis; there
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was a decrease in the amount of nucleic acid and a depression of C14-labeled amino acids incorporation into the protein fraction of the organism. It was suggested that this antibiotic acts as an antimetabolite in nucleic acid biosynthesis and exhibits antimicrobial activities. The possible mode of action of angustmycins against transplantable tumors was thus indicated. Further studies on the effect of angustmycins upon transplantable tumors were made by N. Tanaka et al. (1961b). Azaserine: The recent literature on this antitumor agent was compiled by Duvall (1960). According to Brock and Brock (1961), azaserine reacts irreversibly with cells in phosphate buffer. Phenylalanine antagonizes the action of azaserine. This was believed to be due to a blocking of this binding or uptake of azaserine by the cells. When the cells were treated first with azaserine, phenylalanine had no effect on its action. Ayamycin, a new antitumor antibiotic, was isolated by Tanno (1960) from a culture filtrate of an organism related to Streptomyces flaveolus. Mice bearing Ehrlich ascites tumors were treated by intraperitoneal injection of ayamycin; all tumor cells disappeared and the life span was prolonged; some effect was shown on both body weight and the weight of the spleen. See also Matsuura and Katagiri ( 1961) . Schmidt ( 1961) reported that carzinophilin depressed the DPN level of Yoshida ascites tumor cells by 50% after 5 minutes of incubation. This effect preceded inhibition of glycolysis. Nicotinamide prevented this effect or reversed it if added afterward. Carzinopliilin interfered with oxidative phosphorylation, as evidenced by its effect on the ATP/ADP ratio. This effect occurred after DPN depression and thus was a result and not the cause of inhibition of glycolysis. Carzinostatin was isolated from the culture filtrate of Streptoniyces E-793, related to S. albus (Shoji, 1961; Kumagi, 1962). It was fractionated into B, soluble in methanol, and A, insoluble. When these 2 fractions were combined, a synergistic action against S. lutea was obtained. This activity was considered as an indicator of antitumor activity, almost all of Ehrlich ascites tumor-bearing mice surviving about 30 days, whereas control mice died within 15 days, when treated with three different concentrations of %fold dilutions below the toxic level. In addition to a decrease of tumor cells, the generation of giant cells was observed on treatment with this antibiotic.
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Cervicarcin, a new antitumor agent, was studied by Ohkuma et al. (1962b). Chromomycin gave recovery from basal cell carcinoma in men with an ulcer under the left eye (Uematsu, 1960). Further studies on the antitumor properties of chromomycin have been made by Takaki et al. (1960), and Kuru ( 1961). The effect of crucin and olivomycin on Ehrlichs ascitic carcinoma was examined by Boyko (1962). Cytomycin and blasticidin S, cytosine-containing antibiotics, were found (N. Tanaka et al., 1961c) to exhibit considerable tumor inhibition (50-80% ) against Walker adenocarcinoma 256 in rats, Ehrlich carcinoma and sarcoma 180 in mice. Less activity was observed with adenocarcinoma 755 in C57 BL/6 mice. No significant inhibition was obtained with ascitic tumors, including Yoshida sarcoma, Ehrlich carcinoma and sarcoma 180. Cytomycin was less toxic but less effective than blasticidin S. Distamycin A showed distinct cytotoxic activity ( DiMarco et al., 1962). Duazomycins A, B and C are the same as Diazomycins (Rao, 1961) . Marinamycin, a product of S. mariensis, was found to exhibit antitumor activity ( Soeda, 1959, 1962). Mitiromycin, comprising a mixture of antibacterial and antitumor agents, was isolated from S. verticillatus by Lefemine et al. (1962). Mitomycin C continues to attract considerable attention as an antitumor agent (White, 1959; Frank, 1960; Truhaut, 1960; Cerny et nl., 1961; A. E. Evans, 1961; Reich et al., 1960). The structure of mitomycins A, B and C, as well as of porfiromycin, was studied by Webb et crl. (1962). Gourevitch et al. (1961) reported on the destruction of mitomycin C by S. caespitosus, the organism that produces it. Sugiura (1961) tested the inhibitory action of 34 crystalline or purified antibiotics, mitomycin C being most effective on 31 of 35 various tumors. It had a destructive effect on a number of tumors. The conclusion was reached that “the effectiveness, at least temporarily, of mitomycin C against neoplasms of humans makes one hopeful of the eventual attainment of our goal, the cure of cancer in man.” Mithramycin, a yellow crystalline antibiotic isolated from a species of Streptomyces, was found to be an acid forming a crystal-
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line sodium salt. It exhibited marked activity against gram-positive bacteria and HeLa cells grown in cell culture, as well as some experimental tumors (Rao et al., 1962). Neotsid effect upon patients with tumors was studied by Sharlai et al. (1958). Oncostatin C, an antitumor antibiotic, is produced by Streptomgces sp., a variant of S. chrysomallus (Woznicka et al., 1961). It inhibits the growth of gram-positive bacteria and mycobacteria, but it does not inhibit the growth of gram-negative bacteria, yeasts, and fungi. It was reported (Plociennik et al., 1961) to be closely related to actinomycin C. Pactamycin, an antitumor antibiotic produced by S. pactum var. pactum, was investigated by Bhuyan et al. (19f31), and Bhuyan ( 1962). Maximal growth and production efficiency were obtained at 32°C. Pactamycin was bound to the mycelium in different amounts, depending on the fermentation conditions. It was produced during the later autolytic phase and could be extracted with acetone. It is active in vitro against KB human epidermoid carcinoma cells in tissue culture. It is also active against a variety of gram-positive and gram-negative organisms, as well as against a variety of tumors in mice and hamsters. Peptimycin, a product of S . mawecolor, was shown to have antitumor activity (Murase et al., 1961). Phleomycin was also shown (Bradner and Pindell, 1962) to possess antitumor properties. Polymycin effect upon human cancer cells was studied by Navashin et al. (1961). Porfiromycin (J. S. Evans et aE., 1961) prolonged survival time of mice with L-1210 leukemia or Wistar rats with the H (monocytic) leukemia (solid form). The rats showed a lowered leukocyte level compared to the controls, but only slight prolonged survival time. The antibiotic also inhibited the growth of sarcoma 180 and certain other tumors. It was active when given either parenterally or orally. It was markedly toxic in the dog when given orally, 10 mg./ kg. The effects were less predictable and more moderate at comparable dosages in the rhesus monkey. Primocarcin (Sumiki et al., 1960; Isono and Suzuki, 1962) is soluble in water and lower alcohols, moderately soluble in acetone and pyridine, slightly soluble in ethyl acetate, but hardly soluble in benzene, ether and petroleum ether. It showed a tumor inliibitorv
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activity on ascites types of Ehrlich carcinoma. The daily intraperitoneal injection of 0.25 mg. per mouse for 6 days started at 24 hours after transplantation of two million tumor cells caused marked inhibition of increase of ascitic fluid, and prolongation of survival period. Roseolic acid has antitumor properties (Renn et al., 1962). Sarcomycin derivatives were investigated by Caputo et al. ( 1961 ) as to their antitumor properties. Sarkomin, prepared as a urotropine compound, was studied as an antitumor agent (Iwataru, 1959). Sparsomycin was shown to possess antitumor activity (Owen et al., 1962). Spiramycin was found to have inhibitory activity against various tumors; it possesses low toxicity and produces high tissue levels (Back et al., 1961). S-339 was shown to have an effect upon Ehrlich solid tumor (Hossenlopp and Hata, 1961). Streptonigrin is an antitumor agent produced by S. flocculus (Marsh et al., 1961 ) . Fermentation filtrates were slightly inhibitory to Crocker mouse sarcoma 180 and certain other tumors. Various other antitumor agents, produced by actinomycetes, were listed. It is sufficient to mention E-73 (Rao and Cullen, 1960) and NSCA 649 (Schmitz et al., 1960). Combination of antitumor agents. Krementz ( 1960) discussed the possible use of a combination of an antibiotic with an alkylating agent, in the therapy of sarcomas. The results of the radiation therapy in Wilms’ tumors, neuroblastomas, and some other sarcomas in children in whom the irradiation apparently was potentiated by the use of actinomycin D favored this concept. The radiomimetic effects of the alkylating agent suggested the possibility that by this action actinomycin D could be potentiated in antineoplastic properties. Phenylalanine mustard (PAM) and actinomycin D were found to be well tolerated when used in conjunction. A dosage of 1.4mg./kg. body weight of PAM and %pg./kg. body weight of actinomycin D were used in the perfusion of the lower extremity in patients, and 5mg.//kg. body weight of PAM and the same dosage of actinomycin D for the pelvic perfusions. In eleven patients there was noted a regression of the tumor along with the disappearance of pain and symptomatic difficulties referable to the tumor itself.
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The effect of various antileukemic and anticancer agents was further analyzed by Ohashi (1960); the potentiation of radiation by perfusion of various compounds by Knock (1961). Treatment of neoplasms by Whitmore ( 1961); chemotherapy of malignant disseases in children were examined by Colebatch (1960a). Disintegration of tumors in plants by a Streptomyces was studied by Krassilnikov and Koveshnikov ( 1962).
D. ANTIVIRALAGENTS Among the antiviral agents, mention should be made of the following: Cephalomycin was described as an antiviral agent (Onuma, 1960). Mutomycin, as an antiviral agent, was studied by Blyumberg and Ryabova ( 1962). Myxoviromycin was studied by Horigome ( 1959). Nitromycins isolated in antiviral screening programs by Osato et al. (1960). Olivomycin was studied extensively on transplantable tumors (Mayevsky et al., 1962; Chorin et al., 1962), as well as clinically (Kuchkarev, 1962). Polymycin, belonging to the streptothricins and said to be an antiviral agent, was described by Hermanova and Savelieva ( 1961). Quinomycin (Yoshida et al., 1961) has been used as a prophylactic agent for poliomyelitis in mice (Tsunoda, 1962). Rutilantin has been shown to possess antiphage activity (Asheshov and Gordon, 1961). Violarin B, produced by S. uioluceus, was described by Trakhtenberg et al. (1961).It was compared with antibiotic pigments described in the literature, namely mycetin A and rhodomycin. Violarin M was found to be very close to violarin B; it possesses antibacterial activity and an antiviral activity with respect to the influenza virus in chick embryo experiments and smallpox vaccine virus in vitro.
IV. Mode of Action and Utilization of Antibiotics A. MODEOF ACTIONOF CFXTAIN ANTIBIOTICS The mode of action of different antibiotics has been receiving ever-increasing attention. The inhibition of protein synthesis by antibiotics has been reviewed by Hahn (1960) and by Sazikin
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(1961); effect on amino acid metabolism by Tirunarayanan et al. (1962); on cell wall synthesis by Strominger (1960) and Park ( 1960). Transduction of drug resistance by Watanabe and Fukasawa (1961); sensitivity of Salmonella gallinarum to different antibiotics by Saurat and Lautie (1959); of Clostridium by Bittner et al. (1961); inhibition of growth of bacterial and yeast protoplasts ( Shockman and Lampen, 1962).Kersten and Kersten ( 1962) studied inhibition of cell division in Bacillus subtilis of actinomycin C by sublethal doses; the cells increased in size and length, the production of ribonucleic acid and protein being partially blocked while DNA formation increased up to 100% over the control; the formation of actinomycin-DNA complex was suggested. Ascosin’s mode of action was discussed by Ramachandran et al. (1961); that of filipin by Gottlieb et al. (1961). A comparative study of growth inhibition of Nocardia crassa by nystatin and amphotericin B was made by Kinsky (1962). When added to 48-hour mycelial mats grown in the absence of antibiotics, the mats rapidly lost weight. This was specific for polyene antibiotics; this was observed with candidin and filipin, but not with cycloheximide or viridin. An early and specific effect of polyene antibiotics is a permeability change resulting in loss of cell contents. The mode of action of chloramphenicol was studied by Doudney and Haas (1960), Dagley and Sykes (1960), Witkin and Theil (1960), Levine and Curtiss (1960); on bacteriophage by Thomas (1959); on phosphatases by Korotyaev ( 1962). The effect of chloramphenicol and streptomycin in inducing transformation in Haemophilus influenxae, to give rise to strains possessing resistance or some other property was examined by Goodgal (1959). According to Sypherd et al. ( 1962), chloramphenicol inhibits preferentially the inducible enzymes. Hurwitz and Rosano ( 1962) have shown that when chloramphenicol was added to sensitive Escherichia coli cells at the same time as streptomycin, the lethal effect of the latter was prevented; if the cells received a prior exposure to streptomycin, the bacteria were susceptible to killing by streptomycin in the presence of chloramphenicol. Evidence was presented against the formation of a leaking permeability barrier as being the primary cause of death of cells exposed to streptomycin. The action of cycloheximide upon Fomes annosus was studied by Gundersen ( 1962*).
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The action of erythromycin on flagellates was studied by Ebringer ( 1961), its bleaching effect on Euglena gracilis by Ebringer (1962). Resistance of pneumococcus to erythromycin by Ravin and Iyer (1961). Mikamycin was found to be similar to chloramphenicol in its effect upon protein and nucleic acid metabolism; different steps of such metabolism were attacked, however (Yamaguchi, 1961). Mitomycin C was shown to inhibit in nonkilling doses the transformation of pneumococcus to streptomycin resistance, thus curing a fraction of the bacteria of the effect of the DNA which they had fixed. It was suggested that transforming DNA after its fixation must undergo a modification to attain a genetically active state; this step in transformation is sensitive to mitomycin C (Balassa, 1962). Neomycin affects various bacterial enzymes (Moroz, 1962); its effect on the formation of additional compounds was studied by Hein (1962). Polyene antibiotics were studied by Kinsky (196l), Drouhet et al. ( 1960), Ramachandran ( 1961), Gottlieb and Ramachandran (1961). Scholz et al. (1959) found that the effect of nystatin on yeast resulted in an increase in the content of hexose-phosphates and a decrease of inorganic phosphate; there was an increase in the production of triosephosphate, pyruvate, and malate; permeability of the cell membrane was affected within a few minutes after the addition of the antibiotic. Trichomycin was shown to exert a considerable inhibitory effect upon the deaniino-oxidation of various L-amino acids by Candida alhicans in the concentrations below the minimal effective amount necessary in fungistasis (Tsukahara, 1960); the oxidation of Dalanine and D-serine was completely or nearly completely inhibited by the antibiotic. It was suggested that the antibiotic may act on the flavin enzyme system which is directly connected with oxygen. Riboflavin arrests to some extent the inhibitory action of trichomycin on the oxidation of D- and L-amino acids when the compound is added simultaneously or beforehand, thus neutralizing the inhibitory action of trichomycin on the respiratory metabolism of C . albicans (Tsukahara, 1961). According to Marini (1961), the inhibition of yeast glycolysis by polyene antibiotics with a high number of C (nystatin, amphotericin A and B, candidin, candicidin) can be reversed by adding
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K + or NH4+.This compensated for the loss due to the action of the antibiotics in causing damage to the cell membrane and subsequent leakage, and restored metabolic activity. Bile salts inhibit the anticandidal activity of amphotericin B but not the antibacterial activity of neomycin (Schneierson et al., 1962). Puromycin action was studied by Bosch and Bloemendal ( 1961 ) , Streptomycin action continues to receive wide attention (Zabos, 1960; Niitani, 1960; Miyashita, 1959; Petrovskaia, 1960; Ando, 1960; Imsenecki and Petrova, 1960; Erdos et al., 1960; Hurwitz d al., 1962; Spotts and Stanier, 1961; Spotts, 1962). Katagiri et al. (1961) suggested that a site of action of dihydrostreptomycin upon the susceptible cells of E . coli involve, at least as one mechanism, its influence on a-ketoglutarate metabolism. The interaction of streptomycin with deoxyribonucleic acid was studied by Cohen and Lichtenstein ( 1960). The emergence of streptomycin-resistant bacteria due to anaerobiosis was studied by Farkas-Himsley (1961 ); the physiology of streptomycin-dependent bacteria by Wang et al. ( 1958), Engelberg and Artman (1961). The effect of streptomycin upon the growth of the plasmodium of Physarum polycepthalum by Lazo (1960); its mutagenic effect in Chlamydomonus by Sager and Tsubo (1962); its effect on the greening and biosynthesis in Euglena gracilis by Kirk (1962). The effect of streptomycin and pimaricin on Pythium by Hine (1962). Hancock (1961) reported that the reduction in oxidative activity of Escherichia coZi is parallel to the decrease in growth rate; protoplasts prepared from streptomycin-inhibited organisms showed normal stability and had internal osmotic pressures which differed only slightly from those of the normal cells. These results were interpreted as suggesting that it is unlikely that there was any general breakdown of the permeability barrier during inhibition of growth by streptomycin. According to Bragg and Polglase ( 1962), a streptomycin-dependent strain of E . coli produced large amounts of L-valine, while only trace amounts of this amino acid were produced by streptomycinsensitive strains. Sensitive and dependent mutants also differed in the production by the latter of lactic acid when the gas phase was changed from air to nitrogen. Resistant cultures grown in antibioticfree medium were similar to sensitive cultures, but when strepto-
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mycin was added, the resistant organism produced lactic and pyruvic acids; pyruvate reactions were considered of significance in the mechanism of action of streptomycin. The synergistic action of streptomycin with other antibiotics on Brucelln abortus was discussed by Richardson and Holt ( 1962). Treatment of growing E. coli with penicillin hastened subsequent killing of these cells by streptomycin (Plotz and Davis, 1962). Brief treatment with streptomycin failed to affect subsequent killing by penicillin. The synergism of penicillin with streptomycin was found to depend on the damaging effect of penicillin on the cell membrane, thus promoting further damage by streptomycin and increasing its subsequent access to intracellular sites. According to Tsukamura ( 1961a), streptomycin-dependent strains of Mycobacterium are able to synthesize RNA and protein, and reach the lag phase in the absence of streptomycin, but they cannot reach the logarithmic growth phase and cannot synthesize DNA; in the absence of streptomycin there was an abnormal increase of permeability for sulfate in cell membrane and no cord formation; there was no increase in permeability for phosphate; there was also a change in the growth rate and in the colony morphology in the absence of streptomycin. Speyer et al. (1962) found that streptomycin decreased the polyuridylic acid-dependent incorporation of C14-Iabeled phenylalanine into acid-insoluble products in a cell-free system containing ribosomes of streptomycin-sensitive E . coli and supernatant of either sensitive or resistant cells; ribosomes of streptomycin-resistant E . coli and supernatant of either resistant or sensitive cells showed no inhibition. Streptomycin was found to interfere with ribosomal function in sensitive bacteria, the ribosomes thus being the site of streptomycin sensitivity. D. Perry and Slade (1962) studied the phenomena of transformation to streptomycin resistance of various streptococci by deoxyribonucleic acid (DNA) extracted from streptomycin-resistant streptococci. DNA was incorporated from both homologous and heterologous groups and strains. Certain strains within a group served as DNA donors but not as DNA recipients, and certain strains served as DNA donors for some strains but not for others. Not all strains were transformable within a group, the source of DNA and the DNA recipient strain used being important. Radioisotope studies with P32-labeledDNA showed that nontransformable strains
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incorporated amounts of DNA comparable to the transformable strains. Landman and Burchard (1962) found that under conditions of streptomycin starvation, streptomycin-dependent salmonellae were inhibited in their ability to form septa, and grew out into long filaments. Resupplementation with streptomycin resulted in synchronous division. At low streptomycin levels, 95% of the survivors convert spontaneously to stable L forms, a growth form which transmits the septation disability to its progeny indefinitely. It was concluded that the requirement for streptomycin for septation is a primary one, and that the site of activity of streptomycin is at the bacterial membrane. Klein and Pramer (1961, 1962#)studied further the influence of nutritional and environmental factors on the dissimilation of streptomycin by a pseudomonad isolated from soil. The three constituent moieties of the molecule of streptomycin underwent simultaneous transformation. Washed cell suspensions of the bacteria were capable of oxidation of the various forms of streptomycin, but cellfree sonic extracts did not oxidize streptomycin or any of the derivatives or degradation products of the antibiotic. More than 90% of the amidine-nitrogen of the antibiotic was recovered as urea-nitrogen in culture filtrates; hydrolysis of the guanido groups of the streptidine moiety of the antibiotic was thus indicated, the site of action being the bond between the amidine group and the secondary amino-nitrogen of each guanido group, giving urea and streptamine as the products of the reactions. The effect of streptomycin and other antibiotics on oxygen uptake was found (Fedorov and Segi, 1961) to be due to inhibition of dehydrogenases. These antibiotics were believed to inhibit enzymes responsible for the synthesis of cell substance, which does not result in death of the bacteria, since activity is restored upon transfer of the bacteria to an antibiotic-free medium. Tetracycline-resistant strains of Proteus mirabilis were studied by Guillaume et al. (1961). Vancomycin activity was studied by Reynolds (1961, 1962). The effect of Streptomyces enzymes on the production of yeast protoplasts was studied by Garcia-Mendoza and Villanueva ( 1962); the amidation of aminobenzoic and p-aminosalicylic acids by Thrum and Bocker (1962).
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The development of resistance of various pathogenic bacteria to specific antibiotics has resulted in serious clinical problems. It has been largely responsible for the continuous search for new antibiotics and other antimicrobial agents that would tend to replace or supplement other agents that are no longer active in particular cases. Antagonism among antibiotics was examined in detail by Manten and Meyerman-Wise ( 1962).
B. UTILIZATIONOF ANTEUOTICSIN CLINICAL MEDICINE Antibiotics continue to occupy an important, if not a leading, place in the practice of medicine (Garrod, 1960). This is true particularly of the treatment of infectious diseases caused by bacteria and other microorganisms. Waisbren and Strelitzer (1960) analyzed this rating of antibiotics on the basis of clinical usage. They considered their distribution within the body, toxicity, routes of administration, allergenicity, and many other factors which enter into consideration for their proper evaluation, notably their in vitro activity. The bacteria appear to be constantly changing in regard to antibiotic susceptibility, thus suggesting the necessity of continued investigations. It frequently becomes necessary to change the antibiotic employed; an agent that is not paired with the antibiotic that has already been given is considered. The possibility to select for trial combinations of agents that show the least amount of relationships has been suggested. Cameron and Warner (1961) made up a chart (Table 11) giving the drug of choice and the alternative drugs for various diseases. The effect of antibiotic combinations on Staph. uureus was examined by Tate (1960). Panero and Gambassini (1962) tested the sensitivity of fifty strains of Pseudwmonas aeruginosa to seventeen antibiotics and three chemical chemotherapeutics. The highest percentage of sensitive strains was obtained for neomycin and for colimycin. The frequency of strains resistant to other substances varied from 55.1% for erythromycin and 60% for paromomycin to 100% for most of them. Neomycin is recommended for affections of the gastroenteric tract and localized forms; and colimycin for the therapy of other morbid localizations. Pollard and Tanami (1961) tested six antibiotics on trachoma organism in human tissue cells. Chlortetracycline hydrochloride
IAHLL 11
ANTIBIOTICSOF CHOICEAND ALTERNATIVESIN Disease Pneumonia Meningitis
Venereal disease
Cram-negative rod infection
hlycosis
“Resistant” staphylococcal infections: Enterocolitis Septicemia Bacterial endocarditis
Tuberculosis
Agent
Diplococcus pneumoniae Klebsiella pneumoniue Diplococcus pneumoniae Neisserin meningitidis Hernophilus influenme Treponemu pallidurn Neisseriu gonorrhoeae M . lymphogranulomatis Salmonelk Shigella Bnrcella Actinomyce P Cryptococcus neoformans Triehophyton
THE
TREATMENT OF VARIOUSINFECTIOUS DISEASES~
Drug(s) of choice
Alternatives
Tetracycline Penicillin Streptomycin-chloramphenicol Penicillin, penicillinNot established erythromycin, tetracycline Sulfonamides Penicillin Sulf adiazine-chloramphenicol Tetracyclines Chloramphenicol, tetracycline Penicillin Penicillin Tetracyclines Tetracycline Chloramphenicol Tetracycline-chloramphenicol Tetracyclines Chloramphenicol Tetracyclines Tetracycline-streptomycin Penicillin Sulfonamides Amphotericin B Griseofulvin
-
b E!
