Growth morphology of Streptornyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients M. A. GLAZEBROOK AND L. C .
VINING'
Biology Department, Dalhousie University, Halifax, N.S., Canada B3H 4 J l AND
R. L.
WHITE^
Chemistry Department, Acadia University, Wolfville, N.S., Canada BOP 1 x 0 Received April 29, 1991
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Revision received July 26, 1991 Accepted July 26, 1991 GLAZEBROOK, M. A., VINING,L. C., and WHITE,R. L. 1992. Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients. Can. J. Microbiol. 38: 98-103. Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis. Key words: Streptomyces akiyoshiensis, mycelial aggregation, growth conditions. GLAZEBROOK, M. A., VINING,L. C., et WHITE,R. L. 1992. Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients. Can. J. Microbiol. 38 : 98-103. La plupart des milieux dans lesquels la croissance de cultures submergees et agitees de Streptomyces akiyoshiensis fut examinee, n'ont pas permis la formation de suspensions de myceliums bien disperses. L'investigation des conditions de culture encourageant la croissance dispersee ont montre que le pH du milieu de culture est d'une importance critique; une valeur initiale de 5,5 a minimise l'aggregation du mycelium tout en supportant une production adequate de biomasse. Dans les cultures amorcees a ce pH, les inoculums de spores ont donne une meilleure dispersion du mycelium que les inoculums vegetatifs; avec les inoculums de spores, la morphologie de la croissance a ete aussi moins affectee par le volume de l'inoculum. La composition de la solution nutritive a influence l'etendue de la dispersion du mycelium; une croissance lente a ete associee souvent avec l'agglutination, mais aucune correlation precise n'a pu Etre observee entre la formation de boulettes et la capacite des sources de carbone ou d'azote de supporter une croissance rapide. L'augmentation de la concentration de phosphate de 0,5 a 15 mM a cause une diminution modeste de l'aggregation du mycelium. Les conditions qui encourageaient un mycelium bien disperse, adequat pour l'etude du contr8le physiologique du metabolisme secondaire, ont aussi permis la formation de 5-hydroxy-4-oxonorvaline par S. akiyoshiensis. Mots clks : Streptomyces akiyoshiensis, aggregation du mycelium, conditions de croissance. [Traduit par la redaction]
Introduction Cultures of Streptomyces akiyoshiensis produce an antibiotic with activity against human-type Mycobacterium tuberculosis (Kanazawa et al. 1960). The product was first identified by Miyake (1960) as 5-hydroxy-4-oxonorvaline (HON) and has recently been rediscovered as RI-33 1 in cultures of an unidentified streptomycete during screening for agents active against yeasts and fungi (Yamaguchi et al. 1988). It functions as a metabolic analogue of homoserine and interferes with formation of the protein amino acids threonine, methionine, and isoleucine biosynthesized via this intermediate (Yamaki et al. 1988; Yamaguchi et al. 1990). In animals HON exhibits useful systemic antifungal activity, and because the affected protein amino acids are all essential dietary components, it is nontoxic (Watanabe et al. ' ~ u t h o rto whom all correspondence should be addressed. 2~resentaddress: Chemistry Department, Dalhousie University, Halifax, N.S., Canada B3H 453. Printed in Canada / lmprime a u Canada
1987). In studies directed towards the enzymic synthesis of HON, we have investigated the biosynthesis of the antibiotic in S. akiyoshiensis (White et al. 1988). Here, as in earlier work (Tatsuoka et al. 1961), HON was produced in complex media, which provided only limited information about .the fermentation characteristics of the organism. One of our early goals has been the development of reproducible growth conditions using a defined medium where the onset of antibiotic production could be predicted with confidence and high levels of biosynthetic enzyme activity could be achieved. When S. akiyoshiensis was grown in shaken flask cultures in defined media using the growth conditions described in the literature, the mycelium formed clumps composed of entangled hyphae of different ages, states of nutrition, and stages of development. Similar difficulties have been encountered with other actinomycete species (Hobbs et al. 1989; Doull and Vining 1989). Streptomycetes exhibit considerable variation in their growth morphology in submerged liquid cultures as well as on agar media (Tresner et al. 1967;
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Williams et al. 1974). Mycelial development in submerged cultures is markedly influenced by the growth conditions used (Nikitina and Kalakoutskii 1971; Stastna et al. 1977), and despite the important consequences for culture rheology and oxygen transfer in industrial actinomycete fermentations, the factors responsible have not been clearly defined. We report here the influence of various fermentation parameters on the growth morphology of S. akiyoshiensis in shaken cultures and define the conditions that minimize mycelial aggregation, making possible meaningful studies on the physiology of the organism. Materials and methods Cultures Streptomyces akiyoshiensis was obtained from the American Type Culture Collection as ATCC 13480 and maintained as a stock spore suspension. This was prepared by dislodging spores into water from the surface of a culture grown on MYM agar (Stuttard 1982). The spores were filtered through nonabsorbent cotton, collected by centrifugation, and resuspended in 20% (v/v) glycerol to give a calculated optical density at 640 nm (OD,,) of 10 from measurement of a 1:50 dilution in a round cuvette (1.2-cm internal diameter) with a Bausch and Lomb Spectronic 21 spectrophotometer. Spore suspensions were aliquoted and stored at - 20°C. Cultures in which morphology was examined were grown in 25 mL of liquid media contained in 250-mL Erlenmeyer flasks and incubated on a rotary shaker (3.8-cm eccentricity; 220 rpm) at 27°C. Except in experiments testing inoculum effects, each culture received 1.5% (v/v) of the stock spore suspension as an inoculum. A vegetative inoculum was prepared by incubating 20 mL of MYM medium with 20 pL of stock spore suspension for 24 h under the conditions described above for HON production. Samples of the culture were washed once with water by centrifugation and reconstituted as mycelial suspensions in water at the original volume. Media The complex media tested (composition in %w/v) were MYM (maltose, 0.4; yeast extract, 0.4; malt extract, 1.0), SYM (as for MYM but with starch in place of maltose), and mixtures of starch (3.0) with Bacto-Peptone, Bacto-Tryptone, Bacto-Soytone, BactoCasitone, or casein (0.4) in basal solution at pH 6.5. The basal solution consisted of (L - ') MgSO, .7H20, 0.20 g; 3-(morpho1ino)propane sulfonic acid (MOPS), 21 g; NaCl, 10 mg; CaCl,, 10 mg; FeSO, .7H20, 5 mg; ZnCl,, 180 pg; MnCl, .4H20, 45 pg; CuSO, 5H20, 45 pg; (NH4),Mo,02,~4H20,45 pg; and H3B03, 7.2 pg. Most defined media contained the same basal solution supplemented with 15 mM phosphate (a 5:3 w/w mixture of K,HPO, and KH2P04)and with the carbon and nitrogen sources specified in each experiment. Exceptions occurred when the effects of phosphate concentration and of basal medium components were being tested. Media were adjusted with 1 M HCl or 1 M NaOH to the required initial pH before sterilization. A nalyses Growth was normally measured as increase in cell dry weight. At each sampling, cultures were filtered under vacuum through weighed Whatman No. 5 filter-paper disks; the mycelium collected was washed with water and dried to constant weight at 75°C. Values reported are the averages for cultures grown in duplicate. Where the mycelium was well dispersed, differences were within f 7%. Cultures where severe clumping occurred showed variation as high as _+ 50%. The size of mycelial aggregates in culture broth was measured with a microscope, using a calibrated eyepiece and stage micrometer. Viewing fields were varied in diameter according to the size of the aggregates and were chosen at random to include 10-15 aggregates; for each aggregate the longest and shortest widths were
FIG. 1. Growth morphology of S. akiyoshiensis. (A) After 1 day in MYM medium; (B) after 5 days in a medium containing 3% (w/v) starch, 30 mM proline, 15 mM phosphate, and basal salts at pH 6.5. Both cultures received a 1.5% (v/v) spore inoculum and were photographed at 100 x . measured and averaged to give the aggregate size. The values for all aggregates within a field were averaged to obtain the mean size for the sample. Photographs were taken with a Zeiss Photomicroscope 11. The amount of HON present in clarified culture broths was determined by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde (White et al. 1989).
