[Antagonistic activity of Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 and BIM B-439D strains towards pathogenic bacteria and micromycetes].

In this study the antagonistic activity of strains Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 and BIM B-439D against bacterial and fungal pathogens of agricultural crops has been researched. It is shown that both strains of bacilli demonstrated a high level of antagonism to the vascular bacteriosis pathogen, average level of antagonism to micromycetes--root rot pathogens. To ofiobulez pathogen strain B. amyloliquefaciens subsp. plantarum BIM B-439D was more active. Cultural liquid of this strain effectively inhibited the spore's germination of pathogenic micromycetes Penicillium expansum and Botrytis cinerea. Both strains of bacilli synthesized several hydrolytic exoenzymes: proteases, amylases, ß-glucanases, chitinases and xylanases. The obtained data suggest the possibility of expanding the range of strain B. amyloliquefaciens subsp. plantarum BIM B-439D application for plant protection, as well as the need for further researches of the exometabolites spectrum of strain B. amyloliquefaciens subsp. plantarum IMV B-7404 and their biological activity in order to create an effective bioformulation for crop protection.

[Dehydrogenases oxidizing ethanol and acetaldehide in Rhodococcus erythropolis EK-1].

Four types of alcohol- and acetaldehyde dehydrogenases were found in the cells of strain Rhodococcus erythropolis EK1 grown on ethanol. They are as follows: NAD-, NADP-, pyroquinoline quinone (PQQ)- and 4-nitroso-N,N-dimethyl aniline (NDMA)-dependent enzymes. Activity of NAD- and NADP+ -dependent alcohol dehydrogenases, as well as PQQ and NDMA-dependent acetaldehyde dehydrogenases was low and made up 3-11 nmol x min(-1) x mg(-1) of protein. Ethanol oxidation in the given strain is realized by NDMA-dependent alcohol dehydrogenase, which activity reached its maximum value (up to 15 nmol x min(-1) x mg(-1) of protein) in the early exponential growth phase. Acetaldehyde is oxidized with participation of NAD and NADP-dependent dehydrogenases with optimum pH 9.0-9.5. The results obtained are a basis for development of approaches to intensification of surfactants synthesis by Rhodococcus erythropolis EK-1 strain.

[Effect of cultivation conditions on the physicochemical properties of exopolysaccharide ethapolan].

The physicochemical properties of the complex exopolysaccharide ethapolan (EPS) produced by Acinetobacter sp. 12S during growth on media with various C/N ratios and different concentrations of mineral components and phosphate buffer were studied. Irrespective of the cultivation conditions, the concentrations of carbohydrates (38-44%) and pyruvic acid (3.2-3.7%) in the total EPS, as well as in the acylated (AP) and non-acylated (NAP) polysaccharides obtained from them, were practically the same. The EPS, AP, and NAP were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3 : 2 : 1 : 1. The polysaccharides contained different concentrations of mineral salts (6-28%), uronic acid (3.7-22.0%), and fatty acids (5.8-15.4%); they also differed in the ratio of acetylated and nonacetylated polysaccharides. Due to the differences in the chemical composition and molecular mass (500 kDa - 1.5 MDa), the viscosities of the EPS solutions (in the presence of 0.1 M KCl, in the H(+)-form, and in Cu(2+)-glycine system) were different as well.

[Role of exogenic precursors in formation of surface-active substances during cultivation Rhodoococcus erythiropolis EK-1 on ethanol].

A possibility to intensify synthesis of surfactants of Rhodococcus erythropolis EK-1 under the presence of citrate (lipid synthesis regulator) and fumarate (gluconeogenesis precursor) has been shown. A 40-100% increase of indices of surfactants synthesis with introduction of cytrate (0.1%) and fumerate (0.2%) in the beginning of the stationary producer growth phase is determined by activation of gluconeogenetic branch of metabolism and by intensification of lipids synthesis that was evidenced by the 1.4-1.5-fold and 3.4-3.6-fold increase of isocitrate liase and phosphoenolpyruvate synthetase activities, respectively, as well as by a 1.5-1.6-fold decrease of activity of isocitrate dehydrogenase. The paper is presented in Russian.

[C2 metabolism and intensification of the synthesis of surface-active substances in Rhodococcus erythropolis EK-1 grown on ethanol].

Oxidation of ethanol, acetaldehyde, and acetate in Rhodococcus erythropolis EK-1, producer of surface-active substances (SAS), is catalyzed by N,N-dimethyl-4-nitrosoaniline (DMNA)-dependent alcohol dehydrogenase, NAD+/NADP(+)-dependent dehydrogenases (optimum pH 9.5), and acetate kinase/acetyl-CoA-synthetase, respectively. The glyoxylate cycle and complete tricarboxylic acid cycle function in the cells of R. erythropolis EK-1 growing on ethanol; the synthesis of phosphoenolpyruvate (PEP) is provided by the two key enzymes of gluconeogenesis, PEP carboxykinase and PEP synthetase. Introduction of citrate (0.1%) and fumarate (0.2%) into the cultivation medium of R. erythropolis EK-1 containing 2% ethanol resulted in the 1.5- and 5.3-fold increase in the activities of isocitrate lyase and PEP synthetase (the key enzymes of the glyoxylate cycle and gluconeogenesis branch of metabolism, respectively) and of lipid synthesis, as evidenced by the 1.5-fold decrease of isocitrate dehydrogenase activity. In the presence of fumarate and citrate, the indices of SAS synthesis by strain R. erythropolis EK-1 grown on ethanol increased by 40-100%.

