O. E. Makarov

[Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum].

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indole-3-acetic acid. The synthesis of indole-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.

Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains--Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7--possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas-liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C-P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.

Oxidoreductase activity of sorghum root exudates in a phenanthrene-contaminated environment.

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene on the enzymatic activity of root exudates of the phytoremediating plant Sorghum bicolor (L.) Moench was studied. Analysis of sorghum root exudates allowed us to reveal the activities of oxidase, peroxidase, and tyrosinase. The activities of these enzymes were progressive as the soil phenanthrene concentration increased. Using lyophilized samples, we found that as a result of the enzymatic activity of the root exudates, some of the PAHs and products of PAH degradation were oxidized in the reaction mixture supplemented with the mediating agents (ABTS or DL-DOPA) but that no oxidation was observed in the reaction mixtures without the mediators. The revealed enzymatic activity of the sorghum root exudates may indicate the involvement of the root-released oxidoreductases in rhizospheric degradation of PAHs and/or their derivatives. In addition, from the data obtained, the coupling of plant and microbial metabolisms of PAHs in the rhizosphere may be surmised.

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti.

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways. The first, major pathway is through terminal aromatic ring cleavage (presumably at the C3–C4 bond) producing benzocoumarin and 1-hydroxy-2-naphthoic acid, whose further degradation with the formation of salicylic acid is difficult or is very slow. The second pathway is through the oxidation of the central aromatic ring at the C9–C10 bond, producing 9,10-dihydro-9,10-dihydroxyphenanthrene, 9,10-phenanthrenequinone, and 2,2′-diphenic acid. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites, has been reported for a bacterium of the Ensifer genus.

Versatile peroxidase of Bjerkandera fumosa: substrate and inhibitor specificity.

The inhibitor and substrate specificities of versatile peroxidase from Bjerkandera fumosa (VPBF) were studied. Two different effects were found: NaN(3), Tween-80, anthracene, and fluorene decreased the activity of VPBF, but p-aminobenzoic acid increased it. A mixed mechanism of effector influence on the activity of this enzyme was shown. The catalytic properties of VPBF in the oxidation of mono- and polycyclic aromatic compounds were studied also. 2,7-Diaminofluorene, ABTS, veratryl alcohol, and syringaldazine can be oxidized by VPBF in two ways: either directly by the enzyme or by diffusible chelated Mn(3+) as an oxidizing agent. During VPBF oxidation of 2,7-diaminofluorene, both with and without Mn(2+), biphasic kinetics with apparent saturation in both micromolar and millimolar ranges were obtained. In the case of ABTS, inhibition of VPBF activity by an excess of substrate was observed. Direct oxidation of p-aminobenzoic acid by versatile peroxidase was found for the first time. The oxidation of three- and four-ring PAHs by VPBF was investigated, and the oxidation of anthracene, phenanthrene, fluorene, pyrene, chrysene, and fluoranthene was shown. The products of PAH oxidation (9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone) catalyzed by VPBF were identified.

Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus Pleurotus ostreatus D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the time course of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk’s medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthracene had a smaller effect. Addition of Mn2+ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase forms by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.

Chrysene bioconversion by the white rot fungus Pleurotus ostreatus D1.

The effect of cultivation conditions on chrysene bioconversion by the fungus Pleurotus ostreatus D1 was shown. Under the laccase production conditions, transformation of this polycyclic aromatic hydrocarbon occurs with accumulation of the quinone metabolite. Under both the laccase and versatile peroxidase production conditions, chrysene degradation occurs, with the stages leading to phthalic acid formation and its further utilization. The formation of phthalic acid as a metabolite of chrysene degradation by white rot fungi was revealed for the first time. The data obtained suggest that the laccase revealed on the mycelial surface and the extracellular laccase are probably involved at the initial stages of chrysene metabolism, whereas versatile peroxidase seems to be required for oxidizing the metabolites formed.