Results In preliminary experiments where shaken submerged cultures of S. akiyoshiensis were grown in media containing maltose or starch with a variety of complex nitrogen sources, a well-dispersed mycelial suspension (Fig. 1A) was obtained only during the rapid growth phase in a maltose - yeast extract - malt extract (MYM) mixture. In the late growth phase of cultures in MYM medium and throughout growth in the other media, the mycelium aggregated to form discrete pellets (Fig. 1B). Similar aggregation was encountered when cultures were grown in defined media containing a basal solution of inorganic salts at pH 6.5 and various single sources of carbon and nitrogen. To determine whether aggregation of the mycelium into pellets was due to inorganic constituents in the medium, components of the basal solution were omitted both individually and in groups; the magnesium sulfate concentration was also varied stepwise from 0.01 to 0.5 g .L - Each of the modified basal solutions was supplemented with 3% (w/v) starch, 15 mM proline, and 15 mM phosphate, since these nutrients had supported a high biomass yield in the preliminary experiments. The media were adjusted to pH 6.5 and inoculated (1.5% v/v) with the stock spore suspension. Although growth rates and biomass yield were affected by the differences in inorganic salt composition, average mycelial pellet sizes were consistently large (10.5 mm diameter). The results suggested that mineral nutrition was not the critical factor in mycelial aggregation. Because the growth morphology in MYM medium demonstrated that S. akiyoshiensiscould, under suitable condi-
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TABLE1. Effect of initial pH on broth pH, biomass yield, and mycelial pellet size Culture pH
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Initial
Minimum
Maximum
Maximum dry weifht (g.L -
Maximum pellet size mma
Day
NOTE: The basal solution was supplemented with 3% (w/v) starch, 30 m M proline, and 15 m M phosphate. Cultures were inoculated with 1.5% (v/v) stock spore suspension and analysed daily. T h e p H values reported are the lowest o r highest recorded at any sampling during a 7-day incubation. In all cultures the dry weight increased throughout the sampling period; thus all maxima were recorded o n day 7. 'Average size (0.5(longest + shortest dimension)) and standard deviation of all pellets from that sample observed within the microscope viewing field.
TABLE2. Effect of inoculum on biomass yield and mycelial pellet size Maximum dry weight Inoculum
g - ~ - ' Day
Maximum pellet size mm
Day
Spores (% v/v)
0.1 1.5 4.0
4.9 7.0 6.9
5 5 3
0.17k0.04 0.12k0.03 0.14k0.04
3 3 4
6.0 8.1 7.9
5 3 3
1.61 k0.23 0.87k0.12 0.61k0.09
4 4 4
Vegetative (% v/v)
0.1 2.5 5.O
NOTE:Media were adjusted t o a n initial p H of 5.5 and various inocula were used. Other conditions were as in Note t o Table 1.
tions, proliferate as a dispersed mycelial suspension, further experiments were undertaken to define the conditions supporting dispersed growth and to examine the extent to which they could be varied during testing of the physiological parameters governing HON production.
Influence of pH The basal solution supplemented with 3Vo (w/v) starch, 15 mM proline, and 15 mM phosphate was adjusted to pH values from 5.5 to 7.5 and inoculated with 1.5% (v/v) of stock spore suspension. Throughout the 7-day period in which culture parameters were measured, the pH of each broth remained well buffered at close to its initial value (Table 1). The early growth rates in all cultures were similar and declined after day 2, but biomass continued to accumulate during the observation period. Cultures at pH 5.5 grew more slowly after day 3 than those at pH 6.5 and above; as a result, their maximum biomass yields were appreciably lower. In a separate experiment where the effect of further reductions in initial pH were examined, values at or below pH 5.0 progressively inhibited growth (data not shown). In all cultures the average size of mycelial aggregates increased during rapid growth; this increase in size was faster and more prolonged at pH 6.0 and above. Consequently, the maximum size of the aggregates was 4- to 5-fold smaller in cultures at pH 5.5 than at higher pH values. Visual inspection suggested that the suspension of mycelium was dispersed to the same degree as in MYM medium; this was confirmed
by examining the broth with a microscope, which showed a dispersal similar to that in Fig. 1A. In all subsequent experiments, where the influence of other culture conditions was investigated, the media were buffered at an initial pH of 5.5 To determine whether aggregation of spores used as the culture inoculum might be related to the size of mycelial aggregates that developed during subsequent growth of cultures, spores suspended in samples of the media used in the preceding experiment were examined by light microscopy at 1000-fold magnification. At pH 6.5 and above, the suspensions consisted of well-dispersed single spores. Increasing aggregation occurred as the pH was lowered; this was reflected in both the number of spores in a clump and the stability of the association. At pH 5.5, the aggregates contained an average of five firmly attached spores. Since an initial culture pH of 5.5 improved mycelial dispersal during growth, other factors must be mainly responsible for growth morphology. Their enhancement at pH 5.5 evidently offsets the disadvantage of clumping in the spore inoculum.
Effect of the inoculum Cultures were grown in basal solution supplemented with 3% (w/v) starch, 30 mM proline, and 15 mM phosphate; they were inoculated with various amounts of spore or vegetative mycelial suspension. In all cultures the pH values remained within the 5.4-5.6 range. In those with a spore inoculum, the biomass yield increased when the inoculum size was raised from 0.1 to 1.5% (v/v); the yield did not increase further when the inoculum size was raised to 4.0% (v/v), but the biomass maximum was attained earlier (Table 2). The maximum size of the mycelial aggregates varied only marginally but occurred later with the 4.0% (v/v) inoculum. Use of a 0.1 % (v/v) vegetative inoculum gave cultures with relatively large mycelial pellets. Although the average pellet size decreased as the vegetative inoculum was increased to 2.5 and 5.0% (v/v), it remained much larger than observed with spore inocula. In cultures given a vegetative inoculum the biomass yield increased and was attained earlier when the inoculum was increased from 0.1 to 2.5070, but it showed no further change at 5070 (w/v). The maximum yields with the larger vegetative inocula were somewhat higher than obtained with spore inocula. In all subsequent experiments a 1.5% (v/v) spore inoculum was used. Influence of the nitrogen source The basal solution was supplemented with 3% (w/v)
GLAZEBROOK ET AL.
