]?alia Microbial. 28, 309--328 (1978)

Microbial Growth and Production of Antibiotics Z. VAz~x and K. MIKL-LiK Department of Biogenesis of ~'aturaI Substances, and Del)art~nent of Molecular Biology and Genetics Institute o.f Microbiology, Czechoslovak Acaden~y of Sciences, 142 20 Prague 4 Received January 10, 1978

Antibiotic compounds do not constitute a homogeneous group. From the chemical point of view t h e y are highly heterogeneous and it is difficult to formulate a general hypothesis t h a t would explain the causes of production of these compounds on the basis of d a t a obtained so far (Donovick and Brown, 1965). This difficulty is also reflected b y contradictory assumptions concerning the importance of these compounds for production microorganisms. The view that the production of antibiotics is a protective mechanism facilitating survival of microorganisms in a natural environment is still being discussed (Krassilnikov, 1954; Brian, 1957; Pollock, 1971; Hill, 1972; Gottlieb, 1976). In spite of the fact t h a t full agreement has not yet been reached the view t h a t the production of antibiotics is advantageous in the competition for nutrients is finding more and more supporters (Demain, 1974). The fact t h a t m u t a n t strains of microorganisms t h a t are not impaired in their basic life functions and do not produce antibiotics exist led to an opposite view, viz. t h a t antibiotics are waste products of the cellular metabolism. According to this concept the ability to produce antibiotics is only an incidental property that cannot be correlated with any essential mechanism required for nutrition of microorganisms and their growth (~Vaksman, 1959, 1961). F u r t h e r assumptions were based on the fact that antibiotics can represent degradation products of certain cell macromolecules or components of cell structures, e.g. cell walls (Gwatkin, 1962; Szabo et al., 1965) or spore coats (Bernlohr and Novelli, 1963). These assumptions in their original form were not experimentally verified and are considered to be out-of-date at present. However, it can be imagined that, e.g., the biosynthesis of basic sugar precursors of the cell wall and antibiotics of the group of aminoglycosides is catalyzed by common initial enzymic systems. Thus, the addition of an inhibitor of cell-wall synthesis (penicillin) leads to a n increase of the production of streptomycin in Streptomyces griseus (Barabs et al., 1976; Barabs and Szabo, 1977). A single paper describing the release of streptomycin from enzymically prepared fragments of cells of Streptomyces griseus has been published so far (Barabs et al., 1977) b u t it should be kept in mind that cells producing antibiotics are also equipped with salvage mechanisms for reutilization of compounds originating in the degradation processes in the cell, e.g. free purines and pyrimidines resulting from degradation of nucleotides or other precursors originating from cell structures. These mechanisms can then be involved in the biosynthesis of antibiotic

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compounds under conditions in which no intense growth of microorganisms takes place. The fact that the basic building units of antibiotics are actually important intermediates of general metabolic processes led Bu'Lock (1961) to formulate a new hypothesis (maintenance) that accentuated the importance of metabolic processes occurring after the termination of the multiplication phase for maintenance of further lif'e processes. Thus the close relationship between growth processes and processes leading to the production of the so-called secondary metabolites has first been pointed out here. More sensitive techniques used to follow the biosynthesis of antibiotics made it possible to show that low concentrations of these compounds already are produced at the beginning of the growth phase. Therefore, it is natural that in further papers the antibiotics were assumed to play a role in the control of certain metabolic processes in producing cells. In this case, the synthesis of an antibiotic during growth would be limited by a real need for this inhibitdr in the producing microorganism. Thus, for instance, actinomycin inhibiting the DNA-dependent l~NA synthesis could limit mRNA synthesis in Streptom~/ces antibioticus. The excessive production of the antibiotic during later growth phases would then appear as a derepressed biosynthetic process (Katz, 1967). An even more pronounced regulatory role in cell differentiation, sporulation and germination in particular, is attributed to the peptide antibiotics produced by the genus Bacillus (Hodgson, 1971; Sadoff, 1972). It is assumed that edeins interfering with DNA synthesis influence processes of the first sporulation step, formation of the axial chromatin filament, and that derepression of the transcription mechanism and formation of specific mRNA takes place during the further step (after isomerization of edeins). Bacitracin, an inhibitor of cell wall synthesis, is also involved in the first stages of the sporulation. The antibiotic is released into the cultivation medium prior to the formation of the cortex layer and, thus, it cannot inhibit the synthesis of this mucopeptide layer. A portion of the antibiotic is adsorbed to the 50S ribosomes contained in the cytoplasm of the spore. In Bacillus brevis gramicidins are produced after the termination of the exponential phase of growth and prior to the formation of the free spore. Gramicidins are perhaps involved in the transport of hydrogen; ammonium and potassium ions into and out of the developing spore (Sadoff, 1972). Other authors assume that peptide antibiotics (e.g. tyrocidine) regulate gene transcription during the transient phase by interrupting selectively the expression of the vegetative genes (Sarkar and Paulus, 1972). Polymyxins are produced during the fifth stage of sporulation, i.e. during a highly advanced developmental stage when the spore protoplast is already surrounded by double membranes and the cortex layer and when protein coats of the spore are formed. The maximum quantity of the antibiotic is released to the cultivation medium during lysis of the sporangium. The relationship between the ability to produce antibiotics and sporulation was also described in streptomyeetes. Asporogenic mutants of S. griseus (Sehatz and Waksman, 1945) and S. lavendulae (Waksman and Schatz, 1945; Yegorov et al., 1971) do not produce streptomycin and streptothricin. It can be assumed that this relationship was also observed in other antibiotic producers and is well recognized by industrial scientists. However, it cannot be explained in a simple way (Demain, 1974) and is not generally valid. It is also possible to prepare asporogenic mutants of streptomycetes that retain their ability to produce antibiotics. We trust that this short and certainly incomplete introduction to ideas concerning

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the importance of synthesis of antibiotic compounds for the producing microorganisms proper was necessary. It characterizes the thirty years of development of views on the microbial physiology and mechanisms governing metabolic processes in cells. It also reflects differences in the approach of scientists investigating the succession of microbial communities under natural conditions and authors interested in increasing the production of antibiotics by mutant strains of microorganisms under conditions that never occur in the nature, in other words under conditions when secondary metabolites (Bu'Lock, 1965) become excessive metabolites (Van6k et al., 1973) with completely new metabolic linkages regulating their production. The two-phase character'o/the metabolic process -- a simplified view

