The genes of two ring-hydroxylating dioxygenases (RHDs) of Sphingomonas sp. VKM B-2434 were cloned and expressed in Escherichia coli. The relative values of the RHD specificity constants were estimated for six polycyclic aromatic hydrocarbons (PAHs) based on the kinetics of PAH mixture conversion by the recombinant strains. The substrate specificity profiles of the enzymes were found to be very different. Dioxygenase ArhA was the most specific to acenaphthylene and showed a low specificity to fluoranthene. Dioxygenase PhnA was the most specific to anthracene and phenanthrene and showed a considerable specificity to fluoranthene. Knockout derivatives of Sphingomonas sp. VKM B-2434 lacking ArhA, PhnA, and both dioxygenases were constructed. PAH degradation by the single-knockout mutants was in agreement with the substrate specificity of the RHD remaining intact. Double-knockout mutant lacking both enzymes was unable to oxidize PAHs. A mutant form of dioxygenase ArhA with altered substrate specificity was described.

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.

The ability of the strains-destructors of various aromatic compounds to utilize trinitrotoluene (TNT) up to concentration of 70 mg/l was shown. An increase in the TNT concentration from 100 to 150 mg/l did not inhibit its conversion rate by the Kocuria palustris RS32 strain. The Acinetobacter sp. VT11 strain utilized TNT as a sole substrate for growth; 3,5-dinitro-4-methyl anilide acetate and 2,6-dinitro-4-aminotoluene were identified as intermediates of TNT degradation by active strains of Pseudomonas sp. VT-7W and Kocuria rosea RS51. At the same time, 4-methyl-3,5-dinitroformamide was discovered for the first time upon the TNT destruction by the bacteria strains of Rhdococcus opacus 1G and Rhdococcus sp. VT-7. The active bacterial strains achieved an 82-90% destruction of TNT when they were introduced into the soil.

Aerobic bacterial degradation of PAHs is reviewed. Particular attention is paid to its kinetic aspects (rate and specificity). The general concepts of PAH biodegradation in nature and the role of aerobic bacteria in this process are described. The problem of PAH bioavailability and the mechanism of PAH penetration through bacterial cell wall are discussed. The key role of the reaction of PAH hydroxylation in controlling the rate and specificity of PAH biodegradation process is substantiated. The effects of competitive inhibition, intermediate inhibition, cross induction, and cometabolism are considered. The importance of microbial communities for PAH biodegradation in natural ecosystems is shown. The review contains the list of 138 references.

Показана способность штаммов-деструкторов различных ароматических соединений утилизировать тринитротолуол (ТНТ) в концентрации до 70 мг/л. Увеличение концентрации ТНТ от 100 до 150 мг/л не ингибировало скорость конверсии этого соединения штаммом Kocuria palustris RS32. Штамм Acinetobacter sp. VT11 использовал ТНТ в качестве единственного субстрата для роста. Среди интермедиатов деградации ТНТ активными штаммами Pseudomonas sp. VT-7W и Kocuria rosea RS51 идентифицированы 3,5-динитро-4-метил-анилид уксусной кислоты и 2,6-динитро-4-аминотолуол. При деструкции ТНТ штаммами бактерий Rhodococcus opacus 1G и Rhodococcus sp. VT-7 впервые обнаружен 4-метил-3,5-динитроформамид. Активные бактериальные штаммы при интродукции в почву осуществляли разложение ТНТ на 82–90%.

The proposed method of kinetic analysis of aqueous-phase biodegradation of polycyclic aromatic hydrocarbons (PAH) mixture presupposes representation of kinetic curves for each pair of mixture components, S x and S y , in double-logarithmic coordinates (ln S x ; ln S y ). If PAH mixture conversion corresponds to the multisubstrate model with a common active site, then the graphs in double-logarithmic coordinates are straight lines with the angular coefficients equal to the ratio of respective first-order rate constants \( k_{x}^{y} = {\frac{{V_{y} K_{x} }}{{K_{y} V_{x} }}} \), where K x and K y are half-saturation constants, V x and V y are the maximum conversion rates for substrates S x and S y ; the graph slope does not depend on any concentrations and remains constant during the change of reaction rates as a result of inhibition, induction/inactivation of enzymes or biomass growth. The formulated method has been used to analyze PAH mixture conversion by the culture of Sphingomonas sp. VKM B-2434. It has been shown that this process does not satisfy the multisubstrate model with a single active site. The results suggest that the strain VKM B-2434 contains at least two dioxygenases of different substrate specificity: one enzyme converts phenanthrene and fluoranthene and the other converts acenaphthene and acenaphthylene. The ratios of first-order rate constants have been obtained for these pairs of substrates.