TABLE 3. Effect of the nitrogen source on broth pH, biomass yield, and mycelial pellet size
Maximum dry weight
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Broth pH Nitrogen source
Minimum
Maximum
(NH4)2S04 Isoleucine Proline Aspartate KNO, Glutamate
3.9 4.9 5.3 5.5 5.4 5.5
5.5 5.5 5.5 6.6 6.5 6.5
Maximum pellet size
g . ~ - ' Day
1.7 3.0 7.5 8.3 7.8 8.3
2 7 4 3 4 4
mm
Day
0.07 k0.02 0.10k0.07 0.10k0.03 0.22k0.07 0.22k0.05 0.27 k0.07
2 7 4 2 4 3
NOTE:The basal solution was supplemented with 3% (w/v) starch, 15 mM phosphate, and a nitrogen source supplying 30 mM N . Media were adjusted to pH 5.5 and inoculated with 1.5% (v/v) of stock spore suspension. Values are reported as in Table 1 .
TABLE 4. Effect of the carbon source on broth pH, biomass yield, and mycelial pellet size
Carbon source
Maximum broth pH
Glucose Starch Maltose Galactose Sucrose Fructose Lactose
6.0 6.6 6.0 6.0 5.7 5.8 5.9
Maximum dry weight (g-L - I ) " 1.4 8.3 5.5 1.4 1.1 4.5 1.2
Maximum pellet size (mm) 0.18 +_ 0.06 0.20 k 0.05 0.22 k 0.09 0.24 +_ 0.05 0.28 k 0.14 0.33 k 0.31 0.63 k 0.33
NOTE: The basal solution was supplemented with 30 mM K N 0 3 , 15 mM phosphate, and a carbon source at 3% (w/v). Media were adjusted to pH 5.5 and inoculated with 1.5% (v/v) of stock spore suspension. Values are reported as in Table 1 . 'In cultures with glucose, starch, and fructose, dry weight reached a maximum at 4 days; in all other cultures the maximum was at 7 days. Mycelial pellets increased in size until the growth maxima.
starch and various nitrogenous substances supplying 30 mM nitrogen to the medium. In some media, the buffering capacity of the 0.1 M MOPS present was insufficient to prevent substantial changes in pH (Table 3). That cultures with ammonium sulfate acidified rapidly to growth-inhibitory conditions is also indicated from the low biomass yield and early maximum. Cultures with isoleucine followed a similar pattern, although at a slower rate. Cultures containing aspartate, glutamate, or potassium nitrate all increased in pH and formed mycelial pellets larger than those present in proline-containing medium. Pellets reached their maximum size at or shortly before the growth maximum. The relatively small pellet size with ammonium sulfate and isoleucine may reflect .the early arrest of growth, which would be expected to prevent further enlargement of mycelial aggregates.
Influence of the carbon source Although the use of potassium nitrate as a nitrogen source did not minimize aggregation, it supported production of an abundant well-dispersed mycelial suspension. Because it also provided a carbon-free nitrogen source, it was included at a 30 mM concentration with the basal solution to compare the influence of various carbon sources supplied at 3070 (w/v). During early growth the pH values in these cultures decreased by 0.1-0.3 units from their initial value of 5.5; however, they remained well above the growth-arresting
0
0 1
3
5
7
DAYS
FIG. 2. Growth (0)and HON production ( 0 ) in cultures of S. akiyoshiensis. The medium contained basal solution, pH 5.5, supplemented with 3% (w/v) starch, 30 mM aspartic acid, and 15 mM potassium phosphate. A 1.5% (v/v) spore inoculum was used.
threshold and later showed the increases expected in cultures containing potassium nitrate (Table 4). None of the alternative carbon sources tested gave biomass yields as high as those obtained with starch. Cultures with glucose grew rapidly at the outset, but the rate of biomass accumulation declined after 2 days. With galactose, lactose, and sucrose, growth was very slow. Glucose and starch gave the smallest mycelial aggregates and lactose the largest; there was no consistent relationship between size of mycelial aggregates and the ability of a carbon source to support growth or to maintain a pH value close to 5.5 in the culture.
Influence of phosphate concentration The basal solution was modified to contain potassium phosphate concentrations of 0.5, 2, 5, and 15 mM; each basal solution was supplemented with 3070 (w/v) starch and 30 mM potassium nitrate. In all cultures the pH values increased during late growth, as anticipated; this increase was 0.5-0.6 units greater in cultures with the three lowest phosphate concentrations, presumably reflecting the reduced buffering capacity of these media. Biomass production was similar (7.2-8.0 g .L - I ) in all cultures and was not progressively reduced as the phosphate concentration was lowered;
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however, there was a gradual increase in mycelial aggregation, .the maximum mean pellet sizes being 0.17,O. 19,0.24, and 0.28 mm with 15, 5,2, and 0.5 mM phosphate, respectively. Thus although its effect was not large, the availability of phosphate at the concentrations used in these cultures did influence the growth morphology.
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Production of 5-hydroxy-4-oxonorvaline Cultures of S. akiyoshiensis grown in a starch-aspartate medium initially buffered at pH 5.5 produced HON in amounts exceeding the yield (2.7 mM) obtained by Tatsuoka et al. (1961) from a complex medium (Fig. 2).