-
m
b
3 b
x
e:
i
Staphylococcus pyogenes Staphylococcus pyogencs
Neomycin M ethicillin
Streptococcus ljiridans Streptococcus faecalis Staphylococcus pyogenes
Penicillin-streptomycin Penicillin-streptomycin Methicillh
Xfycobacterium tuberculosis Streptomycin-PAS-INH
Cameron and Warner ( 1961).
cr:
Bacitracin, vancomycin Erythromycin group, novobiocin, ristocetin
-
Penicillin-neomycin, ristocetin Vancomycin, ristocetin, bacitracin Viomycin, pyrazinamide, cvcloserine
5
r!
m
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SELMAN A. WAKSMAN
and tylosin tartrate were found to interrupt the cytochemical sequence which reflected maturation of the organism. Penicillin and sulfanilamide only delayed maturation; streptomycin had no effect. They described a cytochemical procedure for the detection of trachomastatic drugs. Applebaum ( 1961) examined the highly successful results obtained in the treatment of the more common forms of bacterial meningitis. Meningococcic infections are controlled by the sulffonamides; pneumococcic and streptococcic meningitis by penicillin; meningitis due to Haemophilus influenzae by streptomycin, chloramphenicol, and the tetracyclines; staphylococcic meningitis is difficult to treat, because of the high incidence of penicillin-resistant staphylococci, treatment depending on the results of sensitivity tests; tuberculous meningitis is treated by a combination of isoniazid, streptomycin, and paraaminosalicylic acid. Retropharyngeal nocardial infection was controlled by streptomycin and tetracycline ( Adams, 1961) . Kaye et al. (1961) found that the great majority of Proteus strains isolated from patients belong to P. niirabilis. They were most sensitive to kanamycin, the inhibitory effect being increased by its combined use with penicillin. A combination of penicillin with dihydrostreptomycin also showed a synergistic effect. Chloramphenicol, however, showed an antagonistic effect upon both penicillin and kanamycin. Nasal carriers of staphylococci belonging to the same phage type can be controlled by combinations of neomycin with bacitracin or gramicidin (Jarvis and Wigley, 1961). The sensitivities of 621 strains of gram-negative bacteria isolated from patients with clinical infections were assayed by Petersdorf et al. (1961) against a variety of antibiotics. Chloramphenicol proved to be the most effective agent against Escherichia coli. Neomycin or kanamycin against Klebsiellu, polymyxin B against Pseudomonas, and penicillin against Proteus mirabilis. Dameshek (1960) issued a new warning about the use of chloramphenicol. Popken et al. (1960) evaluated 17 antibiotics and one combination of equal parts of two antibiotics against a Mycoplusma gallinamm infection in chick embryos. These antibiotics were classified into the following four groups: Group I. Those that have a median effective dose of less than 0.2 mg./egg. Here belong carbomycin, leucomycin, erythromycin,
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spiramycin, a combination of equal parts of griseoviridin and viridogrisein, and 6-demethyl-chlortetracycline. Group 11. Those that have a median effective dose of 0.2 to 0.4 mg./egg. Here belong tetracycline, hydroxytetracycline, 6-demethyltetracycline, viridogrisein, griseoviridin, oleandomycin, and chlortetracycline. Group 111. Those that have a median effective dose of more than 1mg./egg; these include novobiocin and streptomycin. Group IV. The last group included chloramphenicol and neomycin. No valid calculation of a median dose for chloramphenicol could be made due to lack of data. Neomycin was inactive a t a maximum tolerated dose. McAdams (1960) studied the role of antibiotics in surgical procedures on the colon and came to the conclusion that sterilization of the colon is not essential to good results following colon surgery. When all coliform organisms were eliminated preoperatively in patients prepared with a combination of bacitracin and neomycin, postoperative morbidity was increased. With suppression of coliform organisms, pseudomonas and proteus organisms tend to become pathogenic alone or in combination with others. When sulfathalidine and streptomycin were used orally in preoperative preparation of a group of twenty-five patients, better results were obtained than with sulfathalidine and neomycin administered to a group of eighty-me patients. The oral streptomycin gave certain potential hazards and led to its discontinuance. Certain antibiotics given as a means of prophylaxis increased their resistance from in uitro to in uiuo, thus causing complications postoperatively. The conclusion was reached that lowered incidence of incisional infection was due to improved technique and not to the effect of antimicrobial agents. The extensive use of antibiotics against gonococci led to progressive decrease of sensitivity of the organisms to penicillin and streptomycin (Roiron et al., 1961). Out of 327 strains studied, 72.4% were sensitive to 0.05 g ./ml. of penicillin. Three hundred forty-one strains, or 75.6%, were sensitive to 50 pg./ml. of streptomycin, recovery being obtained in 73.14% of the cases; 22.9% of the strains were resistant to a concentration of lOOOp.g./ml. streptomycin and recovery was observed in 6.1% of the cases only. Strains resistant to streptomycin were less sensitive to penicillin
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WAKSMAN
than nonselected strains (57.9% in the first case, 72.4% in the second). All strains were sensitive to tetracycline and to spiramycin. Akiba et nl. (1960) studied the induction of resistance to antibiotics of drug-sensitive Shigelh strains with cultures of an E. coli strain with multiple resistance. Transfer of resistance occurred without accompanying unselected markers; induced-resistance clones maintained the same biochemical and serological characteristics as those belonging to the sensitive recipient strain. Sensitivity of different strains of Pasteurella to antibiotics was studied by Hejzlar and Lukas (1962). Bliss and Alter (1962) studied the in vitro and in vivo changes of resistance to streptomycin of a sensitive strain of Staphylococcus aureus. Although a rise in the minimal inhibitory concentration of streptomycin in broth was seen infrequently, over half of the cultures from treated mice showed a shift in the population pattern toward greater tolerance for the antibiotic, a phenomenon that did not occur in staphylococci after exposure in wiuo to penicillin or chlortetracycline. The level of population tolerance for streptomycin was believed to be related to the concentration of the antibiotic in the tissues of the host, strains already possessed of a measure of resistance failing to gain in resistance when exposed to streptomycin in viuo. No significant changes in the prothrombin times occurred during administration of neomycin and nystatin, despite the fact that the first causes a disappearance of coliform and urease-positive bacteria with a concomitant increase in yeasts, and that both antibiotics cause a disappearance of yeasts as well as of coliform and ureasepositive bacteria (Weiss et al., 1961). Lullmann and Reuter (1960) demonstrated that neomycin, polymyxin B, streptomycin, and kanamycin inhibit neuromuscular transmission. Acetylcholine contraction of the denervated rat diaphragm could be used to demonstrate the membrane stabilizing action of the antibiotics. Additive inhibition by d-tubocurarine and antibiotics was demonstrated in the intact rabbit by measuring the head-drop dose. Intrabronchial administration of neomycin was discussed by Lorian ( 1961). Candida complications of antibiotic therapy were reviewed by Kashkin et al. (1961). The effect of antibiotics on intracellular Salmonella typhosa was discussed by Hopps et al. (1961) and by Showacre et al. (1961). The therapeutic effective-
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ness of different forms of neomycin and related compounds was discussed by Shapovalova ( 1962). Clauberg ( 1960) emphasized that clinical examples stress the destruction of organisms caused by the application of chemotherapeutic agents. This removes the bacterial stimulus necessary for the formation of specific resistance. As a result of this, immunization is largely or totally retarded; reparative actions serving to overcome a disease necessarily become irregular. According to Dobias and Hazen (1961), nystatin has found its chief use in the treatment of various forms of moniliasis. Infections due to Aspergillus fumigatus and isolated lesions of histoplasmosis also respond to nystatin. Its use in deep mycoses is limited, due to negligible absorption from the gastrointestinal tract and the unsuitability of parenteral routes of administration. Seabury ( 1961) reported that treatment of thirty-nine patients by intravenous amphotericin B showed a therapeutic effect upon blastomycosis, cryptococcosis, histoplasmosis, and sporotrichosis. The ultimate place of this antibiotic in the treatment of systemic coccidioidomycosis and candidiasis is still undetermined. Amphotericin B is suitable for intravenous, intrathecal, and local instillation. The side effects are many and sometimes serious. Severe side effects are usually ameliorated by hydrocortisone or prednisolone. Renal toxicity is accomplished by increased excretion of potassium and by reduced urea clearance. Optimal doses of streptomycin for pulmonary tuberculosis were analyzed by Johnston et al. (1!361). The development of resistant organisms in pulmonary tuberculosis by the use of isoniazid and streptomycin has stimulated the search for other effective antituberculosis drugs. Various secondary drugs, including cycloserine, pyrazinamide, kanamycin, viomycin, and tetracycline, have become available for the retreatment of isoniazid- and streptomycin-resistant cases (T. L. Petty and Mitchell, 1962). Ethionamide, also called thioamide, has been recently added. It is bactericidal in vitro and in vivo against isoniazid- and streptomycinresistant and susceptible organisms. Cross resistance occurs only with the thiosemicarbazones. Ethionamide, in combination with other secondary drugs, may be effective in the retreatment of isoniazid-resistant pulmonary tuberculosis. The use of cycloserine in the treatment of lung and urinary tuberculosis was discussed by Steinitz (1961).
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The phenomena of ototoxicity by dihydrostreptomycin and its use in combination with other antimicrobial agents were discussed by Dholakia and Weinstein (1962). Principles of antiviral chemotherapy were discussed by Tamm ( 1962). Weinstein (1962) warned against the misuse and abuse of antimicrobial agents. UPON ANIMALDISEASES C. EFFECTOF ANTIBIOTICS An extensive literature is rapidly accumulating on the use of antibiotics in the treatment of animal infections. A recent summary of the treatment of poultry for Salmonella typhi-murium infections was published by Hobbs et al. (1960). Mycoplasma gallinarum infections of chickens and turkeys can be treated with tylosin ( Barnes et al., 1960). Blobel and Burch ( 1961) measured the concentrations of dihydrostreptomycin in milk secretions of cow’s given dihydrostreptomycin intramuscularly; the drug was demonstrable in the milk of all cows. At any of the various levels used, dihydrostreptomycin was eliminated from the udder within 48 hours after it was given. The intravenous administration of chlortetracycline also resulted in the excretion of detectable amounts of chlortetracycline through the milk. The levels of this antibiotic persisted up to 48 hours after the intravenous administration.
D. ANTIBIOTICS AND ANIMALGROWTH The exact mode by which antibiotics stimulate growth in animals and in man is still not clearly understood. This action of antibiotics is not entirely due to the changes in intestinal bacterial flora or to the bactericidal and bacteriostatic actions of the antibiotics. The amounts used for supplemental feeding of animals are very minute as compared to the usual dosages considered necessary for therapy in human or animal infectious disease (Ozawa, 1955; Loughlin and Mullin, 1955; Loughlin et al., 1958; Tronstein, 1961; Blake, 1961). Boller et al. (1962) reported that the administration of 50 mg. of chlortetracycline daily to sufferers from various gastric conditions resulted in consistent gains in weight. This was due to improved utilization of protein when gastric secretion was defective.
ACTINOMYCETES AND THEIR ANTIBIOTICS
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E. EFFECTOF ANTIBIOTICSUPON PLANTGROWTH AND PLANTDISEASES Antibiotics are finding extensive uses in sprays by horticulturists; the minute amounts appear to be adequate to increase the growth of plants, such as roses, or for eliminating various bacterial and certain fungus diseases of plants. The effect of antibiotics in the control of plant diseases has been summarized by Pridham (1961).Black rot of crucifers can be controlled by treating seeds with antibiotics (Klisiewicz and Pound, 1961). The effect of antibiotics on tree diseases was reviewed by Wicker and Leaphart ( 1961). Shimomura and Hirai (1959) tested a number of antibiotics for their effect on the multiplication of tobacco mosaic virus. About 30 preparations were tested, some antibiotics showed 20% or more inhibition to virus multiplication under experimental conditions. These were actidione, naramycin, fermicidin, chlortetracycline, tetracycline, dextromycin, kanamycin, and mitomycin C.
F. ANTIBIOTICSIN FOODPRESERVATION The extensive use of antibiotics in preventing bacterial contamination of foodstuffs has now become a general practice. An attempt has also been made to use certain polyenes in order to prevent fungus contaminations. The important aspects of such uses is to be able to destroy the antibiotics prior to the consumption of the food. Hence, only heat unstable antibiotics as tetracyclines and polyenes can be employed. The problem of the action of antibiotics at low temperatures becomes of great importance ( Arpai, 1961) . REFERENCES Ahnratani, I., Kashii, K., Minoina, K., Terai, K., Imankhi, Y., and Sugai, T. (1959). J Osaka City Med. Center 8, 91-98. Adams, J. N., and McClung, N. M. (1962a). J. Gen. Microhiol. 28, 231-241. Adams, J. N., and McClung, N. M. (196213). J. Bucterwl. 84, 206-216. Adams, W. S. (1961). J. Laryngol. Otol. 75, 433-440. Advisory Committee on Med. Lab. Techniques (1960). J. 'Med. Lab. Tech. 17, 133-143. Agre, N. S. (1961). Mikrobiologiya 30, 414-417. Agre, N. S., and Orleansky, V. K (1962). Mikrobiobgiya 31, 95-102. Ahmad, N., Paul, B. B., Majumdar, S . , Islam, M. F., and Ahmad, K. (1958). Ann. Biochem. Exptl. Med. (Calcutta) 18, 17-20.
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Akiba, T., Koyama, K., Ishiki, Y., Kimura, S., and Fukushima, T. (1960). Japan 1. Microbiol. 4, 219-227. Alikhanian, S. I. (1962). Advan. Appl. Mkrobiol. 4, 1-50. Alikhanian, S. I., and Borisova, L. N. ( 1961). J. Gen. Microbiol. 26, 19-28. Alikhanian, S. I., and Tetejatnik, A. F. (1962). M ~ k r o b w ~ g ~31, y u 262-264. Alikhanian, S. I., Iljina, T. S., and Lomovskaya, N. D. (1960). Nature 188, 245-246. Alikhanian, S. I., Mindlin, S. Z., and Zaitseva, Z. M. (1961). Dokl. Akud. Nauk S.S.S.R. 136,468-471. Ando, K. (1960). Nagoya J. Med. Sci. 23, 39-51. Andriiuk, K. I. (1960). Mikrobiol. Zh. Akad. Nauk Ukr. S.S.R. 22, 27-34. Anzai, K., and Suzuki, S. (1961). J. Antibiotics (Tokyo) A14, 253. Anzai, K., Okuma, K., Nagatsu, J., and Suzuki, S. (1962). 1. Antibiotics (Tokyo) A15, 110-111. Applebaum, E. ( 1961 ) . Arch. Pedht. 78, 335-348. Arai, T.,and Kurodn, S. (1959). Symp. Tuxon. Actinomycetes, Tokyo, 1959 pp. 22-30. Arai, T., and Kuroda, S. (1962). J. Bacterwl. 83, 924. Arai, T., Kuroda, S., and Suenaga, T. (1960). Chiba Duiguku Fuliui Kenkyujo Hokoku 13, 32-38. Arai, T., Koyama, Y., Suenaga, T., and Honda, H. (1962a). J. Antibiotics (Tokyo) A15, 231-232. Arai, T., Kuroda, S., and Ito, M. (1962b). J. Bucteriol. 83, 20-26. Arcamone, F., Bizioli, I?., Canevazzi, G., and Grein, A. (1958). German Patent 1,039,198. Arcamone, F., DiMarco, A , , Gaetani, hl., and Scotti, T. (1961). Ciorn. Microbiol. 9, 83-90. Arishima, M., and Sakanioto, J. (1961). Japanese Patent 13,897; Chem. Abstr. 55, 15826c ( 1961). Arpai, J. (1961). Arch. Mikrobiol. 39, 195-208. Artamonova, 0. I., and Krassilnikov, N. A. (1960). Tr. Inst. Mikrobiol., Akud. Nauk S.S.S.R. 8, 275-337. Asheshov, I. N., and Gordon, J. J. (1961). Biochem. I . 81, 101-104, Awa, A. ( 1961). Gunn 52, 49-55. Bachmann, E.,and Zahner, H. (1961). Arch. Mikrobiol. 38, 326-338. Back, N.,Shields, R. R., and Munson, A. E. (1961). Antibiot. Cheniotherupy 11, 652-660. Bailey, W. C., Holadny, W. J., Kontras, S . B., and Clatworthy, TV. IV~,Jr. (1961). A.M.A. Arch. Surg. 82, 943-949 Balassa, G. ( 1962). Ann. Inst. Pasteur 102, 547-555. Baldacci, E. (1961). Advan. Appl. Microbial. 3, 257-278. Baldacci, E.,and Locci, R. (1961). Ann. Microbiol. Enzimol. 11, 19-30. Balitskaya, A. K. (1961). Tr. Inst. Mikrobiol. i Virusol., Akad. Nauk Kazakh. S.S.R. 4. 14-18. Balitskaya, A. K., Vetlugina, L. A,, and Sartbaeva, A. ( 1962). Antibiotiki 7, 99103. Bdl, S. ( 1961 ) , Canadian Patent 613,514.
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Fusel Oil A. DINSMOOR WEBBAND
JOHN
L. INCRAHAM
Department of Viticulture and Enology and the Department of Bacteriology, University of California, Davis, California
I. History ................................................ 11. Characteristics of Fusel Oil Components .................... 111. Analytical Methods ..................................... A. Distillation Methods ................................. B. Chromatographic Methods ............................ C. Chemical Methods .................................. D. Other Analytical Methods ............................ IV. Results of Fusel Oil Analysis ............................. V. Biosynthesis of Fusel Oil Components ...................... A. Fusel Oil Formation from Amino Acids . . . . . . . . . . . . . . . . . B. Fusel Oil Formation from Glucose .................... C. n-Propyl and n-Butyl Alcohols ........................ D. Factors Affecting Fusel Oil Formation . . . . . . . . . . . . . . . . . . References .............................................
317 318 322 323 325 329 330 331 338 338 341 343 346 350
All yeast-fermented aqueous alcoholic mixtures contain small amounts of materials which will separate into a second, oily layer upon removal of most of the ethanol. This oily phase is usually obtained by adding water to a small side stream drawn from a point below the ethanol draw point of the rectification column of a continuous distillation unit. It may also be obtained from residues of simple distillations and from wines or beers by extractions with solvents which are immiscible with the aqueous phase. The separated mixture is designated fusel oil, from the old German word, fousel, meaning bad spirit.
1.
History
Scheele (1785) described the separation of a second phase from a grain spirit of low alcohol content during severely cold weather which he characterized as being “nauseous-smelling” and white in color. This is perhaps the earliest recorded occurrence of fusel oil, although the term “fusel” was not used in his description. He reported that the addition of this material to a fine brandy gave it the unpleasant smell of ordinary grain spirit. J. B. Dumas (1834) analyzed a sample of fousel obtained from 317
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the distillate of fermented potatoes. After repeated water washings and fractional distillations, he obtained a sample boiling from 130" to 132°C. which had the empirical formula, C5H120. But it was Cahours (1839) who recognized that Dumas' compound was "hydrate of amylene" or amyl alcohol, a heavier member of the homologous series which includes methanol and ethanol. Dumas and Stas ( 1840) confirmed Cahours' identification. Pasteur (1855) credits J. B. Biot with the discovery that the fusel oil boiling from 130" to 132°C. is optically active. Since the optical activity of fusel oils from different sources varied, Pasteur concluded that at least two substances were present, one optically active and the other inactive, i.e., fusel oils of differing activities merely reflect the difference in proportions of the two substances. The determination of the structure of the amyl alcohols present in fusel oils resulted from the researches of many workers. Pedler ( 1868), Erlenmeyer and Hell (1871), and Kramer and Pinner (1870) all contributed to characterizing the fusel oil alcohols as isoamyl ( 3-methyl-l-butanol) and active amyl ( 2-methyl-l-butanol ) . Le Be1 (1873, 1876) developed methods for obtaining relatively pure active amyl fractions from the crude fusel oils. He also studied methods for synthesis of related compounds from active amyl alcohol. Somewhat earlier Wurtz (1852) became interested in the alcoholic substances present in fusel oils with boiling points between those of ethyl alcohol and the amyl alcohols. He isolated isobutyl alcohol and prepared a number of derivatives from it. One year later, Chancel (1853) isolated a fourth alcohol, normal propyl, from the fusel oil mixture thus completing the list of primary alcohols which are known today as the principal components of the mixture.
II. Characteristics of Fuse1 Oil Components Dumas (1834) observed that repeated water washings and fractional distillation yielded a fusel oil boiling from 130" to 132°C. with the empirical formula corresponding to amyl alcohol. Pasteur (1855) separated active amyl alcohol from isoamyl alcohol by fractional crystallization of the barium salts of their acid esters of sulfuric acid and reported the boiling point of active amyl alcohol to be between 127" and 128°C. at atmospheric pressure. The boiling
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point of the inactive isoamyl alcohol was reported to be 129°C. under the same conditions. Marckwald and McKenzie (1901) reported active amyl alcohol to boil at 128°C. More recent work by Terry (1960) establishes the boiling point of active amyl alcohol as 128.5"C. and that of isoamyl alcohol as 132.0"C. at atmospheric pressure. Of the other two principal alcohols of fusel oil, n-propyl is listed by Timmermans (1950) as boiling at 97.2"C., while isobutyl alcohol boils at 108.0"C. As Schiipphaus (1892) pointed out, separation of the four principal alcohols from a crude fusel oil by fractional distillation is more difficult than the properties of the pure compounds would indicate because water-alcohol azeotropes with similar boiling points are formed. Horsley (1952) recorded the alcohol-water binary azeotropes of interest in fuse1 oil distillation (Table I ) . InformaTABLE I WATERAZEOTROPES OF CERTAIN FUSELOIL COMPONENTS Azeotrope Alcohol
B.P., " C .
Wt. % water
Ethyl Isopropyl n-Propyl sec-Butyl Isobutyl n-Butyl Isoamyl n-Hexyl
78.17 80.3 87 87.5 89.8 92.7 95.2 97.8
4.0 12.6 28.3 27.3 33 42.5 49.6 75
tion concerning the active amyl alcohol-water azeotrope is not available but it is likely that the properties of this system are similar to those of the isoamyl alcohol-water system. Horsley lists no ternary azeotropes of water with two different alcohols. Bukala et al. (1961) extensively studied azeotropic systems which might be applicable to recovery of higher alcohols from the crude fusel oils obtained in fermented sulfite liquors from paper and pulp mills. A system was developed in which the benzene-ethanol azeotrope is used to isolate the higher aliphatic alcohols in anhydrous condition from the crude fusel oil. No separation of active from isoamyl alcohol is obtained. The formation of binary azeotropes between either isoamyl alco-
320
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
hol or active amyl alcohol and other substances was investigated by Terry (1960) in an attempt to find a system which would permit ready separation of the two amyl alcohols by fractional distillation. The attempt was not successful since in all cases investigated the boiling points of the azeotropes were closer than were the boiling points of the pure alcohols (Table 11). AZEOTROPESO F
TABLE I1 AcavE AMYL ALCOHOLS
ISOAMYL AND
Binary azeotropic mixtures
System n-Octane Active amyl alcohol Isoamyl alcohol Chlorobenzene Active amyl alcohol Isoamyl alcohol Ethylbenzene Active amyl alcohol Isoamyl alcohol Toluene Active amyl alcohol Isoamyl alcohol 2,2,5-Trimethylhexane Active amyl alcohof Isoamyl alcohol o-Fluorotoluene Active amyl alcohol Isoamyl alcohol Diisopropyl ketone Active amyl alcohol Isoamyl alcohol 2-Picoline Active amyl alcohol Isoamyl alcohol 2,6-Dimethylpiperidine Active amyl alcohol Isoamyl alcohol 1,2-Dimethylpiperidine Active amyl alcohol Isoamyl alcohol
B.P. of component ( "C.) 126 138.5 132.0 132
B.P. ( 760 mm. ) ( "C.)
Alcohol (wt. % )
Alcohol (mole %)
117.0 117.0
34 30
40 36
124.4 123.9
43 38
49 44
125.0 125.7
53 49
57 53
109.9 109.7
12 10
12 10
115.5 116.0
29 26
37 34
112.0 112.1
16 14
19 17
124.1 124.5
21 8
26 10
132.8 132.8
49 61
50 62
130.7 132.6
54 76
60 80
Undetermined 81
-
136
111
124
114
125
129
128
128 130.3 132.5
85
32 1
FUSEL OIL
The densities and refractive indexes of the alcohols of interest in fuse1 oils are shown in Table 1II.I TABLE 111 DENSITIES AND REFRACTIVE INDEXES OF FUSELOIL ALCOHOLS Alcohol Ethyl
Isopropyl n-Propyl sec-Butyl Isobutyl n-Butyl Isoamy1 Active amyl n-Hexvl
Density (gm./ml., 20°C.)
0.7893 0.7851 0.8035 4.8109,,, 0.8020 0.8096
0.8059, 0.8154,,. 0.8225,,.
Refractive index ( D , 20°C.)
1.3614 1.3775He y 1.38402,,, 1.3995,,. 1.3959 1.3970,,. 1.4048,,. 1.4088,,.
-
Optical rotary dispersion (rotation of plane-polarized light as a function of wave length) of purified active amyl alcohol was determined by Marckwald and McKenzie ( 1901) . Timmermans (1950) lists values obtained by others. The physical properties of mixtures of isoamyl and active amyl alcohols have been extensively studied. Hafslund and Lovell (1946) found that the density-composition plot was linear; however, Ikeda et al. (1956) found deviations from linearity, particularly in the mixtures of over 70% active amyl alcohol content. The refractive index-composition curve was also found to deviate from linearity for mixtures rich in active amyl alcohol ( Ikeda et d.,1956). Optical rotation has a linear relationship to composition of the two alcohols according to Marckwald and McKenzie ( 1901). Hafslund and Lovell (1946) were also able to fit their data for specific rotation as a function of composition to a straight line, as did Ikeda et al. (1956), using eleven different mixtures. These data have proven quite useful for rapid analyses of mixtures of the two alcohols, e.g., when following the progress of a distillation. The curves for density, optical rotation, and refractive index as a function of the composition of the active amyl-isoamyl mixture are shown in Fig. 1. 1 It is of historical interest that Pasteur (1855) detected the significant difference in density between isoamyl and active amyl alcohols and stated that the difference was nearly 0.01. Actually, the difference is 0.0095g./ml. or 1,165-hundredths of the value, Pasteur’s alcohols must have been very nearly pure.
32
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
0m !I 0
I0
20
30
40
50
60
70
80
90
100
% Active omyl
FIG.1. Per cent active amyl alcohol in isoamyl alcohol plotted against refractive index, density, and observed optical rotation. (From Ikeda et al., 1956).
