[Effect of substances which change the proton-motive force on activity of methane microbe oxygenation].

High extracellular concentration of K+ stimulated methane oxygenation with Methylomonas rubra 15 [Russian character: see text], Methylococcus thermophilus 111 [Russian character: see text] and Methylococcus capsulatus 494 at neutral value of pH. That was determined by K+ arrival to the cells at neutral medium pH that resulted in the increase of pH difference between the exterior and interior sides of the membrane (ApH) and, respectively, in the increase of the methane oxygenation rate. Thus, methane monooxygenation depends on the availability of ion gradients on a membrane. Ionophores valinomycin and monensin inhibited methane oxygenation by the cells of Methylomonas rubra 15 [Russian character: see text] that evidenced for the methane oxygenation dependence on the protone-motive force which could be formed as the result both of protons displacement with oxygenation of methane monooxygenation products and of the gradient of potassium and sodium ions. Protonophore FCCP suppressed completely methane oxygenation in Methylococcus capsulatus 494 and M. thermophilus 111 [Russian character: see text] at neutral pH, and took no effect at the alkaline values of pH. This suggests that FCCP dissipates the proton-motive force and does not inhibit methane monooxygenase activity. The results obtained indicate that the process of methane oxygenation should be combined with energy generation in a form of the transmembrane electric charge (delta psi) and proton gradient (deltapH).

[Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis]. / Nekotorye osobennosti metabolizma étanola u mutantnogo shtamma Acinetobacter sp., ne obrazuiushchego ékzopolisakharidy.

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.

[Ethanol formation by methane-utilizing bacteria at ethane co-metabolism]. / Obrazovanie étanola metanispol'zuiushchimi bakteriiami pri kometabolizme étana.

It was established, that EDTA (1.0 mM) and formamide (100 mM) are inhibitors of methanol dehydrogenase in Methylobacter luteus 12b, Methylomonas rubra 15sh and Methylococcus thermophilus 111p. The investigated strains co-metabolised ethane with the use of formate as the co-substrate. The application of formamide (or EDTA) as inhibitors of methanol dehydrogenase prevented from further transformation of ethanol and resulted in accumulation of extracellular ethanol. It was shown, that M. rubra 15sh accumulated extracellular ethanol under cultivation in a chemostate. The carried out researches have shown a regulation path of co-metabolism process of hydrocarbons by methane utilizing bacteria. Using the specific inhibitors of methanol dehydrogenase and a source of reducing agent (energy) for methane monooxygenase with the help of the cells of methane-oxidizing bacteria it is possible to obtain from ethane or other hydrocarbons the products of their monooxygenation--alcohols.

[Search for methanotrophic producers of exopolysaccharides]. / Poisk metanotrofnykh produtsentov ékzopolisakkharidov.

Bacteria that produce exopolysaccharides (EPS) and use methane as the only source of carbon were selected by studying a collection of methanotroph strains: Methylococcus capsulatus E 494, 874, and 3009; M. thermophilus 111p, 112p, and 119p; Methylobacter ucrainicus 159 and 161; M. luteus 57v and 12b; Methylobacter sp. 100; Methylomonas rubra 15 sh and SK-32; Methylosinus trichosporium OV3b, OV5b and 4e; M. sporium 5, 12, A20d, and 90v; and Methylocystis parvus OVVP. Mesophilic methanotroph strains with the ribulose monophosphate way of C1-compound assimilation synthesized EPS more actively than bacteria operating the serine cycle. The dynamics of EPS synthesis by methanotrophs during chemostat cultivation was studied.

[Ecological consequences of radioactive pollution for soil bacteria within the 10-km region around the Chernobyl Atomic Energy Station]. / Ekologicheskie posledstviia radioaktivnogo zagriazneniia dlia pochvennykh bakterii v 10-km zone ChAES.