Almost all classical papers concerning the metabolism of producing microorganisms include data about the growth and production phases. Chemical changes occurring during the submerged cultivation of S. griseus, a producer of streptomycin, were divided by Dulaney and Perlman (1947) into the phase of crescence and senescence. During the first phase they observed a rapid consumption of glucose, soluble nitrogen and phosphate, rapid growth of the myeelium, high oxygen consumption and only a very low production of the antibiotic. During the second phase the rate of production of the mycelium and oxygen consumption decreased, phosphate and nitrogen were excreted into the medium but the biosynthesis of streptomycin sharply increased. Different variations of this basic finding were then described in a number of other papers concerned with antibiotic producers. Van Dyck and De Somer (1952) in a study of the production of chlortetracycline in S. aureofaciens and Biffi et al., (1954), Boretti et al. (1956), Gubernyev et al. (1956a, b) and Prokofieva et al. (1959) also described two phases. The first phase is again characterized by a rapid consumption of nutrients, multiplication of cells and only a negligible production of the antibiotic. The second phase is characterized by a decrease of the protein and ribonucleic acid content. The content of deoxyribonucleic acids does not practically change whereas the production of antibiotics sharply increases. It is of importance that during this phase a .slow growth of cells can be detected and a decrease in the nueleoprotein content occurring at this time is not due to autolysis and release of nitrogen to the cultivation medium. The above papers describe well the situation when cells of the producer, after the phase of rapid multiplication, enter the phase of decelerated growth, followed by the stationary phase. This latter phase can also be characterized as the phase of "non-growing cells". However, this term is rather inaccurate as during this phase the numbers of multiplying and dying cells are in equilibrium. However, DoskoSil et al. (1958, 1959) described five phases in the development of these producing microorganisms. This classification was based on the analysis of substrate consumption at different intervals of cultivation, production of organic acids, synthesis and degradation of nucleic acids, respiration and morphology of the mycelium of S. aureofaciens and S. rimosus. When developing their ideas about biosynthesis of secondary metabolites Bu'Lock and Powell (1965) reached the conclusion that the whole process of biosynthesis can be divided into two basic parts, the trophophase (trophic--growth phase) and the idiophase (special phase, production phase). However, it is rather too facile to consider the biosynthesis of antibiotics as merely a two-phase event. Already Gatenbeck (1965) studying the biosynthesis of pigments in Penicillium islandicum found that the synthesis of these compounds proceeds at the maximum rate in the middle of the growth phase, thus, it is inadequate to

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divide the development of the organism into the growth and production phases. A similar conclusion was also reached by Ruczaj et al. 0972) who studied the biosynthesis of rifamycin in Nocardia mediterranei. A number of examples can be presented when the g r o w t h and the production phases are strictly separated. However, this separation is often based only on the determination of cell or mycelium dry weight, which does not sufficiently characacterize the dynamics of cell multiplication. The overproduction of an antibiotic during the production phase can reflect basic metabolic changes that. occurred long before the growth phase. It is a serious shortcoming of the two-phase hypotheses of metabolic processes leading to the production of antibiotics that they do not accentuate the transient phases, either at the beginning of the exponential phase (acceleration phase) or at the end of the exponential phase of growth (retardation phase). Furthermore, t h e y neglect completely the preparatory phase which, in some cases, m a y fuse with the accelerating or germination phases (either vegetative young cells or spores can serve as inoculum). I t appears most satisfactory at present to consider processes of growth and production as a series of mutually connected metabolic changes occurring from the time of inoculation of the culture (Van~k et al., 1973) or even earlier during the history of the inoculation material (Calam, 1976) or as a continuous process (Bu'Lock, 1975).

Preparatory phase During development differentiating cells of microorganisms pass through a series of structural and functional alterations. Spores of producing organisms differ in the level of d o r m a n c y and resistance. Endospores of bacilli, exospores of streptomyeetes and sexual and asexual spores of fungi belong to this group. Transition from the d o r m a n t state to vegetative forms was studied with many models. Three phases of the germination process can be discriminated in endospores of the genus Bacillus. Activation which can be induced, e.g., by a temperature shock, is the first phase. I t is a reversible process resulting in changes in tertiary structure of spore macromolecules (Keynan and Evenchik, 1969). Initiation, the second phase, is defined as an irreversible process, during which the enveloping spore layers are degraded and spore exudates are released. Spores become sensitive to chemical and physical agents. Outgrowth, the last phase, includes a sequential activation of macromolccular systems facilitating the production of vegetative forms. Several different approaches were exploited to obtain more data about the transition of the spores to vegetative forms in Streptomycetes. However, the results referred to in the literature show that considerable differences in the germination process exist in spores of different streptomycetes (Kalakoutskii and Agre, 1976). One group of exogenous spores of streptomycetes contains an external sheath which influences their physical properties, especially the wettability. These spores are hydrophobic and float when inoculated into liquid media, which makes impossible to s t u d y their physiology under rigorous experimental conditions. Recent studies employing Streptomyces viridochromogenes exospores (Hirsch and Ensign, 1975) suggest t h a t activation and initiation are involved in the germination process. Activation was reported to be accomplished b y a mild heat shock. On the other hand, germination of S. granatieolor exospores was inhibited b y more than 60 % after the same treatment. A short homogenization of spores to disturb the fibrous sheath (Sfastns 1977) results in a synchronous germination under appropriate cultivation conditions and the timing of various biochemical events m a y be estimated.