111. Analytical Methods Methods which have been developed for analyses of fusel oil are in reality directed toward two different objectives. In one case the desired end is measurement of the total quantity of fusel oil present in an aqueous alcoholic mixture while in the second case the primary interest is in determining the relative amounts of the various components of the fusel oil mixture itself. In some cases, of course, the newer analytical methods have permitted determination of the exact amounts of each of the several fusel oil components actually present in a beverage, thus combining the two objectives. The earliest methods of fusel oil analysis depended upon the fact that the amyl alcohols, and to a lesser extent isobutyl alcohol, have limited solubility in cold aqueous solutions of relatively low ethanol content. The fusel oils were separated from the nonvolatile materials of beer or wine by a simple distillation followed by fractional distillation to remove the bulk of the ethanol. On cooling the fractional distillation residue, fusel oil separates as a second phase. Water solubility of the higher alcohols is decreased by the dissolution of salt in the aqueous phase, and this technique is employed in some of the earlier analytical methods. As the separated fusel
FUSEL OIL
323
oil phase was measured either volumetrically or gravimetrically, relatively large amounts of the wine or beer were required to obtain meaningful results. The solubility of the fusel oil alcohols in water-immiscible solvents such as carbon tetrachloride, chloroform, or petroleum ether has also been the basis for analytical procedures. In most methods of this type, fusel oil and ethanol are extracted from the preIiminary distillate and then the ethanol is removed by a subsequent extraction with saturated aqueous salt solutions. Since the partition coefficients of proply and isobutyl alcohols are similar to that of ethanol, this method, like those described above, tends to miss the lighter alcohols. The measurement thus becomes primarily a determination of the amyl alcohols. Variations of the basic method of volumetric determination of separated fusel oil are numerous. In one case the alcohols are oxidized to acids and estimated by titration while in another a quantitative acetylation is employed. Both methods have difficulties. Quantitative oxidation of alcohols to acids is not practically attainable. Determination of alcohols by acetylation is a rather complicated procedure. The uncertainties in both the oxidation and acetylation techniques can be lessened if the procedures are rigorously standardized. Calibration curves must be run and the composition of the fusel oils being measured cannot be widely variable. There are variations in the completeness of oxidation among the alcohols found in fusel oil mixtures as Lafon and Baraud (1960) have shown. Kepner and Webb (1954) have pointed out the necessity of careful control in the acetylation procedure. The secondary alcohol, 2-butanol, which is present in some fusel oils in minor amounts presents a problem in both procedures. It does not, of course, yield an equivalent amount of acid on oxidation, and it may not behave normally under the acetylation conditions employed for primary alcohols. A.
DISTILLATION
METHODS
Simple distillation is employed in the isolation of the fusel oil from the wine or beer mixture in nearly every analytical method. In the very earliest analyses, crude fractional distillations combined with water washings and chemical drying were used to isolate and determine the fusel oil. The technique employed could only approximate the quantities of amyl alcohols present, and the lower
324
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
molecular weight alcohols were completely lost. With the development of more efficient fractional distillation equipment good separations of propyl alcohol from isobutyl and isobutyl from the amyls has become possible. However, the problem of isolating small amounts of fusel oil from the large quantity of aqueous ethanol remains. There is no simple, quick method for isolating the fusel oil mixture from a wine or beer; neither distillation nor extraction is satisfactory in all respects. Perhaps the process which offers the best compromise between speed and completeness of fusel oil recovery is the following: Four or five liters of wine or beer are distilled slowly in an apparatus fitted with a Vigreaux column to obtain some fractionation. The portion distilling below the boiling point of ethanol is discarded since it contains esters, aldehydes, ethanol, and water only. The alcohol-water-higher alcohol fraction which distills next is collected; distillation is stopped only after the temperature at the head of the column has been at the boiling point of water for some time. This assures that all of the higher alcohol-water azeotropes have been collected. This fraction containing ethanol, water, and fusel oil is then redistilled using a fractionating column of fifty or more theoretical plates. The ethanol fraction is discarded and the distillation is continued until all of the higher alcohol-water azeotropes have been collected. The alcohols are extracted from the aqueous phase of the distillate with diethylether and the ether extract is dried over anhydrous magnesium sulfate which reduces the water content to less than 0.2% by weight (Webb et al., 1952). One part, by weight, of purified anhydrous ethanol is added to ten parts of the dry fusel oil-ether mixture. The resulting mixture is distilled through a high efficiency fractionating column to effect the analysis. The low boiling fractions ( ether, ethanol-water azeotrope, and ethanol ) are discarded, and the propyl, isobutyl, and mixed isoamyl and active amyl fractions are collected separately and weighed. The weights of the smaI1 fractions collected while the head temperature is changing between pure fractions may be divided evenly between the adjacent pure components. Good separation of the active amyl alcohol from isoamyl alcohol is not possible. Therefore, the two alcohols are collected and determined together. Their relative proportions may
FUSEL OIL
325
be determined by optical activity, density, or the refractive index measurements with reference to the curves of Ikeda et al. (Fig. 1 ) . In certain fusel oils small amounts of isopropyl alcohol, 2-butanol, n-butanol, n-pentanol, n-hexanol, and high boiling esters are found. In general, no one of these substances is present in amounts greater than about 5% by weight. The lower boiling alcohols are usually indicated by inflections in the temperature vs. quantity distillation curve while the n-amyl, n-hexyl, and ester portion remain in the distillation pot. Jensen and Rinne (1952) analyzed fusel oils from fifteen Finnish sulfite mills by means of fractional distillation through spinning band columns. Compositions were calculated from the distillation curves after an aliquot of each constant boiling fraction had been positively identified by means of its other physical properties. Ogata and Matsubara (1953) determined the composition of a fusel oil from fermented sweet potatoes by fractional distillation using the Widmer column. The separated fractions were identified by physical measurements and by formation of derivatives.
B. CHROMATOGRAPHIC METHODS Recently various chromatographic techniques have been used in fusel oil analysis. Liquid-liquid column partition chromatography using silicic acid as the stationary phase was adapted by White and Dryden ( 1948) to the analysis of the 3,5-dinitrobenzoate derivatives of alcohols. A fluorescent dye, adsorbed on the silicic acid provided a yellow background when viewed under UV radiation. Against this background the zones of 3,5-dinitrobenzoate appeared as black bands thus allowing ready separation of the various fractions. The White and Dryden method has certain intrinsic difficulties: Formation of the 3,5-dinitrobenzoate is very probably not quantitative, and isolation of the alcohols in an anhydrous condition prior to analysis, is required. Also, active amyl-3,5-dinitrobenzoate is not separable from isoamyl-3,5-dinitrobenzoate by this method. However, the technique is applicable to the estimation of relative amounts of the components of a mixture of primary alcohols since the degree of reaction of the various alcohols and 3,5-dinitrobenzoyl chloride is approximateIy the same. It is likely that liquid-liquid partition systems could be developed for a number of solid derivatives of alcohols such as the p-phen-
326
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
ylazophenylurethanes. Derivatives carrying the azo linkage have the advantage of being colored and hence more easily visible on columns than are the 3,5-dinitrobenzoates under UV light. Also the alcohols could be oxidized to acids and separated as the pphenylazophenacyl derivatives on silicic acid columns. Such techniques however multiply the possibilities of error, since the number of operations is increased. It is also possible that a mixture of fusel oil alcohols could be separated directly on a silicic acid column carrying a polyol as absorbing phase by using a less polar moving phase. An instrumental detection system (e.g., UV absorption or refractive index, etc.) would be required to monitor the effluent stream. Paper chromatographic techniques have been applied to the determination of the alcohols by several investigators but in every case there are reasons for preferring other techniques. Daghetta (1956),for instance, extracted the fusel oil with chloroform, oxidized the alcohols to acids and chromatographed the acids as their ammonium salts using butanol. Sundt and Winter (1957) prepared the 3,5-dinitrobenzoates of the alcohols and chromatographed these on paper. Again paper chromatography systems of the various colored alcohol derivatives such as the 3,5-dinitrobenzoate-a-naphthylamine addition compounds, the p-phenylazophenylurethanes, and the p-phenylazobenzoates appear to hold promise. Also, there are colored acid derivatives which can be separated by paper chromatography. In any case, however, as with the column chromatographic methods, a number of preparative steps are required and methods of measuring the amounts of the separated alcohols or derivatives are imprecise. The authors know of no system of column or paper chromatography which is capable of resolving the active amyl-isoamyl alcohol or derivative systems. The rapid development of the technique of gas-liquid partition chromatography has resulted in many applications to fusel oil analyses. Analyses both to determine the total quantity of higher alcohols in an alcoholic beverage and to measure the relative amounts of various compounds in previously isolated fusel oils have been developed. The problem of determination of the amounts of active amyl and isoamyl alcohols in fusel oil mixtures, so difficult by distillation and as yet unsolved by paper or column chromatography, has proved to be relatively simple by gas-liquid partition chromatography, as will be discussed later.
FUSEL OIL
327
As a method for routine analysis of wine or other alcoholic beverage for fusel oil content, gas-liquid partition chromatography is also quite useful. Since nonvolatile solids are present and the higher alcohols occur only in trace amounts a preliminary distillation is frequently required. For this purpose Bouthilet and Lowrey (1959) described a simple still by which a 20:l concentration of the higher alcohols in brandy samples can be attained. Harold d at. (1961) used steam distillation to separate the fusel oil of beer from the solids and then extracted with ether to separate the alcohols from the large volumes of water. Mecke and de Vries (1959) employed a mixture of two parts ether and one part pentane as extractant and made one back-extraction with water to remove ethanol. The higher alcohols were almost quantitatively recovered and contamination with ethanol was minimized. Webb and Kepner (1961) removed the ethanol and fusel oils from wines in a simple still equipped with a Vigreaux column. The distillate was saturated with salt and the alcohols were extracted with diethyl ether. Then the ether and most of the ethanol were removed by distillation through a Micropodbielniak column leaving an anhydrous fusel oil mixture as pot residue. The presence of relatively large amounts of water and ethanol in samples of higher alcohols to be analyzed by gas chromatography presents a problem. Bouthilet and Lowrey (1959) used a column packed with Flexol and detected the higher alcohols as humps on the trailing side of the large ethanol peak. Zarembo and Lysyj (1959) found that columns packed with Armeen SD, a mixture of straight-chain saturated amines of about 16 carbon atoms chain length, permitted water to pass through the column rapidly, followed by the alcohols approximately in order of increasing molecular weights. Diglycerol and glycerol columns, on the other hand, have high retention times for water. Repeated routine analysis is possible with these columns by back-flushing the water from the fore part of the column between determinations. For most accurate estimates of the quantities of the various alcohols in the mixtures, however, the ethanol content must be reduced to the same order of magnitude as that of the other alcohols to prevent the ethanol peak from obscuring adjacent peaks. The results provided by gas-liquid partition chromatographic analysis of fusel oil mixtures present problems of interpretation. If, on one hand, one is interested simply in the relative amounts
328
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
of the higher alcohols and esters present, these ratios may readily be obtained from the ratios of the areas of the appropriate peaks on the chromatographic chart. If, on the other hand, one wishes to determine the total concentration of fusel oils in the original sample one must decide how many of the higher boiling components separated by the chromatograph are considered to be fusel oil. For example, Harold et al. (1961) report a beer analysis in terms of individual compounds but apparently consider the fusel oil to consist of butyl and amyl acetates as well as the higher alcohols, but most published research describing fusel oil analyses by gas-liquid partition chromatography consider only relative amounts of the four principal alcohols, n-propyl, isobutyl, and the amyl alcohols. Also, in the determination of total fusel oil by gas-liquid phase chromatography, losses during the concentration operations must be kept to a minimum and the volume of the injected sample must be accurately known or some unnatural component must be added in known concentration to serve as an internal standard. After years of experience with gas-liquid partition chromatography Corse and Dimick (1958) stated that there was no packing known which could allow separation of active amyl from isoamyl alcohol. Shortly afterward, Van der Kloot and co-workers ( 1958) discovered that the two alcohols could be readily resolved on columns packed with glycerol. Webb and Kepner (1961)demonstrated that, in addition to glycerol, 1,4-butanediol, 1,2,4-butanetriol, i-erythritol, sorbitol, and diglycerol were capable of resolving the two amyl alcohols, and Prabucki and Pfenninger (1961) report that separation is also possible on columns packed with diethyl-D-tartrate. Baraud ( 1961) obtained good separations using triethanolamine. The detergent Tide was used by Porcaro and Johnston ( 1961) to separate the three amyl alcohols, active amyl, isoamyl, and n-amyl. Kambayashi et al. (1960) using columns of tetraethylene glycol dimethyl ether, polyethylene glycol, or dibenzyl ether and Kuffner and Kallina (1959) using Carbowax 300 were unable to resolve active amyl and isoamyl alcohols. Ingraham and Guymon (1960) used UCON-LB385 for separation of n-propyl, isobutyl, and fermentation amyl alcohols. Using a second aliquot of the sample and a glycerol column, the ratio of active amyl alcohol to isoamyl alcohol was determined, thus permitting a complete determination of the relative amounts of the four main fusel oil alcohols.
FUSEL OIL
329
C. CHEMICAL METHODS As Guymon and Nakagiri (1952)pointed out in their review of fusel oil analytical methods, the colorimetric method based upon the Komarowsky reaction (1903),i.e., the reaction of isobutyl and isoamyl alcohols with aromatic aldehydes in concentrated sulfuric acid solution, is particularly satisfactory for determining fusel oil in alcoholic beverages. As compared with other nonchromatographic techniques it has the great advantages of speed and accuracy. Mathers and Schoeneman ( 1955) suggest that 4-hydroxy-benzaldehyde-3-sulfonic acid sodium salt is the aromatic aldehyde of preference owing to the increased stability of the resulting color complexes. Methods using this aldehyde are less sensitive to minor variations in procedure. The method determines isoamyl, active amyl, tertiary amyl, 1%-amyl,and isobutyl alcohols and is not affected by ethyl, isopropyl, and n-propyl alcohols. The response to 2butanol and to n-butanol, alcohols occasionally present in fusel oils, has not been determined. One would expect, however, that they react similarly to isobutyl alcohol although Stevens (1960) states that, “In general, straight chain alcohols give little or no colour . . . .” Mathers and Schoeneman (1955)showed that the visible spectrum of the color complex formed with n-amyl alcohol is similar to that formed with active amyl alcohol. Both Guymon and Mathers have discussed the feasibility of determining the relative amounts of isobutyl alcohol in the fusel oil mixture by means of absorbance measurements at two wave lengths, the absorbance-wave length curves for the color complex of isobutyl alcohol being significantly different from that of isoamyl alcohol. Ingraham and Guymon (19sO) used this method, as well as gasliquid partition chromatography, in their studies of fusel oil production by mutant yeasts. However, the ratio of the absorbancies of the active amyl alcohol complex at 445 mp and at 560 mp approaches one-half the corresponding ratio for isobutyl alcohol complex ( Mathers and Schoeneman, 1955). Variations in concentration of active amyl alcohol in a fusel oil would therefore interfere with estimations of isobutyl alcohol by this technique, at least in cases where 4-hydroxybenzaldehyde-3-sulfonicacid is the color reagent. An independent measurement of the optical rotatory power of the fusel oil can be made to estimate the active amyl alcohol. However, 2-butanol which is occasionally present interferes.
330
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
Measurement of the color developed upon heating a sample of spirit containing 50% ethyl alcohol with an equal volume of concentrated sulfuric acid is the basis of another analytical method for fusel oil. Aldehydes are removed in a preliminary step, and the color developed after heating is compared with that developed in solutions containing known amounts of isobutyl alcohol. The apparent results are divided by 0.6. Lafon and Couillaud (1955) consider this method to be arbitrary and indicate a preference for techniques using an aromatic aldehyde. Genevois and Lafon (1958) developed a method for the estimation of secondary alcohols in mixtures of alcohols by oxidation to the corresponding ketone and measurement of the ketone by the iodoform reaction. The desirability of prior demonstration of the absence of methyl ketones in the mixture is obvious. While the Rose-Herzfeld (Ehrlich, 1907) method for fusel oil analysis, depending as it does upon measurement of the increase in volume of chloroform as the higher alcohols are extracted into it, is a strictly physical method, the Allen-Marquardt ( A.O.A.C., 1950), Schichtanz and Etienne (1939), Schichtanz et al. (1940) techniques all possess certain chemical phases. In each of the latter methods physical techniques-extractions-are used to isolate the higher alcohols from the wine or spirit. The separated higher alcohols (mainly isoamyl and active amyl ) are determined chemically in the Allen-Marquardt procedure by chromic acid oxidation followed by acidimetric titration and in the Schichtanz-Etienne method by acetylation with acetylchloride followed by titration of the liberated hydrochloric acid.
D. OTHERANALYTICALMETHODS The mass spectrometer was used by Webb and co-workers (1952) to determine active amyl alcohol in mixtures containing isoamyl alcohol as the only other component. It is possible that the technique could be extended to the analysis of four or five component mixtures. With fusel oils as usually isolated, however, the mixture is too complex to permit interpretation of the mass spectrogram. Batsin (1955) has used the polarimeter as a means of estimating fusel oil quantities. The method depends upon there being a constant proportion of active amyl alcohol and 2-butanol (and other optically active substances) in the samples. Batsin collected
FUSEL OIL
331
data from a number of different distilleries and for various distillation fractions. Tables were prepared from which it was possible to approximate fusel oil concentrations closely by polarimeter readings. The specific rotation for active amyl alcohol was assumed to be 4 . 9 0 " at 20°C. and using sodium light. The presence of 2-butanol or other optically active compounds was not considered.
IV. Results of Fuse1 Oil Analysis Numerous researchers have reviewed various phases of production and analyses of fusel oils. Primarily of historical interest are those of Schoen (1937), Schupphaus (1892), and Penniman et al. ( 1937). More recently Brau (1957), Genevois and Lafon ( 1957), Peynaud and Guimberteau (1959), Thoukis (1958a), Baraud (1961), and Stevens (1960) reviewed the field drawing on the findings provided by recent analytical advances. Baraud has made the valuable point that one cannot gain much knowledge about the influence of different yeasts, substrates, or fermentation conditions on fusel oil composition from analysis of fusel oils of industrial origin, because the conditions of isolation and treatment of the fusel oil which vary widely with location and time, have a great influence on composition. Design and operating conditions of the rectification column, for instance, are very probably responsible for the fact that isopropyl alcohol has not been found in all fuel oils. In cases of insufficient plates or of forced operation the isopropyl alcohol does not separate with the other higher alcohols but appears in the product stream. Similarly, it is common industrial practice to minimize loss of ethyl alcohol into the fusel oil fraction by repeated water washes or by adding salt or quicklime to induce separation of a second alcohol-rich phase. Such treatments markedly change the ratios of the propyl and butyl to amyl alcohols. Ikeda et aZ. (1956) consider this problem at length and conclude that it is preferable to work with fusel oil samples from laboratory fermentations in which conditions of isolation and concentration are known and controlled. The number of published fusel oil analyses which truly represent the content of the wine, beer, or distilled beverage is relatively small. Some typical representative analyses are summarized in Table IV. The two analyses of brandies are not comparable with the wine and beer analyses because in the former fusel oil was
w w
TABLE IV CONTENTOF VARIOUS BEVERAGESIN CONGENERS REPORTED) (WT. % OF COMPOUNDS
tQ
Baraud ( 1961) Cognac Compound Methanol Acetal 1-Propanol 2-Butanol Isobutyl alcohol Active amyl alcohol Isoamyl alcohol
fin bois
0.80 3.15 1.65 0.90
15.45 78.05"
Webb & Kepner (1961) Burgundy Montrachet Jerez
Cognac grande
yeast
champagne 0.40 trace 1.70 0.85 17.70 79.35"
a
Pale ale
Strong ale
Stout
Unhopped beer
-
-
-
-
-
-
-
trace
9
1.6
-
-
-
-
25 750
24.2 75.80
21 70"
23 10.9 64.1
Sum of active amyl and isoamyl alcohols.
-
-
yeast
18.2
2.6
20.2
-
Hudson & Stevens (1960) Compound Methanol Acetal 1-Propanol 2-Butanol Isobutyl alcohol Active amyl alcohol Isoamyl alcohol
yeast
-
-
12.4 12.0 57.4
2.7 16.5 78.2
-
-
8.4 4.5 66.9
Harold& al. Van der Kloot ( 1961) Enebo and Wilcox (1959) Australian (1957) beer Beer Beer
-
-
-
-
7 7 7
4
790
8 88"
-
-
25.3 74.7
* EI
5
5 m 4 0
8 >
3 8 Z
c-(
P
8 P
5:
TABLE V COMPOSITIONS OF FUSELOILS FROM VARIOUSSOURCES (WT. % OF COMPOUNDS REPORTED) Reference
Source
1-Propanol
Isobutyl alcohol
Active amyl alcohol
Isoamyl alcohol 79.2 41.0 40.7 38.6
-
2.6
15.0 7.6 15.4
15.7 33.5 37.5
18.2 28.3 18.2 8.5
Thompson Seedless Emperor Muscat Alexandria Mixed
0.8 5.6 1.2 4.9
7.4 10.8 5.5 21.3
16.7 15.4 16.0 11.1
75.1 68.2 77.3 62.7
Webb and Kepner (1961a)
Muscat raisin Zinfandel
0.7 2.3
4.9 15.6
19.3 16.8
75.1 65.3
Kumamoto (1932)
Kaoliang Molasses Sweet potato
6.8 2.8 0
0.7 0 0
19.3 19.1 86.1
73.2 78.1 13.9
Ogata (1953)
Sweet potato
8.4
6.8
46.2
38.6
Enders ( 1938)
Fermented wood sugar
0.3
21.0
34.2
44.5
Boswell and Gooderham (1912)
Beet molasses
0
8
56
36
Hellstrom (1943)
Sulfite liquor
2
23
13
62
Baraud (1961)
Apples Molasses Barley SuKte liquor
Ikeda ( 1956)
w
9M r 0
F
~~
0 0
w
334
A. DINSMOOR WEBB AXD JOHN L. INGRAHAM
certainly lost in the “heads” and “tails” cuts during distillation. The three wine analyses probably reflect the influence of yeast strains as they were aliquots of the same grape juice fermented under identical conditions. The variations in amounts of n-propyl alcohol produced are striking. The beer analyses also show large variations in the leveI of propyl alcohol. Numerous analyses of fusel oils from different sources have been published, These are of interest only in that they do illustrate the variability of fusel oils as they are available from different distilleries. In Table V some typical analyses are listed. Only those in which both the active and isoamyl alcohols were determined are tabulated. The large variation in the ratio of isoamyl to active amyl alcohols is of particular interest. Since these two alcohols have nearly identical properties except for optical activity, they should be influenced to the same degree by the distillation and subsequent washing treatments. Consequently, the variations in quantities of the two must reflect significant influences of substrate or fermentation conditions. The variations in amounts of propyl and isobutyl alcohols can, of course, reflect substrate or fermentation differences but it is much more likely that rectification and washing treatment variations cause the observed differences. Depending on the method of collection, all or part of the ethanolsoluble, water-insoluble compounds with boiling points near to and higher than that of ethanol of the material to be distilled may be found in the fusel oil. A very large number of such compounds have been found. Stevens (1960) listed a number of these compounds according to chemical type. Table VI summarizes literature references in which these various compounds have been definitely identified. The various source materials are tabulated. It is recognized that certain of the compounds listed may have been extracted from wooden containers during aging or may have been produced during distillation; n-propyl, isobutyI, active amyl, and isoamyl alcohols are not listed since they are common to all yeastfermented beverages. Indeed, it is likely that these four alcohols are present in all plant systems. The wide range in types of compounds and the great number of compounds of any particular type present in fusel oil is striking (Table V I ) . As analytical techniques have improved the list of compounds has grown; certainly, it will lengthen in the future.
FUSEL OIL
335
TABLE VI ISOBUTYL ALCOHOL, ACTIVEAMYL COMPOUNDS OTHERTHAN 1-PROPANOL, ALCOHOL, AND ISOAMYL ALCOHOL REPORTED PRESENTIN F u s n Ous FROM VARIOUS SOURCES" Compound Isopropyl alcohol
I-Butanol ( - ) -2-Butanol tert-Butyl alcohol 1-Pentanol
3-Pentanol 3-Methylbutan-2-01 1-Hexanol
1-Heptanol 2-Heptanol 1-Octanol 1-Nonanol 2-Nonanol 1-Decanol 2-Phenethyl alcohol
Borneo1 Fenchyl alcohol
Fermented material found in:a Wine-brandy (3); beer (19); cane molasses (11); beet molasses (3); wood alcohol (12); sweet potato (25) Wine-brandy (38); beer ( 19); cane molasses (27 ); beet molasses ( 5 ) ; sulfite alcohol ( 17 ) Wine-brandy (10) Beer ( 4 ) Wine-brandy (38); beer ( 19); cane molasses (27); sweet potato (24) Beet molasses (3) Wine-brandy ( 3 ) ; cane molasses (3) Wine-brandy (26); cane molasses (11);beet molasses ( 5 ) ; sweet potato (25); potato (29); sulfite alcohol (17) Wine-brandy (26) ; cane molasses (32) ; potato (29) Cane molasses (32); sweet potato (25) Cane molasses (32); potato (29) Cane molasses (32); potato (29) Cane molasses (33) Cane molasses (32) Wine-brandy ( 18); beer ( 1) ; cane molasses ( 34 ) ; sweet potato (35); sake (33); synthetic medium (31) Sulfite alcohol (13) Sulfite alcohol (23)
a The numbers in parentheses indicate the source of data: (1) Ayrapaa, 1961; ( 2 ) Baraud, 1961; ( 3 ) Baraud and Genevois, 1958; ( 4 ) Bavisotto et al., 1961; (5) Boswell and Gooderham, 1912; ( 6 ) Braus and Miller, 1958; ( 7 ) Carrol and O'Brien, 1958; ( 8 ) Chapman and Hatch, 1929; (9) Duhaux and Belien, 1959; (10) Durodie and Roelens, 1942; (11) Dutt, 1938; (12) Enders and Kambach, 1938; (13) Ekstrom, 1932; (14) Enebo, 1957; (15) Gryaznof, 1959; (16) Harold et al., 1961; (17) Hellstrom, 1943; (17a) Hellstrom, 1944; (18) Ikeda et al., 1956; ( 19) Jenard, 1960; (20) Jensen, 1950; (21) Kepner and Webb, 1956; (22) Kepner and Webb, 1961; (23) Komppa and Toluitie, 1931; (24) Kumamoto, 1932; (25) Ogata and Matsubara, 1953; (26) Ordonneau, 1886; (27) Rao, 1938; (28) Rao, 1943; (29) Shoruigin et al., 1933; (30) Siefker and Pollock, 1956; (31) Smith and Coffman, 1960; (32) Swenerton, 1929; (33) Taira, 1933; (34) Taira, 1936; (35) Taira and Masujima, 1934; (36) Ubeda, 1941; (37) Van der Kloot et nl., 1958; (38) Webb et al., 1953; (39) Webb and Kepner, 1962.