Discussion In submerged cultures used for the production of secondary metabolites, adequate dispersal of the mycelium is an important factor in maintaining uniform environmental conditions. Large, dense aggregates create regions of nutrient deprivation within their interior. Since depletion of an essential nutrient can lead to the onset of secondary metabolism (Demain et al. 1983; Doull and Vining 199l), aggregation of the mycelium jeopardizes efforts to establish the reproducible, uniform conditions needed to determine the role of environmental factors on secondary metabolic processes. Evidence of heterogeneous nutritional conditions during clumped growth of S. akiyoshiensis was provided by the formation of a dark-red secondary metabolite (the structure of which has not been determined) at the centre of large pellets, and the absence of this pigment from smaller pellets and dispersed hyphae. A further indication of nutritional heterogeneity was the early initiation of HON production and the extent of its overlap with the growth phase in cultures showing heavy pellet formation (data not shown); in cultures with well-dispersed mycelium, growth and production were distinctly biphasic (see Fig. 2). Extensive aggregation of fungal mycelium can be prevented by adding a water-soluble anionic polymer to the culture (Elmayergi et al. 1973; Trinci 1983), and similar results have been obtained wi.th Streptomyces coelicolor A3(2) (Hobbs et al. 1989). Treatment of fungal spores with the polymer altered their electrokinetic behavior and it was suggested that association of the polymer with spore or cell surfaces imparts similar ionic charges, which cause the surfaces to avoid contact with one another (Jones et al. 1988). The presence of strong surface interactions was proposed by Oh and Nash (1981) to account for clumping of Streptosporangium brasiliense spores at acidic pH and dispersal of the aggregates under a1kaline conditions. Evidence that S. akiyoshiensis spores show a similar pH-dependent aggregation indicates that this property is not limited to Streptosporangium species and thus may not, as tentatively suggested (Oh and Nash 1981), be associated with the maintenance of spore aggregates in sporangial sacs. The results are consistent with a study of the electrokinetic properties of actinomycetes by Douglas et al. (1970), which demonstrated that the spores of most species examined carry a net negative charge above a pH of about 2.5, and that this charge increases up to pH values of 6-7. Where spores form aggregates, their subsequent germination may well lead to initial mycelial entanglement. However, it is apparent that this is not the primary factor in the subsequent formation of pellets in S. akiyoshiensis cultures
where an initial pH of 5.5 in the culture medium promotes well-dispersed mycelial growth. In Penicillium chrysogenurn, Pirt and Callow (1959) observed that raising the pH of the culture medium from pH 6.0 to 7.4 progressively altered the growth morphology of the fungus from sparsely branched, dispersed mycelium to thickened and much-branched filaments that aggregated into dense pellets. In strains of S. coelicolor and Streptomyces alboniger, morphological differentiation to form spores is inhibited below pH 7 (Liu et al. 1985), and thus it is likely that actinomycete as well as fungal growth morphology is influenced by pH. As in P. chr-ysogenum, the extent of this influence may be species or strain dependent and may be modified by other factors such as nutrition (Pirt and Callow 1959). In cultures of S. coelicolor A3(2), which form pellets under most growth conditions, faster and more dispersed growth was obtained as the size of the spore inoculum was increased (Doull and Vining 1990). The survey of actinomycete growth morphology by Lawton et al. (1989) also revealed numerous species that formed dense pellets with a small spore inoculum, but more open aggregates or dispersed mycelium when large inocula were used. In cultures of Streptomyces vinaceus and Streptomyces viridochromogenes, for example, the morphology changed from compact pellets to spiky oblong pellets and eventually to dispersed mycelium as the size of the spore inoculum was increased. However, the influence of inoculum size varied between species and even between strains of the same species. While the relative insensitivity of S. akiyoshiensis morphology to the size of the spore inoculum used may mean that all concentrations saturated the response, it is also possible that this species is not strongly affected by inoculum size. Nevertheless, as in S. coelicolor A3(2) (Doull and Vining 1990), aggregation of S. akiyoshiensis mycelium was more prevalent in cultures given a vegetative inoculum and was particularly severe if this inoculum contained only a small number of foci for growth. The failure of vegetative or small spore inocula to give dispersed mycelial growth has also been observed with several neutrophilic and acidophilic streptomycetes (Flowers and Williams 1977), and with Streptomyces aureofaciens (Tresner et al. 1967). Although medium composition is generally considered to be an important factor in determining the growth morphology of actinomycetes, few detailed studies have been described. Tresner et al. (1967) reported that the fermentation medium used for S. aureofaciens strongly influenced the formation of characteristic arthrosporic complexes. Increasing the nitrogen source concentration favoured mycelial fragmentation. However, here as in most other studies of growth morphology in submerged cultures, complex media were used. In providing the conditions for dispersed growth of S. akiyoshiensis, the composition of the nutrient medium was less critical than pH but did influence the extent of mycelial aggregation. As with S. coelicolor A3(2) (Doull and Vining 1989), starch proved to be beneficial as a carbon source in promoting dispersed growth. Some carbon and nitrogen sources were associated with increased pellet formation, but this characteristic was not well correlated with the ability to support fast or slow growth, nor was production of abundant biomass invariably associated with dispersed growth. In media that favoured mycelial dispersal, decreasing the phosphate concentration caused a
GLAZEBROOK ET AL.
distinct increase in aggregation. A similar effect has been observed in cultures of S. coelicolor A3(2) (Doull and Vining 1989).
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Acknowledgement This research was supported by a Strategic Research Grant from the Natural Sciences and Engineering Research Council of Canada. Demain, A.L., Aharonowitz, Y., and Martin, J.-F. 1983. Metabolic control of secondary biosynthetic pathways. In Biochemistry and genetic regulation of commercially important antibiotics. Edited by L.C. Vining. Addison Wesley, Reading, MA. pp. 49-72. Douglas, H.W., Ruddick, S.M., and Williams, S.T. 1970. A study of-the electrokinetic properties of some actinomycete spores. J. Gen. Microbiol. 63: 289-295. Doull, J., and Vining, L.C. 1989. Culture conditions promoting dispersed growth and biphasic production of actinorhodin in shaken cultures of Streptomyces coelicolor A3(2). FEMS Microbiol. Lett. 65: 265-268. Doull, J., and Vining, L.C. 1990. Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate. Appl. Microbiol. Biotechnol. 32: 449-454. Doull, J., and Vining, L.C. 1991. Regulation of secondary metabolism. Proceedings of the International Symposium on Industrial Biotechnology, Hyderabad, Nov. 18-20, 1990. Edited by P . Sridhar and V.P. Malik. Elmayergi, H., Sharer, J.M., and Moo-Young, M. 1973. Effect of polymer additions on fermentation parameters in a culture of Aspergillus niger. Biotechnol. Bioeng. 15: 845-859. Flowers, T.H., and Williams, S.T. 1977. Measurement of growth rates of streptomycetes: comparison of turbidimetric and gravimetric techniques. J. Gen. Microbiol. 98: 285-289. Hobbs, G., Frazer , C.M., Gardner, D .C. J., et al. 1989. Dispersed growth of Streptomyces in liquid culture. Appl. Microbiol. Biotechnol. 31: 272-277. Jones, P., Moore, D., and Trinci, A.P.J. 1988. Effects of Junlon and Hostacerin on the electrokinetic properties of spores of Aspergillus niger, Phanerochaete chrysosporium and Geotrichum candidum. J . Gen. Microbiol. 134: 235-240. Kanazawa, K., Tscuhiya, K., and Araki, T. 1960. A new antituberculous amino acid (6-hydroxy-y-0x0-L-norvaline). Am. Rev. Respir. Dis. 81: 924. Lawton, P., Whitaker, A., Odell, D., and Stowell, J.D. 1989. Actinomycete morphology in shaken culture. Can. J. Microbiol. 35: 881-889. Liu, C.-J., Pogell, B.M., and Redshaw, P.A. 1985. Regulation of "conditional" aerial mycelium mutants of streptomycetes. J . Gen. Microbiol. 131: 1015-1021.