Biodegradation of hydrophobic organic contaminants is often accompanied by the accumulation of hydrophobic intermediates. This paper is devoted to the experimental evaluation of electron balance between the hydrophobic intermediates and the other biodegradation products. The ratio of the fraction of electrons that are not held in the hydrophobic intermediates to the total fraction of electrons released from the substrate during its conversion is called a degradation level (DL). Under certain conditions, the DL value is equal to the angular coefficient of the plot in the coordinates (1−(S/S0);1−(COD/COD0)), where S and COD are current values of substrate concentration and chemical oxygen demand of the ethylacetate extract from the culture; S0 and COD0 are the respective initial values. This approach has been used for analysis of the conversion of PAHs by Sphingomonas sp. VKM B-2434. The DL values obtained correspond well to most published data on PAH metabolism by this strain. The DL values significantly increased as a result of addition of the strains consuming the products of conversion of PAHs by Sphingomonas sp. VKM B-2434. The proposed method seems useful for characterizing biodegradation of hydrophobic pollutants by both pure and mixed microbial cultures.

The strain Sphingomonas sp. VKM V-2434 converts the mixture of seven polyaromatic compounds (PACs): fluorene, dibenzothiophene, carbazole, phenanthrene, anthracene, fluoranthene, and pyrene. The effect of each of the above PACs on the rate of mixture conversion was determined. The following two strains, which utilize the substances inhibiting the studied process, were added to the culture: strain FON-11 utilizing 9-fluorenone (fluorene metabolite) and strain CBZ-21 utilizing carbazole. In the case of the mixed culture of three strains, conversion rates were 1.5 and 1.2–3.8 times higher for the PAC mixture and its individual components, respectively, than the rates for Sphingomonas sp. VKM V-2434 monoculture. The degree of degradation of PAC conversion products increased from 32 to 44%. The rate of PAC conversion by the mixed culture exceeded the sum of conversion rates for the individual component strains; this cooperative effect was particularly marked for anthracene and pyrene.

Consumption of 1-hydroxy-2-naphthoic acid by strain Arthrobacter sp. K3 was investigated. Drastic increase in the substrate concentration in flow culture was shown to induce the lag phase of growth in case the initial substrate concentration in the medium was not saturating; the culture originally saturated with the substrate ( S K S ) was resistant to the concentration increase. In accordance with the constructed kinetic model, lag phase results from an accumulation of intermediates in the metabolic system.

A versatile bacterial strain able to convert polycyclic aromatic hydrocarbons (PAHs) was isolated, and a conversion by the isolate of both individual substances and PAH mixtures was investigated. The strain belonged to the Sphingomonas genus as determined on the basis of 16S rRNA analysis and was designated as VKM B-2434. The strain used naphthalene, acenaphthene, phenanthrene, anthracene and fluoranthene as a sole source of carbon and energy, and cometabolically oxidized fluorene, pyrene, benz[ a ]anthracene, chrysene and benzo[ a ]pyrene. Acenaphthene and fluoranthene were degraded by the strain via naphthalene-1,8-dicarboxylic acid and 3-hydroxyphthalic acid. Conversion of most other PAHs was confined to the cleavage of only one aromatic ring. The major oxidation products of naphthalene, phenanthrene, anthracene, chrysene, and benzo[ a ]pyrene were identified as salicylic acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid, o -hydroxyphenanthroic acid and o -hydroxypyrenoic acid, respectively. Fluorene and pyrene were oxidized mainly to hydroxyfluorenone and dihydroxydihydropyrene, respectively. Oxidation of phenanthrene and anthracene to the corresponding hydroxynaphthoic acids occurred quantitatively. The strain converted phenanthrene, anthracene, fluoranthene and carbazole of coal-tar-pitch extract.

The transformation of phenanthrene and anthracene by Rhodococcus rhodnii 135, Pseudomonas fluorescens 26K, and Arthrobacter sp. K3 is studied. Twenty-one intermediates of phenanthrene and anthracene transformation are identified by HPLC, mass spectrometry, and NMR spectroscopy. P. fluorescens 26K and Arthrobacter sp. K3 are found to produce a wide range of intermediates, whereas R. rhodnii 135 oxidizes phenanthrene, resulting in the formation of a sole product, 3-hydroxyphenanthrene. Putative transformation pathways of phenanthrene and anthracene are proposed for the three bacterial strains studied. These strains can be used to obtain valuable compounds (such as hydroxylated polycyclic aromatic hydrocarbons) that are difficult to produce by chemical synthesis.

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.

Of the four investigated Rhodococcus strains (R. rhodochrous 172, R. opacus 4a and 557, and R. rhodnii 135), the first three strains were found to be able to completely transform fluorene when it was present in the medium as the sole source of carbon at a concentration of 12-25 mg/l. At a fluorene concentration of 50-100 mg/l in the medium, the rhodococci transformed 50% of the substrate in 14 days. The addition of casamino acids and sucrose (1-5 g/l) stimulated fluorene transformation, so that R. rhodochrous 172 could completely transform it in 2-5 days. Nine intermediates of fluorene transformation were isolated, purified, and structurally characterized. It was found that R. rhodnii 135 and R. opacus strains 4a and 557 hydroxylated fluorene with the formation of 2-hydroxyfluorene and 2,7-dihydroxyfluorene. R. rhodochrous 172 transformed fluorene via two independent pathways to a greater degree than did the other rhodococci studied.