336
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
TABLE VI (Continued) Compound Guaiacol d-Citronellol dl-a-Terpeniol Formic acid Acetic acid Butyric acid Isobutyric acid Isovaleric acid Caproic acid Enanthic acid Caprylic acid Capric acid Pelnrgonic acid Lauric acid Salicylic acid Methyl salicylate Ethyl formate Ethyl acetate Ethyl propionate Ethyl isobutyrate Ethyl caproate Ethyl enanthate Ethyl caprylate Ethyl pelargonate Ethyl caprate Ethyl laurate Ethyl myristate Ethyl pentadecanoate Ethyl palmitate Ethyl lactate Ethyl succinate Ethyl malate Propyl valerate Butyl acetate Butyl valerate Isobutvl acetate a
Fermented material found in:a Whiskey ( 6 ) ; wood alcohol (12); sulfite alcohol ( 1 7 ) Sweet potato (25) Sweet potato ( 2 5 ) Wood alcohol ( 1 2 ) Wine-brandy (22); whiskey ( 12) Wine-brandy (38); wood alcohol (12) Wine-brandy ( 2 2 ) Wine-brandy ( 2 2 ) Wine-brandy ( 22); cane molasses (28) Wine-brandy ( 22 ) Wine-brandy ( 22 ) ; cane molasses ( 28 ) ; wood alcohol (12) Wine-brandy ( 22) ; cane molasses ( 28) ; wood alcohol ( 12) Wine-brandy ( 9 ) ; cane molasses ( 2 8 ) Cane molasses ( 2 8 ) Cane molasses ( 2 8 ) Wine-brandy (38) Beer ( 1 4 ) ; whiskey ( 7 ) Wine-brandy ( 3 9 ) ; beer ( 14); whiskey ( 7 ) ; raw spirit (15) \Vine-brandy ( 2 ) Wine-brandy (39); cane molasses ( 11) Wine-brandy (38); cane molasses ( 2 8 ) ; potato ( 2 9 ) Wine-brandy ( 2 ) Wine-brandy ( 22) ; cane molasses ( 28 ) ; potato (29) Wine-brandy ( 22 ); cane molasses (28) Wine-brandy ( 22 ) ; cane molasses ( 28 ) ; potato (29) Wine-brandy ( 22) ; cane molasses ( 28 ) ; potato (29) Wine-brandy ( 22 ) ; potato ( 29 ) Wine-brandy (22) Wine-brandy ( 2 2 ) ; potato ( 2 9 ) Wine-brandy (39); synthetic medium (31 ) Wine-brandy (39) Wine-brandy ( 3 9 ) Kaoliang ( 24 ) Beer (19) Kaoliang (24) Wine-brandy ( 2 ) ; beer (16)
For sources, see footnote on page 335.
FUSEL OIL
337
TABLE VI f Continued) Compound Isobutyl caprylate Isobutyl caprate sec-Butyl acetate Isoamyl acetate Isoamyl valerate Isoamyl isovalerate Isoamyl caproate Isoamyl caprylate Isoamyl caprate Isoamyl laurate Isoamyl lactate Isoamyl palmitate Active amyl caproate Active amyl caprylate Active amyl caprate Active amyl laurate Hexyl acetate Hexyl valerate 2-Phenethyl acetate 2-Phenethyl caproate y-Butyrolactone Diacetyl Dimethyl sulfide Pyridine Trimethylpyrazine Tetramethylpyrazine Diethylpyrazine Methyltriethylpyrazine Formaldehyde Acetaldehyde Isobutyraldehyde Hexanol 2-Hexenal Acetal Acetone Furfural p-Methylguaiacol p-Ethylguaiacol Vanillin Phenol 4-OH-3-CH,O-1propylbenzene 2-Butanone Limonene Camphene a
Fermented material found in: a Wine-brandy ( 38) Wine-brandy (38) Beer (16) Wine-brandy ( 2 ) ; beer (19); raw spirit (15) Kaoliang (24) Wine-brandy (39) Wine-brandy (38) Wine-brandy ( 38) Wine-brandy (38); kaoliang ( 12) Wine-brandy ( 38 ) Wine-brandy (39) Kaoliang ( 1 2 ) Wine-brandy (38) Wine-brandy (38) Wine-brandy (38) Wine-brandy (38) Wine-brandy (39) Kaoliang (24) Wine-brandy (22 ) Wine-brandy (39) Wine-brandy (39); synthetic medium (31) Wine-brandy (21 ); beer ( 4 ) Beer ( 4 ) Beet molasses (34) Beet molasses ( 8 ) ; potato (29) Beet molasses ( 8 ) ; potato (29) Beet molasses ( 8 ) ; potato (29) Potato (29) Beer (30) ; whiskey ( 7 ) Beer ( 37); whiskey ( 7 ) ; synthetic medium (31) Wine-brandy (21) Wine-brandy (21 ) Wine-brandy (21) Wine-brandy (38); cane molasses (11) Beer ( 3 7 ) ; whiskey ( 7 ) Wine-brandy (36); beer (30); cane molasses ( 11) Whiskey ( 6 ) Whiskey ( 6 ) Whiskey ( 6 ) Whiskey ( 6 ) Sulfite alcohol (20) Wine-brandy ( 4 ) Sulfite alcohol (17a) Sulfite alcohol (23)
For sources, see footnote on page 335.
338
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
V. Biosynthesis of Fuse1 Oil Components In the introduction to the chemical section of this review fusel oil was defined in an operational sense, i.e., that liquid which separates as a second phase from distillates of yeast-fermented media. In this section we will discuss all the monohydric alcohols, other than ethanol, which yeast produce. This latter definition is more restrictive in that only alcohols are considered and less restrictive in that nonvolatile alcohols are included. A. FUELOIL FORMATION FROM AMINOACIDS Speculations concerning the possible origin of fusel oil components began late in the 19th century when it was proposed that these compounds might result from bacterial contamination or from the reduction of fatty acids (Emmerling, 1905; Pierre and Puchot, 1872; Pringsheim, 1905a, by 1906, 1907, 1908). But the first experimental approach to the problem was that of Felix Ehrlich, who in a series of carefully planned experiments, established that amino acids can serve as precursors of fusel oil. Ehrlich was probably led to investigate this possibility as a result of his studies on the “leucines.” In 1904 (Ehrlich, 1904) he isolated and characterized isoleucine. The structural similarity between leucine and isoamyl alcohol on the one hand and between isoleucine and active amyl alcohol on the other suggested a metabolic relationship between these amino acids and aliphatic alcohols. Accordingly, Ehrlich ( 1906a, 1907) carried out resting-cell fermentations of glucose by yeast to which leucine and isoleucine had been added, and showed that their addition increased the amount of fusel oil 7- to 8-fold, and that about two-thirds of the leucine disappearing could be accounted for by the fusel oil produced. On the basis of these experiments, Ehrlich proposed that leucine and isoleucine are split by a “hydrating” enzyme to form isoamyl and active amyl alcohols, respectively, in addition to COZ and ammonia, i.e., CH,
CH- CH,-
CH(NH,)-COOK
iH,O
CHf
-
CHw CH- CH,CHf
Leucine
CH,OH
+ CO, + NH,
Isoamyl alcohol
and
CH,-CH,-CH-CH(NH,)-COOH
I
CH,
Isoleucine
+
H,O-CH,-CH,-
7CHSH- CH,OH + CO, + NH, Active amyl alcohol
FUSEL OIL
339
However, Ehrlich was not able to detect the presence of free ammonia during the fermentation, an observation which lead him to conclude that the ammonia was immediately incorporated into the yeast protein (Ehrlich, 1907). This viewpoint was strengthened when he‘was unable to obtain fusel oil formation by acetone-dried powders and pressed yeast juice, which were able to catalyze alcoholic fermentation, i.e., a condition in which protein synthesis did not occur (Ehrlich, 1906a). Ehrlich also showed that addition of ammonium salts and asparagine inhibited the formation of fusel oil. Together these observations of Ehrlich form the basis for the concept that higher molecular weight aliphatic alcohols are byproducts of “alcoholic fermentation of amino acids” (Ehrlich, 1907) by yeasts. If readily utilizable forms of nitrogen such as asparagine or ammonium are present, they are preferentially used, but if leucine, isoleucine, and valine must be metabolized to satisfy nitrogen requirements for growth, fusel oil results. Ehrlich was disturbed by his observation that yeast cells fermenting sucrose without added nitrogen produce about 20% as much fusel oil as is produced in a fermentation to which 0.6% leucine is added. But he suggested that autolysis of yeast protein supplies the amino acids to produce fusel oil under these conditions, and he supported this hypothesis by citing the observation that slow fermentations, in which there is more time for autolysis, produce more fusel oil than rapid fermentations. Ehrlich also showed that yeasts produced tyrosol (p-hydroxyphenyl ethanol) if tyrosine was added to the fermenting mixture (Ehrlich, 1907, 1911) and tryptophol was produced if tryptophan was added to the fermentation (Ehrlich, 1912). Ehrlich’s contributions to our understanding of fusel oil formation remain, after almost 60 years, the most significant that have been made. One is particularly impressed by these contributions if he considers the techniques available to Ehrlich. In his early experiments he concentrated the amyl alcohols by fractional distillation, oxidized them to the corresponding carboxylic acids and made their silver salts. In his later experiments he determined the increase in volume of a chloroform layer after extracting the fusel oil from a specifically diluted alcohol solution. Neubauer and Fromherz (1911) investigated the pathway of fusel oil formation from amino acids in greater detail. They set about to prove that a-keto acids were intermediates in the process
340
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
by showing that ( 1 ) a-keto acids can be produced from amino acids and that ( 2 ) added a-keto acids can be converted to alcohols with one less carbon atom. Since a-keto acids could not be isolated when naturally occurring amino acids were added to yeast fermentation, they added the unnatural phenylaminoacetic acid and were able to isolate the corresponding keto acid, phenylglyoxylic acid, in addition to benzyl alcohol and mandelic acid. Also p-hydroxyphenylethyl alcohol could be isolated from fermentations to which p-hydroxyphenylpyruvic acid was added, but it was present in only trace amounts in fermentations to which p-hydroxyphenyllactic acid was added. On the basis of these observations they modified Ehrlichs scheme as follows: R I yNH, COOH
NH,
L ~
R I c=o I
COOH
=
R I CHO
+
-
R I CH,OH
CO,
The essence of the observation of Ehrlich and Neubauer and Fromherz were reconfirmed by numerous investigators over the next several decades (Buchner and Meisenheimer, 1906; Lampitt, 1919; Houssian, 1937; Thorne, 1937; Zalesskaya, 1940; Yamada 1932; Genevois, 1952; Vogt, 1952; Ribkreau-Gayon et al., 1955; Spanyer and Thomas, 19%; Antoniani et at., 1958; Yoshizawa et al., 1961): but nothing basically new was added until 1958 when SentheShanmuganathan and Elsden ( 1958) reinvestigated the problem in the light of modern knowledge using modern techniques. SentheShanmuganathan and Elsden studied tyrosol formation because they found that this product can be easily estimated by the Folin and Ciocalteu (1927) reagent after separation from interfering materials by an ether extraction under alkaline conditions. They obtained a cell-free system which catalyzed the conversion of tyrosine to tyrosol if supplemented with pyridoxal phosphate and a-ketoglutarate. Tyramine was eliminated as an intermediate since it could not be converted to tyrosol either by intact cells or by the cell-free system, while p-hydroxyphenylpyruvic acid and p-hydroxyphenylacetaldehyde were converted to tyrosol if DPNH is made available. On the basis of their experiments, it seems clear that the pathway of tyrosol formation by Saccharomyces cerevisiae is:
341
FUSEL OIL
( 1 ) tyrosine
+
a-ketoglutarate
( 2 ) p-hydroxyphenylpymvic acid
glutamic acid + p-hydroxyphenylpyruvic acid
transaminase
,p-hydroxyphenylacetaldehyde + co,
carboxylase
( 3 ) p-hydroxyphenylacetaldehyde + DPKH
, tyrosol +
alcohol dehydrogenase
DPN
In a later paper SentheShanmuganathan (1960a) showed that crude preparations of S . cerevisiae were similarly capable of transferring the amino groups of aspartic acid, leucine, norleucine, isoleucine, valine, norvaline, methionine, phenylalanine, and tryptophan to a-ketoglutaric acid. He ( SentheShanmuganathan, 1960b) purified the tyrosine transaminase 100-fold, and showed it to be specific for a-ketoglutarate. The crude preparations were also capable of decarboxylating the resulting a-keto acids. It appears from the results of SentheShanmuganathan that isobutyl, isoamyl, and active amyl alcohol can be formed from valine, leucine, and isoleucine by the scheme suggested above for tyrosol formation, and from his results and others (Neuberg and Kariczag, 1911) that yeast carboylase catalyzes the decarboxylation step. SentheShanmuganathan suggests that from his observations and those of Barron and Levine (1952) and Ebisuzaki and Barron (1957) that the third step in the formation of fusel oil from amino acids is catalyzed by either the classic alcohol dehydrogenase or by alcohol dehydrogenase I1 ( Barron and Levine, 1952), or both. B. FUSEL OILFORMATION FROM GLUCOSE From the foregoing research it is clear that the major fusel oil components can be synthesized from amino acids by transamination, decarboxylation, and reduction, and the results strongly suggest that the components of fusel oil are by-products of the anaerobic nitrogen metabolism of yeast. However a number of observations are not satisfactorily explained by this theory, namely: (1) Significant levels of fusel oil including phenylethyl alcohol and tyrosol (Stevens, 1960) are formed by resting yeast cells in the absence of added amino acids. ( 2 ) In media containing low levels of amino acids, there is not a good correlation between the amino acid composition of the medium and the composition of the resulting fusel oil. ( 3 ) The kinetics of amino acid utilization and fusel
342
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
oil formation during alcohol fermentation in complex media are not complementary, i.e., amino acids are quickly removed from the medium, but the rate of fusel oil formation is no greater during the period of rapid amino acid uptake than it is later when the medium is essentially free of amino acids (Fig. 2). In fact,
1
200)
I
I
Time in hours
i
25
50
75
100 125 150 Time in hours
f
175
200
FIG. 2. The course of amino acid disappearance, yeast cell multiplication, and formation of ethanol and fusel oil during fermentation of grape juice (Castor and Guymon, 1952). (Reproduced from Science by permission.)
fusel oil formation appears to parallel ethanol production not amino acid utilization. (4) Certain fusel oil components, e.g., n-propanol, a major component, and n-butanol, a minor component, do not correspond to any natural amino acids. As stated earlier, Ehrlich suggests that autolysis of yeast protein accounted for fusel oil formation in the absence of exogenous amino acids. The inadequacy of this explanation was proven by Thoukis ( 1958a) who carried out nine successive resting-cell fermentations of sucrose with the same batch of yeast cells and obtained a cumulative yield of about 0.5 gm. of fusel oil from 7 gm. of yeast (dry weight). Clearly, the fusel oil was formed from sucrose and not from yeast protein. Genevois and Lafon (1956) showed that C14-labeled acetate is
FWSEL OLL
343
incorporated into isoamyl alcohol formed during fermentation in the presence of unlabeled amino acids thus establishing beyond any doubt that not all of this alcohol is formed from exogenous leucine. Since Saccharomyces cerevisiae has the ability to synthesize all amino acids, one might expect that fusel oil which is not derived from exogenous amino acids is synthesized at least to the keto acid stage by the same route as that by which the corresponding amino acid is synthesized. Ingraham and Guymon (1960) produced strong evidence to support this hypothesis by their study of amino acid auxotrophic yeasts. Resting-cell fermentations by yeast strains incapable of synthesizing a particular amino acid were found to be completely incapable of synthesizing the corresponding component of fusel oil, i.e., strains requiring leucine, isoleucine, or valine for growth did not produce isoamyl, active amyl, or isobutyl alcohols, respectively.
C. ~-PROPYL AND ~ - B U T YALCOHOLS L The pathway of synthesis of n-propyl and n-butyl alcohols by yeasts have been elucidated only recently. The first attempts were those of Kepner et al. (1954) who reasoned that n-propyl alcohol might be formed from a-aminobutyric acid by the conventional Ehrlich pathway. However, when they added this compound to a resting-cell fermentation of glucose by yeast, they obtained only a 0.03% yield of propyl alcohol from this nonprotein amino acid in contrast to a nearly 100% conversion of leucine to isoamyl alcohol. A likely precursor of n-propyl was a-ketobutyric acid, a known intermediate in the synthesis of isoleucine (Willson and Adelberg, 1957) and hence also an intermediate in the synthesis of active amyl alcohol. Guymon et al. (1961a) showed this to be the case. They added gradated levels of a-aminobutyric acid, which is known to serve as a source of a-ketobutyric acid in yeasts, to nitrogen-free fermentations of glucose. The amounts of n-propyl and active amyl alcohols which were produced by these fermentations increased linearly with the amount of a-aminobutyric acid added to the fermentation, while the amounts of isobutyl alcohol remained essentially constant and that of isoamyl alcohol decreased slightly at high concentrations of the added amino acid (Fig. 3 ) . These authors further showed that in the presence of a-aminobutyric acidl-C14 there was no significant labeling of any fusel oil component,
344
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
while the addition of a-aminobutyric a ~ i d - 2 - Cbrought ~~ about the selective labeling of n-propyl and active amyl alcohols. As expected from the proposed scheme the n-propyl alcohol produced contained essentially all of the labels of the carbinol carbon atom. Yamada c?t al. (1962) have also shown that the addition of a-aminobutyric acid stimulates n-propyl alcohol formation.
.;-/-;
OL=
lsoomyl alcohol
004
006
008
10
% o-omino butyric acid
FIG.3. The effect of addition of a-aminobutyric acid to resting-cell fermentation of glucose by Saccharomyces cereoisiae on the production of isoamyl, active amyl, isobutyl, and n-propyl alcohols (Guymon et al., 1961a). (Reproduced from Archives of Biochemistry and Biophysics by permission.)
Among the amino acid auxotrophic mutants studied by Ingraham et al. (1961)one required both isoleucine and valine for growth. Since leucine is synthesized from the immediate precursor of valine, this mutant is incapable of synthesizing all three branched chain amino acids. As expected, resting-cell fermentations of glucose by this mutant produced no isoamyl, active amyl, or isobutyl alcohols. But, quite unexpectedly large amounts of n-butyl and higher than normal amounts of n-propyl alcohols were produced. Tracer experiments indicated that n-butyl alcohol is synthesized by this mutant from a-keto-n-valeric acid which in turn is synthesized from a-ketobutyric acid by a sequence of reactions analogous to those known to be responsible for the conversion of keto valine to keto leucine (Strassman et at?., 1956) (see Fig. 4 ) . That is, addition of 2-labeled acetate results in predomi-
1 Active Amy1 Alcohol]
I rz-Propyl Alcohol] it
t
t
-Propanaldehyde + CO,
t I
Threonine _c a -Ketobutyrate
-
a -Acetohydroxybutyrate
-
a
Methylbutyraldehyde + CO, ~
~
~ -a ~
Active Acetaldehyde
t
Pyruvate
1
-L
ci -Acetolactate
a ,O-Dihydroxy-
_t.
isovalerate
a-Ketoisovalerate
Valine
w
r 7
+ CoA
-
a -Ketovalerate n-Butyraldehyde F A c e t y l = CoA 0-Carboxy-0-hydroxycaproate
1-
n -Butyl Alcohol
Alcohol
I
I a -Hydroxy-8-carboxycaproate
t ci-Hydroxycaproate
-
n -Valeraldehyde
I
-
Isobutyl Alcohol
-
Isovaleraldehyde
+
-p -carboxy isocaproate
a -Hydro-
-
a -Ketoisocaproate
co, n -Amy1 Alcohol
FIG.4. Pathways of formation of major fuse1 oil components and n-butyl and n-amyl alcohols.
3 F
Pisocaproate -Carboxy 4-hydroxy+ CoA
Isobutyraldehyde,
t -Hydroxy-8-carboxyvalerate
I
-
I I
{Acetyl-CoA
[ 4-Carboxy-0-hydroxyvalerate]
1
+ ~ Isoleucine ~ ”
_-
[Acetyl-CoA
a
t
~-Keto-B-rn&hylvalerate ~ : ~ ~
8
-
+ CO,
1I4I
Leucine W
A
cn
346
A. DINSMOOR WEBB AND JOHN L. WGRAHAM
nate l-labeling of n-butyl alcohol; 2-labeled a-ketobutyrate results in %labeling of n-butyl alcohol, while l-labeled a-ketobutyrate and l-labeled acetate does not result in significant activity in n-butyl alcohol. Since a-keto-n-valeric acid was a suspected intermediate in the production of n-butyl alcohol, norvaline was added as a source of the keto acid to resting-cell fermentations of both the auxotrophic mutant and its wild type parent. As predicted, the production of n-butyl alcohol by both strains was stimulated by the addition of the amino acid. Surprisingly, however, under these conditions, the mutant was found to produce n-amyl alcohol in addition to n-butyl and n-propyl alcohols. Tracer experiments indicated that n-amyl alcohol is synthesized by the mutant from a-ketovaleric acid by a pathway analogous to that by which n-butyl alcohol is synthesized from a-ketobutyric acid, i.e., by way of a-ketocaproic acid (Fig. 4 ) . The enzymes of this reaction pathway which has the net result of converting an a-keto acid to an alcohol with the same number of carbon atoms, appear to be nonspecific since a-ketoisovaleric, a-keto-n-butyric, and a-keto-n-valeric acids can serve as primary substrates for isoamyl, n-butyl, and n-amyl alcohols respectively. It was suggested by Guymon et al. (1961a) that some n-propyl alcohol may be made by this pathway from pyruvate since threonine auxotrophs which cannot make a-ketobutyric acid were found to produce this alcohol. There are limits, however. Norleucine is known to be converted to a-keto-n-caproic acid by Saccharomyces cerevisiae. Yet addition of this amino acid to a resting-cell fermentation of glucose by the mutant, does not bring about the production of n-hexyl alcohol. It now appears that the pathways of formation of the various fusel oil components are known. The major components, isoamyl, active amyl, isobutyl, and n-propyl alcohols and the minor component, n-butyl alcohol, are formed from intermediates in the synthesis of the three branched chain aliphatic amino acids : leucine, isoleucine, and valine (Fig. 4 ) .