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Germination of aerial spores of S. granaticolor has two ~norphologically and functionally distinguishable phases: swelling, observed after 60 min of cultivation, and germ tube formation after 120--140 rain. During the first phase RNA and protein synthesis proceeds but no net DNA synthesis takes place. The germination process continues through a sequence of time-ordered events. A rapid uptake of labelled uridine by spores begins after 20 s whereas incorporation of precursor molecules into RNA was observed after a 3-min lag. Protein synthesis is measurable after a 4-min lag and DNA synthesis can be detected after 60--70 min of cultivation (Mikullk et al., 1977). This temporal sequence of synthesis of RNA, proteins and DNA was also demonstrated in experiments with endospores of the genus Bacillus (Armstrong and Sueoka, 1968; l~ana and Halvorson, 1972; Nickerson et al., 1975) indicating that the sequential activation of transcription, translation and DNA replication systems is a general mechanism accompanying germination of spores in prokaryotic organisms. In fungal spores, activation is defined as a treatment leading to an increase in germination. In mycological terminology, germination represents the first irreversible stage which is morphologically, cytologically and physiologically distinct from the dormant forms (Sussman, 1975). During germination of the fungal spore various classes of RNA are synthesized sequentially but the sequence varies with the fungal species. Evidence has been presented that fungal spores contain an active functional RNA polymerase (Afman et al., 1972). Similar results were obtained in experiments with dormant spores of B. subtilis (Kerjan, 1973) and with aerial spores of S. granaticolor (Mikulik et al., 1977). Contradictory results and speculations have been published on the structure and function of RNA polymerase from dormant spores and vegetative cells. In previous reports it could be shown that the ~ subunit of RNA polymerase from dormant spores of B. subtilis is replaced with a polypeptide with a lower molecular weight (Maia et al., 1971; Cohen et al., 1972). These changes in specificity of promoter recognition could account for the selective gene expression during germination. The above suggestion was. revised when RNA polymerase was isolated in the presence of powerful inhibitors of proteoly~ic activity and the purified enzyme was found to be structurally similar to the vegetative RNA polymerase (Szulmajster, 1973). Moreover, it was found that RNA polymerase is not synthesized de novo in spores until at the end of outgrowth (Ben Z~iev et al., 1975). The present knowledge of the structure of RNA polymerase during development poses a series of questions to be answered. How are the germination phases controlled at the transcription level? Factors affecting R57A polymerase (co, ~, ~, • a, and others) (Fukuda and Doi, 1977) could be involved in a positive control in differentiating cells. After the induction of germination all components of the system required for transcription of phage DNA are present in dormant spores (Cohen et al., 1973). However, de novo synthesis of proteins immediately after germination is required for the initiation of sequential transcription of the spore genome (Cohen et al., 1975). The small number of proteins synthesized can be responsible for activation of specific promoter regions and/or for depolymerization and removal of certain compounds suppressing transcription of specific genes from DNA. Many spores contain a broad spectrum of compounds, mostly unidentified, that are bound to DNA, RNA and proteins. Tewfik and Bradley (1967) showed that DNA from spores of Streptomyces venezuelae differs from the mycelial species in many physical properties, e.g. buoyant density, thermal denaturation temperature and absorption spectrum in the visible region. In further experiments it was shown

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(Enquist and Bradley, 19"71) that changes are associated with the age of the spores. Spore D N A was found to bind 15--20 ~ proteins. It was also shown that spore D N A is complexed with a pigment that can be released from D N A with ethanol or acetone. In this respect the D N A preparation from spores was almost identical with that of vegetative cells. I t was shown that D N A from dormant spores differs from t h a t of vegetative cells in sensitivity to irradiation (Stafford and Donnellan, 1968; Tanooka and Sakakibara, 1968). Further experiments demonstrated that the spore-specific properties of D N A are not due to the nature of D N A itself b u t rather to the in vivo state of D N A (Sakakibara et al., 1969). Compounds promoting sporulation were isolated from different sporogenic strains of B. subtilis, B. cereus, B. megaterium and B. papilliae (Srinivasan, 1965; Aubert et al., 1961). One of them was identified as N-succinylglutamic acid. The others remain unidentified. They are heat-stable and their activity is n o t destroyed after treatment with mild acid, alkali, DNase, RNase or trypsin. The factor was found to bleach alkaline permanganate, indicating that an oxidizable substance is involved here. Attempts to identify such compounds have shown t h a t m a n y mutations and inhibitors can prevent sporulation. Recent experiment of Mitani et al. (1977) demonstrated t h a t limited starvation for purine nucleotides promoted b y decoyine o r hadacidin, induces sporulation in B. subtilis even in the presence of rapidly metabolizable sources of carbon, nitrogen and phosphate. Another low-molecular weight factor associated with sporogenesis was described in S. venezuelae (Scribner et al., 1973). These results suggest that binding of small molecules to D N A (e.g., antibiotics, pigments, peptides, nucleotides), as well as their liberation from the D N A complexes m a y be involved in regulation of specific genes. Studies of translation systems of vegetative cells and spores revealed many structural and functional differences. Controversial data concerning the presence of polysomes in spores were obtained (Deutscher et al., 1968; Lavallg and De Hauwer, 1970). Results from many laboratories indicate that extracts from spores are less active in in vitro amino acid incorporation than analogous extracts from vegetative cells. Idriss and Halvorson (1969) found that ribosomes from dormant spores of B. cereus and B. megaterium are inactive in protein synthesis in vitro. An extract from spores of B. subtilis was reported to incorporate only 20 ~/o of phenylalanine with respect to the vegetative extract (Bishop et al., 1969). The cell-free extract of S. granaticolor spores was found to incorporate only 8 ~ of phenylalanine as compared with the same extract from vegetative cells (Mikulik et al., 1976). These functional alterations of in vitro spore systems were explained b y changes in t R N A content (Vold and Minatogawa, 1972) and b y the existence of defective ribosomes (Kobayashi, 1972; Fortnagel et al., 1973): It was of interest to investigate whether the low activity of the in vitro proteinsynthesizing system of spores is caused b y structural alterations of ribosomes from dormant spores (Mikulik et al., 1975). Extracts of spores were subjected to analytical centrifugation and compared with ribosomes of the vegetative forms. Differences in sedimentation properties between extracts of spores and of vegetative cells, as well as other features, such as composition of ribosomal proteins, suggest that most of ribonucleoprotein particles present in dormant spores are ribosomal precursors. The development of the spore is multipotent. The spore can differentiate into the vegetative cell and, if the cultivation medium is not suitable, only a part of the genome, the activity of which leads to the formation of the sporangium without a previous division is transcribed (microcycle). Occasionally, under different condi-