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Miyake, A. 1960. 6-Hydroxy-y-0x0-L-norvaline, a new antitubercular antibiotic. 1. Structural studies. Chem. Pharm. Bull. (Tokyo), 8: 1071-1073. Nikitina, E.T., and Kalakoutskii, L.V. 1971. Induction of nocardioform growth in actinomycetes on media with D-fructose. Z. Allg. Mikrobiol. 11: 601-606. Oh, Y.K., and Nash, C.H. 1981. Influence of pH on spore aggregation of Streptosporangium brasiliense. Folia Microbiol. (Prague), 26: 164-166. Pirt, S.J., and Callow, D.S. 1959. Continuous-flow culture of the filamentous mould PeniciNium chrysogenum and the control of its morphology. Nature (London), 184: 307-3 10. Stastna, J., Caslavska, J., Wolf, A., Vinter, V., and Mikulik, K. 1977. Origin and morphology of atypical forms of Streptomyces granaticolor. Folia Microbiol. (Prague), 22: 339-345. Stuttard, C. 1982. Temperate phages of Streptomyces venezuelae: lysogeny and host specificity shown by phages SVl and SV2. J . Gen. Microbiol. 128: 115-121. Tatsuoka, S., Miyake, A., Hitomi, H., et al. 1961. HON, a new antibiotic produced by Streptomyces akiyoshiensis nov.sp. J . Antibiot. 14: 39-43. Tresner, H.D., Hayes, J.A., and Backus, E.J. 1967. Morphology of submerged growth of streptomycetes as a taxonomic aid. Appl. Microbiol. 15: 1185-1191. Trinci, A.P.J. 1983. Effect of Junlon on morphology of Aspergillus niger and its use in making turbidity measurements of fungal growth. Trans. Br. Mycol. Soc. 81: 408-412. Watanabe, S., Numata, K., Omura, S., and Yamaguchi, H. 1987. Fungal formulations. Jpn. Kokai Tokkyo Koho J P . 61,243,018 (86,243,018). Chem. Abstr. 106: 72944r. White R.L., Demarco, A.C., and Smith, K.C. 1988. Biosynthesis of the unusual amino acid 5-hydroxy-4-oxonorvaline. J. Am. Chem. Soc. 110: 8228-8229. White, R.L., Demarco, A.C., and Smith, K.C. 1989. Analysis of o-phthalaldehyde derivatives of acidic and polar amino acids in fermentation broths by high-performance liquid chromatography. J . Chromatogr. 483: 437-442. Williams, S.T., Entwhistle, S., and Kurylowicz, W. 1974. The morphology of streptomycetes growing in media used for commercial production of antibiotics. Microbios, 11A: 47-60. Yamaguchi, H., Uchida, K., Hiratani, T., et al. 1988. RI-331, a new antifungal antibiotic. Ann. N.Y. Acad. Sci. 544: 188-190. Yamaguchi, M., Yamaki, H., Schinoda, T., et al. 1990. The mode of antifungal action of (S)2-amino-4-oxo-5-hydroxypentanoic acid, RI-331. J. Antibiot. 43: 411-416. Yamaki, H., Yamaguchi, M., Nishimura, T., et al. 1988. Unique mechanism of action of an antifungal antibiotic. Drugs Exp. Clin. Res. 14: 467-472.
VINING'
Biology Department, Dalhousie University, Halifax, N.S., Canada B3H 4 J l AND
R. L.
WHITE^
Chemistry Department, Acadia University, Wolfville, N.S., Canada BOP 1 x 0 Received April 29, 1991
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Revision received July 26, 1991 Accepted July 26, 1991 GLAZEBROOK, M. A., VINING,L. C., and WHITE,R. L. 1992. Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients. Can. J. Microbiol. 38: 98-103. Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis. Key words: Streptomyces akiyoshiensis, mycelial aggregation, growth conditions. GLAZEBROOK, M. A., VINING,L. C., et WHITE,R. L. 1992. Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients. Can. J. Microbiol. 38 : 98-103. La plupart des milieux dans lesquels la croissance de cultures submergees et agitees de Streptomyces akiyoshiensis fut examinee, n'ont pas permis la formation de suspensions de myceliums bien disperses. L'investigation des conditions de culture encourageant la croissance dispersee ont montre que le pH du milieu de culture est d'une importance critique; une valeur initiale de 5,5 a minimise l'aggregation du mycelium tout en supportant une production adequate de biomasse. Dans les cultures amorcees a ce pH, les inoculums de spores ont donne une meilleure dispersion du mycelium que les inoculums vegetatifs; avec les inoculums de spores, la morphologie de la croissance a ete aussi moins affectee par le volume de l'inoculum. La composition de la solution nutritive a influence l'etendue de la dispersion du mycelium; une croissance lente a ete associee souvent avec l'agglutination, mais aucune correlation precise n'a pu Etre observee entre la formation de boulettes et la capacite des sources de carbone ou d'azote de supporter une croissance rapide. L'augmentation de la concentration de phosphate de 0,5 a 15 mM a cause une diminution modeste de l'aggregation du mycelium. Les conditions qui encourageaient un mycelium bien disperse, adequat pour l'etude du contr8le physiologique du metabolisme secondaire, ont aussi permis la formation de 5-hydroxy-4-oxonorvaline par S. akiyoshiensis. Mots clks : Streptomyces akiyoshiensis, aggregation du mycelium, conditions de croissance. [Traduit par la redaction]
Introduction Cultures of Streptomyces akiyoshiensis produce an antibiotic with activity against human-type Mycobacterium tuberculosis (Kanazawa et al. 1960). The product was first identified by Miyake (1960) as 5-hydroxy-4-oxonorvaline (HON) and has recently been rediscovered as RI-33 1 in cultures of an unidentified streptomycete during screening for agents active against yeasts and fungi (Yamaguchi et al. 1988). It functions as a metabolic analogue of homoserine and interferes with formation of the protein amino acids threonine, methionine, and isoleucine biosynthesized via this intermediate (Yamaki et al. 1988; Yamaguchi et al. 1990). In animals HON exhibits useful systemic antifungal activity, and because the affected protein amino acids are all essential dietary components, it is nontoxic (Watanabe et al. ' ~ u t h o rto whom all correspondence should be addressed. 2~resentaddress: Chemistry Department, Dalhousie University, Halifax, N.S., Canada B3H 453. Printed in Canada / lmprime a u Canada
1987). In studies directed towards the enzymic synthesis of HON, we have investigated the biosynthesis of the antibiotic in S. akiyoshiensis (White et al. 1988). Here, as in earlier work (Tatsuoka et al. 1961), HON was produced in complex media, which provided only limited information about .the fermentation characteristics of the organism. One of our early goals has been the development of reproducible growth conditions using a defined medium where the onset of antibiotic production could be predicted with confidence and high levels of biosynthetic enzyme activity could be achieved. When S. akiyoshiensis was grown in shaken flask cultures in defined media using the growth conditions described in the literature, the mycelium formed clumps composed of entangled hyphae of different ages, states of nutrition, and stages of development. Similar difficulties have been encountered with other actinomycete species (Hobbs et al. 1989; Doull and Vining 1989). Streptomycetes exhibit considerable variation in their growth morphology in submerged liquid cultures as well as on agar media (Tresner et al. 1967;