Azospirillum brasilense Exopolysaccharide Complexes, their Possible Involvement in Bacteria-Wheat Roots Interactions and the Suggested Nature of these Interactions

Two exopolysaccharide complexes hereafter termed lipopolysaccharide-protein complex and polysaccharide-lipid complex were isolated by means of washing-off with physiological solution from the cell surface of Azospirillum brasilense Spl07, Sp245, S17, 8, 15, 55, 75 and 80 of a complex biopolymer mixture followed by separation. On exposure to wheat seedling root hairs (Triticum aestivum cv. Saratovskaya 29) LPPCs and PSLCs were shown to cause root hair deformations similar to morphological changes of root hairs under the effect of bacterial cells. The data indicating the ability of complexes to bind to WGA, localized on wheat seedling roots were obtained by means of studying LPPCs and PSLCs inhibition in agglutination test under the effect of WGA. PSs containing GlcN, specifically interacting with WGA, were isolated from PSLCs.

Relationship between the Carbohydrate Specificity of Lectins and the Carbohydrate Composition of the Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] Mycelium at Different Stages of Its Morphogenesis

The specificity of lectins from the basidiomycete Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] during solid-state cultivation was found to be maximum to carbohydrates detected in the pyri-dine-soluble fraction of mycelium during the brown mycelial film stage. The carbohydrate composition of the mycelium (i.e., its content of maltose, rhamnose, mannitol, and inositol) was found to be different upon normal fruiting body formation and when the nonpigmented mycelium produced defective fruiting bodies.

Gas-liquid chromatography-obtained differences in the dissolution enthalpy between two positional isomers in a polar stationary phase: a measure of the inter- or intramolecular hydrogen bond energy?

Previous GLC work with several 2- and 4-substituted phenols and anilines, as well as with a pyrrolizidine alcohol, had determined the difference between the heats of dissolution of two positional isomers in a strong polar stationary liquid phase; one of these isomers forms an intramolecular hydrogen bond (intra-HB) and the other has no such bond for steric reasons. The energies of the intermolecular hydrogen bonds (inter-HBs), ΔH(inter-HB), formed by the 1,2- and 1,4-isomers with the molecules of a polar phase had been assumed approximately equal, so the difference between them could be ignored. The same assumption had been made for the energies of nonspecific interactions (NSIs), ΔH(NSI). It had been concluded that the found difference can be considered as an intra-HB energy (enthalpy), ΔH(intra-HB), when the energies (enthalpies) of inter-HBs formed by the 1,2- and 1,4-isomers under study with the molecules of a polar phase are much greater in absolute value than ΔH(intra-HB). And, conversely, when |ΔH(intra-HB)|>|ΔH(inter-HB)|, an inter-HB enthalpy will result. With the same assumptions, we here obtained an extended thermodynamic equation and corrected this above conclusion on the basis of a general consideration of the dissolution thermodynamics for two isomers of a molecule in a polar phase. Account was taken of the coefficients of isomer partitioning between the liquid and the gaseous phase at the experimental temperature. The conclusion made previously was adjusted for ΔH(NSI) and formulated as follows: The GLC method determines the intra-HB energy at |ΔH(intra-HB)|≤|ΔH(inter-HB)+ΔH(NSI)|. If |ΔH(intra-HB)|>|ΔH(inter-HB)+ΔH(NSI)|, the method yields the values of ΔH(inter-HB)+ΔH(NSI). This new conclusion was illustrated with virtual (numerical) experiments in which various ΔH(intra-HB), ΔH(inter-HB), and ΔH(NSI) values were postulated and results were obtained that would have been achieved by GLC if it had been done. Using a capillary column with the PEG 20M stationary phase, we measured the differences between the heats of dissolution for six pairs of isomers of phenolic compounds and for seven pairs of disubstituted benzene derivatives, which have similar structures but cannot form an intra-HB. The benzene derivatives served to make an approximate experimental estimate of the difference between the energies of NSIs of the isomers under study with a polar phase, ΔH(NSI).