D. FACTORS AFFECTINGFUSEL OIL FORMATION The major components of fusel oil are formed by a number of yeasts under a wide variety of physiological conditions. Guymon et al. (1961b) showed that aerobic conditions do not inhibit the formation of fusel oil; in fact, more fusel oil is formed under aerobic
347
FUSEL OIL
conditions by a factor of about 4 in spite of the fact that under these conditions yeasts are capable of reutilizing fusel oil components (Mertz and Ingraham, 1961). The fusel oil mixture produced aerobically contains up to three times as much isobutyl alcohol as the mixture produced anaerobically, although it is not known whether this enrichment is caused by greater production of this alcohol or by lesser reutilization. Naturally occurring strains of wine yeasts do not vary greatly in ability to produce fusel oil (Castor, 1954), although there is considerable variation among different genera of yeast. In a study (Guymon et al., 1961a) of nine yeast species including fermentative and nonfermentative types, all were found to produce fusel oil (Table VII) regardless of their ability to produce ethanol. Fusel oil production appears to be a general property of yeasts. TABLE VII PRODUCTION OF FUSEL OIL BY YEASTS OF DIFFERENT FERMENTATION CHARACTER UNDER AERATED AND NONAERATED CONDITIONS Fusel oil Ethyl alcohol formed (vol. % ) Yeast
Pichia kluyveri Pichia membranafaciens Hansenulu anomala Schwanniomyces occidentalis Candida albicans Candida rugosa Kloeckera magnu Debaryomyces kloeckeri Saccharomyces beticus
mg./100 ml.
mg./100 gm. (sugar used)
Aerobic
Anaerobic
Aerobic
Anaerobic
Aerobic
Anaerobic
1.2 0.4 0.2
4.9 0.1 4.3
50.2 3.1 16.8
6.4 0.3 4.3
437 27 142
72 7 57
3.5 1.9 Tr. 0.2 Tr. 10.8
0.8 2.9 Tr. 4.2 0.2 12.3
21.9 67.5 7.6 4.4 35.0 20.8
3.6 3.8 0.3 3.2 1.1 4.7
219 614 84 52 350 95
109 12 53 31 26
-
The effect of temperature of fermentation on fusel oil formation was studied by Lindet in 1888 (Lindet, 1888). He found that about 10% less fusel oil was formed in the range of 8"-1OoC. than in the range of 25"-27"C. More recently, similar results were obtained by Castor (1954). The effect of a decrease in fusel oil formation at low temperature is too small to be of industrial significance. It is commonly observed that the presence of suspended solids during fermentation increase the yield of fusel oil. The evidence
348
A. DINSMOOR WEBB AND JOHN L. INGRAHAM
indicates that this effect has a physical rather than a chemical basis, since the addition of animal charcoal has been reported to increase fusel oil production 2.5 times ( Dietrich and Klammerth, 1941). It is possible that suspended solids exert their effect by creating aerobic conditions during the early stages of fermentation. Fusel oil produced under such conditions would not be reutilized during the later anaerobic stages. The effect of the addition of amino acids which are not direct precursors of fusel oil components on the formation of fusel oil has been studied extensively (Yamada et al., 1962; Antoniani et d., 1958; Peynaud and Guimberteau, 1958). Certain observations are easily explained on the basis of present knowledge; for example, the effect of addition of leucine, isoleucine, and valine on the stimulation of production of the corresponding higher alcohols. Also the stimulation of active amyl alcohol production by the addition of threonine (Yoshizawa et al., 1961) is to be expected since this amino acid is an intermediate in the formation of active amyl alcohol ( Fig. 4 ) . Similarly, the observed stimulation of isobutyl alcohol production by the addition of alanine (Yamada et aE., 1962) might be expected since pyruvate is a precursor of valine. Asparagine undoubtedly exerts its depressive effect on higher alcohol production (Ehrlich, 1907) by serving as a readily available nitrogen source as does ammonium ion (Ehrlich, 1907). Addition of other amino acids has led to conflicting results (Antoniani et al., 1958; Peynaud and Guimberteau, 1958). The essentiality of glucose to fusel oil formation from amino acids by intact yeast cells has been known since Ehrlich and considered by him to substantiate his view that fusel oil is a by-product of the process by which the cell obtains nitrogen for growth. SentheShanmuganathan and Elsden (1958) have pointed out two obvious requirements for glucose and suggested a third. Glucose metabolism is required to supply a-ketoglutarate to accept the amino group and to supply DPNH to reduce the aldehyde precursor of the particular fusel oil component in question. They also showed that sodium azide and 2,4-dinitrophenol inhibit tyrosol formation from tyrosine indicating that the process requires energy. They suggest that the energy-requiring step might well be the entry of tyrosine into the cell. Fusel oil production is a normal activity of yeast. All strains examined carry out the process. Production occurs under aerobic
FUSEL OIL
349
as well as anaerobic conditions; in complex media in which exogenous amino acids are plentiful and in minimal media in which they are absent; in media in which glucose concentration is high and in media in which glucose concentration is low. Fuse1 oil is produced in glucose-limited chemostat culture ( Mertz and Ingraham, 1961), a condition of minimal glucose concentration. In media high in fermentable carbohydrate, fusel oil is produced during the period of active growth as well as the period of metabolism following the cessation of growth. Competition in the biological world permits only the most efficient organisms to survive. Consequently we are able to find “purpose” in most biochemical events. One cannot help but wonder, in this vein, about the significance of fusel oil formation by yeasts. Its significance under anaerobic conditions in certain complex media is probably just as Ehrlich and others have suggested, namely: fusel oil formation is an inevitable consequence of the cell’s use of amino acids as a source of nitrogen. But this explanation cannot account for the process under other conditions. According to SentheShanmuganathan ( 1960) the last two steps in the formation of fusel oil components are catalyzed by the same enzymes as those which catalyze the last two steps in ethanol production. If keto acids are present, therefore, it seems inevitable that they be converted to the homologous alcohol. Possibly this is the answer. It is interesting that the major components of fusel oil are byproducts of isoleucine-valine-leucine metabolism. Since it is known that the synthesis of these amino acids is controlled by multivalent repression in Escherichia coli (Freundlich et al., 1962) it is tempting to speculate that control is inadequate in yeasts thus permitting higher alcohols to be produced by a process of “shunt metabolism” (Foster, 1949). However, it is also possible that the formation of propyl, amyl, and butyl alcohols is favored merely as a result of the specificity of carboxylase. The energy which is apparently wasted in the formation of fusel oil is not great. Utilization of DPNH in the last step of the process cannot be considered to be a loss of energy under anaerobic conditions; the only energy loss is the expenditure of one acetyl-CoA in the synthesis of isoamyl alcohol. Considering the very small amounts of fusel oil formed the net energy loss is insignificant.
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A. DINSMOOR WEBB AND JOHN L. INGRAHAM
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AUTHOR INDEX Numbers in italics show the page on which the complete reference is listed.
A Abbott, M., 31, 45 Abelson, P. H., 101, 103, 131, 133 Abraham, E. P., 11,45 Aburatani, I., 268, 293 Adam, J., 277, 303 Adams, G. A., 141, 146, 148, 153,154 Adams, J. N., 240,293 Adams, I?. A,, 209, 214 Adams, W. S., 288,293 Adelberg, E. A,, 343,353 Agatov, P. A., 270, 298 Agre, N. S., 247, 255, 293 Ahmad, K., 264, 293 Ahmad, N., 264, 293 Aisenberg, A. C., 68, 70, 71,91 Aito, K., 275, 305 Ajello, L., 240, 298 Akiba, T., 290, 294 Akira, M., 261, 311 Albert, A., 10, 14, 20, 23, 24, 25, 26, 34, 40, 42, 43, 44, 45 Albright, C., 29, 49 Aldridge, W., 33, 45 Alexander, M., 23, 24, 26, 47, 256, 300 Alexandrowa, S., 335, 352 Alikhanian, S. I., 200, 213, 241, 242, 245, 294, 305 Allen, D. W., 17, 45 Altenbern, R. A., lS0, 185 Alter, B. M., 290, 295 Ambler, R., 4, 45 Ames, B. N., 34, 45 Amici, A. M., 240, 311 Amsterdam, D., 283, 310 Anand, N., 27, 45 Anastassiadis, P. A., 138, 141, 143, 146, 153 Anderson, B., 44, 45 Anderson, D. L., 199, 214, 241, 244, 296 Anderson, E. S., 292, 300
Anderson, L. E., 259, 299 Ando, K., 283,294 Andriiuk, K. I., 237, 294 Anslow, W. K., 266, 295 Antoni, S., 281, 295 Antoniani, C., 340, 348, 350 Antonov, V. K., 236,252,262, 311 Anzai, K., 262, 263, 271, 294, 306 Appelman, A. W. M., 199,214 Applebaum, E., 288, 294 Arai, T., 240, 241, 244, 260, 294 Araki, T., 249, 266, 313 Arcamone, F., 250, 273, 277, 294, 297 Argoudelis, A. D., 268, 309 Arima, K., 2163, 312 Arishima, M., 252, 260, 294, 309 Arman, P., 33, 45 Armstrong, J., 4, 45 Arnow, P., 32, 48, 269, 303, 305 Arpai, J., 293, 294 Artamonova, 0. I., 246,294 Artman, M., 270,283,298 Asano, K., 251, 301 Asbeshov, I. N., 280, 294 Ashino, K., 262, 307 Asselineau, J., 5, 8, 45 Astrachan, L., 16, 50 Aurele, A. J., 292, 304 Auro, M. A., 172,185 Avernheimer, A. H., 139, 154 Avery, 0. T., 190, 213 Awa, A., 275,294 Ayrapiia, T., 335, 350 Ayre, N. C., 246, 302
B Bablanian, R., 37, 38, 50 Bachmann, E., 251, 257, 261, 294, 306, 315 Back, N., 279,294 Backus, E. J., 247,255,313,314 Baddiley, J,, 4, 45, 257, 295 Bahadur, K., 137, 141, 149,153
355
356
AUTHOR INDEX
Baikina, V. M., 280, 313 Bailey, W. C., 275,294 Balassa, G., 282, 294 Baldacci, E., 244, 249, 294 Balfour, B., 43, 45 Balitskaya, A. K., 264, 274, 294 Ball, E. C., 30, 45 Ball, S., 251, 294 Barabas, G., 242, 245, 246, 312 Baraud, J., 323, 328, 331, 332, 333, 335, 350, 352 Barbatschi, F., 277, 303 Bamard, R. E., 273,295 Bamer, H., 20, 46, 77,91 Bames, E. M., 292,300 Bames, L. E., 292, 295 Bamett, L., 38,46 Barnwell, J. L., 138, 139, 141, 143, 144, 149, 152,154,155 Baron, F., 45 Baroni, C., 239, 313 Barrachini, O., 161, 185 Barron, E. S. G., 341, 350 Bartholomew, W. H., 166, 167, 185, 186 Basilio, C., 16, 19, 49, 193, 214, 215, 284, 311 Bassler, G., 242, 245, 246, 312 Batsin, M. K., 330, 350 Batt, R. D., 254, 295, 305 Bauer, D. J., 37, 45,49, 50 Bavisotto, V., 328, 335, 350, 353 Beadle, G. W., 197, 213 Beckett, A. H., 13, 44, 45 Bedrynska-Dobek, M., 244, 295 Bekhtereva, M. N., 238, 256, 295 Belien, H., 335, 350 Belikova, A. P., 259, 295 Belova, Z. N., 265, 295 Below, W., 335, 352 Belozersky, A. N., 6, 45, 86, 92, 263, 306 Benedict, R. G., 162, 164, 172, 185 Bennett, S. I., 262, 315 Berendsen, G. W., 199, 214 Beretta, G., 250, 304 Bergel, F., 68, 70, 73, 91 Bergelson, D. D., 236, 252, 262, 311 Berger, J., 251, 260, 295, 299
Bergstrom, L., 5, 50 Bergy, M. E., 259, 295 Berkoz, B., 32, 45 Bemheim, B. C., 290, 300 Bemstein, J., 37, 47 Berthelot, K., 338, 350 Bessell, C. J., 286, 295 Bezborodov, A. M., 253, 256, 295 Bhat, J. V., 254, 295 Bhate, D. S., 251, 285, 295 Bhatt, V. V., 249, 261, 313 Bhuyan, B. K., 278, 295 Bianchi, M. L., 240, 311 Bickel, H., 7, 45, 259, 260, 295, 298 Bilimoria, M. H., 254, 295 Birch, A. J., 258, 270, 295 Birkinshaw, J. H., 30, 46 Birlova, L. V., 280, 313 Bishop, M., 28, 48 Bitteeva, M. B., 265, 295 Bittner, J. J., 281, 295 Bizioli, F., 250, 294 Blackwood, A. C., 138, 139, 140, 141, 143, 144, 145, 146, 147, 153, 154 Blake, J. T., 292, 295 Blinov, N. O., 264,295 Bliss, E. A., 290, 295 Blobel, H., 292, 295 Block, S. S., 44, 46 Bloemendal, H., 283, 295 Blom, R. H,, 150, 153 Blumsom, N. L., 257, 295 Blyumberg, N. A., 280, 295, 296 Bocker, H., 285, 313 Bohonos, N., 277, 303 Bolduan, 0. E. A., 115, 133 Boller, R.,292, 295 Bolton, E. T., 101, 103, 131, 132, 133 Bondareva, A. S., 280, 305 B o d , G. J., 46 Boniece, W. S., 263, 304 Boothe, J., 26, 47 Boothroyd, B., 263, 309 Boretti, G., 257, 297 Borisova, A. I., 253, 302 Borisova, C. N., 268, 310 Borisova, L. N., 241, 294 Boroff, C. D., 151, 153 Borowski, E., 268, 303
357
AUTHOR INDEX
Bosch, L., 283, 295 Boska, J. A,, 270, 307 Boswel, M. C., 333, 335, 350 Bourse, R., 252, 262, 305 Bouthilet, R. J., 327, 350 Bower, R. R., 240, 252, 260, 307 Boyko, V. I., 277, 295 Bradley, D., 24, 49 Bradley, S. G., 199, 214, 241, 243, 244, 255, 265, 295, 296, 301 Bradner, W. T., 273, 278, 296, 310 Braendle, D. H., 199, 214 Bragg, P. D., 283, 296 Brau, H. M., 331, 350 Braus, D. H., 335, 350 Brazhnikova, M. G., 262, 264, 296, 304 Brenner, S., 16, 36, 46 Brierley, M. R., 166, 167, 168, 187 Britten, R. J., 101, 103, 131, 133 Brock, M. L., 276, 296 Brock, T. D., 276, 296 Brockmann, H., 265,296 Brookes, P., 27, 46 Broschard, R. W., 26, 50, 277, 314 Brown, D. J., 19, 46 Brown, D. M., 27, 45, 46 Brown, G. L., 17, 49, 192, 214 Brown, M. E., 237, 296 Brown, W. E., 139, 141, 147, 153 Brown, R., 261, 296 Browne, F. L., 233 Browning, C. H., 42, 46 Brunner, R., 236, 296 Bryanskaya, A. M., 238, 301 Buchanan, B. B., 239, 296 Buchanan, J., 4, 45 Buchanan, J. M., 27,48 Buckman, S. J., 221, 231, 233 Biicher, T., 282, 310 Biichner, E., 340, 350 Bukala, M., 319, 350 Bu’Lock, J. D., 257, 296 Bungay, H. R., 165, 179, 185 Buonassisi, V., 209, 214 Burch, C. W., 292, 295 Burch, M. R., 206, 311 Burchard, W., 285, 303 Burchenal, J. H., 275, 312
Burhzyk, B., 319, 350 Burdette, W. J., 274, 296 Burg, R. W., 266, 299 Burkholder, P. R., 247, 313 Bums, R. O., 349, 351 Burris, R. H., 166, 187 Burt, A. M., 161, 185 Burton, K., 190, 199, 214 Burvill, M., 25, 26, 45 Butkevich, N. V., 110, 132 Butkevich, V. S., 110, 132 Butte, J. C., 258, 301 BUU-HOT,N., 18, 19, 46 Buyanovskaya, I. S., 262, 310 Bychkova, M. M., 262, 310
C Cahours, A., 318, 350 Caimey, T. L., 262, 315 Callow, D. H., 145,153 Calvalitto, S., 11, 46 Calvin, M., 199, 215 Cameron, D. G., 286, 287, 296 Cameron, D. W., 258, 295 Camiener, W., 251, 279, 307 Campbell, A. H., 266, 295 Canetti, G., 270, 299 Canevazzi, G., 250, 294 Canfield, J. H., 63, 64 Capriotti, A., 252, 296 Caputo, A., 279, 296 Carilli, A., 182, 185 Carriker, A. W., 117, 132 Carroll, R. B., 335, 350 Carss, B., 45 Carter, H. E., 266, 268, 270, 281, 296, 299, 309 Casas-Campillo, C., 255, 296 Castor, J. G. B., 69, 93, 342, 343, 347, 350, 351 Cemi, V., 277, 296 Chaiet, L., 28, 48, 263, 305 Chain, E. B., 166, 167, 172, 182, 185 Chaix, P., 68, 92 Chancel, G., 318, 350 Chandhuri, N., 46 Chang, L., 283, 314 Chapman, A. C., 335, 350 Charalampous, F. C., 256, 257, 296
358
AUTHOR INDEX
Chargaff, E., 27, 49, 76, 92 Chase, M., 35, 47 Chattejee, A. N., 265, 299 Cheng, L., 270, 311 Cherchess, B. Z., 257, 307 Cherenkova, L. V., 262, 310 Chemukh, A. M., 268, 296 Chiba, T., 272, 301 Chidester, C. G., 259, 268, 311 ChiIton, W. S., 268, 309 Chimenes, A. M., 69, 91 Chochlov, A. C., 236, 252, 262, 311 Chorine, V. A., 273, 280, 296 Chow, A. W., 262, 315 Christian, W., 30, 50 Christie, A. O., 239, 296 Chu, E. H. Y., 209, 214 Ciegler, A., 208, 214 Ciferri, O., 255, 296 Ciocalteu, V., 340, 351 Clark, J. B., 254, 296 Clarke, P. H., 4, 46 Clatworthy, W. W., Jr., 275, 294 Clauberg, K. W., 291, 296 Clemente, J., 288, 308 Clendenning, J. R., 64, 117, 119, 121, 123, 125, 126, 132 Clendenning, K. A., 139, 141, 151, 153 Cleveland, L. R., 39, 46 Cluzel, R., 267, 296 Cluzel-Nigay, M., 267, 296 Coetzee, J. N., 270, 296 Coffey, G., 221, 233 Coffman, J. R., 335, 353 Cogan, D., 36, 46 Coghill, R. D., 138, 141, 155 Cohen, A. I., 209, 214 Cohen, B., 52, 55, 64 Cohen, G. N., 6,413 Cohen, S. S., 20, 35, 46, 50, 77, 91, 283, 296 Colebatch, J. H., 275, 280, 296 Collins, J. F., 10, 46 Collins, R. J., 18, 46 Conway, S. J., 240, 314 Cooper, C. M., 163,185 Cooper, P. D., 10, 46, 49, 161, 185
Corcoran, J, W., 258, 296, 301 Cori, G. T., 30, 50 Corman, J., 162, 164, 172, 185 Corse, J., 328, 350 Cosslett, Y,, 13, 49 Costich, E. W., 180, 186 Cosulich, D. B., 26, 50, 277, 314 Cotta-Ramusino, F., 266, 297 Couillard, P., 330, 352 Cowie, D. B., 101, 103, 131, 132, 133 Crathom, A., 6, 46 Craveri, R., 260, 297 Cravetz, H., 222, 233 Crick, F. H. C., 15, 36, 46, 50 Cross, T., 270, 297 Crosse, R., 263, 309 Crowell, E. A., 343, 344, 346, 347, 351 Cullen, W. P., 278, 279, 308 Cummins, C. S., 3, 28, 46, 240, 297 Curtin, J. A., 127, 128, 129, 132, 288, 307 Curtiss, R., 281, 303
D Daghetta, A., 326, 350 Dagley, S., 281, 297 Dale, R. F., 166, 167, 169, 184, 187 Ddia Maia, M. H., 247, 297 Dameshek, W., 288, 297 Danauskas, J. X., 290, 300 Daniels, E. E., 270, 296 Dann, M., 277, 303 Dann, O., 19, 46 Dargeon, H. W., 275, 312 Dark, F., 2, 49 Davey, M., 23, 24, 45 Davidek, J., 264, 297 Davies, M. C., 247, 313 Davis, B. D., 27, 45, 284, 308 Davis, E. V., 181, 169, 186, 187 Davis, G. H. C., 241, 297 Davis, J. B., 52, 56, 64, 128, 132, 254, 297, 308 Davison, A., 4, 45 DeBoer, C., 250, 261, 305 DeBruyn, P. P., 23, 42, 46, 76, 92 Deindoerfer, F. H., 182, 185, 236, 300
AUTHOR I N D E X
de la Haba, G. L., 17, 50 Delaney, J. E., 109, 133 DelDuca, M. G., 56, 64 De Ley, J., 5, 46 Delova, I. D., 244, 311 De Mars, R., 36, 46 Demeter, M., 283, 298 Demny, T. C., 238,297 DeMorais, J. 0. F., 247, 297 Dknes, G., 23, 46 Denisova, S. I., 246, 305 Denisova, V. F., 278, 310 deVries, M., 327, 352 Dewey, V., 20, 47 Dholakia, G. R., 292, 297 Dierickx, L., 254, 298 Dietrich, K. R., 348, 350 Dietz, A., 244, 250, 251, 259, 261, 278, 279, 295, 297, 305, 307 DiMarco, A., 257, 273, 277, 294, 297 Di Menna, M. E., 238, 297 Dimick, K. P., 328, 350 Dion, W. M., 182, 185 Divekar, P. V., 260, 297 Djerassi, C., 32, 45 Dmitrieva, V. S., 262, 310 Dobias, B., 291, 297 Dobrzanski, W. T., 270, 297 Docky, R., 5, 46 Domercq, S., 266, 308 Domoto, K., 275, 297 Donker, H. J. L., 135, 154 Donovick, R., 37, 47, 263, 297 Doskocil, J., 204, 215 Doskocilova, D., 259, 297 Doty, P., 191, 192, 214, 215 Doudney, C. O., 281, 297 Doudoroff, M., 7, 46 Dougherty, R., 34, 46 Dourmashkin, R., 34, 46 Dowler, W. M., 281, 308 Drescher, R. F., 219, 233 Droubet, E., 282, 297 Druzhinina, E. N., 268, 302 Dryden, E. C., 325, 353 Drysdale, R. B., 190, 192, 214 Dubin, D. T., 34, 45 Dubinin, N. P., 245, 297
359
DuBoy, H. G., 290, 311 Duhaux, E., 335, 350 Dumas, J. B., 317, 318, 350 Dumbell, K., 37, 45 Dunitz, J. D., 18, 46 Dunn, D., 20, 36, 46 Durel, P., 289, 309 Durodi6, J., 335, 350 Dutcher, J. D., 11, 50, 263, 297 Dutt, S., 335, 350 Dutta, G. P., 39, 48 Duvall, L. R., 276, 297 Dworschack, R. G., 162, 164, 172, 185, 260, 297 Dyer, J. R., 270, 296