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tions, thick-walled cystae resembling cells are formed. The o p t i m u m time for induction of the sporu]ation microcycle falls in the interval, when D N A replicates during growth. In addition to the genus Bacillus, the microcycle was described in aspergilli and pennicillia. However, the above examples are in fact pronounced interventions with the programming of life processes of the ceil. I t is apparent that during this period the cell can, under more suitable conditions, adapt or coordinate its metabolic processes with the cultivation medium b y means of subtile corrections, e.g. by changing the quantitative ratio between the glycolytic p a t h w a y and the hexose monophosphate shunt, etc. Cultivation media used for production of streptomycin, chlortetracycline, ergot alkalids, where a higher concentration of phosphate ions influenced unfavourably the yield of antibiotics or alkaloids, m a y serve as example here. I t was demonstrated t h a t a higher concentration of phosphate ions increases the proportion of the glycolytic p a t h w a y in metabolic processes (Hogfs 1964a, b, c). During growth the cells are extraordinarily sensitive to changes of cultivation conditions. In S. aureofaciens a short interruption of aeration between 6 and 12 h of cultivation resulted in a drastic decrease of production of chlortetracycline, whereas metabolic processes leading to an increase of biomass remain unchanged (Matelovs et al., 1955). Stimulation of biosynthesis of eephalosporin C b y methionine (Drew and Demain, 1973; Gr6ger, 1976), induction of production of ergot alkaloids b y t r y p t o p h a n or its analogues (Robbers and Flos, 1976) and stimulation of production of alkaloids in PeniciUium cyclopium b y phenylalanine (Dunkel et al., 1976) m a y serve as examples, when compounds stimulating the production had to be added at the beginning of cultivation. These examples are interesting, due mainly to the fact that "stimulators" or "effectors" are simultaneously precursors of the compounds studied. T r y p t o p h a n added to the culture of Claviceps at the end of the exponential phase of growth is incorporated as a precursor into the ergolin ring of ergot alkaloids b u t it does not increase their production. The same holds true for methionine serving as a donor of sulphur of the ring of cephalosporic acid (Niiesch et al., 1973). Methionine added at the beginning of the cultivation brings about remarkable morphological and metabolic changes in the mycelium of cephalosporia and it is thus a question whether only a stimulation of enzymes involved in the synthesis of cephalosporin C, a hypothetica ! synthase of the ~-lactam ring, takes place here. Similarly, induction of biosynthesis of ergot alkaloids b y t r y p t o p h a n can hardly be explained only as derepression of enzymes synthesizing alkaloids. In this connection it is of interest that t r y p t o p h a n added at the beginning of cultivation decreases partially the unfavourable effect of phosphate ions on the production of alkaloids. Phosphate ions apparently exhibit a manifold effect on metabolism and it should also be kept in mind t h a t t h e y function as a determining factor of the rate of A T P synthesis (Robbers and Floss, 1976). Our results and conclusions reached when studying the biosynthesis of tetracycline compounds (Van~k et al., 1973) are in agreement with the assumption that these compounds influence the overall metabolic t y p e of the production microorganism, the course of metabolic differentiation. The basic pattern of the developmental programme and the sequence of individual metabolic steps are determined during the initial hours of cultivation in agreement with the conditions of the external medium (Nover and Luckner, 1976; Dunkel et al., 1976). When studying the production of antibiotics it was repeatedly demonstrated that the addition of different compounds to the cultivation medium at the beginning of

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cultivation, even of compounds that are not precursors, m a y influence their yield to a considerable extent. Thus, stimulation of biosynthesis of chlortetracycline b y fluoride (Di Marco et al., 1956), p-chlorophenoxyacetic acid, phenylacetic acid, ~-indoleacetic acid, ~-naphthylacetic acid and other compounds in a low-production strain of S. aureofaeiens (Van~k, 1958) and b y benzyl isothiocyanate in high-production strains (Hogfs et al., 1958) was described. Additional enzymological studies showed that also these compounds can be considered as determining the metabolic type of the production microorganisms. It is apparent that also the strain specificity of the production microorganisms plays a role here. Discovery of bioregulators influencing the development of actinomycetes producing streptomycin was of utmost importance. Addition of a low-molecular weight compound (C13H2204), called the A factor, to the cultivation medium of non-production m u t a n t s during the first hours of growth restores the production of streptomycin. The A factor exhibits a multiple effect: it induces regeneration of the aerial mycelium, submerged spores, pigment, influences the course of glucose metabolism and formation of a number of enzymes (Khokhlov et al., 1976). The factor does not remain in the cultivation medium. It was also demonstrated that the factor does not induce genetically fixed changes. Its role is clearly regulatory, determining the reading of DNA. Another interesting "cosynthetic factor" was found in a number of streptomyeetes producing oxytetracycline. Added to the cultivation medium of S. aureofaciens S-1308 (producing mainly 5a, lla-dehydrochlortetracycline), the factor was found to raise the production of chlortetracycline from 100--400 ~g/ml to 5000 ~g/ml. So far, the chemical structure of the cosynthetic factor h a s not been clarified b u t it m a y assumed t h a t it is a compound related with pteridines or flavins. This would indicate t h a t the m u t a n t S. aureofaeiens S-1308 exhibits a vitamin deficiency (Miller et al., 1960).

Growth The important observation made b y Schaechter et al. (1958) studying the growth of Salmonella typhimurinrn should be briefly mentioned at the outset. T h e y found the cell mass to increase exponentially with increasing growth rate, whereas the average amount of I~NA per cell increased at a slightly higher rate, indicating that the proportion of R N A with respect to the total cell mass also increased with increasing growth rate. However, the amount of D N A per cell was found to increase at a lower rate than the cell mass so that the proportion of D N A to the cell mass decreased. It could be deduced on the basis of the above experiments that the growth rate is a variable influencing the size of cells and their composition (Pritchard, 1974). Formation of the product and cell mass is very closely related to the amount of R N A present in the cell and it appears that the R N A content in individual cells in a batch culture begins to decrease roughly in the middle of the exponential phase of growth. Thus, in the batch culture more than one-half of cells contain less than the maximum possible R N A quantity. A more e x t e n s i v e source 'of infbrmation about rate-limiting steps in ribosome formation in vivo has come from the analysis of the flow of labelled precursors into r-RNA and, heflce, into ribosomal paricles during exponential growth. During the balanced growth of bacterial cultures, the number of ribosomes per cell depends on the growth rate of the culture. In experiments of H a r v e y (]953) it could be shown that the number of polysomal ribosomes decreases with decreasing growth rate. Furthermore, measurements of the amount of nascent pep-