The diversity of aerobic chemoorganotrophic (capable of growing on nutrient agar) bacteria in radioactive soil (0.3-17.0 microCi/kg soil) sampled in the 10-km zone around the Chernobyl Nuclear Power Plant (CNPP) was found to be lower than that observed in control, uncontaminated soil with a radioactivity of 0.002-0.006 microCi/kg soil. All the radioactive soil samples contained the bacteria Bacillus cereus and Methylobacterium extorquens or M. mesophillicum, which exhibited a high tolerance to 0.3-1.0 M hydrogen peroxide, whose action can to a certain extent simulate the effect of ionizing radiation. Some of the contaminated soil samples contained other species of chemoorganotrophic bacteria with a low tolerance to H2O2. The survival of bacteria in the Chernobyl accident zone is probably due to the functioning of mechanisms efficiently neutralizing peroxide compounds and repairing radiation-damaged DNA. The population of cellulolytic, nitrifying, and sulfate-reducing bacteria in contaminated soil was found to be 1-2 orders of magnitude less than in control soil, indicating the unfavorable effect of anthropogenic radiation on the abundance and diversity of soil bacteria.

[The biology of bacteria that assimilate C1--C2 compounds and the biotechnology aspects of their use]. / Biologiia bakterii, assimiliruiushchikh C1--C2-soedineniia, i biotekhnologicheskie aspkety ikh ispol'zovaniia.

Main directions of the research work of the Department of Biology of Gas-Oxidizing Microorganisms are described in the paper. Fundamental studies concern ecology, selection, taxonomy, physiology, biochemistry, genetics of bacteria utilizing C1--C2 compounds, mathematical simulation of microbiological processes. Applied studies are devoted to development of scientific basis of biotechnologies for synthesis of important products (single cell protein, exopolysaccharides, food ingredients, biogas) from non-food substrates, search for the hydrocarbon deposits and protection of the environment.

[Role of exogenous carbon dioxide in the metabolism of methane-oxidizing bacteria]. / Rol' ékzogennoi uglekisloty v metabolizme metanokisliaiushchikh bakterii.

The object of this work was to study the ability of methane oxidizing bacteria to use CO2 as an acceptor of electrons liberated in methane oxidation and the role of CO2 fixation in the constructive metabolism of the bacteria. All of the studied methane oxidizing bacterial cultures were found to be capable of fixing the 14C of hydrocarbonate. The activity of the process was shown to be similar in different strains. Up to 30% of the carbon in the biomass composition could originate from the carbon of HCO3-. Methane oxidizing bacteria that assimilated C1-compounds via the hexulose phosphate and serine pathways had the same level of HCO3- fixation. No differences were found among strains of the same species, among species, or among genera. The assimilation of HCO3- was catalyzed by PEP-carboxylase (i. e. in a heterotrophous way) or, in some cultures, by ribulose-1,5-diphosphate carboxylase, the key enzyme in the autotrophous pathway of CO2 assimilation. The enzymological mechanisms of HCO3- assimilation are discussed. The biological role of CO2 fixation in the metabolism of methane oxidizing bacteria that use the hexulose phosphate and serine pathways of methane assimilation may be different. The process can either play the role of anapleurotic reactions in the tricarboxylic acid cycle, or be an element of the serine pathway of methane assimilation. Calculations have shown that the extent to which a substrate to be metabolized is reduced seems to determine the activity of exogenous CO2 fixation. The contribution made by HCO3- fixation into the carbon metabolism of methane oxidizing bacteria confirms that they are related to lithotrophous organisms.

[Theoretical evaluation of necessity of carbon dioxide assimilation by microorganisms during growth on various substrates]. / Assimiliatsiia uglekisloty mikroorganizmami pri roste na razlichnykh substratakh.

The biological role of exogenous carbon dioxide during substrate assimilation with a various degree of reductivity is evaluated. The investigation of metabolic pathways of carbon dioxide incorporation into the metabolic processes of methaneoxidizing bacteria shows that the HCO3- ion assimilation is catalyzed by phosphoenolpyruvate carboxylase and in certain strains also by the key enzyme of autotrophic pathway of the carbon dioxide assimilation, ribulose-1,5-diphosphate carboxylase. The theoretical calculations and experimental studies indicate that exogenous carbon dioxide is a necessary participant of the metabolic processes of methane or methanol assimilation. It is also an acceptor of the excess electrons of these compounds. It is the degree of reductivity of the substrate metabolized that determines the activity of the exogenous carbon dioxide fixation by microorganisms. The carbon dioxide fixation by heterotrophic microorganisms must be considered, therefore, as a process which is mostly due to the elementary composition of the source of carbon under conversion.