E Easterbrook, K. B., 46 Ebert, M., 22, 50 Ebisuzaki, K., 341, 350 Eble, T. E., 259, 295 Ebringer, L., 282, 297 Ecker, R. E., 160, 162, 164, 174, 175, 176, 177, 185, 186 Edwards, 0. F., 240, 241, 297 Efrom, A., 150, 153 Egawa, Y., 262, 266, 307 Eggers, H., 38, 46 Egorova, S. A., 244, 303 Ehrlich, F., 330, 338, 339, 348, 350 Ehrlich, J,, 259, 260, 298, 299 Ehrlich, R., 119, 132 Eigner, J., 192, 214 Eisenberg, G. M., 290, 314 Ekstrom, P. G., 335, 350 Elbein, A. D., 256, 297 Ellis, B., 18, 46 Ellis, L. F., 292, 295 Elpiner, I. E., 253, 255, 298, 308 Elsden, S. R., 340, 348, 353 Elsworth, R., 174, 186 Emery, T., 7, 46 Emilianowicz-Czerska, W., 267, 268, 297 Emmerling, O., 338, 350 Emmons, C. W., 260, 298 Enders, C., 333, 335, 350 Enebo, A. P., 332, 335, 351
360
AUTHOR INDEX
Engelberg, H., 270, 283, 298 Ephrussi, B., 23, 46, 69, 91 Erdos, T., 27, 46, 283, 298 Erlcnrneyer, E., 318, 351 Ethiraj, S., 237, 308 Etienne, A. D., 330, 352 Evans, A. E., 277, 298 Evans, J. S., 278, 298 Everett, H. J,, 180, 186
F Fadeeva, N. P., 253, 255, 298, 308 Fairbrother, R., 9, 46 Falconer, C., 28, 48 Fantes, K. H., 263, 309 Fantini, A. A,, 207, 214 Farkas-Himsley, H., 283, 298 Farr, R., 23, 42, 46 Federico, L., 340, 348, 350 Fedor, W. S., 152, 154 Fedorov, M. V., 285, 298 Feger, V. H., 162, 184, 172, 185 Feitelson, B., 18, 46 Feldman, L. I., 255, 298 Femstrom, G. A., 163, 185 Few, A., 5, 47 Fiala, A., 66, 71, 91 Fiala, S., 66, 71, 91 Filipposyan, S. T., 280, 296 Finn, R. K., 160, 161, 163, 164, 167, 179, 180, 186 Flaks, J., 20, 46, 77, 91 Fleischmann, L., 340, 348, 350 Fletcher, D. L., 286, 295 Flippin, H. F., 290, 314 Fliick, V., 251, 262, 310 Foght, J. L., 268, 309 Foley, G. E., 209, 214, 272, 298 Folin, O., 340, 351 Folkers, K., 28, 37, 38, 48, 50 Folkes, J. P., 18, 47, 74, 91 Foluitie, Y., 335, 351 Fomina, I. P., 272, 275, 278, 306 Forbes, E., 196, 215 Ford, J., 31, 46 Foster, J. W., 349, 351 Fowler, G., 71, 92 Fox-Hulme,P., 37, 45
Frady, J., 254, 296 Frajola, W. J., 199, 214 Frank, H., 4, 50 Frank, W., 277, 298 Franklin, R. M., 26, 49, 76, 80, 92, 265, 309 Frazier, W. R., 270, 307 Freeman, C. G., 137, 139, 141, 142, 143, 144, 145, 146, 147, 148, 154 Freundlich, M., 349, 351 Frohardt, R. P., 260, 298 Fromherz, K., 339, 352 Frush, H. L., 102, 132 Fujino, M., 261, 311 Fujita, H., 274, 301 Fujiwara, T., 261, 298 Fukasawa, T., 195, 215, 281, 314 Fukuda, D. S., 255, 304 Fukushima, T., 290, 294 Fuller, W., 17, 49 Fulmer, E. I., 138, 141, 143, 149, 154 Fulrnor, W., 26, 50, 277, 314 Fulton, J. D., 41, 46, 47 Funaki, M., 263, 309 Furth, J., 23, 24, 26, 47 Furukawa, T., 340, 348, 353 Furushiro, K., 261, 298 Fuscoe, J. M., 56, 64 Fusillo, M. H., 1.60, 186
G Gaden, E. L., 156, 172, 182, 185, 186 Gaetani, M., 273, 277, 294, 297 Caeumann, E., 259, 260, 281, 295, 298 Galanina, L. A., 270, 298 Calasso, G. J., 272, 298 Gale, E. F., 6, 18, 47, 74, 79, 91 Gambassini, L., 286, 307 Garcia-Mendoza, C., 285, 298 Gamer, H. R., 256, 297 Garretson, A. L., 279, 305 Garrett, E. R., 270, 298 Garrett, G., 9, 46 Garrod, L. P., 286, 298 Carside, J. S., 292, 300 Cattani, M. L.. 263, 298 Gaudy,-E., 281, 299
361
AUTHOR INDEX
Cause, G. F., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 262, 271, 298 Gavrilova, 0. A., 255, 302 Geiger, F., 265, 310 Genevois, L., 330, 331, 335, 340, 342, 350, 351 Genoese, V., 261, 298 Georg, L. K., 240, 298 Georgi, C. E., 138, 141, 144, 146, 147, 154 Georgiadis, M. P., 268, 309 Gerke, J. R., 269, 298 Gershanovich, V. N., 72, 73, 92 Gesheva, R. Z., 246, 303 Ghosh, B. K., 265, 299 Ghuysen, J. M., 254, 298 Giardinello, F. E., 161, 186 Gibson, F., 866, 298 Gibson, M. I., 43, 45 Gilardi, E., 244, 298 Gilby, A. R., 5, 47 Ginsberg, D. M., 177, 186 Ginzburg, G. M., 262, 310 Giolitti, G., 259, 266, 304, 310 Giovanella, B., 279, 296 Giuffre, N. A., 274, 298 Giuliano, R., 279, 296 Glauert, A. M., 241, 299, 300 Goldacre, R., 23, 24, 43, 45 Goldat, S. U., 200, 213 Goldberg, I. H., 265, 299, 309 Goldberg, M. W., 251, 295 Goldner, B. H., 63, 64 Goldstein, A,, 252, 307 Goll, M., 221, 234 Golyakov, P. N., 269, 299 Gooderham, J. L., 333, 335, 350 Goodgal, S. H., 281, 299 Goodman, J. J., 270, 299 Gordon, J. J., 280, 294 Gordon, R. E., 240, 299 Gordon, R. F., 292, 300 Goret, P., 239, 308 Gorskaya, S. V., 256, 310 GottIieb, D., 244, 266, 281, 282, 299, 308
Gourevitch, A., 11, 47, 273, 277, 296, 299, 310 Gowdy, B., 119, 132 Grace, N. H., 152, 155 Gracheva, I. V., 256, 310 Grasser, R., 239, 299 Graessle, 0. E., 261, 299 Grant, P. T., 41, 46, 47 Gray, J. E., 278, 298 Green, A. A., 30, 50 Greenberg, G., 45 Greenfield, R. B., 201, 214 Gregory, K. F., 253, 299 Grein, A., 250, 294 Greve, F., 251, 262, 310 Griffin, G. J., 238, 299 Grimstone, A. V., 38, 47 Grisebach, H., 22, 50 Gros, F., 19, 48 Gross, F., 261, 309 Grosshard, E., 238, 299 Grossberg, S. E., 288, 307 Grosset, J., 270, 299 Grove, J., 31, 45 Grunberg, E., 260, 299 Gryaznof, V. P., 335, 351 Gualandi, G., 166, 167, 172, 185 Guda, H. E., 199, 214 Guillaume, J., 285, 299 Guimberteau, G., 331, 348, 352 Gundersen, K., 31, 47, 266, 281, 299 Gunner, J,, 18, 46 Guymon, J. F., 328, 329, 342, 343, 344, 346, 347, 350, 351
H Haas, F. L., 281, 297 Hackett, D. P., 253, 256, 306 Hafslund, E. R., 321, 351 Hagemann, G., 251, 260, 299 Hahn, F. E., 18, 19, 47, 280, 299 Haines, H. J., 240, 241, 297 Haldar, D., 265, 299 Hall, G., 7, 45 Hamamoto, K., 241, 306 Hamill, R. L., 263, 299 Hamner, C., 31, 46 Hampton, A., 43, 45
362
AUTHOR INDEX
Hamrk, D., 37, 47 Hancock, R., 18, 47, 283, 299 Handler, A. H., 209, 214 Haney, M. E., Jr., 263, 299 Hansen, S., 18, 46 Harada, K., 195, 214 Harada, Y., 269, 314 Harbers, E., 26, 47 Harden, A., 135, 154 Harington, J. S., 254, 299 Harmsen, G. W . , 138, 139, 141, 143, 144, 149, 154 Harold, F. V., 327, 328, 332, 335, 351 Harris, H., 3, 4, 28, 46 Harris, J. O., 52, 64 Harris, R. J,, 34, 46 Harrison, V. R., 102, 103, 106, 107, 108, 115, 132 Hasimoto, M., 313 Haskell, T. H., 259, 299 Hata, T., 279, 300 Hatch, F. A., 335, 350 Hausmann, W. K., 277, 303 Hawrylewicz, E., 119, 132 Hayes, W., 65, 92, 194, 214 Hazen, E. L., 291, 297 Heden, C. G., 174, 177, 186 Heidelberger, C., 46 Heim, A. H., 64, 107, 108, 117, 119, 121, 123, 125, 126, 127, 128, 129, 132 Hein, H., 282, 299 Heinemann, B., 279, 310 Heinemann, K. E., 273, 310 Heinisch, B., 335, 350 Heinrich, M., 20, 47 Hejzlar, M., 290, 299 Hell, C., 318, 351 Hellstrom, N., 333, 335, 351 Henderson, K., 35, 47 Hendrickson, R., 137, 154 Herbert, D., 174, 186 Hkretier, P., 23, 46 Herman, H., 267, 268, 297 Hermanova, K. I., 280, 299 Hembdi, F., 238, 271, 299, 314 Herold, M., 238, 31 1 Herr, L. J., 237, 300
Herr, R. R., 259, 295 Hershey, A. D., 35, 36, 47 Hess, W. C., 102, 103, 105, 106, 107, 108, 109, 115, 132 Hesseltine, C. W., 242, 299 Heuser, L. J., 263, 270, 297, 307 Hewitt, L. F., 54, 64, 166, 186 Hichens, M., 268, 309 Hickey, R. J,, 136, 146, 149, 151, 152, 155 Higa, A., 265, 303 Higashide, E., 249, 251, 261, 262, 263, 264, 300, 301, 306, 311 Higgens, C. E., 263, 304 Hikiji, T., 250, 262, 278, 280, 305, 307 Hildebrand, R. P., 327, 328, 332, 335, 351 Hill, L. R., 244, 298 Hill, R. L., 244, 300 Hine, R. B., 283, 300 Hirai, T., 293, 311 Hiraiwa, F., 249, 266, 313 Hirsch, H. M., 87, 92 Hirsch, P., 240, 256, 258, 300, 306 Hirsch, U., 257, 304 Hirte, W., 237, 300 Hirth, L., 282, 297 Hitchings, G., 37, 50 Hitchings, G. H., 22, 47 Hitomi, H., 249, 261, 262, 263, 264, 266, 300, 311, 313 Hixson, A., 166, 186 Hlavka, J,, 26, 47 Hoagland, M. B., 14, 16, 47 Hoan, N., 18, 19, 46 Hoare, D. S., 3, 47 Hobbs, B. C., 292, 300 Hodes, M., 27, 49 Hodge, H. M., 172, 185 Hoehn, M. M., 252, 260, 263, 304, 312 Hoeksema, H., 268, 300 Holaday, W. J., 275, 294 Holloway, P. W., 258, 295 Holper, J. C., 252, 307 Holt, J. N., 284, 309 Honda, H., 260, 294 Hook, E. W., 288, 301, 307
AUTHOR INDEX
Hooper, I. R., 273, 279, 310 Hopkins, J. W., 19, 47 HOPPS,H. E., lC8, 47, 290, 300,311 Hopwood, D. A., 200, 214, 241, 242, 299, 300 Horigome, E., 280, 300 Horii, S., 263, 264, 300, 313 Horne, R., 13, 49 Horowitz, J,, 76, 92 Horsley, L. H., 319, 351 Horst, R. K., 237, 300 HorvAth, I., 63, 64, 241, 300 Hosler, P., 167, 179, 184, 185, 186 Hossenlopp, C., 279, 300 Hosty, T. S., 240, 298 Hotchkiss, M., 240, 241, 297 Hottinguer, H., 23, 46, 69, 91 Houssian, A., 340, 351 Howell, A., 239, 308 Howell, C., 23, 45 Hsu, T. C., 209, 214 Huber, G., 250, 261, 262, 304 Hudson, J. R., 332, 351 Huebener, H. J., 252, 306 Hiitter, R., 244, 24.5, 246, 251, 259, 261, 266, 295, 298, 300, 306, 315 Hughes, D. E., 5, 47 Hughes, M. S., 263, 313 Hulyalkar, R. K., 251, 265, 295 Humphrey, A. E., 236, 300 Hunter, G., 6, 46 Hunvitz, C., 281, 283, 300 Hunvitz, J., 23, 24, 26, 47
I Ichinose, K., 241, 312 Ikawa, M., 3, 47, 49 Ikeda, A., 183, 187 Ikeda, Hatsuko, 269, 301 Ikeda, Hiroshi, 269, 301 Ikeda, R. M., 321, 324,330,331, 333, 335, 351, 353 Ikeda, Y., 215 Ikegami, R., 274, 301 Iljina, T. S., 241, 294 Imakawa, S., 183, 187 Imanishi, M., 263, 215, 300, 301 Imanishi, Y., 268, 293
363
Imsenecki, A. A,, 283, 300 Ingraham, J. L., 328, 329, 343, 344, 346, 347, 349, 351, 352 Inoue, M., 261, 311 Inouye, S., 271, 310 Intile, J. A., 240, 300 Intoni, R., 266, 297 Isbell, H. S., 102, 132 Ishida, N., 259, 262, 272, 301, 303, 305 Ishiki, Y., 290, 294 Islam, M. F., 264, 293 Isono, K., 252, 269, 278, 300, 306, 312 Issaguljanz, W., 335, 352 Itakura, C., 251, 277, 306 Ito, E., 9, 49 Ito, M., 244, 272, 294, 301 Ito, T., 8, 47 Ito, Y., 263, 300 Ivanitskaia, L. P., 70, 87, 90, 92, 273, 300 Iwasaki, E., 249, 306 Iwasaki, H., 249, 251, 261, 262, 266, 300, 301, 313 Iwataru, K., 279, 300 Iyer, V. N., 282, 308
J Jackson, E. B., 290, 300 Jackson, P. W., 274, 298 Jackson, R. W., 162, 164, 172, 185, 260, 297 Jacob, A., 248, 300 Jacob, F., 16, 46, 80, 92, 193, 194, 214, 215 Jacquignon, P., 18, 19, 46 Jagger, J., 177, 186 Jagnow, G., 237, 300 Jakubov, G. Z., 264, 295 Janirek, G., 264, 297 Jannasch, H. W., 109, 112, 132 Jarai, M., 257, 302 Jarvis, A. w., 288, 301 Jeener, R., 36, 47 Jenard, H., 335, 351 Jeney, A., 238, 271, 299, 314 Jensen, W., 325, 335, 351
364
AUTHOR INDEX
Joel, C. D., 30, 45 Johnson, A. C., 240, 314 Johnson, M. H., 10, 48, 164, 165, 167, 169, 186 Johnson, hl. J., 141, 143, 145, 146, 147, 148, 154 Johnston, R. N., 291, 301 Johnston, V. D., 328, 352 Jones, A. C., 57, 64 Jones, G. E., 109, 112, 132 Jones, L. A., 199, 214, 243, 244, 255, 296, 301 Jones, O., 16, 48 Jones, R., 31, 47 Jones, R., Jr., 273, 301 Joubert, L., 239, 308 Junk, R., 252, 306
K Kambach, K., 333, 335, 350 Kagino, K., 271, 310 Kalakutski, L. V., 239, 243, 301 Kallina, D., 328, 351 Kallio, R. E., 57, 64 Kalowsky, A., 269, 314 Kalyuzhnaya, L. D., 238, 301 Kambayashi, A., 328, 351 Kamijo, S., 275, 297 Kanai, R., 256, 258, 301 Kanao, M., 280, 307 Kanazawa, K., 249, 266, 313 Kanda, N., 251, 301 Kaneda, T., 258, 301 Kanzaki, T., 249, 251, 261, 264, 300, 301, 306, 311 Karasawa, K., 259, 305 Kariczag, L., 341, 352 Karow, E. O., 166, 167, 185, 186 Kashii, K., 268, 293 Kashkin, P. N., 290, 301 Kasparova, J., 204, 215 Katagiri, H., 270, 283, 301 Katagiri, K., 262, 263, 267, 276, 280, 303, 305, 311, 315 Katayama, M., 269, 301 Kato, H., 272, 301 Kato, S., 241, 312 Katsuta, K., 277, 312
Katz, E., 264, 265, 301, 314 Katznelson, H., 140, 154 Kaufman, H. E., 36,47 Kavanagh, F. W., 178, 186 Kawaguchi, H., 261, 305, 307 Kawamata, J., 75, 92, 255, 274, 275, 301, 306 Kawato, M., 250, 256, 301, 311 Kaye, D., 288, 301 Kaz, G. I., 72, 73, 92 Kaz, L. N., 241, 308 Keleman, M., 4, 45 Kellenberger, E., 5, 47 Keller-Schierlein, W., 7, 45, 261, 298, 300 Kellner, B., 90, 92 Kelly, H., 27, 50 Kelly, S. E., 162, 164, 172, 185 Kelsh, J. M., 160, 185 Kent, L., 2, 49 Kepner, R. E., 321, 323, 324, 327, 328, 330, 331, 332, 333, 335, 343, 351, 353 Kersten, H., 26, 47, 264, 274, 281, 301, 308 Kersten, W., 26, 47, 264, 274, 281, 301, 308 Kessler, G., 28, 48 Kessler, W. B., 273, 301 Kesterson, J. W., 137, 154 Keynan, A., 265, 303 Khoi, N., 18, 19, 46 Khokhlov, A. S., 257, 262, 307, 310 Khokhlova, J. M., 264, 295 Kidder, G., 20, 47 Kimura, S., 290, 294 Kingsburg, D. W., 265, 301 Kinsky, S. C., 32, 33, 47, 281, 282, 301 Kinsolving, C., 29, 49 Kirby, K. S., 87, 92 Kirillova, N. F., 244, 246, 302 Kirk, J. M., 264, 302 Kirk, J. T. O., 283, 302 Kirschfeld, S., 20, 50 Kiser, J. S., 288, 308 Kivman, G. Y., 268, 296 Klammerth, O., 348, 350
AUTHOR INDEX
Klein, D., 285, 302 Klens, P. F., 221, 233 Klisiewicz, J. M., 293, 302 Klomparens, W., 31, 46 Klosowska, T., 238, 302 Klouwen, H. M., 199, 214 Knock, F. E., 280, 302 Knothe, H., 260, 302 Knox, R., 181, 186 Kobayashi, K., 256, 302 Kochetkova, G. V., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92 Kohler, H., 268, 302 Koening, M. G., 288, 301 Koepsell, H. J., 162, 154, 172, 185, 201, 215 Koffler, H., 256, 297 Kolesinska, J., 266, 302 Kolesnikova, I. G., 238, 295 Kollar, S. J., 257, 302 Kolosov, N. H., 236, 252, 262, 311 Komarowsky, A., 329, 351 Komoda, H., 344, 348, 353 Komppa, G., 335, 351 Kondo, E., 255, 302 Kondo, H., 260, 2161, 304, 307 Kondo, S., 252, 260, 302, 309 Konev, Y. E., 245, 252,302 Konova, I. V., 253, 302 Konovalov, S. A., 353 Konstantinova, N. V., 262, 296 Kontras, S. B., 275, 294 Kontratieva, I. K., 252, 303 Kooi, E. R., 138, 141, 143, 149, 154 Korchagin, V. B., 268, 302 Koreniako, A. I., 244, 245, 246, 252, 255, 302, 303, 306 Kornfeld, E., 31, 47 Korobitskaya, A. A., 268, 302 Koroleva, V. G., 275, 278, 306 Korotyaev, A. I., 281, 302 Korzybski, T., 236, 252, 302 Kosaki, M., 344, 348, 353 Koshiyama, H., 261, 307 Kosmatchev, A. E., 248, 302 Kotelev, V. V., 252, 302 Koveshnikov, A. D., 252, 280, 303
365
Kovsharova, I. N., 262, 296 Kowszyk-Gindifer, Z., 278, 308 Koyama, K., 290, 294 Koyama, Y., 260, 294 Kozloff, L., 35, 47 Kradolfer, F., 261, 309 Kramer, G., 318, 351 Krassilnikov, N. A., 236, 243, 244, 245, 246, 252, 280, 290, 294, 301, 302, 303, 306 Krazinski, H., 26, 47 Krebs, E., 30, 47 Krementz, E. T., 279,303 Kruglyak, E. B., 262,296 Krumperman, P. H., 221, 233 Kucera, C. J., 161, 186 Kuchkarev, R. N., 280,303 Kudryavina, N. A,, 259,295 Kuehl, F., 28, 48 Kiister, E., 243, 303 Kuffner, F., 328, 351 Kumagi, K., 276, 303 Kumamoto, S., 333, 335, 352 Kurioka, S., 252, 310 Kuroda, S., 240, 241, 244, 294 Kuroya, M., 262, 272, 301,303 Kuru, M., 277,303 Kurylowicz, W., 236, 252, 302 Kusnezov, V. D., 243,303 Kutchayeva, A. G., 246,303 Kutscher, A. H., 271, 303 Kutzner, H. J., 255, 257, 270, 303, 304 Kuziela, T., 271, 307 Kuznetsova, V. S., 265, 303
L Lackner, H., 265, 296 Lacks, S., 19, 48 Lafon, J., 330, 340, 352 Lafon, M., 323, 330, 331, 342, 351, 352 Lafon-Lafourcade, S., 266, 308 Laiko, A. V., 75, 76, 80, 81, 82, 91, 92,273,303 Lampen, J. O., 32, 48, 268, 269, 281, 282, 303, 305, 310, 311 Lampitt, L. H., 340, 352
366
AUTHOR INDEX
Li, M. C., 274,304 Lancaster, J. E., 277, 314 Lichtenstein, J., 20, 46, 77, 91, 283, Landau, J. V., 283,300 296 Landau, N. S., 83, 84, 85, 86, 88, 89, Liebmann, J., 139, 151, 154 92 Liebscher, W., 292, 295 Landman, 0. E., 285, 303 Lilly, M. D., 4,46 Lane, D., 192,214 Lindegren, C. C., 70,92, 208, 214 Lang, J. F., 221, 233 Lindenfelser, L. A., 237, 247, 304 Laurence, D. J. R., 96, 132 Lindet, L., 347, 352 Lautie, R., 281, 310 Lindner, F., 250, 261, 262, 304 Lavrova, M. F., 264,304 Lingappa, Y.,238, 304 Lawley, P. D., 27, 46, 48 Linnane, A., 29, 48 Lawshe, E. I., 331, 352 Lipman, F., 17, 48 Lam, W. R., 283,303 Lisenko, 2. A., 238, 301 Leapheart, C. D., 293, 314 Lister, A. J., 161, 186 Le Bel, J. A., 318, 352 Locci, R., 249, 294 Lebeurier, G., 282, 297 Lechevalier, H. A., 236, 242, 263, Locke, L. A., 344,353 Lockhart, W. R., 160, 161, 162, 164, 268, 303, 314 174, 175, 176, 177, 185, 18G, 291, Lederberg, J., 8, 48 301 Lederer, E., 5, 8, 45 Ledingham, G. A., 138, 139, 140, 141, Lockwood, J. L., 238,304 143, 144, 145, 146, 147, 148, 153, Lockwood, L. B., 138, 155 Loeb, M., 20, 46, 77, 91 154, 155 Lomakina, N. N., 264, 304 Lee, S. B., 145, 154 Lomovskaya, N. D., 241,294 Lefemine, D. V., 277, 303 Long, S. K., 137, 140, 141, 143, 144, Leider, M., 275, 303 145, 146, 148, 150,154 Lein, J., 11, 47, 273, 277, 279, 299, Longfellow, D., 87, 93 310 Loomis, W. F., 164, 186 Lemmon, R. M., 199, 215 Lengyel, P., 16, 19, 49, 193, 214, 215, Lorian, V., 290, 304 Lorkiewicz, Z., 78, 80, 92 284, 311 Loughlin, E. H., 292, 304 Lepeshkina, G. N., 280, 296 Loutovitch, E. B., 238, 312 Lerman, L. S., 24, 48, 199, 214 Leslie, J. D., 138, 140, 141, 143, 144, Louverture, A., 292,304 Lovell, C. L., 321, 351 145, 146, 153, 155 Lowery, W., 327,350 Lessel, E. F., 240, 303 Ludwig, H., 250, 304 Lessler, M. A., 199, 214 Liillmann, H., 290, 304 Lessner, H. E., 273, 301 Lugli, A. M., 259, 266, 304, 310 Lester Smith, E., 10, 49 Lukas, B., 290, 299 Lettre, H., 71, 92 Lummis, N. E., 267, 311 Levenberg, B., 27, 48 Levin, G. V., 64, 102, 103, 105, 106, Lute, M., 35, 47 107, 108, 109, 115, 117, 121, 123, Luzzati, V., 24, 48 Lwoff', A., 35, 48 125, 126, 127, 128, 129, 132 Lyashenko, V. A,, 273,296 Lcvine, M., 281, 303 Lyons, A. J., Jr., 244, 246, 304, 308 Levine, S., 341, 350 Lysikowska, L., 271, 307 Levinthal, C., 265, 303 Lysyi, I., 327, 353 Leyh-Bouille, M., 254, 298
AUTHOR INDEX
M Maass, E., 10, 48 McAdams, A. J., 289,304 McCall, K. B., 138, 141, 144, 146, 147, 154 McCarthy, J. A., 109, 133 McCarty, M., 190, 213 McClung, N. M., 240, 256, 293, 304 McCormick, J. R. D., 257, 304 McCoy, E., 201, 215 McDaniel, L. E., 268, 314 MeDougall, B., 266, 298 McDurmont, C. I., 240, 242, 298, 303 McFarlane, M., 5, 48 McGonagle, M. P., 263, 309 McGuire, J. M., 252, 260, 261, 263, 304, 312 Mach, F., 245, 255, 304, 308 Machado, M. P., 255, 296 Machek, G., 236,296 McKensie, A., 319, 321, 352 Mackenzie, H., 18,46 MacLeod, C. M., 190, 213 McLimans, W. F., 161, 169, 186,
187 MacMillan, J., 31, 48 McNary, J. E., 270, 296 McQuillan, K., 5, 47 McVeigh, I., 251, 304 Madigan, M. E., 269,298 Maeda, K., 260,261,304,307,312 Mahler, H., 34, 48 Maiko, I. I., 238, 301 Maitra, P. K., 253, 256, 304 Majumdar, S. K., 257, 264, 270, 293, 304 Makoto, M., 328, 351 Malamy, M., 23, 24, 26, 47 Malikukov, V. A., 278, 310 Mallett, G. E., 255, 304 Malmborg, A. S., 174, 177,186 Mandel, H. G., 20,48 Mandels, G. R., 222, 233 Mandelstam, J., 2, 4, 10, 48, 49 Manire, G. P., 272, 298 Mann, E. K., 274, 304
367
Mann, R. L., 256, 261,297, 304 Mann, W., 283,314 Manten, A., 286, 304 Marckwald, W., 319, 321, 352 Margalith, P., 251, 304 hlarini, F., 32, 48, 269, 282, 304, 305 Markham, R., 20, 48 Marmur, J., 191, 192, 214, 215 Marsden, J. P., 37, 48 Marsh, W. S., 279, 305 Martin, G., 16, 48 Martin, H., 4, 50 Marton, M., 237, 238, 246, 305, 312 Mason, D. J., 6, 48, 250, 259, 261, 305 Mason, H. S., 57, 64 Masson, F., 24,48 Masujima, T., 335, 353 Masumoto, K., 250, 271, 305 Masuo, E., 255,302 Mathaei, J. H., 16, 48, 193, 214 Mathers, A. P., 329, 352 Mathews, J., 244, 297 Matrishin, M., 270, 299 Matsubara, H., 272, 301 Matsubara, Y., 325, 333, 335, 352 Matsumoto, K., 272, 301 Matsuura, S., 262, 276, 303, 305 Matsuyama, A., 253, 306 Matsuzaki, M., 261, 307 hlatthews, R. E. F., 20, 36, 48 Maurer, P. R., 254, 305 Mauzy, W. L., 160,185 Mayama, M., 244, 251, 262, 303, 306 Mayevsky, M. M., 280, 305 Mazaeva, V. G., 280,305 Mecke, R., 327, 352 Mehrotra, B., 34, 48 Meier, K. E., 266, 305 Meisenheimer, J,, 340, 350 hlejia, M. J., 240, 308 Melechen, N., 36,47 Melnick, I.. 27. 48 Menon, S . k, 251,252, 260, 261, 265, 295, 31.7 Menshikov, G. P., 246, 305 Merritt, C., 29, 49 Mertz, C., 347, 349, 352
