Activity of alkanmonooxygenase alkB gene in strains of hydrocarbon-oxidizing bacteria isolated from petroleum products.

Alkanmonooxygenase enzymes AlkB and Cyp153 are responsible for the aerobic degradation of n-alkanes of petroleum and petroleum products. To prove the usage of n-alkanes from oil and petroleum products by hydrocarbon-oxidizing bacteria isolated from aviation kerosene TS-1 and automobile gasoline AI-95, the detection of the key genes alkB, Alk1, Alk2, Alk3 and Cyp153 encoding alkanmonooxygenases AlkB and Cyp153 (responsible for the oxidation of hydrocarbons with a certain chain length) was carried out. It was found that bacterial strains isolated from TS-1 jet fuel, except Deinococcus sp. Bi7, had at least one of the studied n-alkane degradation genes. The strains Sphingobacterium multivorum Bi2; Alcaligenes faecalis Bi3; Rhodococcus sp. Bi4; Sphingobacterium sp. Bi5; Rhodococcus erythropolis Bi6 contained the alkB gene. In the strains of hydrocarbon-oxidizing bacteria isolated from gasoline AI- 95, this alkanmonooxygenase gene was not detected. Using the real-time PCR method, the activity of the alkB gene in all bacterial strains isolated from petroleum products was analyzed and the number of its copies was determined. By real-time PCR using a primer with a different sequence of nucleotides to detect the alkB gene, its activity was established in all bacterial strains isolated from gasoline AI-95; besides, the strain Paenibacillus agaridevorans Bi11 was assigned to the group with a high level of its activity (1290 copies/ml). According to the assessment of the growth of isolated hydrocarbon-oxidizing bacteria on a solid Evans mineral medium with the addition of the model mixture of hydrocarbons, the strains were divided into three groups. The distributions of strains of hydrocarbon-oxidizing bacteria in the groups based on the activity of the alkB gene and groups formed based on the growth ability and use of the model mixture of hydrocarbons and petroleum products were found to be consistent. The results obtained indicate that we need to use a complex of molecular and physiological methods for a comprehensive analysis of the distribution of the studied genes in bacteria and to assess their activity in the strains of hydrocarbon-oxidizing bacteria capable of biodegradation of petroleum hydrocarbons.

[Structure of Epiphytic Bacterial Communities of Weeds].

Dynamics of the taxonomic structure of epiphytic bacterial communities of the rhizosphere and phyllosphere of seven weed species was studied. The major types of isolated organisms were identified using phenotypic and molecular biological approaches. Dispersion analysis revealed that the ontogenesis stage and plant organ were the factors with the greatest effect on the taxonomic structure of the communities. The dominant microorganisms of weeds were similar to those of cultivated plants. The minor components revealed in the spectra of bacterial communities of weeds belonged to poorly studied genera of chemolithotrophic proteobacteria.

[Characterization of the Structure of the Prokaryotic Complex of Antarctic Permafrost by Molecular Genetic Techniques].

A prokaryotic mesophilic organotrophic community responsible for 10% of the total microbial number determined by epifluorescence microscopy was reactivated in the samples ofAntarctic permafrost retrieved from the environment favoring long-term preservation of microbial communities (7500 years). No culturable forms were obtained without resuscitation procedures (CFU = 0). Proteobacteria, Actinobacteria, and Firmicutes were the dominant microbial groups in the complex. Initiation of the reactivated microbial complex by addition of chitin (0.1% wt/vol) resulted in an increased share of metabolically active biomass (up to 50%) due to the functional domination of chitinolytics caused by the target resource. Thus, sequential application of resuscitation procedures and initiation of a specific physiological group (in this case, chitinolytics) to a permafrost-preserved microbial community made it possible to reveal a prokaryotic complex capable of reversion of metabolic activity (FISH data), to determine its phylogenetic structure by metagenomic anal-ysis, and to isolate a pure culture of the dominant microorganism with high chitinolytic activity.

[Prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: detection by cultural and direct microscopic techniques].

The heterotrophic mesophilic component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. Application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved form subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of preserved fossil soil and permafrost samples collected in the Arctic zone. The differences in response to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.

[Ecophysiological Characteristics of actinomycetes of desert soils of Mongolia].

