D. O. Egorova

Degradation of chlorinated biphenyls and products of their bioconversion by Rhodococcus sp. B7a strain

Strain Rhodococcus sp. B7a isolated from artificially polluted soil destructs mono- and di-substituted ortho- and/or para-chlorinated biphenyls with utilization of chlorinated benzoic acids and shows high degradation activity as regards trichlorinated biphenyls. It is shown that p-hydroxybenzoic and protocatehoic acids are the products of p-chlorobenzoic acid catabolism.

Degradation of 4-chlorobiphenyl and 4-chlorobenzoic acid by the strain Rhodococcus ruber P25

The strain Rhodococcus ruber P25 utilizes 4-chlorobiphenyl (4CB) and 4-chlorobenzoic acid (4CBA) as sole carbon and energy sources. 4CB degradation by washed cells of strain P25 was accompanied by transient formation of 4CBA, followed by its utilization and release of equimolar amounts of chloride ions into the medium. The strain R. ruber P25 possessed active enzyme systems providing 4CBA degradation via the stages of formation of intermediates, para-hydroxybenzoate (PHBA) and protocatechuic acid (PCA), to compounds of the basic metabolism. The involvement of protocatechuate 4,5-dioxygenase in 4CBA degradation by rhodococci was revealed. It was established that the initial stage of 4CBA degradation (dehalogenation) in the strain R. ruber P25 was controlled by the fcbA and fcbB genes encoding 4-CBA-CoA ligase and 4-CBA-CoA dehalogenase, respectively. The genes encoding 4CBA dehalogenase components have not been previously detected and characterized in bacteria of the genus Rhodococcus.

Destruction of mixture of tri-hexa-chlorinated biphenyls by Rhodococcus genus strains

Destruction of polychlorinated biphenyls (PCBs) by strain-destructors Rhodococcus sp. B7a and Rhodococcus sp. G12a has been studied. It was shown that these strains destruct 78–95% of PCB mixture containing tri-hexa-chlorinated biphenyls. Rhodococcus destruct all components of the mixture of tri-, tetra-, penta-, and hexa-chlorinated biphenyls without accumulation of toxic chlorinated metabolites. The studied bacteria destruct PCB that are the most stable for oxidation, such as 2,5,2′,5′-CB; 3,4,3′,4′-CB; and 2,4,5,2′,4′,5′-CB. The most perspective strains are R. rubber P25, Rhodococcus sp. B7a and Rhodococcus sp. G12a whose metabolic potential can be used for biotechnological refinement of the environment from highly toxic pollutants.

An interdisciplinary approach to the problem of neutralization of man-made polychlorinated biphenyls

19 Currently, biological methods of degradation of manmade polychlorinated biphenyls (PCBs) are becoming increasingly important in connection with the selection of new efficient PCB degrading strains. The most successful studies in the field of biodegrada tion of PCBs are associated with the use of low chlo rinated PCB congeners [1, 2]. It has been found that the biodegradability of PCB congeners increases with decreasing number of chlorine atoms in the initial sub strates. At the same time, interest persists in the search for surfactants capable of transferring more hydropho bic medium and high chlorinated PCB congeners into an aqueous medium in order to create stable emulsions available for efficient operation of bacterial strains [3, 4].

Molecular biological characterization of biphenyl-degrading bacteria and identification of the biphenyl 2,3-Dioxygenase α-subunit genes

Bacterial isolates from soils contaminated with (chlorinated) aromatic compounds, which degraded biphenyl/chlorinated biphenyls (CB) and belonged to the genera Rhodococcus and Pseudomonas, were studied. Analysis of the 16S rRNA gene sequences was used to determine the phylogenetic position of the isolates. The Rhodococcus cells were found to contain plasmids of high molecular mass (220–680 kbp). PCR screening for the presence of the bphA1 gene, a marker indicating the possibility for induction of 2,3-dioxygenase (biphenyl/toluene dioxygenase subfamily), revealed the presence of the bphA1 genes with 99–100% similarity to the homologous genes of bacteria of the relevant species in all pseudomonad and most Rhodococcus isolates. A unique bphA1 gene, which had not been previously reported for the genus, was identified in Rhodococcus sp. G10. The absence of specific amplification of the bphA1 genes in some biphenyl-degrading bacteria (Rhodococcus sp. B7b, B106a, G12a, P2kr, P2(51), and P2m), as well as in an active biphenyl degrader Rhodococcus ruber P25, indicated the absence of the genes encoding the proteins of the biphenyl/toluene dioxygenase subfamily and participation of the enzymes other than this protein family in biphenyl/CB degradation.