368
AUTHOR INDEX
Meryman, H. T., 210, 214 Meselson, M., 16, 46 Meyer, J., 257, 308 Meyer, W. E., 26, 50, 277,314 Meyerman-Wisse, M. J., 286, 304 Michel, B. J., 181, 186 Mickelson, M. N., 147, 154 Mihm, J. M., 240,299 Mikhailova, G. R., 245, 305 Miller, B. J., 181, 186 Miller, F. D., 335,350 Miller, I. M., 238, 263, 297, 305 Miller, M. W., 236, 305 Miller, R. E., 30, 48 Miller, S. A., 163, 185 Mindlin, S. Z., 200, 213, 245, 294, 305 Minoura, K., 268, 293 Minton, S., 37, 50 Mironova, I. B., 265,303 Mitchell, P., 2, 5, 6, 14, 48 Mitchell, R. S., 291, 308 Mitsugi, T., 255, 302 Mitsuhashi, S., 195, 214 Mitton, H. E., 141, 155 Miura, T., 261, 312 Miura, Y., 183, 187 Miyachi, S., 256, 258, 301 Miyagi, K., 241, 312 Miyairi, N., 267, 275, 305, 312 Miyaka, T., 261, 305 Miyakawa, T., 252, 302 Miyake, A., 249, 251, 261, 262, %63, 264, 266, 300, 301, 306, 313 Miyashita, Y.,283, 305 Miyazawa, S., 266,305 Mizuno, K., 261,311 Modarski, M., 273, 305 Moller, E., 19, 46 Moller, F., 244, 305 Molkov, Y. N., 280, 305 Momose, S., 275, 305 Monier, R., 68, 92 Monnier, J., 252, 262, 305 Monnikendam, P., 277,303 Monod, J., 6, 46, 80, 92 Montag, B., 46 Moorehead, P. S., 209, 214 Morey, A. V., 62, 63, 64
Mori, R., 238, 305 Morieson, A. S., 327, 328, 332, 335, 351 Morkubo, Y., 280, 307 Moroz, A. F., 282, 305 Morrell, S. A,, 139, 154 Morris, H., 70, 91 Morris, M. E., 238, 309 Morrison, R. I., 137, 141, 142, 145, 148, 154 Morthland, F. W., 76, 92 Mortimer, A. M., 286, 295 Moss, F., 174, 186 Moualim, R., 18, 46 Mowat, J. H., 26, 50, 277, 314 Moyle, J., 2, 5, 8, 14, 48 Mueller, M., 251, 295 Miiller, W., 28, 47 Muirhead, D. R., 138, 139, 141, 143, 144, 149, 154 Mullin, W. G., 292, 304 Munson, A. E., 279,294 Murase, M., 250, 262, 278, 305 Murphy, D., 137, 138, 139, 141, 143, 144, 145, 147, 149, 154 Murray, P. J., 327, 328, 332, 335, 351 Musilek, V., 255, 257, 305 Musilkova, M., 257, 305 Musser, E. A., 278,298 Mustakas, G. C., 150, 153
N Nachmansohn, D., 52, 64 Nagai, H., 70, 92 Nagai, S., 70, 92 Nagatsu, J., 251, 271, 277, 278, 294, 306, 312 Najjar, V., 30, 47 Nakagiri, J., 329, 351 Nakajima, K., 251, 306 Nakamura, G., 250, 251, 262, 271, 305 Nakamura, S., 259, 262, 305, 306 Nakao, Y., 253,315 Nakata, A., 255, 306 Nakazawa, K., 249, 251, 261, 262, 263, 264, 266, 300, 301, 306, 311, 313
AUTHOR INDEX
Namiki, M., 253, 306 Nath, V., 39, 48 Nathans, D., 17, 48 Naumova, I. B., 263,306 Navashin, S. M., 272,275, 278, 306 Neelameghan, A., 273, 306 Nefelova, M. V., 265, 269, 306 Neilands, J. B., 7, 8, 46, 47 Neipp, L., 261, 298, 309 Neish, A. C., 139, 141, 147, 151, 152, 153, 154 Nekachalov, V. Y., 290, 301 Nemes, M., 37, 38, 50 Nemeth, L., 90, 92 Nesemann, G., 250, 252, 304, 306 Neubauer, O., 339, 352 Neuberg, C., 341, 352 Neuhaus, F., 4, 45 Newton, B. A,, 41, 48 Neyasaki, T., 252, 310 Nickerson, W. J,, 28, 48 Niederpruem, D. J., 253, 256, 306 Niemczyk, H., 278, 308, 314 Niida, T., 241, 250, 306 Niinome, Y., 262,303 Niitani, H., 283, 306 Nikitina, N. I., 244, 245, 246, 252, 302, 303, 306 Nimmo-Smith, R., 37, 50 Nirenberg, M. W., 16, 48, 193, 214 Nishimura, H., 244, 251, 261, 306 Nishimura, T., 267, 2716, 277, 312 Nitla, K., 250, 262, 278, 305 Noda, Y.,313 Nogatsu, J., 252, 269, 306 Nolof, G., 240, 306 Nomi, R., 257, 305 Nomine, G., 251,260,299 Nordbring-Hertz, B., 44, 48 Nowland, P., 29, 48 Nuesch, J., 261, 306 Nunoko, N., 256,302 Nyiri, L., 242, 245, 306
0 Oata, Y., 20, 49 O’Brien, E., 201, 214 O’Brien, L. C., 335, 350
369
Ochoa, S., 16, 19, 49, 193, 214, 215 Oda, T., 261, 312 Officer, J., 37, 50 Ogasawara, M., 250, 306 Ogata, K., 253, 315 Ogata, Y., 325, 333, 335, 352 Ogawa, N., WS, 302 Ogur, M., 86,92 Ohashi, S., 261, 307 Ohashi, T., 280, 306 Ohkuma, K., 251, 263, 271, 277, 306 Ohmori, T., 261, 307 Ohmura, E., 253, 315 Ohtsuka, H., 251, 306 Okami, Y., 250, 260, 261, 862, 278, 305, 307, 312 Okamoto, S., 251, 306 Okanishi, M., 261, 307 Okazawa, Y., 253, 306 Okuda, T., 262, 266, 307 Okuma, K., 271,294 Oldshue, J. Y.,180, 181, 186 Olive, L. S., 207, 214 Oliver, T. J., 240, 252, 280, 261, 307 Olson, B. H., 141, 143, 145, 146, 147, 148, 154,248, 310 Ono, H., 328, 351 Ono, T., 69, 92 Onuma, M., 280, 307 Ordonneau, C., 335,352 Orezzi, P., 277, 297 Orleansky, V. K., 247, 293 Orlova, N. V., 257, 307 Orlova, T. I., 265,303 Ormerod, W. E., 40, 41, 48 Osato, T., 280, 307 Osawa, S., 20, 49 Ose, E. E., 292, 295 Osono, T., 280, 307 Osteux, R., 285, 299 Ostrowska-Krysiak, B., 256, 307 O’Sullivan, D., 38,48 Otaka, E., 20, 49 Otero Abalo, R., 252, 307 Otsuka, H., 267, 311 Otto, L. A., 63, 64 Otto, R. H., 240, 252, 260,307 Ouchi, T., 252, 307
370
AUTHOR INDEX
Overrein, L., 256, 300 Owen, S. P., 251, 279, 307 Owen, W. L., 137,154 Ozaki, M., 255,311 Ozawa, E., 292, 307 Ozeretskovsky, N. A., 262, 307 P Pagano, J. F., 263, 297 Palkina, N. A., 72, 73, 92 Panero, C., 286, 307 Park, J. T., 3, 10, 18, 47, 49, 74, 93, 281, 307 Parker, T., 31, 47 Parks, R., 20, 47 Pamas, J., 271, 307 Partilla, H., 292, 295 Pasternak, V., 30, 48 Pasteur, L., 318, 321, 352 Paszkiewicz, A., 278, 308, 314 Patki, S. J., 13, 45 Patrick, J., 26, 50, 277, 314 Patrick, R., 137, 140, 141, 143, 144, 145, 146, 148, 150,154 Paul, B. B., 264,293 Pawlowska, K., 238, 302 Peacocke, A. R., 23, 49, 190, 192, 214 Pedlar, A., 318, 352 Penasse, L., 251, 260, 299 Penniman, W. B. D., 331,352 Peppler, H. J., 166, 187 Perkin, M. P., 167, 180, 181, 186 Perkins, E. S., 36, 49 Perkins, H. R., 74, 92 Pedman, D., 141, 143, 144, 145, 147, 148, 154, 201, 214, 270, 274, 283, 298, 307, 310 Perry, D., 284, 307 Perry, J. J., 255, 256, 307 Petersdorf, R. G., 288, 307 Petit, J., 68, 92 Petrova, K. Z., 283, 300 Petrovskaia, V. G., 283, 307 Petrow, V., 18, 46 Pettijohn, 0. G., 138, 141, 155 Petty, M. A., 266, 307 Petty, T. L., 291, 308
Peynaud, E., 266, 308, 331, 340, 348, 352 Pfenninger, H., 328, 352 Phaff, H., 69, 93 Phares, H. F., 261, 299 Phillips, D. H., 164, 165, 167, 169, 186 Phillipsborn, W. V., 268, 309 Pho, D., 256,313 Pidacks, C., 26, 50, 277, 314 Piechowska, M. A., 256, 307 Pier, A. C., 240, 308 Pierpont, H., 274, 308 Pierre, J., 338, 352 Pinclell, M. H., 278, 296 Pine, L., 239, 256, 296, 308 Pinner, A., 318, 351 Pirt, S. J., 145, 153, 162, 174, 186 Pitot, H. C., 69, 92 Pittillo, R. F., 259, 260, 273, 298, 299, 308, 310 Plociennik, Z., 278, 308 Plotz, P. H., 284, 308 Polgar, L., 23, 46 Polglase, W. J., 283, 296 Pollard, M., 286, 308 Pollock, G. E., 335, 353 Ponnamperuma, C. A., 199,215 Pontecorvo, G., 196,215 Popken, F. E., 288,308 Popova, L. A., 269, 308 Porcaro, P. J,, 328, 352 Porteous, J. W., 239, 296 Porter, J. N., 238, 308 Portnov, S. M., 238, 301 Portolks, A., 266, 313 Post, R., 29, 49 Postgate, J., 62, 64 Potter, M. C., 52, 63, 64 Potter, V. R., 67, 68, 69, 92 Pound, G. S., 293, 302 Powelson, D. M., 6, 48 Pozmogova, I. N., 269, 306 Prabucki, A. L., 328,352 Pramer, D., 285, 302 Prauser, H., 257, 308 Prelog, V., 7, 45, 260, 261, 298 Preobrazhenskaya, T. P., 252, 312
AUTHOR INDEX
Prevot, A. R., 239,249, 308,310 Price, K. E., 273, 310 Pridham, T. G., 237, 244, 245, 246, 247, 260, 293, 297, 304, 308 Pringsheim, H. H., 338, 352 Prockop, D., 264, 301 Prokofieva-Belgovskaya, A. A., 241, 308 Prokop, J. F., 240, 260, 2.61, 307 Prominskaia, T. B., 238, 312 Proshlyakova, V. V., 262, 296 Pruess, L. M., 255, 298 Prusoff, W. H., 36, 49 Puchot, E., 338,352 Pugh, L. H., 264,301 Pursiano, T. A., 11, 47, 277, 299
Q Quinnelly, B. G., 273, 308 R Rabinowitz, M., 265, 299, 309 Radkin, N., 3,49 Raghunatha Rao, Y. N., 335,352 Raistrick, H., 30, 49 Rake, G. W., 161,186 Ramachandran, S., 281, 282, 299, 305 Rampan, Y. I., 259, 295 Rane, L., 273,301 Ranganayaki, S., 137, 141, 149,153 Rangaswami, G., 237,308 Rao, K. V., 277,278, 279,308,309 Raper, K. B., 208, 214 Rasetti-Nicod, G., 289, 309 Rauen, H. M., 26, 47, 264, 274, 308 Raut, C., 70, 92 Rautenstein, Y. I., 245, 253, 255, 256, 298,308 Ravin, A. W., 282, 308 Raymond, R. L., 254,297, 308 Reed, D. L., 150,153 Rees, C., 26, 45 Rees, M., 4, 45 Reese, E. T., 222, 233 Reeves, J. C., 292, 300 Regueiro Varela, B., 252, 307 Rehm, H. J., 237,309
371
Reich, E., 26, 49, 76, 80, 85, 92, 265, 267, 277,309 Reichenthal, J., 257, 304 Reiner, B., 27, 49 Renfroe, H. B., 268, 309 Renis, H. E., 256, 297 Renn, D. W., 279,309 Renner, U., 281,313 Reusser, F., 201, 209, 212, 215, 238, 309 Reusser, P., 261, 309 Reuter, H., 290, 304 Reyes, C. R., 251, 304 Reynolds, P. E., 285, 309 Rhine, P., 325, 351 Rhodes, A,, 263,309 Rhodes, R. P., 172,186 Rhone-Poulenc, 251, 262, 309 Rhuland, L., 75, 92 Ribereau Gayon, J,, 340, 352 Richards, J. W., 179, 186 Richardson, M., 284, 309 Richert, J. H., 240, 300 Richmond, M. H., 10, 46 Rickards, R. W., 258, 295 Rigler, N. E., 255, 298 Riklis, E., 63, 64 Rinehart, K. L., Jr., 268, 309 Ritchie, R. T., 291, 301 Rittenberg, D., 63, 64 Robbins, P. W., 266, 299 Roberts, P., 10, 49 Roberts, R. B., 101, 103, 131, 133 Robertson, F. M., 139, 152, 154 Robertson, R., 23, 42, 46 Robinson, A., 13, 44, 45 Robinson, F., 37, 38, 50 Robinson, H. J., 261, 299 Roch, L. A., 335, 350 Rodionovskaya, E. I., 262, 310 Rodriguez-Villanueva, J., 240, 309 Roelens, E., 335, 350 Rogers, H. J., 10, 49, 74, 92 Roiron, V., 289, 309 Romanenko, E. A., 280,305 Romansky, M. J., 240, 314 Roper, J. A., 196, 215 Rosano, C. L., 281, 283, 300
372
AUTHOR INDEX
Sato, Y., 256, 302 Saukkonen, J. J., 11, 49 Saurat, P., 281, 310 Savage, G. M., 201,206,215 Savelieva, A. M., 280, 299 Sazykin, I. O., 268, 280, 310 Scardovi, V., 266, 310 Schabel, F. M., Jr., 273, 308, 310 Schadewald, L. K., 259,311 Schaeffer, P., 193,214 Schaffner, C. P., 268,270,296,303,309 Scheele, C. W., 317, 352 Schell, P., 27, 46 Schicktanz, S. T., 330, 352 Schildkraut, C., 192,214 Schillings, R. T., 268, 309 Schissler, D. O., 57, 64 Schmid, J., 251, 262, 310 Schmidt, C. G., 276, 310 S Schmidt, U., 265, 310 Schmidt-Kastner, G., 251, 282, 310 Sachsenmaier, W., 71, 92 Schmidt-Thomi., J., 250, 252, 304, 306 Sackmann, W., 259,261,295,309 Schmitz, H., 273, 279, 282, 296, 310 Sacks, T. G., 270,296 Schneierson, S. S., 283, 310 Sadana, J. C., 62, 63, 64 Schneller, A,, 26, 47 Sadler, P. W., 37, 38, 45, 48 Schoen, M., 331,352 Safferman, R. S., 238,309 Schone, R., 247,310 Sager, R., 283,309 Schoeneman, R. L., 329,352 Sahay, B. N., 241,244, 309 Schonwalder, H., 242, 264, 310 Saito, H., 200, 215, 242, 309 Scholz, R., 282, 310 Saito, R., 263, 309 Schorigin, P., 335, 352 Sakagami, Y., 265,277,309, 312 Schramm, G., 27,49 Sakai, H., 261, 263, 298, 312 Sakamoto, J. M. J., 252, 260, 294, Schiipphaus, R. C., 319, 331, 352 Schuster, H., 27, 49 302, 309 Schuurmans, D. M., 248, 310 Salser, J. S., 256, 304 Schwyzer, R., 14,49 Salton, M. R. J., 4, 13, 49 Scott, D. S., 141, 145, 155 Sampey, J. R., 275,310 Scott, s., 8, 9, 49 San Clemente, C. L., 248, 310 Scotti, T., 273, 277, 294, 297 Sanders, P., 152,154 Seabury, J. H., 291,310 SBndor, L., 277, 296 Sears, M. L., 36, 49 Sands, N. K., 101,132 Sebald, M., 249, 310 Santoro, T., 256, 304 Seeler, G., 240, 310 Sarachek, A., 71, 92 Segi, I., 285, 298 Sarhaeva, N. A., 72,92 Seguin, L., 271, 303 Sartbaeva, A., 264,294 Sekiguchi, M., 255, 306 Satake, K., 252, 310 Sekizawa, Y.,258, 270, 271,310 Sato, G., 209, 214 Selbie, F., 43, 45 Sato, K., 262, 303
Rose, D., 141, 154 Rosen, G., 86, 92 Rosini, M. P., 241, 309 Ross, R. T., 222, 233 Rosseels, J., 36,47 Rossolimo, 0. K., 280, 296 Roth, G. D., 240, 309 Roth, N. G., 172, 185 Rothwell, F. M., 219, 233 Routien, J. B., 264, 309 Rowley, D., 10, 49 Roy, S. C., 253,256,304 Rubbo, S., 23, 24, 25, 26, 43, 45 Ruczah, Z., 278, 308 Rudaya, S. M., 244,245,309,312 Rushton, J. H., 180, 186 Ryabova, I. D., 280,295 Ryter, A., 5, 47
AUTHOR INDEX
Semenov, S. M., 268, 302 Semenova, V. A,, 246,262,310 SentheShanmuganathan, S., 340, 341, 348, 349, 352, 353 Sermonti, G., 182, 185, 196, 200, 207, 215 Sevag, M., 30,48 Severina, V. A., 256,310 Sexton, W. A,, 257, 310 Sfat, M. R., 166, 167, 185,186 Sgarzi, B., 259,266, 304, 310 Shafikova, F. A., 263,306 Shamina, Z. B., 241, 310 Shapovalova, S. P., 291,310 Sharlai, R. I., 278, 310 Sharp, C., 34,48 Shatkin, A. J., 26, 28, 49, 76, 80, 85, 92, 285, 267, 277, 309 Shavel’zon, R. A., 245,297 Shaw, W. H. C., 266,295 Sheffield, F. W., 49 Shemar, J. B., 270, 307 Shemiakin, M. M., 236, 252, 062, 311 Shepherd, C. J., 47, 76, 92 Sherman, F., 85,92 Sherris, J. C., 161, 185 Shiba, S., 75, 92 Shibata, M., 238, 249, 250, 251, 261, 262, 263, 208, 300, 301, 306, 311, 313 Shields, R. R., 279, 294 Shimada, Y., 250, 2433, 311 Shimaoka, N., 251,306 Shimizu, K., 261,298 Shimohira, M., 251, 306 Shimomura, T., 293, 311 Shinobu, R., 250, 251, 256, 263, 301, 311 Shirasaka, M., 255, 311 Shiratori, O., 262, 272, 301, 303 Shitara, J., 344, 348, 353 Shockman, G. D., 281, 311 Shoji, J., 262, 263, 267, 276, 303, 311 Shotwell, 0. L., 260, 297 Showacre, J. L., 290, 311 Shrimpton, D. H., 292, 300 Shugaeva, N. V., 86,92 Shunk, C., 37, 38,50
373
Shyu, W., 253,299 Siefker, J. A., 335, 353 Sih, C. J., 255, 311 Sikyta, B., 204, 215, 238, 311 Silaev, A. B., 265,303 Silvestri, L. G., 244, 300 Silvestri, T. L., 247, 311 Simon, E. H., 22, 49 Simon, R., 43, 45 Simons, C. F., 179,185 Simpson, F. J., 138, 140, 141, 143, 144, 145, 146, 147, 149, 153, 15-2, 155 Simpson, W., 70, 92 Sinclair, A. C., 261, 307 Sisler, F. D., 53, 64 Sjolander, O., 257, 304 Skerrett, J. N. H., 23, 49 Skipper, H., 27, 50 Skriabin, G. K., 246,303 Slade, H. D., 284, 307 Slezak, J., 238, 311 Sloneker, J. H., 281, 299 Smadel, J. E., 290, 300, 311 Smith, C. G., 209, 215, 268, 278, 295, 300 Smith, C. L., 209, 215 Smith, D. C., 331, 352 Smith, D. E., 335, 353 Smith, D. H., 291, 301 Smith, J., 20, 36, 46, 48 Smith, N. H., 76, 92 Smith, R. L., 266, 311 Smith, R. M., 250, 259, 305 Smith, S. M., 195,215 Sneath, P. H. A., 193,215 Snell, E., 3, 47, 49 Snell, J. F., 270, 295, 311 Sobin, B. A., 278,308 Soeda, M., 250,277,311 Soder, A., 250, 304 Sokoloff, B., 273, 311 Sokolova, A. I., 246, 252, 302 Sokolski, W. T., 259, 266, 267, 268, 311 Solomons, G. L., 167, 180, 181, 186 Solotorovsky, M., 242, 303
374
AUTHOR INDEX
Solovieva, N. K., 244, 245, 262, 309, 310, 311,312 Spada-Sermonti, I., 200, 21 5 Spalla, C., 240, 257, 297, 311 Spanyer, J . W., 340, 353 Spencer, M., 17, 49 Speyer, J. F., 16, 19, 49, 193, 214, 215, 284, 311 Spielman, M., 11, 50 Spinner, E., 23, 45 Spirin, A. S., 6, 45, 86, 92 Spotts, C. R., 27, 49, 82, 93, 283, 311 Squires, R. W., 167, 178, 182, 184, 186 Stacchini, A., 266, 297 Staehlin, M., 27, 49 Stamper, M., 263, 299 Stanier, R. Y., 7, 27, 46, 49, 82, 93, 140, 141, 148, 154, 283, 311 Stanislavskaya, M. S., 280, 296 Stapley, E. O., 263, 269, 305, 314 Stark, W . M., 252, 256, 260, 263, 266, 297,304,311,312 Stas, J. S., 318, 350 Stauffer, J. F., 166, 187 Stauss, V. L., 102, 105, 107, 108, 109, 132 Stavely, H., 11, 50 Steel, R., 163, 166, 167, 168, 179, 187 Steigler, A., 250, 304 Steinert, G., 39, 49 Steinert, M., 39, 46, 49 Steinitz, K. H., 291, 312 Stepanova, N. E., 269, 308 Stephenson, O., 18, 46 Stephenson, S., 49 Sternbach, L . H., 251, 295 Stevens, R., 329, 331, 332, 334, 341, 351, 353 Stevenson, D. P., 57, 64 Stevenson, M., 16, 47 Stewart, J. E., 57, 64 Stickings, C., 30, 49 Stier, T., 69, 93 Stitt, W. D., 221, 231, 233 Stocker, B. A. D., 195,215 Stone, A., 24, 49 Stone, J., 23, 24, 45
Storck, R., 5, 49 Stout, J. D., 238, 312 Stoyanovski, A. F., 238, 312 Straffon, R. A., 275, 312 Strange, R., 2, 49 Stranks, D. W., 137, 138, 139, 141, 143, 144, 145, 146, 147, 149, 154 Strassman, M., 344, 353 Strauss, N., 281, 312 Strelitzer, C., 286, 314 Strohm, J., 166, 167, 169, 184, 187 Strominger, J. L., 3, 8, 9, 11, 49, 74, 93, 281, 312 Sturgeon, B., 18, 46 Suenaga, T., 241, 260, 294 Sueoka, N., 191, 215 Sugai, T., 268, 293 Sugano, Y., 241, 312 Sugawara, S., 255, 31 1 Sugi, Y., 277, 312 Sugino, Y., 253, 315 Sugiura, K., 277, 312 Suhara, I., 253, 315 Sumiki, Y., 236, 251, 261, 277, 278, 306, 312 Sundt, E., 326, 353 Surikova, E. I., 256, 312 Suzuki, M., 262,266,307 Suzuki, S., 251, 252, 262, 263, 269, 271, 277, 278, 294, 300, 306 Suzuki, Y., 270, 283, 301 Sveshnikova, M. A., 262, 298 Swaby, R., 44, 45 Sweeley, C. C., 270, 296 Swenerton, J,, 335, 353 Sykes, J., 281, 297 Sypherd, P. S., 281, 312 Szabo, G., 242, 245, 246, 252, 271, 312, 314 Szabo, I., 238, 246, 305, 312 Szumski, S. A., 270, 312 Szybalski, W., 76, 78, 80, 92, 93, 199, 214
T Taber, W. A., 260, 297 Tadenuma, M., 340, 348, 353 Taguchi, T., 75, 92, 262, 312
AUTHOR INDEX
Taig, M. M., 244, 245, 309, 312 Taira, T., 335, 353 Takahashi, T., 277, 312 Takahashima, M., 263, 312 Takaki, R., 277, 312 Takamiya, A., 256, 258, 301 Takemura, S., 27, 49 Takeuchi, T., 250, 261, 262, 278, 305, 312 Takewaka, T., 261, 301 Talyzina, V, A., 280, 305 Tamatoshi, K., 263, 300 Tamboline, F. R., 141, 155 Tamm, C., 27, 49 Tamm, I., 37, 38, 46, 50, 292, 312 Tan, T. C., 275, 312 Tanaka, K., 253,261, 312 Tanaka, N., 259, 267, 275, 276, 277, 305,312, 313 Tanaka, T., 260, 307 Tanami, Y., 286, 308 Tanno, K., 272, 276, 301,313 Taptikova, S. D., 246, 303 Tate, L. S., 286, 313 Tatsuoka, S., 249, 264, 266, 313 Tatum, E. L., 26, 28, 49, 76, 80, 85, 92, 197, 213, 265, 207, 277, 309 Taubman, S. B., 258, 301 Tawara, K., 244,251, 306 Taylor, E. R., Jr., 115, 133 Taylor, E. W., 111, 133 Taylor, J. F., 30, 50 Tejerina, G., 266, 313 Telling, R. C., 174, 186 Tendler, M. D., 247, 313 Tengerdy, R. P., 167, 184,187 Tenney, R. I., 328, 335,353 Terai, K., 268, 293 Terawaki, A., 75, 92 Terentieva, T. G., 272, 306 Terry, T. D., 319, 320, 353 Teterjatnik, A. F., 242, 294 Theil, E. C.,281, 314 Thirumalachar, M. J., 249, 252, 260, 261, 313 Thoai, N., 256, 313 Thomas, A. J., 344, 353 Thomas, A. T., 340, 353
375
Thomas, C. G. A., 161,186 Thomas, H. A., Jr., 109, 133 Thomas, R., 281, 313 Thomas, W. J., 169, 187 Thome-Beau, F., 256, 313 Thompson, M. F., 64, 117, 119, 121, 123, 125, 126,132 Thompson, R. E., 74, 93 Thompson, R. L., 37, 50 Thompson, R. Q., 263,313 Thomson, P. J., 270, 295 Thorne, R. S. W., 340, 353 Thoukis, G., 331, 342, 353 Thurn, A. N., 240, 309 Threnn, R. H., 8, 9, 11, 49 Thrum, H., 285,313 Timmermans, J., 319, 321,353 Timofeevskaya, E. A., 280,305 Tirunarayanan, M. O., 281, 313 Titsworth, E., 260, 299 Tochikura, T., 270, 283, 301 Tomesanyi, A., 283, 298 Tomisek, A., 27, 50 Tomkins, R. V., 141, 145, 155 Tomlinson, A., 26, 45 Toropova, E. G., 90, 93 Torre, B. D., 239, 313 Trakhtenberg, D. M., 262, 280, 310, 313 Trebst, A., 22, 50 Treffers, H. P., 281, 312 Trejo, W. H., 246, 313 Tresner, H. D., 247, 255, 313, 314 Tronstein, A. J., 292, 313 Truhaut, R., 277,313 Truumees, I., 279, 309 Tsubo, Y., 283, 309 Tsuchiya, F., 263, 309 Tsuchiya, H. M., 162, 164, 172, 185 Tsuchiya, K., 249, 266, 313 Tsuji, K., 269, 313 Tsukahara, T., 282, 313 Tsukamura, M., 270, 284,313 Tsukiura, H., 261, 305 Tsunoda, A., 280, 313 Turner, W., 37, 50 Turri, M., 244, 298 Tyson, R. S., 141, 155