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Williams et al. 1974). Mycelial development in submerged cultures is markedly influenced by the growth conditions used (Nikitina and Kalakoutskii 1971; Stastna et al. 1977), and despite the important consequences for culture rheology and oxygen transfer in industrial actinomycete fermentations, the factors responsible have not been clearly defined. We report here the influence of various fermentation parameters on the growth morphology of S. akiyoshiensis in shaken cultures and define the conditions that minimize mycelial aggregation, making possible meaningful studies on the physiology of the organism. Materials and methods Cultures Streptomyces akiyoshiensis was obtained from the American Type Culture Collection as ATCC 13480 and maintained as a stock spore suspension. This was prepared by dislodging spores into water from the surface of a culture grown on MYM agar (Stuttard 1982). The spores were filtered through nonabsorbent cotton, collected by centrifugation, and resuspended in 20% (v/v) glycerol to give a calculated optical density at 640 nm (OD,,) of 10 from measurement of a 1:50 dilution in a round cuvette (1.2-cm internal diameter) with a Bausch and Lomb Spectronic 21 spectrophotometer. Spore suspensions were aliquoted and stored at - 20°C. Cultures in which morphology was examined were grown in 25 mL of liquid media contained in 250-mL Erlenmeyer flasks and incubated on a rotary shaker (3.8-cm eccentricity; 220 rpm) at 27°C. Except in experiments testing inoculum effects, each culture received 1.5% (v/v) of the stock spore suspension as an inoculum. A vegetative inoculum was prepared by incubating 20 mL of MYM medium with 20 pL of stock spore suspension for 24 h under the conditions described above for HON production. Samples of the culture were washed once with water by centrifugation and reconstituted as mycelial suspensions in water at the original volume. Media The complex media tested (composition in %w/v) were MYM (maltose, 0.4; yeast extract, 0.4; malt extract, 1.0), SYM (as for MYM but with starch in place of maltose), and mixtures of starch (3.0) with Bacto-Peptone, Bacto-Tryptone, Bacto-Soytone, BactoCasitone, or casein (0.4) in basal solution at pH 6.5. The basal solution consisted of (L - ') MgSO, .7H20, 0.20 g; 3-(morpho1ino)propane sulfonic acid (MOPS), 21 g; NaCl, 10 mg; CaCl,, 10 mg; FeSO, .7H20, 5 mg; ZnCl,, 180 pg; MnCl, .4H20, 45 pg; CuSO, 5H20, 45 pg; (NH4),Mo,02,~4H20,45 pg; and H3B03, 7.2 pg. Most defined media contained the same basal solution supplemented with 15 mM phosphate (a 5:3 w/w mixture of K,HPO, and KH2P04)and with the carbon and nitrogen sources specified in each experiment. Exceptions occurred when the effects of phosphate concentration and of basal medium components were being tested. Media were adjusted with 1 M HCl or 1 M NaOH to the required initial pH before sterilization. A nalyses Growth was normally measured as increase in cell dry weight. At each sampling, cultures were filtered under vacuum through weighed Whatman No. 5 filter-paper disks; the mycelium collected was washed with water and dried to constant weight at 75°C. Values reported are the averages for cultures grown in duplicate. Where the mycelium was well dispersed, differences were within f 7%. Cultures where severe clumping occurred showed variation as high as _+ 50%. The size of mycelial aggregates in culture broth was measured with a microscope, using a calibrated eyepiece and stage micrometer. Viewing fields were varied in diameter according to the size of the aggregates and were chosen at random to include 10-15 aggregates; for each aggregate the longest and shortest widths were
FIG. 1. Growth morphology of S. akiyoshiensis. (A) After 1 day in MYM medium; (B) after 5 days in a medium containing 3% (w/v) starch, 30 mM proline, 15 mM phosphate, and basal salts at pH 6.5. Both cultures received a 1.5% (v/v) spore inoculum and were photographed at 100 x . measured and averaged to give the aggregate size. The values for all aggregates within a field were averaged to obtain the mean size for the sample. Photographs were taken with a Zeiss Photomicroscope 11. The amount of HON present in clarified culture broths was determined by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde (White et al. 1989).
Results In preliminary experiments where shaken submerged cultures of S. akiyoshiensis were grown in media containing maltose or starch with a variety of complex nitrogen sources, a well-dispersed mycelial suspension (Fig. 1A) was obtained only during the rapid growth phase in a maltose - yeast extract - malt extract (MYM) mixture. In the late growth phase of cultures in MYM medium and throughout growth in the other media, the mycelium aggregated to form discrete pellets (Fig. 1B). Similar aggregation was encountered when cultures were grown in defined media containing a basal solution of inorganic salts at pH 6.5 and various single sources of carbon and nitrogen. To determine whether aggregation of the mycelium into pellets was due to inorganic constituents in the medium, components of the basal solution were omitted both individually and in groups; the magnesium sulfate concentration was also varied stepwise from 0.01 to 0.5 g .L - Each of the modified basal solutions was supplemented with 3% (w/v) starch, 15 mM proline, and 15 mM phosphate, since these nutrients had supported a high biomass yield in the preliminary experiments. The media were adjusted to pH 6.5 and inoculated (1.5% v/v) with the stock spore suspension. Although growth rates and biomass yield were affected by the differences in inorganic salt composition, average mycelial pellet sizes were consistently large (10.5 mm diameter). The results suggested that mineral nutrition was not the critical factor in mycelial aggregation. Because the growth morphology in MYM medium demonstrated that S. akiyoshiensiscould, under suitable condi-
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TABLE1. Effect of initial pH on broth pH, biomass yield, and mycelial pellet size Culture pH
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Initial
Minimum
Maximum
Maximum dry weifht (g.L -
Maximum pellet size mma
Day
NOTE: The basal solution was supplemented with 3% (w/v) starch, 30 m M proline, and 15 m M phosphate. Cultures were inoculated with 1.5% (v/v) stock spore suspension and analysed daily. T h e p H values reported are the lowest o r highest recorded at any sampling during a 7-day incubation. In all cultures the dry weight increased throughout the sampling period; thus all maxima were recorded o n day 7. 'Average size (0.5(longest + shortest dimension)) and standard deviation of all pellets from that sample observed within the microscope viewing field.
TABLE2. Effect of inoculum on biomass yield and mycelial pellet size Maximum dry weight Inoculum
g - ~ - ' Day
Maximum pellet size mm
Day
Spores (% v/v)
0.1 1.5 4.0
4.9 7.0 6.9
5 5 3
0.17k0.04 0.12k0.03 0.14k0.04
3 3 4
6.0 8.1 7.9
5 3 3
1.61 k0.23 0.87k0.12 0.61k0.09
4 4 4
Vegetative (% v/v)
0.1 2.5 5.O
NOTE:Media were adjusted t o a n initial p H of 5.5 and various inocula were used. Other conditions were as in Note t o Table 1.
tions, proliferate as a dispersed mycelial suspension, further experiments were undertaken to define the conditions supporting dispersed growth and to examine the extent to which they could be varied during testing of the physiological parameters governing HON production.