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tides which can be released from ribosomes by puromycin also indicate that the number of active ribosomes decreases with the growth rate. The turnover of cell proteins was measured under conditions where a rapid equilibrium between the internal amino acid pool and the growth rate was achieved. The turnover of a few percent of proteins with a half-life of about 1 h at 37 ~ could be detected at a wide range of growth rates. At very low growth rates the half-life decreased to about 30 min. Therefore, it is doubtful, whether the turnover of completed protein chains can contribute to a decrease of the average polypeptide chain growth rate. It is most likely that the number of active ribosomes decreases with decreasing growth rate which indicates that there is no simple correlation between the control of ribosome synthesis and the function of the ribosomes in polypeptide formation. Excluding a regulatory function of nucleotide triphosphates on RNA synthesis. Kjeldgaard (1974) suggests two opposite mechanisms of control to explain the variation of the rate of RNA synthesis: regulation at the level of initiation of new rRNA chains, or regulation at the level of breakdown of nascent rRNA. It was found when studying synthetases of gramicidin S in a chemostat under limitation by carbon, nitrogen, phosphorus and sulphur sources that the growth rate, rather than the nature of the limitation was the major controlling factor regulating the level of the enzymes (Matteo et al., 1976). In a batch culture an explanation of the effect of certain experimental conditions on the cells is highly complicated by a permanently changing composition of the nutrient medium, which is unavoidable. Nevertheless, the basic picture of the r a p i d multiplication of cells and growth at the expense of readily accessible nutrients of the cultivation medium, followed by a slow growth and production of antibiotics after their exhaustion, was observed in all biochemical studies, e.g. in the case of penicillin (Hockenhull, 1959), streptomycin (Hockenhull, 1960), actinomycin (Katz et al., 1958), and tetracyclines (DoskoSil et al., 1958; Di Marco and Pennella, 1957). Exhaustion of a certain substrate (e.g., glucose) from the cultivation medium of S. antibioticus induces the production of actinomycin, exhaustion of nitrogen from the cultivation medium of Penicillium islandicum induces the biosynthesis of compounds of the oligoketide type (with malonylCoA as the building unit). A certain suitable ratio between the sources of carbon and nitrogen in the fermentation medium is considered to be most important for the optimum production of some microbial products. Slow growth, conversion of Gram-positive thick filaments of th so-called primary mycelium to thin filaments of the secondary mycelium, a decrease of the protein and RNA content (Prokofieva et al., 1959) are the features characterizing the transient phase which is often described as the end of the exponential phase of growth or beginning of the stationary phase (non-balanced growth) etc. Various misunderstandings follow from the fact that the exponential phase of growth of production microorganisms, as measured by the dry weight of cells or growth of the cell mass, does not coincide with curves obtained when determining protein synthesis by means of incorporation of 14C-leucine intoproteins. According to the results obtained when studying protein synthesis in S. aureofaciens, a producer of chlortetraeycline (Mikulik et al., 1971a), protein synthesis already begins after 4 h of cultivation and reaches its maximum between 12 and 16 h. The content of cy~ochromes, QO2 values, intracellular ATP level and the number of enzymes reach their maxima after 12 h of cultivation (Hotfhlek and VanSk, 1973). During this time, the synthesis of chlortetracyeline already takes place. Protein synthesis begins to decrease after 18 h of cultivation. However, the beginning of the exponential growth of the culture of S. aureofaciens falls in this time period.

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When following the sensitivity of young (4 h) cells of the production and nonproduction strains of S. aureofaciens to ehlortetracyeline, considerable differences in the incorporation of 14C-leucine into proteins and of 14C-thymine into D N A were detected. The addition of 500 ~g TC/ml to a 4-h-old culture producing TC caused a 37 % inhibition of protein synthesis and a complete inhibition of TC production. The incorporation of 14C-leucine into proteins of the production strain was fully inhibited under the same experimental conditions. When 500 ~g TC/ml were added to a 16-h-old culture producing TC the antibiotic production was not inhibited at all. Polysomes and ribosomes of S. aureofaciens were stable at a concentration of 5 ~g/ml. Aggregates were formed at higher concentrations of 200--400 ~g/ml (Mikulik et al., 1971b). Two t y p e s of bonds, reversible and irreversible, were detected b y means of 8H-tetracycline: It was found t h a t about 320 tetracycline molecules are reversibly bound to the 70S ribosome, whereas only one tetracycline molecule is b o u n d irreversibly (Mikulik et al., 1969, 1971c). Remarkable differences were also found in the accumulation of tetracycline. Cells of the non-production strain were found to accumulate 23 % more tetracycline t h a n cells of the production strain. It can be assumed that resistance to tetracycline in the production strain is associated not only with the protein-synthesizing system b u t also with modification of the cell membrane (Mikultk et al., 1971a). Previous results obtained when studying the translation mechanisms in S. aureofaciens are in agreement with those obtained when investigating the mechanisms regulating the biosynthesis of actinomycin in S. antibioticus (Katz and Weissbaeh, 1963; Collett and Jones, 1974). Protein synthesis in S. antibioticus already decreases 9 h after the inoculation. The exponential growth of the myeelium begins with the 12th h of cultivation and is terminated after a b o u t 36 h of cultivation (medium with glutamic acid). Up to t h a t time about 70 ~ of the cell mass is formed and the concentration of the antibiotic reaches roughly 15 to 20 ~g per ml medium. This is already a sufficient concentration for growth inhibition to set in. However, the synthesis of macromolecules never decreases to zero. Basal protein and R N A synthesis remains preserved for several days, irrespective of the fact t h a t the cells actively synthesize actinomycin (Jones, 1976, 1977). It is difficult to s t u d y factors causing a decrease of R N A synthesis in the culture of S. antibioticus long before the onset of synthesis of aetinomyein. The same holds for mechanisms facilitating the synthesis of a certain small amount of I~NA, even in the presence of a strong inhibitor of D ~ A - d e p e n d e n t R N A polymerase. Jones studying the transcription mechanisms in S. antibioticus found t h a t the decrease of R N A synthesis in ac~inomyein-producing cultures is at least partially caused b y specific proteins that bind to D N A and ~hus inhibit transcription catalyzed b y DNA-dependent R N A polymerase. The persistence of a certain residual R N A synthesis is associated with the existence of other proteins that bind to actinomycin and with a peculiar property of the transcription complexes formed between D N A and R N A polymerase, facilitating transcription of some sequences of DNA, even in the presence of bound actinomycin. Thus, it can be said that investigation of the logarithmic phase of growth in aetinomycetes (by determining the dry weight) reflects only a part of the proteinsynthesizing activity of young cells from the initial hours of cultivation (8--12 h). I m p o r t a n t metabolic changes and synthesis of specific protein molecules take place during the cultivation, long before the overproduction of antibiotic compounds is observed.