It is shown that the actinomycete complex in steppe-desert light brown salty soil of desert steppes of Mongolia is represented by the genera Streptomyces and Micromonospora. The species diversity of the genus Streptomyces, which dominates the complex, decreases with increasing osmolarity of the medium. The influence of environmental factors--temperature and osmolarity of medium--on the development of metabolically active members of the phylum Actinobacteria in the domain Bacteria of the prokaryotic microbial soil community was established. The proportion of metabolically active bacteria belonging to Actinobacteria increases with increasing osmolarity and incubation temperature of soil. The dominance of the filamentous metabolically active members of the phylum Actinobacteria over the unicellular organisms was shown. The halotolerant actinomycetes isolated from the steppe-desert soils were alkalotolerant, xerophilic, and thermotolerant and exhibited antimicrobial activity with respect to Gram-positive bacteria and actinomycetes.

[Effect of mechanical grinding of Sphagnum on the structure and physiological state of bacterial communities].

The microcosm method was used to demonstrate an increase in bacterial numbers and drastic changes in the taxonomic structure of saprotrophic bacteria as a result of mechanical grinding of Sphagnum moss. Ekkrisotrophic agrobacteria predominant in untreated moss were replaced by hydrolytic bacteria. Molecular biological approaches revealed such specific hydrolytic bacteria as Janthinobacterium agaricum and Streptomyces purpurascens among the dominant taxa. The application of kinetic technique for determination of the physiological state of bacteria in situ revealed higher functional diversity of hydrolytic bacteria in ground moss than in untreated samples. A considerable decrease of the C/N ratio in ground samples of living Sphagnum incubated using the microcosm technique indicated decomposition of this substrate.

[Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils].

With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.

[Monitoring of the chitinolytic microbial complex of the phylloplane].

A comprehensive study of chitinolytic microbial complexes of the phylloplane from cultured and forest plants has been conducted. An increase of the number and biomass of metabolically active cells of the representatives of the domain Bacteria and a decrease in fungal biomass in the experimental microcosms have been shown to occur after the introduction of chitin. The characteristic features of the taxonomic structure of metabolically active chitinolytic complexes of the phylloplane of the plants studied have been elucidated. Representatives of the phyla Proteobacteria, Bacteroidetes, and Verrucomicrobia have been shown to play important roles in the chitinolytic complexes of green leaf samples, while mycelial actinobacteria of the phylum Actinobacyteria played a similar role in needles of coniferous trees. A collection of chitinolytic microorganism cultures isolated from the phylloplane of different plant species has been created.

[Temperature as a factor of development of psychrotolerant mycelial bacteria complexes in soils of north regions].

It has been demonstrated that complexes of mycelial bacteria (actinomycetes), in which the amount of psychrotolerant actinomycetes reaches hundreds of thousands of CFU/g of the soil (frequently exceeding the portion of mesophilic forms), are developed in peat and podzolic soils of the tundra and taiga at low temperatures. As actinomycetes grow and develop in cold soils, their mycelium increases in length. Use of the molecular in situ hybridization method (fluorescent in situ hybridization, FISH) demonstrated that the portion of metabolically active mycelial actinobacteria exceeds the portion of unicellular actinobacteria in the Actinobacteria phylum. Specific peculiarities of psychrotolerant populations in relation to the spectrum of consumed substrates (histidine, mannitol, saccharose) were established by the method of multirespirometric testing.

[Thermophilic chitinolytic microorganisms of brown semidesert soil].

In brown semidesert soil, thermophilic prokaryotic organisms identified as Streptomyces roseolilacinus and Silanimonas lenta were shown to play the main role in chitin transformation at 50 degrees C. The phylogenetic positions of the isolated dominant chitinolytic microorganisms were determined on the basis of 16S rRNA gene sequencing. The consumption of chitin as a source of carbon and nitrogen by both the bacterium and the actinomycete was shown by considerable biomass accumulation, high emission of carbon dioxide, and presence in the medium of the chitinase exoenzyme.

[Methodological aspects of assessing chitin utilization by soil microorganisms].