Influence of pH The basal solution supplemented with 3Vo (w/v) starch, 15 mM proline, and 15 mM phosphate was adjusted to pH values from 5.5 to 7.5 and inoculated with 1.5% (v/v) of stock spore suspension. Throughout the 7-day period in which culture parameters were measured, the pH of each broth remained well buffered at close to its initial value (Table 1). The early growth rates in all cultures were similar and declined after day 2, but biomass continued to accumulate during the observation period. Cultures at pH 5.5 grew more slowly after day 3 than those at pH 6.5 and above; as a result, their maximum biomass yields were appreciably lower. In a separate experiment where the effect of further reductions in initial pH were examined, values at or below pH 5.0 progressively inhibited growth (data not shown). In all cultures the average size of mycelial aggregates increased during rapid growth; this increase in size was faster and more prolonged at pH 6.0 and above. Consequently, the maximum size of the aggregates was 4- to 5-fold smaller in cultures at pH 5.5 than at higher pH values. Visual inspection suggested that the suspension of mycelium was dispersed to the same degree as in MYM medium; this was confirmed
by examining the broth with a microscope, which showed a dispersal similar to that in Fig. 1A. In all subsequent experiments, where the influence of other culture conditions was investigated, the media were buffered at an initial pH of 5.5 To determine whether aggregation of spores used as the culture inoculum might be related to the size of mycelial aggregates that developed during subsequent growth of cultures, spores suspended in samples of the media used in the preceding experiment were examined by light microscopy at 1000-fold magnification. At pH 6.5 and above, the suspensions consisted of well-dispersed single spores. Increasing aggregation occurred as the pH was lowered; this was reflected in both the number of spores in a clump and the stability of the association. At pH 5.5, the aggregates contained an average of five firmly attached spores. Since an initial culture pH of 5.5 improved mycelial dispersal during growth, other factors must be mainly responsible for growth morphology. Their enhancement at pH 5.5 evidently offsets the disadvantage of clumping in the spore inoculum.
Effect of the inoculum Cultures were grown in basal solution supplemented with 3% (w/v) starch, 30 mM proline, and 15 mM phosphate; they were inoculated with various amounts of spore or vegetative mycelial suspension. In all cultures the pH values remained within the 5.4-5.6 range. In those with a spore inoculum, the biomass yield increased when the inoculum size was raised from 0.1 to 1.5% (v/v); the yield did not increase further when the inoculum size was raised to 4.0% (v/v), but the biomass maximum was attained earlier (Table 2). The maximum size of the mycelial aggregates varied only marginally but occurred later with the 4.0% (v/v) inoculum. Use of a 0.1 % (v/v) vegetative inoculum gave cultures with relatively large mycelial pellets. Although the average pellet size decreased as the vegetative inoculum was increased to 2.5 and 5.0% (v/v), it remained much larger than observed with spore inocula. In cultures given a vegetative inoculum the biomass yield increased and was attained earlier when the inoculum was increased from 0.1 to 2.5070, but it showed no further change at 5070 (w/v). The maximum yields with the larger vegetative inocula were somewhat higher than obtained with spore inocula. In all subsequent experiments a 1.5% (v/v) spore inoculum was used. Influence of the nitrogen source The basal solution was supplemented with 3% (w/v)
GLAZEBROOK ET AL.
TABLE 3. Effect of the nitrogen source on broth pH, biomass yield, and mycelial pellet size
Maximum dry weight
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Broth pH Nitrogen source
Minimum
Maximum
(NH4)2S04 Isoleucine Proline Aspartate KNO, Glutamate
3.9 4.9 5.3 5.5 5.4 5.5
5.5 5.5 5.5 6.6 6.5 6.5
Maximum pellet size
g . ~ - ' Day
1.7 3.0 7.5 8.3 7.8 8.3
2 7 4 3 4 4
mm
Day
0.07 k0.02 0.10k0.07 0.10k0.03 0.22k0.07 0.22k0.05 0.27 k0.07
2 7 4 2 4 3
NOTE:The basal solution was supplemented with 3% (w/v) starch, 15 mM phosphate, and a nitrogen source supplying 30 mM N . Media were adjusted to pH 5.5 and inoculated with 1.5% (v/v) of stock spore suspension. Values are reported as in Table 1 .
TABLE 4. Effect of the carbon source on broth pH, biomass yield, and mycelial pellet size
Carbon source
Maximum broth pH
Glucose Starch Maltose Galactose Sucrose Fructose Lactose
6.0 6.6 6.0 6.0 5.7 5.8 5.9
Maximum dry weight (g-L - I ) " 1.4 8.3 5.5 1.4 1.1 4.5 1.2
Maximum pellet size (mm) 0.18 +_ 0.06 0.20 k 0.05 0.22 k 0.09 0.24 +_ 0.05 0.28 k 0.14 0.33 k 0.31 0.63 k 0.33
NOTE: The basal solution was supplemented with 30 mM K N 0 3 , 15 mM phosphate, and a carbon source at 3% (w/v). Media were adjusted to pH 5.5 and inoculated with 1.5% (v/v) of stock spore suspension. Values are reported as in Table 1 . 'In cultures with glucose, starch, and fructose, dry weight reached a maximum at 4 days; in all other cultures the maximum was at 7 days. Mycelial pellets increased in size until the growth maxima.
starch and various nitrogenous substances supplying 30 mM nitrogen to the medium. In some media, the buffering capacity of the 0.1 M MOPS present was insufficient to prevent substantial changes in pH (Table 3). That cultures with ammonium sulfate acidified rapidly to growth-inhibitory conditions is also indicated from the low biomass yield and early maximum. Cultures with isoleucine followed a similar pattern, although at a slower rate. Cultures containing aspartate, glutamate, or potassium nitrate all increased in pH and formed mycelial pellets larger than those present in proline-containing medium. Pellets reached their maximum size at or shortly before the growth maximum. The relatively small pellet size with ammonium sulfate and isoleucine may reflect .the early arrest of growth, which would be expected to prevent further enlargement of mycelial aggregates.
Influence of the carbon source Although the use of potassium nitrate as a nitrogen source did not minimize aggregation, it supported production of an abundant well-dispersed mycelial suspension. Because it also provided a carbon-free nitrogen source, it was included at a 30 mM concentration with the basal solution to compare the influence of various carbon sources supplied at 3070 (w/v). During early growth the pH values in these cultures decreased by 0.1-0.3 units from their initial value of 5.5; however, they remained well above the growth-arresting
0
0 1
3
5
7
DAYS
FIG. 2. Growth (0)and HON production ( 0 ) in cultures of S. akiyoshiensis. The medium contained basal solution, pH 5.5, supplemented with 3% (w/v) starch, 30 mM aspartic acid, and 15 mM potassium phosphate. A 1.5% (v/v) spore inoculum was used.
threshold and later showed the increases expected in cultures containing potassium nitrate (Table 4). None of the alternative carbon sources tested gave biomass yields as high as those obtained with starch. Cultures with glucose grew rapidly at the outset, but the rate of biomass accumulation declined after 2 days. With galactose, lactose, and sucrose, growth was very slow. Glucose and starch gave the smallest mycelial aggregates and lactose the largest; there was no consistent relationship between size of mycelial aggregates and the ability of a carbon source to support growth or to maintain a pH value close to 5.5 in the culture.