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Transient phase When analyzing the growth and production curves of different antibiotics and the so-called secondary metabolites it can be clearly demonstrated that it is often impossible to draw a sharp b o u n d a r y between the growth phase and the transient phase. A relatively high number of antibiotics that can be classified in the group of compounds, the intense production of which is associated with growth, is rather surprising. The relationship between growth of microorganisms and production of antibiotic compounds was also studied b y applying problems of the so-called non-balanced growth of microbial populations, which is still subject to intense studies b y microbial physiologists (Woodruff, 1966). It is based on the assumption that regulatory mechanisms cannot prevent overproduction of some primary metabolites after the end of balanced growth. These primary metabolites are then utilized for the synthesis of secondary metabolites (antibiotics). In the view of microbial physiologists, non-balanced growth reflects a situation when the cell does not synthesize all of its components at the "same rate". The non-balanced growth occurs at the beginning of the stationary phase or as a result of a "shift-down" (cells growing in a rich nutrient medium are transferred to a minireal medium), when the synthesis of ribosomes is stopped and replication of the chromosome is completed (new synthesis does not begin). Synthesis of some proteins, reserve compounds or the cell wall can still proceed for a certain time interval. The optical density of the culture usually increase as well. This state can also be induced b y adding antibiotic compounds. In the presence of chloramphenicol the synthesis of the cell wall and nucleic acids continues, incomplete ribosomes accumulate b u t protein synthesis is stopped and the population does not multiply. On the contrary, penicillin interrupts the synthesis of the cell wall, whereas protein synthesis can continue under suitable cultivation conditions. I t is a question whether the term non-balanced growth is suitable in this context. ]t characterizes well certain experimental conditions b u t its application to the developmental phases of microorganisms appears to be less accurate. Transition from one regulated state of development of the microbial population to another state which is also regulated, as the same metabolic situation is always observed under the same cultivation conditions, cannot be considered as unnatural, non-balanced or comparable to metabolic catastrophes. The pioneering value of papers on non-balanced growth mainly consists in the fact that the importance of metabolic processes in the so-called transient phase, between the phase of exponential growth and the stationary phase, is accentuated. These papers made it possible to understand the fragility of balance of new metabolic relations and regulatory systems in high-production mutants, a situation, when the predominating part of metabolism is directed towards synthesis of an excessive metabolite (Van~k et al., 1973). The method of "shift-down" resembles that of "replacement cultures", which was previously used in studies of biosynthesis of antibiotic compounds. A pregrown cell population was transferred to a fresh medium, which did not facilitate further growth b u t the microbial cells retained their ability to synthesize secondary metabolites. Under these conditions the resting cells were to transform a higher a m o u n t of a substrate to secondary metabolites at a higher rate. Examples, when biosynthesis of secondary metabolites continues even if overgrown cells are suspended in water, were also described. In these cases precursors of secondary metabolites are produced from compounds accumulated in the cells during previous growth. Bio-

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synthesis of penicillin m a y serve as an example. The mycelium t h a t has already started the production of penicillin (but not younger) continues to synthesize the antibiotic, especially after the addition of a precursor of the side chain. Synthesis of penicillin proceeds even more intensely when the mycelium is transferred to a minimal medium containing an energy source (Bu'Lock, 1961). Experiments with substitution of the original nutrient medium b y a new medium were based on the fact that synthesis of secondary metabolites is fully or partially suppressed in the actively multiplying culture whereas in the resting or stationary culture it proceeds at the m a x i m u m rate. Several groups of investigators tried to verify experimentally the assumptions t h a t the phase of rapid growth can be separated from the production phase in which only a negligible or no growth of microorganisms occurs. Studies concerned with biosynthesis of penicillin are particularly interesting in this respect (Jarvis and Johnson, 1947; Maxon, 1955; Gaden, 1959). However, what does in fact the term "negligible growth" mean from the quantitative point of view? Pirt and Righelato (1967) studying the effect of growth rate on the synthesis of penicillin in Penicillium chrysogenum in a chemostat under glucose limitation found that the specific rate o f penicillin production is to a considerable extent independent of the specific growth rate. However, this specific growth rate must not decrease below an eighth of its m a x i m u m value. Similar conclusions were reached b y Upiter et al. (1973) and Makarevich et al. (1976), who studied growth and production of tetracycline in S. aureofaciens. It was demonstrated t h a t the development of S. aureofaciens and production of tetracycline depend on the optimum ratio of carbon and nitrogen in the nutrient medium. Optim u m levels of nutritional and energy sources for intense growth were determined and, after accumulation of biomass, optimum conditions for the retarded growth were found. Prolongation of this t y p e of growth was shown to lead to an increased production of the antibiotic in the cultivation medium. However, the fact that the synthesis of tetracycline does not proceed without growth of the mycelium of S. aureofaciens was the most important finding obtained in these experiments. I t would certainly be inaccurate to generalize that the synthesis of all secondary metabolites proceeds only during the transient phase or the phase of retarded growth. However, the accumulated experimental material indicates that in the batch culture of microorganisms this phase has to be distinguished fl'om the stationary phase.

