Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

Production of Calcium Gluconate by Penicillium chrysogenum in Submerged Culture

The waste mycelium of Penicillium chrysogenum HA-10 (obtained at the end of penicillin fermentation), or a 24-hr-old freshly grown vegetative inoculum of this organism, was found to utilize glucose for the production of calcium gluconate by submerged fermentation in shake flasks. After 72 to 96 hr of fermentation at 24 C, 90 to 95% of the reducing sugar from the 15% glucose medium was converted to calcium gluconate. Reuse of the mycelium for successive experiments reduced the fermentation period to 72 hr or less because of an enhancement of glucose utilization. Ten successive batches of 15% glucose medium were fermented by the reuse method. Fermentation media containing up to 30% glucose could be used, provided boric acid was added to prevent the precipitation of calcium gluconate formed. We found that 30% hydrol (a by-product of glucose manufacture containing 50 to 55% reducing sugar), when used in place of glucose in the fermentation medium, inhibited the rate of glucose utilization. However, this effect was partially reversed by pretreatment of hydrol with 2 to 4% activated charcoal before addition to the fermentation medium.     

Respiratory Activity of the Mycelium of Eremothecium ashbyii

The respiratory activity of the mycelium of Eremothecium ashbyii from submerged cultures was manometrically determined at different stages of its development and the results were statistically analyzed. The experiments were performed in a manner designed to diminish the endogenous respiration without affecting the response to the addition of an exogenous substrate. Lactose was the carbohydrate tested that produced the lowest oxygen consumption. The oxidation of maltose, which was high at 24 hr, decreased by more than 50% at 48 and 55 hr. Glucose and sucrose were actively oxidized by mycelium of three ages. From the intermediates of carbohydrate metabolism, 24-hr mycelium did not produce oxygen consumption with malate, lactate, citrate, fumarate, and α-ketoglutarate. At 48 hr, mycelium did not oxidize either lactate or citrate; 55-hr mycelium showed oxygen consumption with all intermediates tested. Acetate and pyruvate always produced high oxygen consumption. Ethyl alcohol produced high oxygen consumption with mycelium of all tested ages.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Production and purification of statins from Aspergillus terreus strains

Lovastatin, mevastatin, pravastatin and monacolin J were produced using Aspergillus terreus strains. Mevastatin (170 mg/l) was obtained at 14 days from the A1 strain, lovastatin (256 mg/l) at 21 days from the A2 strain and pravastatin (270-300 mg/l) at 14 days from both the A1 and A2 strains grown on defatted soybean flour. Similar yields of monacolin J (5-10 mg/l) were detected for both strains. Fermentation carried out by adding glycerol to A1 7-d old cultures gave 244 mg lovastatin/l at 14 days employing whole soybean flour. A new extraction procedure was applied to an A2 19-d old culture on the mycelium and the culture filtrate separately. Recovery yield showed that 83% lovastatin was associated with the mycelium and 17% was free in the culture filtrate. © Rapid Science Ltd. 1998     

Isolation of fatty acids from the mycelium ofPenicillium stipitatum thom and their effect on ehrlich ascites carcinoma cells

A total of 5 fractions were isolated from the mycelium ofPenicillium stipitatum Thom obtained by submerged cultivation. Three of them inhibited the incorporation of¹⁴C-labelled precursors into Ehrlich ascitic cells (EAC) at concentrations lower than 100 μg/ml. The fraction M-72-3, inhibiting mainly adenine incorporation, was further separated into 6 fractions. The highest effect on EAC cells was exerted by subfraction IV which consisted of free fatty acids; its main effective components were oleic linoleic and linolenic acid. Their cyto-inhibitory effect on EAC cells was confirmed by their application in a pure form.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Studies on the Biosynthesis of Streptomycin

Myo-inositol, especially in combination with arginine, enhances streptomycin production. Compounds which show structural relationship with myo-inositol are ineffective.