[Metabolism characteristics of Acinetobacter sp. IMS B-7005 grown on the mixture of C2,-C6-substrates].

It has been established that under the conditions of mixotrophic growth of Acinetobacter sp. B-7005 on the mixture of sodium acetate and glucose the activity of acetyl-KoA-synthetase and that of phosphoenolpyruvate synthetase were 10 times lower and indices of synthesis of microbial exopolysaccharide (EPS) ethapolan are almost 2 times lower than on the mixture of potassium acetate and glucose. It is supposed that the positive role of sodium cations may consist in their participation in the active transport of acetate to the cells of Acinetobacter sp. B-7005. An increase of ethapolan synthesis when the producer is grown on the mixture of energy-deficient (acetate + glucose) and energetically nonequivalent (ethanol + glucose) substrates is determined by the intensification of gluconeogenesis, that is evidenced by the functioning of two anaplerotic paths (glioxylate cycle and pyruvate-carboxylase reaction), as well as the increase of activity of phosphoenolpyruvate synthetase compared with cultivation of bacteria on the corresponding monosubstrates.

[Synthesis of the exopolysaccharide ethapolan on a mixture of energy-deficient growth substrates].

Intensification of the synthesis of the microbial exopolysaccharide ethapolan by Acinetobacter sp. B-7005 was shown to occur on a mixture of energy-deficient growth substrates (acetate + glucose). When the bacterium grew on the substrate mixture, both substrates were utilized simultaneously; acetate was taken up by means of active transport at the expense of the energy of the proton-motive force. When acetate was present in the form of a sodium salt, the activities of acetyl-CoA synthetase and phosphoenolpyruvate synthetase (the key enzyme of gluconeogenesis) were tenfold higher than in the presence of potassium acetate, and the indexes of ethapolan synthesis were two times higher. The positive effect of Na+ on ethapolan synthesis is supposed to consist in the creation of ion gradients on the membrane, necessary for the generation of the proton-motive force. Simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, as well as an increase in the activity of isocitrate lyase, malate synthase, and phosphoenolpyruvate synthetase, provide evidence of increased gluconeogenesis in the presence of the acetate + glucose mixture (as compared to gluconeogenesis on the corresponding monosubstrates).

[Formation of the exopolysaccharide ethapolan by Acinetobacter sp. IMV B-7005 on a fumarate-glucose mixture].

Our studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4:1 resulted in a 1.3-2.2-fold increase of the EPS amount synthesized and in a 1.3-2-fold increase of the EPS yield relative to the biomass compared to monosubstrate cultivation. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.

[Synthesis of microbial exopolysaccharide ethapolan on ethanol and molasses mix].

A possibility to change glucose, when cultivating exopolysaccharide (EPS) producer etapolan Acinetobacter sp. B-7005 on a mix of C2-C6-compounds, by the inexpensive substrate--molasses has been shown. The highest indices of EPS synthesis were observed under the conditions of preliminary hydrolysis of molasses, availability of growth factors (yeast autholisate and calcium pantothenate) in the medium, lack of the mineral source of nitrogen nutrition and use of inoculation material grown on acetate. In such conditions of cultivation of bacteria on the mix of ethanol (0.75% in volume) and molasses (0.75 wt. % as to carbohydrates) the amount of synthesized EPS reached 10 g/l, EPS-synthesizing capacity--5 g of EPS/g of biomass, the EPS yield from substrate--74% that is 1.3-1.5 times more than in cultivation on molasses. The increase of EPS synthesis on molasses, as well as on etanol and molasses mix with the use of inoculate grown on C2--substrates (compared with the use of inoculate obtained on the medium with molasses) is determined by the induction of gluconeogenesis that was evidenced by the decrease of isocytrate dehydrogenase activity, increase of activity of enzymes of glyoxylate cycle and key enzyme of gluconeogenesis of phosphoenolpyruvate synthetase.

[Regulation of C2-metabolism under mixotrofic growth conditions of Acinetobacter sp. B-7005 on ethanol and glucose mixture].

The possibility of elimination of C2-metabolism limitation under Acinetobacter sp. B-7005 (producer of exopolysaccharide ethapolan) cultivation on ethanol-glucose mixture was shown. Cultivation of these bacteria on the nonbuffered medium was accompanied by pH decrease (5.6-4.7), acetate accumulation (25-45 mM), drop in the biomass and exopolysaccharide concentration. The decrease of the ammonium nitrate content (to 0.3 g/l) in the medium, its replacement by nitrate, the increase of the pantotenate calcium concentration to 0.0009%, potassium and magnesium cations to 100 and 5-10 mM, application of the inoculum grown on acetate or ethanol, the decrease of the substrates initial concentration to 0.25% permitted a prevention of acetate accumulation and realization of ethanol synthesis on ethanol-glucose mixture on the inbuffered medium.

[Effect of acetate on synthesis of exopolysaccharide ethapolan during Acinetobacter sp. B-7005 cultivation on the medium with ethanol].

A possibility of removal of C2-metabolism limiting and increasing of activity of acetyl-CoA-synthesis in Acinetobacter sp. B-7005--producer of exopolysaccharide (EPS) ethapolan with acetate introduction in the medium with ethanol. When adding potassium acetate 0.1% of into the medium without sodium cations containing 1% ethanol and 0.0009% calcium pantotenate at the stage of obtaining inoculate and at EPS biosynthesis, it is possible to realize the process of ethapolan production on nonbuffered medium in which total content of salts is reduced 4 times without decreasing the synthesis indices and rheological properties.