Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002.

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55 degrees C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. No variation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50 degrees C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.

Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus.

Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl(2) 2H(2)O enhanced amylase production over that on Ca -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2.

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.

Factors limiting the microbial conversion of sterols to 17-ketosteroids in the presence of metal chelate inhibitors.

Bioconversion of sterols to 17-ketosteroids by an Arthrobacter species occurred in the presence of hydrophobic metal-chelating agents but the production of 17-ketosteroids (17-KS) was seriously limited by the rapid loss of the viability of cells in the presence of these inhibitors. Besides, the conversion was inhibited by 17-KS at concentrations of 500 ppm or more. The 17-KS formed consisted exclusively of 1,4-androstadiene-3,17-dione (ADD) and 4-androstene-3,17-dione (AD) and these were found in the extracellular medium predominantly in bound form or as molecular aggregates which may limit their accumulation. It was concluded that enhanced production of 17-KS could be achieved by protecting the viability of cells and by removing the steroid metabolites from the site of inhibition.

Mode of uptake of insoluble solid substrates by microorganisms. I: Sterol uptake by an arthrobacter species.

The mode of uptake of sterols, which are nearly insoluble in water by an Arthrobacter species, was studied on the basis of substrate transfer via the aqueous phase (solubilization/pseudosolubilization) and through direct contact with sterol particles. Growth of the organism, on stero powder was predominantly in nonlogarithmic in character, indicating a possible limitation of substrate transfer. Soluble sterol was shown to be the preferential form of the substrate for assimilation by the organism. Evidence was obtained for increased solubilizition of beta-sitosterol and cholesterol during microbial growth on these substrates. But the rate of solubilization of beta-sitosterol (3.06 mg L(-1) h(-1)) was too inadequate to account for the observed substrate uptake rare (107 mg L(-1) h(-1)) during growth. A cholesterol solubilization rate of 44 mg L(-1) h(-1) could, however, account to an appreciable extent for the observed cholesterol uptake rate of 140 mg L(-1) h(-1) during growth. Increasing attachement of cells to sterol particles during growth was observed by microscopic examination, indicating that growth may take place over the surface of sterol particles. By using the synthetic surfactant HYOXYD AAO (alkyl aryl polyglycol ether), which prevented attachment of cells to sterol particles without affecting the metabolic integrity of the cells, it was shown that growth indeed took place predominantly on the surface of the sterol particles. Increased generation of finer particles of sterol, which provides increased substrate surface area during growth, was demonstrated. It was concluded that with beta-sitosterol, growth takes place almost entirely by attachement, whereas with cholesterol, about 30% of the growth take place on solubilized substrate and the rest through attachament.

Mode of uptake of insoluble solid substrates by microorganisms. II: Uptake of solid n-alkanes by yeast and bacterial species.

Using two species of yeast and one of bacterium, evidence has ben obtained which indicates that the microbial uptake of solid alkane powders occurs primarily through a substrate solubilization mechanism. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited the growth of these organisms on alkane powder; the inhibition could be removed vai a supply of artificially solubilized alkane. One of the yeast strians, which was a mutant incapable of growing on solid alkane powder and liquid alkane, could grow very well on artifically solubilized alkanes. It was demonstrated that the solid alkane solubilization rate during microbial growth could satisfactorily account for the maximal alkane uptake rate actully observed during growth. The specificity of solubilization for the solid alkane used as the growth substrate was demonstrated.

Isolation and functional characterization of hydrocarbon emulsifying and solubilizing factors produced by a Pseudomonas species.

Pseudomonas PG-1 cultivated on pristane produced in good amount a heat-stable polymeric substance which showed strong hydrocarbon emulsifying and solubilizing properties. The substance was isolated in crude form and was found to contain 34% protein, 16% carbohydrate, and 40% lipid. The hydrocarbon solubilizing activity of the isolate was strongly inhibited by EDTA but the chelating agent had no effect on the hydrocarbon emulsifying activity. Both activities of the isolate were strongly inhibited by chymotrypsin treatment indicating the importance of the protein moiety for its activity. Hydrocarbon solubilization by the isolate showed a certain degree of specificity to pristane in modest agitation generally used in microbial cultivation, but this specificity was lost by vigorous agitation in a Waring blender. It was proposed that in the first case, solubilization was effected by a solubilizing factor specific to pristane, whereas in the latter case, nonspecific solubilization occurred due to the action of the emulsifying factor. The rate of pristane solubilization by heat-treated culture broth under the conditions of agitation used in cultivation (rotary shaker, 120 rpm) was found to be ca. 750 mg L(-1) h(-1) which was much larger than the maximal pristane uptake rate of 170 mg L(-1) h(-1) observed during microbial growth on the substrate. It was concluded that hydrocarbon solubilization could satisfactorily account for the substrate uptake and growth.

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons.

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.

Effect of carbon and nitrogen sources on neutral proteinase production by Pseudomonas aeruginosa.

A strain of Pseudomonas aeruginosa from soil produced large quantities of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the protinase synthesis.