Stationary 1)base The microbial cell can cease multiplying exponentially due to various reasons. Exhaustion of nutrients or energy sources from the cultivation medium is most commonly involved. Even an insufficient aeration can be responsible for this effect in very dense suspensions in rich complex media. Organic acids (pH unsuitable for further growth) or other metabolites formed during the cultivation, including antibiotics, belong to metabolites suppressing regressively growth of the population. An increased turnover of proteins and ribonucleic acids is perhaps the most typical manifestation of non-growing cells. Protein turnover in growing cells of bacteria does not usually exceed 1 % per hour, in fact it is mostly substantially lower. The turnover of R N A is usually limited to mRNA. Protein turnover increases to 3 2/0 to 7 % and to 15 2/0 per hour in non-growing cells and during sporulation, respectively. The rate of degradation of proteins and their synthesis are roughly in equilibrium so that their content in the cell does not change. In non-growing cells some turnover of ribosomal R N A can also be observed. As

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the synthesis of ribosomes is simultaneously decreased, the content of ribosomes in cells decreases and m a y completely disappear. The rate of degradation of r R N A is usually 5 ~ per hour b u t can often be much higher. No turnover of D N A could be demonstrated. Apparently, the integrity of the chromosome remains preserved in non-growing cells. Protein turnover is assumed to play a more important role in the non-growing culture than the turnover of RNA, as adaptation of the organism to new conditions requires production of new enzymes. A permanent turnover of m R N A existing both in growing and in non-growing cells could itself provide the essential nucleotides for the production of new molecules of m R N A . Therefore, degradation of ribosomal R N A occurring after the decay of ribosomes is not considered as prerequisite for the synthesis of new R N A molecules. In rather supplies the metabolic pool with a material that is utilized as a source of energy and low-molecular weight metabolites for oxidoreduction processes. I t is generally valid that the content of ribosomes in non-growing cells is decreased to a certain basal level characteristic for the stationary phase. This decrease occurs due to the fact that 4egradation of ribosomes proceeds at a higher rate than their synthesis which is inhibited as a result of a "shift-down". The content of ribosomes can decrease even below this basal level when the culture starves for Mg 2+ or phosphate. The mechanisms leading to the interruption or decrease of synthesis of ribosomes i~ non-growing cells cannot be accurately clarified at present. The level of free amino acids plays a role here since amino acids m a y influence indirectly the level of nucleoside triphosphates. To preserve its biological activity the non-growing cell requires a certain, even if very low, supply of energy designated as the energy of maintenance. Without this supply the number of dead cells in the population rapidly increase. The ceil can obtain this energy both via oxidation of exogenous substrates and by oxidation of intracellular reserve compounds. Low-molecular weight components obtained b y hydrolysis of proteins and R N A (amino acids, purines, pyrimidines, ribose( can be utilized as energy sources.

Miscellaneous A close relationship between the organism and the outer environment exists in every fermentation. Changes in metabolic processes and physiological properties occur, depending on the w a y vof cultivation. These are reflected b y the variability of shape and the size of cells (Sfastns et al., 1977). Cells in various stages of development are present in a batch culture and the measured values always represent average of a very heterogeneous population. Unfortunately, the biosynthesis of biologically active compounds in streptomycetes and molds has not yet been studied with synchronized cultures (Ss ]977). Production of antibiotic compounds proceeds differently in surface and in submerged cultures. The two-phase character is usually not observed in the surface culture and antibiotics are produced already during the growth phase. For instance, during the surface cultivation of S. griseus (Waksman et al., 1946) production of streptomycin proceeds in parallel with growth. Roughly similar relationships were observed during the production of other excessive metabolites. During production of citric acid b y Aspergillus niger in a surface culture specific factor activating the fixation of carbon dioxide to phosphoenolpyruvate must first be synthesized in some strains. This anaplerotic reaction makes it possible to increase the production of citric acid and prolong the production ability to the stationary phase (Fencl et al., 1963). During submerged cultivation and at a high production of citric acid (70 o/ /O

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with respect to the initial sugar concentration) the synthesis proceeds with maximum intensity in the so-called transient phase and the production decreases during the stationary phase. The fact that relatively high yields, e.g. of penicillin (Jarvis and Johnson, 1947) can be reached under conditions when the production organism grows very slowly, are often discussed in the literature. Similar results were also obtained by Malik and Vining (1970) who studied metabolism of chloramphenicol in S. venezuelae in synthetic and complex media. The synthetic medium facilitated only very slow growth but chloramphenieol was produced intensely during the whole growth phase. In a complex medium synthesis of chloramphenicol proceeds at the end of the exponential growth of the culture. In some respects thus these results resemble the experience of collection microbiologists using an alternation of poor and rich media for revival of collection cultures. Alteration of media was found to be successful also during production of granatiein (~fastns et al., 1977). Repeated cultivations in rich media apparently lead to a weakening of certain metabolic systems which is also manifested by a lower production of antibiotic compounds. Production of compounds during different time stages of development of a certain microorganisms need not be caused only by different cultivation conditions. Studying the biosynthesis of 6-methylsalicylic acid in Penicillium p a t u l u m Light {1967) isolates strains, in which the synthesis of MSA occurred in two different developmental phases. It began shortly after transfer of the mycelium from the inoculation medium (germination medium) to the Czapek-Dox medium in one strain and started only after 24 h of cultivation in another strain. The growth curves of the two strains were identical. It is rather difficult to explain these observations. It can be assumed that a repressor was changed (in the poor medium) or that the two cultivations differed in the synthesis of an inducer or corepressor. It is also possible that a precursor of MSA (e.g. polyketothioester) is metabolized to other cell components. A block in this pathway would then lead to the production of MSA. Naturally, all these speculations have to be verified experimentally. Nevertheless, the above examples show that the production of some metabolites is not strictly associated with a certain phase of development of microorganisms and that the genetic equipment of the studied microbial strains, the composition of the medium and the way of cultivation play an important role. Excessive metabolites It is clear in many cases that t h e amount of the produced antibiotic compounds depends on the excessive production of suitable precursors by corresponding metabolic pathways of primary metabolism. For instance, when comparing some enzymic activities of a low-production strain of Streptomyces hygroscopicus producing the macrolide antibiotic turimycin it was found that the production strains exhibit a much higher activity of the enzyme systems synthesizing the basic precursors of this antibiotic. AeetylCoA carboxylase, propionylCoA carboxylase, pyruvate dehydrogenase and NADPH-regenerating enzymes, e.g., glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were more active; on the other hand, the activity of acyl-thioesterase decreased considerably (Gr/~fe et al., 1975a, b). Similar results showing that a high production of the so-called secondary metabolites depends on the excessive supply of primary metabolites were obtained when studying the production of nourseothricins in Streptomyces noursei JA 3890/b (Gr~fe et al., 1977). The production of nourseothricins in this strain depends on the presence