Degradation of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 isolated from waste products of a salt-mining plant

Degradation of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 isolated from technogenic mineral waste products of the BKRU1 “Uralkalii” plant was investigated (Berezniki, Perm krai). The R. wratislaviensis KT112-7 was shown to utilize increased concentrations of o-phthalic (o-PA) (8 g/L) and benzoic (BA) (3.4 g/L) acids. The strain grows with o-PA, BA, and biphenyl at a NaCl content of up to 75, 90 and 100 g/L in the culture medium, respectively. Based on an analysis of the metabolic profile and nucleotide sequences of the bphA1, benA, and phtB genes, the strain KT112-7 was found to degrade o-PA via the formation of 3,4-dihydroxyphthalic and 3,4-dihydroxybenzoic acids. Degradation of biphenyl proceeds via the formation of BA and then at low concentrations of NaCl (up to 50 g/L) via the formation of 4-hydroxybenzoic acid with its subsequent oxidation, while at high concentrations of NaCl (over 60 g/L)-via the direct oxidation of benzoic acid to resultant catechol. These data indicate that the Rhodococcus wratislaviensis KT112-7 is a promising strain in the development of new biotechnologies directed for utilization (transformation) of aromatic compounds, including the conditions of increased mineralization.

Bacterial degradation of a mixture obtained through the chemical modification of polychlorinated biphenyls by polyethylene glycols

Polychlorinated biphenyls (PCBs), also known by the trade name Sovol, are toxic industrial wastes. They have been subjected to chemical treatment by polyethylene glycols (PEGs) and potassium hydroxide. As a result of the interaction of the Sovol with various molecular mass PEGs (MMPEG-4 ∼ 200, MMPEG-22 ∼ 1000), water-soluble mixtures M1 and M2 containing mono(polyethylene glycol)oxy-derivatives (PCB-PEG-4 and PCB-PEG-22), polychlorobiphenylols, and unreacted PCB congeners (PCB 44, PCB 47, PCB 49, PCB 52, and PCB 66) were obtained. It was shown for the first time that mixtures M1 and M2 are susceptible to bacterial degradation without their fractionation. According to the gas-liquid chromatography with flame-ionization and mass-spectrometric detection, the Rhodococcus wratislaviensis KT112-7 strain degraded all of the chemical compounds occurring in the mixtures. In a 5-day experiment, it was found that the KT112-7 strain decomposes mono(polyethylene glycol)oxy-derivatives completely (by 100%) and polychlorobiphenylols and PCB congeners by 90–95% in the M1 and M2 mixtures. The culture medium did not contain transformation products, whereas free chlorine ions were accumulated (72–94% of the maximum possible amount). Thus, the use of the chemical modification and consecutive bacterial degradation provided an effective destruction of technical PCB mixtures with a high content of highly chlorinated congeners.

Structural and functional rearrangements in the cells of actinobacteria Microbacterium foliorum BN52 during transition from vegetative growth to a dormant state and during germination of dormant forms

Cellular organization of the cystlike cells (CLC) of non-spore-forming heterotrophic actinobacteria isolated from soils contaminated with chemical plant waste (Bereznyaki, Russia) and identified as Microbacterium foliorum BN52 was studied. CLC were obtained in laboratory conditions in limited or starving cultures. Two morphotypes of CLC capable of reversion to the vegetative growth were revealed and characterized in detail. The morphological, ultrastructural, and physiological peculiarities of germination and transition to the vegetative growth were studied at first for the dormant forms of M. foliorum BN52. Germination of morphotype II CLC was associated with gradual recovery of the cell shape and subcellular structures. In contrast to the germination of morphotype II CLC, during the first hours of germination of morphotype I CLC cell volume increased significantly with the subsequent formation of very large cell with sizes several times larger than the typical vegetative cells of the strain. The obtained cells were characterized by polynucleoidity, being polyploids undergoing fission at the next stage of germination, resulting in formation of numerous small and ultrasmall viable cell forms. Formation of cyst-like dormant cells, germinating in the form of polyploid cells is assumed to be the basis of survival and adaptation strategies of heterotrophic bacteria, which are incapable of toxicant degradation in natural habitats, under the conditions of the toxic pressure.

Belliella buryatensis sp. nov., isolated from alkaline lake water

Two bacterial isolates from water of the alkaline brackish Lake Solenoe (Buryatia, Russia), 2C and 5CT, were characterized by using a polyphasic taxonomic approach. The strains were small, non-motile, Gram-stain-negative rods that formed small orange-red colonies on the surface of marine agar. Studies based on 16S rRNA gene sequences showed that the strains were related closely to Belliella pelovolcani CC-SAL-25T (98.7 % sequence similarity). The G+C content of the DNA was 38-40 mol%. DNA-DNA hybridization values between strains 2C and 5CT and B. pelovolcani CC-SAL-25T were 56-58 mol%. A menaquinone with seven isoprene units (MK-7) was the major respiratory quinone. The fatty acid profiles were slightly different from that of B. pelovolcani CC-SAL-25T. The novel strains could be distinguished from the phylogenetically closest species B. pelovolcani CC-SAL-25T based on matrix-assisted laser desorption ionization time-of-flight mass spectra of whole cells and a range of physiological and biochemical characteristics. The data obtained suggest that strains 2C and 5CT represent a novel species of the genus Belliella, for which the name Belliella buryatensis sp. nov. is proposed. The type strain is 5CT ( = VKM B-2724T = KCTC 32194T).