Myo-inositol decreases the incorporation of C¹⁴-glucose into streptomycin, particularly into streptidine. This effect suggests that myo-inositol is a precursor of the streptidine ring.

Methionine stimulates antibiotic production in a synthetic medium but proves to be unfavorable in a complex medium.

The γ- and δ-isomers of hexachlorocyclohexane inhibit streptomycin formation.

The formation of streptomycin by washed mycelium was studied. Essentially the same results were here obtained as with growing cultures.

     

Production and optimization of microbial lipase

Production of lipase by the newly isolated Pseu-domonas species has been optimised. Various parameters like initial pH, temperature, incubation period, effect of agitation, inoculum age, inoculum concentration were optimised. It was observed that modified GYP media with 72 hrs incubation, pH 5.5, at 37?°C in agitation conditions were optimum for growth and production of lipase. While optimising the effect of some additional carbon and nitrogen sources, 7% (v/v) olive oil concentration, and 1% (w/v) mannose were found to be the best. In between prediction of the activities has been done through computer programming.     

Effect of Sclerotinia sclerotiorum on the disease development,growth, oil yield and biochemical changes in plants of Mentha arvensis

Experiment was carried out to determine the effect of Sclerotinia sclerotiorum on the disease development, growth, oil yield and biochemical changes in the plants of Mentha arvensis. With the increase in initial inoculum levels of S. sclerotiorum a corresponding decrease in plant fresh and dry weights were recorded. The maximum reduction in the shoot-roots/suckers fresh weight and shoot-roots/suckers dry weights (39.8%, 43.6%, 40.3% and 42.9%), respectively, was observed at the highest initial inoculum level of 12 g fungal mycelium/5 kg soil as compared to uninoculated control. The infection of roots and suckers due to S. sclerotiorum increased with increasing initial inoculum levels. At the lowest initial inoculum (1.0 g mycelium/5 kg soil), infection was observed 18.0% and at the highest (12 g mycelium/5 kg soil), it was 80.2%. Significant (P ⩽ 0.01) reduction in oil yield, total chlorophyll, total phenol and total sugar content of M. arvensis plants was observed at the lowest inoculum level as compared to uninoculated control.     

Biosynthesis of Streptomycin

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Biosorption of Baftkar textile effluent

Decolourization of wastewater from a textile plant by a marine Aspergillus niger was studied. The fungus was previously isolated from Gorgan Bay in the Caspian Sea. The kinetics of decolourization was studied by varying energy sources. The best decolourization was achieved when sucrose was used as source of carbon and energy. NH4+ ion was demonstrated to be the best nitrogen source. Color reduction was found to increase from 80-97% as inoculum concentration increased from 0.04-1.0 g/L. A minimum inoculum of 0.2 g/L is necessary to achieve decolourization. The optimal temperature for the growth of A. niger on Baftkar wastewater is found to be 30 degrees C. 90-96% colour reduction is achieved in 19-20 hr of contact of mycelium cell with the wastewater. Colour reduction in a continuous column reactor of 70% was obtained using treated mycelium (NaOH, 90 degrees C) after 1 hr.     

Production and regulation of extracellular chitinase from the entomopathogenic fungus Isaria fumosorosea

Extracellular chitinase production by the entomopathogenic fungus, Isaria fumosorosea IF28.2 was studied by using submerged fermentation. Maximum chitinase production (178.34±3.91 mU/mL) was obtained when fermentation was carried out at 25°C for 120 h using 72-h-old mycelium in a medium. The effect of inoculum size on chitinase activity was also observed and maximum chitinase activity (159.41±2.91 mU/mL) was obtained with an inoculum size of 3 discs while an incubation period of 96 h proved the most active inducer of chitinase production yielding a chitinase activity of 186.14±3.81 mU/mL. Colloidal chitin (1.5%, w/v) proved to be the best concentration. The optimum pH for chitinase production was 5.7 while 25°C proved to be the best temperature for chitinase production. Supplementation of additional carbon source like 1.5% N-acetylglucosamine (GlcNAc) showed further enhancement in chitinase production. The divalent metal salts, CaCl₂, MgCl₂ and ZnSO₄, inhibited chitinase activity at 10 and 100 mM concentration, whereas inhibition of chitinase activity by KCl, FeSO₄ and EDTA was observed only at higher concentrations. The results presented in this study increase the knowledge on chitinase production in I. fumosoroseus opening new avenues for the study of the role of this enzyme in virulence against different insect pests during the infection process.     