Influence of phosphate concentration The basal solution was modified to contain potassium phosphate concentrations of 0.5, 2, 5, and 15 mM; each basal solution was supplemented with 3070 (w/v) starch and 30 mM potassium nitrate. In all cultures the pH values increased during late growth, as anticipated; this increase was 0.5-0.6 units greater in cultures with the three lowest phosphate concentrations, presumably reflecting the reduced buffering capacity of these media. Biomass production was similar (7.2-8.0 g .L - I ) in all cultures and was not progressively reduced as the phosphate concentration was lowered;
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however, there was a gradual increase in mycelial aggregation, .the maximum mean pellet sizes being 0.17,O. 19,0.24, and 0.28 mm with 15, 5,2, and 0.5 mM phosphate, respectively. Thus although its effect was not large, the availability of phosphate at the concentrations used in these cultures did influence the growth morphology.
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Production of 5-hydroxy-4-oxonorvaline Cultures of S. akiyoshiensis grown in a starch-aspartate medium initially buffered at pH 5.5 produced HON in amounts exceeding the yield (2.7 mM) obtained by Tatsuoka et al. (1961) from a complex medium (Fig. 2).
Discussion In submerged cultures used for the production of secondary metabolites, adequate dispersal of the mycelium is an important factor in maintaining uniform environmental conditions. Large, dense aggregates create regions of nutrient deprivation within their interior. Since depletion of an essential nutrient can lead to the onset of secondary metabolism (Demain et al. 1983; Doull and Vining 199l), aggregation of the mycelium jeopardizes efforts to establish the reproducible, uniform conditions needed to determine the role of environmental factors on secondary metabolic processes. Evidence of heterogeneous nutritional conditions during clumped growth of S. akiyoshiensis was provided by the formation of a dark-red secondary metabolite (the structure of which has not been determined) at the centre of large pellets, and the absence of this pigment from smaller pellets and dispersed hyphae. A further indication of nutritional heterogeneity was the early initiation of HON production and the extent of its overlap with the growth phase in cultures showing heavy pellet formation (data not shown); in cultures with well-dispersed mycelium, growth and production were distinctly biphasic (see Fig. 2). Extensive aggregation of fungal mycelium can be prevented by adding a water-soluble anionic polymer to the culture (Elmayergi et al. 1973; Trinci 1983), and similar results have been obtained wi.th Streptomyces coelicolor A3(2) (Hobbs et al. 1989). Treatment of fungal spores with the polymer altered their electrokinetic behavior and it was suggested that association of the polymer with spore or cell surfaces imparts similar ionic charges, which cause the surfaces to avoid contact with one another (Jones et al. 1988). The presence of strong surface interactions was proposed by Oh and Nash (1981) to account for clumping of Streptosporangium brasiliense spores at acidic pH and dispersal of the aggregates under a1kaline conditions. Evidence that S. akiyoshiensis spores show a similar pH-dependent aggregation indicates that this property is not limited to Streptosporangium species and thus may not, as tentatively suggested (Oh and Nash 1981), be associated with the maintenance of spore aggregates in sporangial sacs. The results are consistent with a study of the electrokinetic properties of actinomycetes by Douglas et al. (1970), which demonstrated that the spores of most species examined carry a net negative charge above a pH of about 2.5, and that this charge increases up to pH values of 6-7. Where spores form aggregates, their subsequent germination may well lead to initial mycelial entanglement. However, it is apparent that this is not the primary factor in the subsequent formation of pellets in S. akiyoshiensis cultures
where an initial pH of 5.5 in the culture medium promotes well-dispersed mycelial growth. In Penicillium chrysogenurn, Pirt and Callow (1959) observed that raising the pH of the culture medium from pH 6.0 to 7.4 progressively altered the growth morphology of the fungus from sparsely branched, dispersed mycelium to thickened and much-branched filaments that aggregated into dense pellets. In strains of S. coelicolor and Streptomyces alboniger, morphological differentiation to form spores is inhibited below pH 7 (Liu et al. 1985), and thus it is likely that actinomycete as well as fungal growth morphology is influenced by pH. As in P. chr-ysogenum, the extent of this influence may be species or strain dependent and may be modified by other factors such as nutrition (Pirt and Callow 1959). In cultures of S. coelicolor A3(2), which form pellets under most growth conditions, faster and more dispersed growth was obtained as the size of the spore inoculum was increased (Doull and Vining 1990). The survey of actinomycete growth morphology by Lawton et al. (1989) also revealed numerous species that formed dense pellets with a small spore inoculum, but more open aggregates or dispersed mycelium when large inocula were used. In cultures of Streptomyces vinaceus and Streptomyces viridochromogenes, for example, the morphology changed from compact pellets to spiky oblong pellets and eventually to dispersed mycelium as the size of the spore inoculum was increased. However, the influence of inoculum size varied between species and even between strains of the same species. While the relative insensitivity of S. akiyoshiensis morphology to the size of the spore inoculum used may mean that all concentrations saturated the response, it is also possible that this species is not strongly affected by inoculum size. Nevertheless, as in S. coelicolor A3(2) (Doull and Vining 1990), aggregation of S. akiyoshiensis mycelium was more prevalent in cultures given a vegetative inoculum and was particularly severe if this inoculum contained only a small number of foci for growth. The failure of vegetative or small spore inocula to give dispersed mycelial growth has also been observed with several neutrophilic and acidophilic streptomycetes (Flowers and Williams 1977), and with Streptomyces aureofaciens (Tresner et al. 1967). Although medium composition is generally considered to be an important factor in determining the growth morphology of actinomycetes, few detailed studies have been described. Tresner et al. (1967) reported that the fermentation medium used for S. aureofaciens strongly influenced the formation of characteristic arthrosporic complexes. Increasing the nitrogen source concentration favoured mycelial fragmentation. However, here as in most other studies of growth morphology in submerged cultures, complex media were used. In providing the conditions for dispersed growth of S. akiyoshiensis, the composition of the nutrient medium was less critical than pH but did influence the extent of mycelial aggregation. As with S. coelicolor A3(2) (Doull and Vining 1989), starch proved to be beneficial as a carbon source in promoting dispersed growth. Some carbon and nitrogen sources were associated with increased pellet formation, but this characteristic was not well correlated with the ability to support fast or slow growth, nor was production of abundant biomass invariably associated with dispersed growth. In media that favoured mycelial dispersal, decreasing the phosphate concentration caused a
GLAZEBROOK ET AL.
distinct increase in aggregation. A similar effect has been observed in cultures of S. coelicolor A3(2) (Doull and Vining 1989).
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