Biodegradation of Petroleum Hydrocarbons by Actinobacteria and Acinetobacteria Strains Producing Biosurfactant.

Environmental pollution with petroleum hydrocarbons has become one of the most urgent problems worldwide. The effectiveness of bioremediation of oil pollutions is significantly affected by the inherent capabilities of microorganisms and their specific adaptive mechanisms of hydrocarbon assimilation. In this study the hydrocarbon biodegradation efficiency and the surface-active properties of the hydrocarbon-oxidizing strains of Gordonia rubripertincta IMB Ac-5005, Rhodococcus erythropolis IMB B-7012 and Acinetobacter calcoaceticus IMB B-7013 were determined. These strains showed high efficiency of biodegradation of n-hexadecane (82.1­86.7 %), kerosene (72.5­80.3 %), diesel fuel (70.1­74.3 %) and crude oil (63.5­68.9 %). The mixed culture of these strains completely assimilated n-alkanes C9­C21 as well as iso-alkanes C8­C17 and significantly decreased (150­230 times) the amount of n-alkanes C22­C26 in the process of cleaning up water from raw oil. The utilization of hydrocarbons by G. rubripertincta IMB Ac-5005 and R. erythropolis IMB B-7012 with low initial hydrophobicity index (2.4 and 9.6 % respectively) was accompanied by the increase in cell surface hydrophobicity (4 and 25 times respectively) and by the synthesis of cell-bound biosurfactants forming stable emulsions of "oil-in-water" type. The dominating components of these biosurfactants were glycolipids: mono- and dimycolates of trehalose. The assimilation of hydrocarbons by highly hydrophobic A. calcoaceticus IMB B-7013 with the initial hydrophobicity index 99.4 % resulted in the decrease of this value by 1.4 times and the synthesis of extracellular biosurfactant forming stable emulsions of "water-in-oil" type. The biosurfactant of A. calcoaceticus IMB B-7013 is similar to emulsans according to its chemical composition and ratio of the main components (carbohydrates, proteins and lipids). The experimental data provide grounds for efficient using of these strains in the process of bioremediation of oil-polluted water and soils.

[EFFECT OF CALCIUM AND IRON IONS ON THE LEVEL OF DIESEL FUEL UTILIZATION BY ACTINOBACTERIA STRAINS].

It has been established that the presence in the medium Ca2+ and Fe2+ ions in 1.2 - 1.3 times increases the assimilation of hydrocarbon by actinobacteria. The possibility of joint growth of binary actinobacteria cultures on mineral medium with diesel fuel without reducing the assimilation of this substrate compared to monocultures it was shown. In the conditions of co-batch culture Rhodococcus erythropolis IMV B-7277 and Dietzia maris IMV B-7278 strains in the laboratory fermenter "Biotec" (stirring speed 500 r.p.m., degree of aeration 0.3 1/1 per min, sulfite number 1.5 g O2 /1 h,) on the medium with 1.0% diesel oil in the presence of 4 mg/l Ca2+, Fe2+ and 1 g/l yeast extract for 30 hours the substrate assimilation rate was 87.8% and the concentration of biomass - 2.0 g/l.

[MICROBIAL DESTRUCTION MINERAL (OIL) MOTOR OIL].

In a review information is presented about composition of mineral motor oils and their negative impact on the environment and the ability of microorganisms, in particular actinobacteria, to assimilate hydrocarbon oil components. The role of bacteria is described in the process of cleaning up polluted environments motor oils and the prospect of their use in biotechnology, environmental clean-up of these pollutants.

[The ability of actinobacteria to assimilate n-alkanes under nitrate-reducing conditions].

It has been established that the oil-oxidizing strains of actinobacteria--components of the preparation "Ekolan-M" are able to assimilate n-alkanes during microaerobic cultivation in nitrate-reducing conditions. After 7 days of growth in these conditions, the level of biodegradation of n-hexadecane of the investigated strains was 52.0%, which is 1.5 times less than for the same period in the aerobic cultivation. n-Hexadecane utilized by cells was completely mineralized to carbon dioxide, the amount of which in the gas phase reached 1.6% on the 9th day of growth.

[Efficiency of the preparation "Ekolan-M" for purification of oil polluted soil].

The efficiency of purification of oil contaminated loamy chernozem by the preparation "Ekolan-M" was investigated. During 12 months a complex soil bioremediation using the preparation and alfalfa, as the land-improving plant, at the final stage of purification resulted in the reduction of hydrocarbon content by 97.0%, and without the preparation - by 65.5 %. In the version of experiment with the preparation a 100% decrease of soil phytotoxicity was achieved and a significant stimulation of plant growth and development was observed. The process of soil purification was accompanied by intensive development of hydrocarbon-oxidizing microorganisms, the amount of which during the process of oil concentration gradually decreased, approaching the level in the control uncontaminated soil.

[The serological properties of saprophytic corynebacteria studied by immunoenzyme analysis]. / Issledovanie serologicheskikh svoistv saprofitnykh korinebakterii s pomoshch'iu immunofermentnogo analiza.