A computational method for estimating specific activity of chitin decomposition by microorganisms is proposed. Spectrophotometric and gas chromatographic methods have been used to determine the rates of chitinase production, biomass accumulation, and carbon dioxide emission by pure cultures of microorganisms grown on a chitin-containing medium. Among dominants of the chitinolytic community of chernozem (Trichoderma viride, Stretomyces albolongus, Alcaligenes, and Arthrobacter), the highest chitinolytic activity is characteristic of prokaryotes. In brown desert-steppe soil, the main destructor prokaryotes are actinomycetes (S. roseolilacinus). The biomass of the fungus T. viride growth on the chitin-containing medium markedly exceeds that of prokaryotes, but the specific activity of respiration and chitinase production in actinomycetes S. roseolilacinus and S. albolongus is an order of magnitude higher than in T. viride.

[The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels].

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of -1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = -10 atm to P = -0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).

[Temperature as an autoecological factor of chitinolytic microbial complex formation in soils].

The dynamics of carbon dioxide emission from soil was studied during chitinolytic succession induced by humidification and chitin introduction at different temperatures (5, 27, and 50 degrees C) using gas chromatography. The abundance and biomass of the chitinolytic bacterial and actinomycete complex in soil were evaluated by luminescent microscopy. Active development of the chitinolytic microbial complexes was observed at all studied temperatures. The most active growth of chitinolytic microorganisms was observed at high temperature during early succession and at low temperature during late succession. High and low temperatures provided for active development of the chitinolytic microbial complex in soils confined to warm climatic zones (brown desert-steppe soil) and soils of temporary zones (gray forest soil). Actinomycetes demonstrated the most active growth among chitinolytic microorganisms in the studied soil samples both at low and high temperatures..

[Microbial transformation of chitin in soil under anaerobic conditions].

Anaerobic chitinolytic complex was studied in three soil types: chernozem, gray forest soil, and chestnut soil. The abundance and biomass of anaerobic chitinolytic microbial complex of fungi, bacteria, and actinomycetes were evaluated by luminescent microscopy. The dynamics of methane emission from soil during chitinolytic succession was studied by gas chromatography. All three studied microbial groups proved to participate in chitin transformation in soil under anaerobic conditions. The highest biomass growth was observed among prokaryotes, particularly actinomycetes, whose biomass doubled. The increase in methane emission during chitinolytic succession was most pronounced in soils with low organic matter content.

[Succession of chitinolytic microorganisms in chernozem soil].

The chitinolytic prokaryotic and eukaryotic microbial complex of chernozem soil has been investigated in the course of a succession initiated by the introduction of chitin and humidification. The dynamics of the cell numbers of chitinolytic microorganisms and of their biomass was assessed by fluorescent microscopy and by inoculation of selective media. Emission of carbon dioxide and nitrous oxide, as well as dinitrogen fixation, was assessed by gas chromatography. It was found that, when the succession was initiated by the introduction of both chitin and humidification, it resulted in greater cell numbers and biomass of chitinolytic microorganisms and higher levels of CO2 and N2O emission and of nitrogen fixation than when the succession was initiated by humidification alone. As compared to the control samples, a significant (twofold) increase in the prokaryote cell number and biomass was found on the fourth day of the succession initiated by humidification and introduction of chitin. One week after the initiation of succession, the fungal biomass and length of mycelium were twice as high as those in the control samples. These results led to the conclusion that chitin utilization in chernozem soil starts during the initial stages of succession and is performed by both prokaryotic and eukaryotic microorganisms.

[A chitinolytic actinomycete complex in black soil]. / Khitinoliticheskii aktinomitsetnyi komplex chernozema.

A chitinolytic actinomycete complex in chernozem soil has a specific taxonomic composition, which differs from that of the actinomycete complex which is typically isolated on standard nutrient media containing sugars and organic acids as carbon sources. The actinomycete complex that was isolated by using nutrient media with chitin as the source of carbon and nitrogen was dominated by representatives of the genus Streptosporangium, and the actinomycete complex that was isolated by using nutrient media with sugars and organic acids as the carbon sources was dominated by representatives of the genus Streptomyces. The confirmation to the ability of actinomycetes to utilize chitin as a sole source of carbon and nitrogen came from the augmented length and biomass of the mycelium, the increased number and biomass of the actinomycete spores, the production of carbon dioxide, and the accumulation of NH4+ ions in the culture liquid of the actinomycetes that are grown in the nutrient media with chitin.