Conditions of cultivation, composition, and biological activity of mycelium of Flammulina velutipes (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karat. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5-10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6-78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages--central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Low dosage of 131iodine effects on chromosomes

The aim of this study was to evaluate the effect on human lymphocyte chromosomes of the (131)I dosage used in scintigraphy on thyroid patients. Until now, there has been as absence of conclusive reports on the effects produced by such dosage. Samples were obtained from 21 patients, and the blood was collected in two occasions: Twenty-four hours prior (control) and after administration of the radionuclide (test). Cells were placed in 1640 RPMI medium with bovine calf serum and incubated with phytohaemagglutinin for 48 and 72 hr at 37 degrees C. Chromosomes were stained with Giemsa Gurr (2 percent, pH = 6.8), and analyzed by two independent investigators by optical microscopy. Of the 6,300 metaphases analyzed from the 48- and 72-hr cultures, 1,146 and 216 gaps and 682 and 52 breaks were found in the test group, respectively. Of the 6,300 metaphases analyzed from the control group, 291 gaps and 119 breaks were observed in the 48-hr cultures whereas in the 72-hr cultures, 10 gaps, and no breaks were found. Our results show that (131)I is responsible for the observed chromosome alterations (paired t-test, p <.05). We suggest re-evaluating the use of (131)I and replacing it with the (123)I, mainly on those patients at fertile age.     

Analysis of human T cell responses to group A streptococci using fractionated Streptococcus pyogenes proteins

Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 narrow range molecular weight fractions by blot-elution from SDS-polyacrylamide gels. Eluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononuclear cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cell extract proteins were revealed by fractionation, the degree of response to each fraction varying between donors. Unfractionated culture supernatant proteins elicited proliferative responses by PBMC from all individuals examined. Responses to culture supernatant fractions containing 25–33 kDa proteins could be attributed to known superantigens. Furthermore, samples from culture supernatants containing higher molecular weight fractions (>45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall antigens. The efficacy of using electroeluted samples to identify T lymphocyte stimulatory proteins was confirmed by demonstrating that a known superantigen of S. pyogenes Manfredo strain, streptococcal pyrogenic exotoxin C (SPEC), could be fractionated successfully using this method and its activity recovered. Our results show that human T cell responses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.     

Fermentative Production of Exocellular Glucans by Fleshy Fungi

Two specimens of higher fungi produced exocellular β-1, 3-glucans when their mycelial forms were cultivated under submerged aerobic conditions. Plectania occidentalis NRRL 3137 consumed up to 6% glucose or xylose with about 30% conversion to polymer in a medium composed of hydrolyzed soy protein, salts, and thiamine. A 5% inoculum was used in a 10-day shaken fermentation. After dilution of the culture liquors and partial disruption of mycelia with a blender, solids were removed by centrifugation, and the polymer was precipitated by the admixture of 2 volumes of ethyl alcohol. A second polymer was formed in 40 to 65% yield by fermentation with Helotium sp. NRRL 3129, which in the imperfect stage would be identified as Monilia sp. It consumed up to 4% glucose, fructose, mannose, or sucrose in 60 to 72 hr. A 2% inoculum in a medium composed of commercial defatted soy flakes, phosphate, and thiamine in tap water gave a satisfactory fermentation. This polymer was precipitated by the addition of 0.5 volume of ethyl alcohol. Both organisms have a broad pH optimum on the slightly acidic side and did best at about 25 C.