The degree of serological similarity of saprophytic corynebacteria have been studied using immunoassay ELISA analysis, that is seven collection strains, belonging to Corynebacterium glutamicum (3 strains), C. ammoniagenes (1 strain), C. vitarumen (1 strain), C. variabilis (2 strains) and three industrial strains-lysine producers. Intact and heated bacteria cells have been used as antigens. It has been shown that industrial strain C. glutamicum 22L and collection strains C. glutamicum IMV AC-715, IMV AC-714, IMV AC-733 have the highest degree of serological relationship. C. vitarumen IMV AC-718, C variabilis IMV AC-716 as well as Corynebacterium sp. E531 and VNIIgenetics 90 are close to them according to their serological properties. C. ammoniagenes IMV AC-732 and C. variabilis IMV AC-717 strains have the lowest degree of similarity with other saprophytic corynebacteria which have been studied.

Taxonomic study of the genus Brachybacterium: Brachybacterium nesterenkovii sp. nov.

A new species, Brachybacterium nesterenkovii, is proposed for a group of coryneform bacteria that were isolated from milk products. These organisms have morphological, biochemical, and chemotaxonomic characteristics that are peculiar to the genus Brachybacterium. In contrast to strains of the only previously described species of the genus, Brachybacterium faecium, the representatives of the new species lack glycine in their peptidoglycan, although the peptidoglycan is of the same general type, and have large amounts of rhamnose in their cells. The strains of B. nesterenkovii exhibit no serological relationship with strains of B. faecium; in a numerical phenotypic analysis the two species were easily separated and formed clear-cut clusters. DNA-DNA hybridization between the type strains of B. faecium and B. nesterenkovii showed a level of homology of 22%. Strain 35 (= IMV Ac-752) is the type strain of B. nesterenkovii.

[Amino acid composition of peptidoglycans isolated from cells of Coryneform bacteria by various methods]. / Aminokislotnyi sostav peptidoglikanov, vydelennykh razlichnymi metodami iz kletok korinepodobnykh bakterii.

A comparative study has revealed the identity of the amino acid composition of the peptide part of peptidoglycans obtained from the intact cells (the first method) and of the amino acid composition of peptidoglycans isolated from cell walls (the second method). This evidences for the possibility of using the first method when determining types of peptidoglycans for diagnosis of the coryneform bacteria genera.

[Free mycolic acids of the cells of coryneform and Nocardia-like bacteria]. / Svobodnye mikolovye kisloty kletok nekootorykh korinepodobnykh i nokardiopodobnykh bakterii.

The composition of free mycolic acids was studied in the cells of Brevibacterium ammoniagenes ATCC 6871, B. flavum 22, B. stationis ATCC 14403, Corynebacterium divaricatum ATCC 14020 and Rhodococcus maris IMV 195. The acids are a mixture of saturated and unsaturated compounds with the total number of carbon atoms from 32 to 36 and the number of C atoms in the alpha-chain from 10 to 15.

[Taxonomic position of the lysine producer Brevibacterium flavum]. / O sistematicheskom polozhenii produtsenta lizina Brevibacterium flavum.

Brevibacterium flavum 22 and 22L producing lysine and glutamic acid should be reclassified as Corynebacterium glutamicum on the basis of their chemotaxonomic characteristics: the IV type of the cell wall, corynomycolic acids C32--C34, 57.8% of GC in DNA.

[Development cycles of coryneform and Nocardia-like bacteria]. / Tsikly razvitiia nekotorykh korinepodobnykh i nokardiopodobnykh bakterii.

The growth cycles and the types of cell separation were studied in a microchamber with the collection strains of Brevibacterium ammoniagenes ATTC 6871, B. helvolum ATCC 19239, B. linens CCM 47 and ATCC 9174, B. maris VKM B-464 and B. stationis ATCC 14403, as well as with the strains of the genus Rhodococcus isolated from soils, viz. R. maris sp. nov. IMB 283 and R. luteus sp. nov. IMB 385. According to the increasing complexity of cellular morphological transformation in the life cycle, the organisms may be arranged in a series: R. maris -- B. ammoniagenes -- B. stationis -- B. linens -- B. helvolum -- R. luteus. The first three organisms are characterized by the snapping type of separation of short rod-like daughter cells. The cells of B. linens separate by both the snapping and bending types. The coccoid cells of B. helvolum ATCC 19239 produce many buds which are transformed into rod-like cells in the course of growth. In the log phase of growth, both true and false branching of the cells is observed; the latter is the result of a peculiar growth of the ends in the separated cells of B. helvolum. The cells of R. luteus form a rudimentary, rapidly fragmenting mycelium whose rod-like elements divide then by binary fission; the daughter cells separate the bending and snapping types.

[Chemotaxonomic markers of several coryneform bacteria and the "Rhodochrous" group]. / Khemotaksonomicheskie priznaki nekotorykh korinepodobnykh bakterii i gruppy "Rhodochrous".

Chemotaxonomic characters (the monosaccharide composition and diaminopimelic acid (DAP) of cell hydrolysates, the type of lipid LCN-A) were studied with bacteria of the "rhodochrous" complex and the Arthrobacter genus isolated from natural substrates as well as with collection strains of the genera Arthrobacter, Brevibacterium and Corynebacterium. If arabinose, galactose and meso-DAP were present in cell hydrolysates of these bacteria, they always contained lipid LCN-A. Bacteria of the "rhodochrous" complex can be differentiated from Corynebacterium sensu stricto by location of lipid LCN-A spots on chromatograms, and from the genera Brevibacterium (B. linens) and Arthrobacter, by the presence of arabinose, meso-DAP and lipid LCN-A in the cells. The representatives of two latter genera do not contain arabinose and lipid LCN-A in their cells.