[Comparative characteristics of the enzymatic systems of methane-utilizing bacteria that oxidize NH2OH and CH3OH]. / Sravnitel'naia kharakteristika fermentnykh sistem metanispol'zuiushchikh bakterii, okisliaiushchikh NH2OH i CH3OH.

Methane hydroxylase (MH) from the obligate methane assimilating culture of Methylococcus thermophilus catalyses oxygenation of both CH4+ and NH4+; therefore, we studied the specificity of enzyme systems catalysing the subsequent oxidation of compounds produced upon the oxygenation of these substrates (CH3OH and NH2OH). CH3OH and NH2OH were shown to be oxidized by different enzymes, viz. methanol dehydrogenase (MD) and hydroxylamine oxidase (HO), respectively. Similar to MH, MD is characterized by the absence of strict substrate specificity, and catalyses oxidation of primary alcohols other than methanol, rather than hydroxylamine. HO catalyses oxidation of hydroxylamine rather than methanol and possesses the activity of hydroxylamine:cytochrome c oxidoreductase. The constitutive character of HO from the methane assimilating bacteria and the substrate specificity of the enzyme suggest that a lithotrophic pathway for producing energy operates in these bacteria. The HO of Methylococcus thermophilus is similar in certain properties to the HO of the nitrifying bacterium Nitrosomonas europaea.

[Lithotrophic metabolic elements in the obligate methylotroph, Methylococcus thermophilus]. / Elementy litotrofnogo metabolizma u obligatnogo metilotrofa Methylococcus thermophilus.

Methane oxidizing bacteria oxidize ammonium via hydroxylamine to nitrite. Electrons liberated upon oxidation of hydroxylamine are transported, depending on the conditions, by the components of the respiratory chain either to oxygen or to pyridine nucleotide. In the former case, the process is coupled with ATP synthesis which occurs at the level of terminal oxidase; in the latter case, NAD+ is reduced by the energy-dependent reversed electron flow in the respiratory chain. The level of nitrite accumulation in the culture liquid of methane oxidizing bacteria suggests that the process of ammonium nitrification is a necessary step of their metabolism. Therefore, oxidation of the mineral component of the growth medium (i. e. ammonium) is an additional source of metabolic energy for the obligate methane oxidizing bacterium Methylococcus thermophilus. Operation of the lithotrophic type of mechanism for energy production at the account of ammonium oxidation makes methane oxidizing bacteria similar to nitrifying microorganisms.

[Transformation of substrates not used for growth by the immobilized methane-oxidizing bacteria]. / Prevrashchenie substratov, ne ispol'zuiushchikhsia dlia rosta, immobilizovannymi metanokisliaiushchimi bakteriiami.

The article deals with the theoretical problems of obtaining certain substrates transformation products using immobilized cells of the methane-oxidizing bacteria. This process is based on the ability of these bacteria to cometabolize the substrates which are not used for growth. The methane-oxidizing bacteria are shown to utilize ethane during growth on methanol. Cometabolism of ethane and methanol occurs due to the presence of conjugated points of these substances oxidizing reactions as well as of the oxidation products assimilation reactions. The optimal concentrations of ethane and methanol are established for the methane-oxidizing bacteria growth and for formation of the ethane oxidation products by means of the immobilized cells.

[Mechanism of methane oxidation by intact cells of obligate methylotrophs]. / O mekhanizme sookisleniia étana intaktnymi kletkami obligatnykh metilotrofov

Ethane and propane were found to be competitive inhibitors of methane oxidation. Obligate methylotrophs can oxidize ethane only in the presence of methane, i.e. co-oxidation of ethane takes place. Studies on the kinetics of simultaneous oxidation of ethane and methane have shown that their oxidation is catalysed by one and the same enzyme system, within one and the same enzyme active site; therefore, the absolute substrate specificity is absent. The reactions of methane and ethane oxidation are coupled since ethane can be oxidized only after the reduced coenzyme had been provided by the reaction of methane oxidation.