376
AUTHOR INDEX
U Ubeda, F. B., 335, 353 Udenfriend, S., 264, 301 Uematsu, K., 277, 313 Uemura, S., 237, 313 Ueyanagi, J., 249, 251, 262, 266, 300, 306, 313 Ujhhzy, V., 277, 296 Ukholina, R. S., 262, 298 Ullmann, A., 283, 298 Ulrich, H., 19, 46 Umbarger, H. E., 349, 351 Umbreit, W. W., 166, 187 Umezawa, H., 250, 259, 260, 261, 262, 267, 272, 275, 276, 277, 278, 280, 304, 305, 306, 307, 312, 313 Underhill, S., 18, 46 Underkofler, L. A,, 136, 138, 141, 143, 146, 149, 151, 152, 154, 155 Urazova, A. P., 280,305 Uzu, M. S., 269, 314
V Vahora, A., 44, 45 Valdimizov, A. V., 200, 213 Valu, G., 238, 299 VBlyi-Nagy, T., 238, 242, 245, 248, 271, 299, 312, 314 van Andel, 0. M., 33, 50 Van der Kloot, A. P., 328, 332, 335, 353 Van Lanen, J. M., 151, 153 van Tamelen, E. E., 270,296 Vanushin, B. F., 86, 92 Vaurs, R., 267, 296 Verner, M., 267, 296 Vetlugina, L. A., 264, 294, 295 Vickerman, K., 39, 50 Villanueva, J. R., 285, 298 Villax, I., 250, 314 Vining, L. C., 260, 268, 297, 314 Vinogradova, K. A,, 246, 303 Vischer, E., 7, 45, 259, 260, 261, 295, 298 Vischer, W. A., 281,313 Vitols, E., 29, 48 Vizir, P. E., 257, 314 Vladimirov, A. V., 245, 305
Vladimirova, G. B., 70, 71, 72, 73, 75, 76, 83, 84, 85, 86, 87, 89, 90, 91, 92 Vogt, E., 340, 353 Voinescu, V., 281, 295 Volkin, E., 16, 50 Vondracek, M., 259, 297 Voser, W., 259, 295 Vyazova, 0. I., 280, 305
W Wachsman, J. T., 5,49 Wacker, A., 20,22,50 Wadstein, T., 31, 47 Wain, R. L., 33, 45 Waisbren, B. A., 286, 314 Wakae, M., 261,305 Wakaki, S., 269, 314 Wakazawa, T., 241, 312 Waksman, S. A,, 236, 238, 263, 314 Wallhauser, K. H., 250, 261, 262, 304 Walpole, G. S., 135, 154 Walters, E. W., 240, 314 Walton, R. B., 268, 314 Wang, Y.,283, 314 Warburg, O., 30, 50, 68, 69, 73, 93 Ward, G. E., 138, 141, 155 Warner, D. T., 210, 215,265,314 Warner, H. A., 286,287,296 Warren, H. B., Jr., 261, 307 Watanabe, T., 195, 215, 281, 314 Watson, J, D., 15, 50 Watson, R. W., 138, 139, 141, 143, 144, 149, 152,154,155 Watson, S. J., 239, 308 Wattel, F., 285, 299 Watts-Tobin, R., 38, 46 Weaver, R. N., 161, 186 Webb, A. D., 321, 323,324, 327, 328, 330, 331, 332, 333, 335, 343, 351, 353 Webb, J. S., 26, SO, 277, 314 Webley, D. M., 161,187 Weed, L. L., 87,93 Weibull, C., 5, 50 Weidel, W., 4, 50 Weidenmiiller, H. L., 250, 304
AUTHOR INDEX
Weinblum, D., 20, 50 Weinding, R., 255, 314 Weinhouse, S., 344, 353 Weinstein, L., 292, 297, 314 Weiss, D. J., 160, 186 Weiss, W., 290, 314 Weissbach, H., 264,265, 301, 314 Welch, A. D., 36, 49 Welsch, M., 264, 314 Werkman, C. H., 147, 154 Wesel, E. M., 279, 305 West, C. A., 270,296 Westfall, B. B., 209, 215 Westhead, J., 263, 304 Wettstein, A., 7, 45, 259, 295 Weygand, F., 22, 50 Whaley, H. A., 270, 296 Wheat, J. A., 138, 140, 141, 143, 144, 145, 146, 147, 153, 155 Whelton, R., 69, 93 Whiffen, A. J., 206,215 White, F. R., 277,314 White, J. W., 325, 353 Whitfield, G. B., 266, 269, 311, 314 Whitmore, W. F., Jr., 280, 314 Wicker, E. F., 293, 314 Wigley, R. D., 288, 301 Wilcox, F. A., 332, 353 Wiley, P. F., 263, 299 Wilhelm, J. J., 238, 308 Wilhelm, K. H., 166, 167,185 Wilkins, M., 17,49 Willers, E. H., 240, 308 Williams, A. M., 201, 215 Williams, D. E., 269, 314 Williams, D. R., 160, 185 Williams, R. P., 26, 50, 277, 314 Willson, C. D., 343, 353 Wilson, A. N., 269, 314 Wilson, J. N., 161,185 Winfield, A. F., 261, 307 Winkler, A., 277, 296 Winter, M., 326, 353 Wintersteiner, O., 11, 50 Wise, W. S., 166, 167, 180, 187 Wisseman, C.L., 18, 47 Witek, S., 319, 350
377
Witkin, E. M., 281, 314 Wolf, C. F., 26, 50, 277, 314 Wolf, F. J., 269, 314 Wolf, J., 256, 307 Wolfe, A., 19,47 Wolfe, R. N., 252, 260, 263, 304, 312 Wollman, E. L.,193, 194, 214, 215 Wood, R. M., 36,49 Woodruff, H. B., 238,268,297, 314 Woods, D. D., 89, 93, 254, 295 Woolley, D. W., 88, 93 Work, E., 3, 5, 47, 50 Woznicka, W., 278, 308, 314 Wright, D. E., 151, 153 Wu, L. C.,281,299 Wurtz, A,, 318,353 Wyatt, G., 35, 50
Y Yagashita, K., 262, 306 Yamada, M., 340, 344,348,353 Yamaguchi, H., 267, 276, 282, 312, 313, 314 Yamaguchi, T., 249, 263, 264, 266, 300, 313 Yamaki, H., 267,277, 312, 313 Yamamoto, H., 249, 251, 261, 262, 263, 264, 300, 301, 311 Yamamoto, K., 249, 251, 261, 306, 314 Yamazaki, S., 280, 307 Yanagishima, N., 70, 92 Yano, K., 280, 307 Yarbrough, H. F., 52, 56, 64 Yarmolinsky, M. B., 17, 50 Yeager, R. L., 268, 311 Yee, G. S., 115, 133 Ykeda, 200 Yokozawa, S., 272, 280, 301, 315 Yoneda, M., 253, 315 Yonehara, H., 259,305 Yoshida, F., 183, 187 Yoshida, H., 344, 348, 353 Yoshida, T., 262,280, 303, 315 Yoshizawa, H., 290, 314 Yoshizawa, K., 340, 344, 348, 353 Young, J. L., 330,352
378
AUTHOR INDEX
Young, R. M., 274, 315 Yumoto, H., 252, 260, 302, 309 Yurina, M. S., 264, 304
Z Zabos, P., 283, 315 Zahner, H., 251, 257, 259, 261, 294, 295, 298, 300, 306, 315 Zaitseva, Z. M., 245, 257,294, 307 Zajdela, F., 68, 92 Zalesskaya, M. I., 353
Zamecnik, P. C., 16, 17, 45, 47 Zamenhof, S., 27, 49 Zarembo, J. E., 327, 353 Zbinovsky, V., 277, 303 Zhevchenko, A. A., 257, 314 Ziegler, D. W., 169, 187 ZifTer, J., 262, 315 Zimenkova, L. P., 88, 89,92 Zurilla, R. W., 56, 64 Zyganov, V. A., 252,302 Zygmunt, W. A., 268, 315
SUBJECT INDEX A Ability to produce spores, 204 Absorption rate constant, K,a, 162 Abundance of actinomycetes in soils, 236 Acclimatization of cultures, glycol production, 142 Accuracy of radioisotope tests, 110 for water coliforms, 106 Acetylglucosamine, 2 Acetylmuramic acid, 2 Acridine orange, 23 Acridines, 23, 24, 42 AcriRavine, 23 Actidion, 31 Actinobolin, 273 Actinogen, 273 Actinomycetes, 235 anaerobic, 239 autotrophic, 258 cell walls, 240 classification of, 242 continuous culture of, 238 enzymes from, 252 genetics of, 241, 242 metabolism of, 256 microaerophilic, 239 new species, 249 nutrition, 256 occurrence in nature, 236 pathogenic, 239 pigments of, 255 in sea water, 238 steroid oxidation by, 255 structure, 240 thermophilic, 247 transduction, 241 Actinomycin, 26, 80, 84, 274 Actinomycin C, 76 Actinophages, 255 Active amyl alcohol, 318 Aeration, 157 control of, 158, 175 efficiency of, 163 methods for evaluation, 168 production of glycol, 146
rates of, determination, 165 smoke clearance test for, 165 Aerobactor aerogenes, 136, 139 Air sterilization, 177 Altemaria, 219 Aminacrine, 24 Amino acids as source of fuse1 oil, 338 Aminoacridines, 23, 25, 35 p-Aminobenzoic acid, 22 a-Aminobutyric acid, 343 Aminopterin, 22 Aminoquinoline, 24 p-Aminosalicylic acid, 22 Amphotericin B, 33, 269 Analysis of fuse1 oil, 322 Animal cells in culture, 161 Antibiotics, 235 animal growth, 292 antifungal, 269 clinical medicine, 286 effect on animal diseases, 292 food preservation, 293 isotope tests for activity, 127 mode of action, 280 new, 259 nutrition and biogenesis of, 257 plant diseases, 293 plant growth, 293 polyene, 32 polypeptide, 12 resistance transfer, 195 therapeutic, selection of, 99 Antimycin, 26 Antitumor substances, 271 Antiviral agents, 271, 280 Antrycide, 40 Arsenicals, 40 Artificial mutation, 205 Atebrin, 42 Aureomycin, 26 Automatic radioisotope microbiological monitoring, 131 Ayamycin, 276 Azaguanine, 20, 36 Azaserine, 26, 276
379
380
SUBJECT INDEX
Azeotropes, 320 fusel oil, 319
B Bacillus polyrnyxa, 136, 139 Bacitracin, 8 Bacterial degradation of paint, 227 Bacterial growth in paint films, 225 Bacterial membrane, 5 Bacterial mitochondria, 5 Bacteriological warfare, 99 defense, 115 Barium metaborate, 231 Benzimidazoles, 37 Benzylpenicillin, 10 Binder, 230 Biochemical fuel cells, 52 Biological half cell, 58 Biosynthesis, fusel oil, 338 Bisthiocyanates, 33 Blasticidin S, 277 Bromodeoxyuridine, 78 5-Bromouracil, 20 Bromouracil, 36, 78 Butadiene, 136 Butanediol, 135 n-Butyl alcohol, 343 Butylene glycol, 135 antifreeze, 151 fermentation, future of, 152 substrates for, 136 recovery of, 149 uses of, 150 2J-Butylene glycol, 135
C Cancer, 66 chemotherapy of, 273 deletion hypothesis, 68 molecular biology of, 65 Warburg theory of, 69 Candida albicans, 268 Captan, 29 Carzinostatin, 276 Catalase, 73 Cell wall( s ) , 2 of actinomycetes, 240 synthesis in respiratory deficient mutants, 74
Cephalosporins, 11 Cereal grains, 138 Cetyltrimethylammonium bromide, 13 Chalking, 230 Characteristics of paint films, 228 Chelation, antimicrobial activity, 43 Chemical inhibitors in paints, 231 Chemical preservatives, paints, 230 Chemistry of biocides, 1 Chiniofon, 40 Chitin, 28 Chitinase, 31 ChloramphenicoI, 18, 36, 79, 84, 265 Chloranil, 29 Chlorinated phenolic compounds, 231 Chloromycetin, 26 Chloroquine, 42 Chlorouracil, 36 5-Chlorouracil, 20 Chlortetracycline, 74, 84 Chromatin bodies, 6 Chromatographic fusel oil analysis, 325 Citric acid, 208 Citrus press juice, in glycol production, 137 Cladosporium, 219 Colicinogenic factors, 193, 195 Coliform organisms, 97 CO, required, 130 inhibition by membrane filters, 105 test for, 102 Combination of antitumor agents, 279 Compounds present in fusel oils, 335 Concentration of dissolved oxygen, 162, 163 of sugar for glycol production, 147 Confirmed test, fecal coliform organisms, 103 Congeners in alcoholic beverages, 317, 332 Conjugation, 194 Continuous culture of actinomycetes, 238 Continuous processes, 212 Control of aeration, 161, 169
SUBJECT INDEX
of microorganisms in the paint industry, 229 Controlled aeration, 158 Copper compounds, 231 Corrosion cells, 62 Critical oxygen level, 163 Culture degeneration, 189, 204 Culture stability, 189 Cycloheximide, 31 Cycloserine, 8 Cytomycin, 277 Cytoplasmic membrane, 5 of protozoa, 39
D Defense against bacteriological warfare, 99 Degeneration of a streptomyces, 201 of cultures, 140 Degradation of paint binders, 221 Degranol, 76 Deletion hypothesis of cancer, 68 Detection of air contamination, 115 Diamidines, 40 Diaminopimelic acid, 3 Diaminopteridines, 22 Dihydrostreptomycin, 269 Dihydroxybenzoylglycine, 7 Diploid fusion, 196 Dissolved oxygen concentration, 162, 163 Disulfidase, 28 DNA, 6, 14, 190 DNA-protein in normal and mutant organisms, 86 DON, 26 Drug resistance transfer, 195 Drying oils, 222 Durability of paint films, 224
E Electrometric techniques for oxygen determinations, 166 Emetine, 40 EMF due to microbial activity, 54 Emulsion paints, 230 Endoplasmic reticulum in fungi, 29 Enzymes as electrical generators, 55
381
Enzymes from actinomycetes, 252 Episomes, 193 Erythromycin, 26, 267 Escherichia coli, as electrical generators, 58 Ethidium, 41 Euflavine, 23 Evaporation rate, 165 Exobiology, 117 Extraterrestrial biological forms, 118
F F-factor, 193, 194 Factors affecting fuse1 oil formation, 346 Ferrichrome, 7 Ferricyanide in microbial fuel cells, 59 Filipin, 32 Fluvobacterium murinum, 219 Flowmeters, 175 Fluoro-2-deoxyuridine, 72 Fluorodeoxyuridine, 77 Fluorouracil, 36, 76, 84 5-Fluorouracil, 20 Folic acid, 20 Food, 98 Freezing of living cells, 210 Frozen food, 99 Fuel cells, 52, 55 Fungal cell wall, 28 Fungal fermentations, 205 Fungi, endoplasmic reticulum in, 29 in paint deterioration, 217 Fungizone, 33 Fuse1 oil(s), 317 analysis of, 322 composition of, 333, 335 formation from amino acids, 338 factors affecting, 346 from glucose, 341 pathways of, 345 variability of, 334 G Gas, 128 Generation of electricity by microbial action, 51
382
SUBJECT INDEX
Genetic maps, 195 Genetic recombination, actinomycetes, 242 Genetics of actinomycetes, 241 Glucan, 28 Gluconic acid, 208 Glucose, source of fuse1 oil, 341 GIycerol phosphate, 4 Glycol fermentation, 136 Glycopeptides, 2 Glyodin, 29 Glyoxals, 27 Gramicidin S, 14 Griseofulvin, 31 Guanidinoanthracene, 26 Gulliver, 117
H Halogenated pyrimidines, 76 Hapioidization, 197 Heating, effect on mutants, 80, 85 Helminthosporium, 237 Heritable respiratory deficiency in yeast, 71 Heterokaryosis, 198 Hexylresorcinol, 13 Hfr, 194 High oxygen tensions, 161 Hydrazine, 27 Hydrocarbons in microbial fuel cells, 56
Hydroxylamine, 27 8-Hydroxyquinoline, 40
I Impellers, 170 Inhibition of coliforms by membrane filters, 105 Inositol, 28, 257 Instahility of microorganisms, 190 Instruments to detect life, 117 Iododeoxyuridine, 36 5-Iodouracil, 20 Ionizing radiation, 199 Isatin thiosemicarbazone, 37 Isoamyl alcohol, 318 Isobutyl alcohol, 318 Isoleucine, 338 Isotope hazards, 112
K Kanamycin, 266 Karyogamy, 196 u-Ketobutyric acid, 343 a-Keto-n-valeric acid, 346 Kreis reaction, 223
L Lead carbonate, 228 Leucine, 338, 344 Life detection experiment, 119 Linseed oil, 222 Lipid, 5 Lipoprotein, 4 Lyophilization, 21 1 Lysine, 3 Lysogenic bacteriophages, 193, 194 Lysozyme, 2
M Macrolides, 266 Mammalian cells in tissue culture, 209 Mannan, 28 Mars, 118 Measuring aeration, 162 Mechanisms of genetic recombination, 190 Meiotic process, 197 Membrane, fungi, 28 Membrane enzymes, 6 Membrane filter( s ) , 98 inhibition of coliforms by, 105 toxicity of, 109 Mepacine, 42 Messenger RNA, 6, 15, 80, 193 Metabolism of actinomycetes, 256 Methacillin, 11 Methods for evaluating aeration, 168 Methylisatin-B-thiosemicarbazone, 37 Microaerophilic actinomycetes, 239 Microbial fuel cells, 52 Microbial inhibitors in paint, 222 Microbial models of cancer, 70, 87 Microbial utilization of linseed oil, 223, 224 Microbiology of paint films, 217 h4icrocolorimeter method for dissolved oxygen, 164
383
SUBJECT INDEX
Microflora in paint films, 219 Micromonospora, 243 Micropolyspora, 243 Microstructure of microorganisms, 1 Mineral oil slants, 211 Mitochondria, carcinogenesis, 71 in fungi, 29 Mitomycin, 26, 84 Mitomycin C, 75, 277 Mitotic nondisjunction, 197 Mode of action, antibiotics, 280 Molasses, in glycol production, 137 Molecular biology of cancer, 65 Muramic acid, 2 Mutagenic chemicals, 199 Mutants with impaired respiration, 72 Mutation, 199
Oxygen, 157 absorption rate ( O A R ) , 162, 172 analyzer, 166 availability of, 159, 161, 171 concentration of dissolved, 162 critical level, 163 demand for, 159 determination, 166 dissolved, Winkler method for, 164 partial pressure of, 162 tension of, 161, 162 transfer rates, 168 Oxytetracycline, 74
P
Paint, 217 deterioration, 217, 225, 227 durability, 231 N films, characteristics of, 228 Neomycin, 267 microbiology of, 217, 219 Neurospora, 197 peeling problems, 227 preservatives for, 231 New antibiotics, 259 raw materials for, 229 New species of actinomycetes, 249 solvent-thinned, 218 Nitrogen mustards, 27, 76 substrate for microbial growth, 221 Nitrogen sources for glycol fermenwater-thinned, 218 tation, 148 Paludrine, 41 Nitrous acid, 27 Parasexual cycle, 196 Nocardia, 240 Pathogenic actinomycetes, 239 Norvaline, 346 Pathways of formation, fuse1 oil, 345 Novobiocin, 8, 268 Penicillin, 8, 9, 74, 84, 207 fermentation, 204 as acylating agent, 10 Nuclear membrane, protozoa, 39 Nucleic acid formation in mutants, 75 Permeability of bacteria, 6 Nutrition and biogenesis of anti- Permeases, 6 Petroleum, 128 biotics, 257 pH, glycol fermentation, 144 of actinomycetes, 256 Phage, 35 Nystatin, 269 Phenanthridines, 40 0 Phenols, 12 Oil, 128 Phenylethyl alcohol, 341 Organisms producing 2,3-butylene Phenylmercury compounds, 231 glycol, 138, 141 P h o m glomerata, 219 Over-aeration, 161 Phospholipid, 5, 28 Oxacillin, 11 Physical methods, to determine aerOxamycin, 8 ation rates, 165 Oxidative polymerization of drying Pigments of actinomycetes, 255 oils, 222 Plastics from butylene glycol, 152
384
SUBJECT INDEX
Polarographic methods for oxygen determination, 188 Poly-P-hydroxybutyrate, 7 Polyene, 268 antibiotics, 32 Polymycin, 13 Polypeptide antibiotics, 12 Power for agitation, 181 Presumptive test, 102 Prevention of culture degeneration, 210 Primers, 228 Primoquine, 42 Proflavine, 23 Proguanil, 41 Propyl alcohol, 318 n-Propyl alcohol, 343 Prospecting for petroleum and gas via isotope techniques, 128 Protein synthesis, 14, 80 in respiration deficient mutants, 74 Protoplast, 2 Protozoa, 38 Pseudofolic acid, 22 Pseudomonas aeruginosa in paint, 219 Pteridines, 20 Pteroylglutamic acid, 21 Pulluluria pullukns, 219 Purge meters, 175 Puromycin, 17 Pyrimethamine, 22, 41
Q Quantitative bacteriological determination, 100 Quaternary amines, 12, 13, 31 Quinacrine, 42
R Radioisotopes, 95 Radioisotope technique, test for coliforms, 102 Rapid bacteriological determinations, 98 Rapid identification of bacterial infection, 99
Rapid microbiological determinations, 95 Raw materials, glycol production, 141 Raw water quality, 98 Recovery of butylene glycol, 149 Respiration of “dead” organisms, 110 Respiratory deficient mutants of E. coli, 87 Staph. auras, 71 Staph. fermentans, 82 yeast, 89 Reynolds number, 181 Ribitol phosphate, 4 Ribosomes, 6, 80 RNA, 8, 192
S Schiff reaction, 223 Selection of antibiotics for treatment, 99 preferred chemotherapeutic agent, 99 Sequential blocking, 14 Serial transfers, 204 Sex factor, 194 Sexual cycle, 197 Shake flasks, 170, 171 Sideramines, 7 Sideromycins, 27 Smoke clearance test, for aeration rates, 165 Soil cultures, 211 Soluble RNA, 16 Solvent-thinned paints, 218 Somatic crossing-over, 198 Source of biological inhibitors, 87 Source of electric power, 53 Space craft, 117 Sparged vessels, 174 Spargers, 170 Stability of cultures, 140 Standard methods, 97 Steroid oxidation by actinomycetes, 255 Stilbamidine, 40 Stirred vessels, aeration, 177 Strain stability, 208 Streptomyces, 199, 237
385
SUBJECT INDEX
Streptomycin, 27, 82, 269 biosynthesis of, 258 Structure, actinomycetes, 240 Styrylaminoquinoline, 25 Substrates for butylene glycol formation, 136 Sulfite oxidation, 163 Sulfite waste liquor in glycol production, 137 Sulfonamides, 21 Sulfur, 33 Suramin, 40 Swimming pools, 98 Synnematin B, 207
T Teichoic acids, 4 Temperature effects on glycol production, 144 Tetracyclines, 26, 84, 268, 270 Tetraoxyanthracene, 26 Thermoactinomyces, 243 Thermophilic actinomycetes, 247 Thiosemicarbazones as antiviral drugs, 36 Thiouracil, 36 Time factor in bioassay, 96 Tissue culture, 169-209 Total bacteria test, 113 Toxicity, 131 of filter membranes, 109 Transduction, 195 actinomycetes, 241 Transfer RNA, 16
Transforming agent, 190 Transmethylation, 259 Tributyl tin, 33 Triethylene melamine, 76 Trypaflavine, 76 Trypanosomes, 39 Trypanosomiasis, 40 Tryptophol, 339 Turbulence, 183 and aeration, 180 Tyrosol, 339, 340, 341
V Variability of fuse1 oils, 334 “Vibrating” platinum electrodes, 166 Viruses, 34
W Waksmania, 243 Warburg, theory of cancer, 69 Water supplies; bacteriological control. 97 Water-thinned Daints. 218 Ways to increase oxygen availability, 171 “Winkler” method for dissolved oxygen, 164 Wood, 227 “Wrist-action’’ shakers, 173 I
,
Y Yeast, 208
Z Zinc oxide, 231
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