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of o-aminobenzoic acid or related compounds in the fermentation medium. These effectors influence processes in the respiratory chain, which is indicated by a roughly five-fold increase of the N A D H - - N A D + ratio during the early phases of cultivation (15--30 h).Insuffieiency of NAD + in the mycelium decreases the catabolism of alanine (alanine dehydrogenase) and other amino acids. A decreased level of NH § does not support the activity of glutamine ~synthetase, but, on the contrary, the activity of NADP+-dependent glutamate dehydrogenase sharply increases: It can be assumed that in the presence of OABA or its analogues, the metabolism of the strain is shifted from amino acid catabolism (alanine) to amino acid biosynthesis during the growth phase. This change stimulates an excessive supply of precursors for the biosynthesis of nourseothricins. The increased production of the antibiotic begins during the last third of the growth phase, when the biomass ceases to increase. A rapid decrease in the production of the antibiotic occurs when growth of the mycelium stops completely. It is possible to present further examples of studies, in which enzymic activities of non-production and production strains synthesizing antibiotic compounds were compared. Toropova et al. (1972) found that high-production strains producing nystatin excrete three- to four-fold greater quantities of volatile acids (formic acid, acetic acid, butyric acid) into the medium during the growth phase. These compounds were found to be rapidly assimilated during the production phase. The intense synthesis is characterized by a rapid decrease of the level of pyruvic and lactic acid (Rafalski and Raezynska-Bojanowska, 1972). This decrease is accompanied by an increase of specific activities of acetylCoA earboxylase, methylmalonylCoA carboxylase and phosphoenolpyruvate carboxylase (Raezyfiska-Bojanowska, 1974). Similar resutts were also obtained when investigating enzymes of the hexose monophosphate shunt and of the gtyeolytic pathway (Toropova et al., 1974, 1975). The definition of excessive metabolites is not based only on the quantitative aspects of the known enzymic pathways. This derives from the fact that a change of production of a secondary metabolite in a wild strain, e.g. from 50 ~g/ml to 10 000 tzg/ml and even more in the improved mutants is clearly due to an interference with the regulatory mechanisms of practically all metabolic pathways and all the life processes of the microbial cell (Van~k et al., 1971). This fact can be observed especially well in microbial strains producing antibiotic compounds, as in this case the mechanisms that make it possible that the production cell survives concentrations of toxic compounds never occurring in the nature, also have to be developed. Thus the study of the secondary metabolism does not coincide with the study of the metabolism leading to excessive production. In addition, the theory of excessive metabolites does not depend exclusively on the assumption that only a secondary metabolite formed after the phase of the exponential growth may become the excessive metabolite. Any metabolite, even an essential metabolite required for growth and multiplication of the microorganism may become excessive if suitable genetic and physiological manipulation is applied. REFERENCES AD~ R., SCHULTZ L. D., HALL B. D.: Transcription in yeast: separation a n d properties of muitiplo R N A polymerases. Proc. N a t . Acad. Sci. U . S . A . 69, 1702 (1972). A~MSTBO~G R. L., SUEO~A N.: Phase transition in ribonucleic acid synthesis during germination of Bacillus subtilis spores. Proc. N a t . Acad. Sci. U . S . A . 59, 153 (1968). AU~EBT J. P., MILLET J., p~E.ttT E., MIL~AUD G. : L(+)-N-suecinyl glutamie acid in Bacillus m egaterium during sporulation. Biochim. Biophys. Acta 51, 529 (1961).

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BAB~B~S G., OTTENBERQER A,, SZAB6 J., ERDEI J., SZAB0 G.: The biological role of aminoglucoside. Intern. Symposium on Nocardia and Streptomyces in Warsaw, 1976. BAR~2S G., SZAB6 G. : Effect of penicillin on streptomycin production by Streptomyces griseus. Antimicrob. Agents Chemoth. 11,392 (1977). BARA~S G., SZAB5 J., SZAB6 G.: Enzymically released cell wall fragments with antibiotic activity from aminoglucoside producing Streptomycetes. 10th International Congress of Chemotherapy, Sept. 18--23, 1977 (Zurich, Switzerland). BEN-Zt~IEV H., HATTORI J., SILBERSEIk'r Z., TESONE C., TORRIANI A.: Ribonucleic acid polymerase from dormant and germinating spores of Bacill?~s cereus T, in Spores VI. Gerhardt P., Costilow It. N. and Sadoff H. L. (eds.), Amer. Soc. Microbiol., p. 472 (1975). B~-R~LOHR R. W., NOVET.LI G. D. : Bacitracin biosynthesis and spore formation. The physiological role of an antibiotic. Arch. Biochem. Biophys. 103, 94 (1963). BIFF~ G., BOlCE~TI G., Di MARCO A., PEIgNELLA P.: Metabolic behavior and chlortetracycline production of Streptomyces aureofaciens in liquid culture. Appl. Microbiol. 2, 288 (1954). BIsgoP H. L., MIGITA L. K., DO~ R. H. : Peptide synthesis by extracts from Bacillus subtilis spores. J. Bacteriol. 99, 771 (1969). BORETTI G., Di MARCOA., JULITA P., RAGGI F., BARD[ U.: Presenza dogli enzimi della via eso monofosfato ossidativa mello Streptomyces aureofaciens. Giorn. Mierobiol. |, 406 (1956). BR~A~ P. W.: The ecological significance of antibiotic production. Syrup. Soc. Gen. Mierobiol. 7, 168 (1957). Bu'LocK J. D. : Intermediary metabolism and antibiotic synthesis. Advances Appl. Mierobiol. 3, 293 (1961). Bu'LOcK J. D.: The biosynthesis of natural compounds. An introduction to secondary metabolism. McGraw-Hill Publishing Company Limited, London (1965). Bu'LocK J. D.: T h e two-faced microbiologist: contributions of pure and applied microbiology to good research. Develop. Industrial ,~Iicrobiol. 15, 11 (1975). Bu'LocK J. D., POVCEV.LA. J.: Secondary metabolism: an explanation in terms of induced enzyme mechanisms. Experientia 21, 55 (1965). CALA.:VIC. T.: Starting investigational and production cultures. Process Biochemistry, p. 7 (1976). Co/