E. V. Mamaeva

Diversity of cultivable bacteria isolated from the water column and bottom sediments of the Kara Sea shelf

In this work, the results of microbiological and molecular genetic investigation of the microorganisms inhabiting the Kara Sea and the adjacent Yenisei and Gydanskii Bays are presented. The microorganisms isolated from the samples collected in the studied area belonged to 4 phyla and 11 genera. Bacteria of two phyla, Firmicutes and Actinobacteria, prevailed; representatives of the Gammaproteobacteria and Bacteroidetes were isolated as well. According to their phenotypic properties, the obtained pure cultures were classified with the genera Streptomyces, Rhodococcus, Micrococcus, Bacillus, Pseudomonas, Acinetobacter, Flavobacterium, and Marinococcus. Analysis of the obtained nucleotide sequences of the 16S rRNA genes confirmed that the isolates belonged to the genus Bacillus. One strain was reidentified as Brevibacillus laterosporus, and two strains were identified Aeromonas piscicola and Plantibacter sp. The results of the study of the enzymatic activity of the obtained pure psychrotolerant cultures suggest that the microbial community is actively involved in the destruction processes occurring in the studied area.

Diversity of Archaea in Bottom Sediments of the Discharge Areas With Oil- and Gas-Bearing Fluids in Lake Baikal

ABSTRACT Using massively parallel sequencing (the Roche 454 platform) we have studied the diversity of archaeal 16S rRNA gene sequences in oxic and anoxic sediments at six sites in Lake Baikal with oil- and gas-bearing fluids discharge. Archaeal communities appeared to be represented mainly by five phyla: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Bathyarchaeota (miscellaneous Crenarchaeotic group), and Woesearchaeota (deep sea hydrothermal vent group 6). Among them we detected sequences of methanogens of the orders Methanomicrobiales, Methanosarsinales, Methanococcales, as well as representatives of the following uncultured archaeal lineages: Group C3, Marine Benthic Group D, and Terrestrial Miscellaneous Group. We have also identified sequences of ammonia-oxidizing archaea of the phyla Crenarchaeota and Thaumarchaeota. Phylogenetic analysis showed the presence ANME-2d-related sequences. However, the analysis of mcrA genes libraries has not revealed typical representatives of ANME groups. Comparison of amplicon libraries 16S rRNA gene fragments from different samples proved the widespread presence of previously detected Baikal archaeal lineages, which are members of the phylum Crenarchaeota and Thaumarchaeota (formerly Group C3 of Crenarchaeota).

Metagenomic Analysis of Microbial Communities of the Sediments of the Kara Sea Shelf and the Yenisei Bay

Microbial diversity in the sediments of the Kara Sea shelf and the southern Yenisei Bay, differing in pore water mineralization, was studied using massive parallel pyrosequencing according to the 454 (Roche) technology. Members of the same phyla (Cyanobacteria, Verrucomicrobia, Actinobacteria, Proteobacteria, and Bacteroidetes) predominated in bacterial communities of the sediments, while their ratio and taxonomic composition varied within the phyla and depended on pore water mineralization. Increasing salinity gradient was found to coincide with increased share of the γ-Proteobacteria and decreased abundance of α- and β-Proteobacteria, as well as of the phyla Verrucomicrobia, Chloroflexi, Chlorobi, and Acidobacteria. Archaeal diversity was lower, with Thaumarchaeota predominant in the sediments with high and low mineralization, while Crenarchaeota predominated in moderately mineralized sediments. Microbial communities of the Kara Sea shelf and Yenisei Bay sediments were found to contain the organisms capable of utilization of a broad spectrum of carbon sources, including gaseous and petroleum hydrocarbons.

Phylogenetic diversity of microbial communities of the Posolsk Bank bottom sediments, Lake Baikal

Massive parallel sequencing (the Roche 454 platform) of the 16S rRNA gene fragments was used to investigate microbial diversity in the sediments of the Posolsk Bank cold methane seep. Bacterial communities from all sediment horizons were found to contain members of the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, Nitrospirae, Chloroflexi, Proteobacteria, and the candidate phyla Aminicenantes (OP8) and Atribacteria (OP9). Among Bacteria, members of the Chloroflexi and Proteobacteria were the most numerous (42 and 46%, respectively). Among archaea, the Thaumarchaeota predominated in the upper sediment layer (40.1%), while Bathyarchaeota (54.2%) and Euryarchaeota (95%) were predominant at 70 and 140 cm, respectively. Specific migration pathways of fluid flows circulating in the zone of gas hydrate stability (400 m) may be responsible for considerable numbers of the sequences of Chloroflexi, Acidobacteria, and the candidate phyla Aminicenantes and Atribacteria in the upper sediment layers and of the Deinococcus-Thermus phylum in deep bottom sediments.

Microbial uncultured community of bottom sediments from the bays of Gydan and Yenisei of the Kara Sea

Using methods of molecular biology (PCR and cloning), we studied the diversity of microorganisms in the surface layers of bottom sediments from the bays of Gydan and Yenisei of the Kara Sea, which have different component composition of the pore water and mineralization level. Representatives of the domains Bacteria and Archaea were identified based on the analysis of the 16S rRNA gene fragment nucleotide sequences. The composition of the community of microorganisms in the bottom sediments changed with the changing salinity gradient of the pore waters. The phylogenetic analysis of the nucleotide sequences showed that the composition of the microbial communities in the southern parts of these bays was affected by fresh-water flows from rivers and streams from the lakes within the catchment area, whereas that in the northern parts was influenced by sea waters. The results indicate the presence of bacteria in the bottom sediments that are capable of using a wide range of substrates as a carbon source including hydrocarbons and organochlorine and aromatic compounds. These data can also indicate the presence of different pollutants in the sediments of these areas and the potential ability of bacteria to degrade chemical compounds that enter the waters and bottom sediments of the Kara Sea.

Anaerobic Methane Oxidation in Enrichment Cultures from Deep Sediments of a Mud Volcano Peschanka (South Baikal)

Under laboratory conditions, the microbial communities of bottom sediments of a mud volcano Peschanka (Lake Baikal) were found to carry out anaerobic methane oxidation (AOM). After 16 days of anaerobic cultivation of the enrichment cultures, methane content in the gas phase decreased, and microbial consortia were established. The content of carbon, nitrogen, and oxygen determined by energy dispersive X-ray spectroscopy (EDS) was higher than in the nearby sediment particles. The presence of bacteria of the NC10 phylum and archaea of the ANME-2d cluster was established by fluorescent in situ hybridization (FISH).

Composition of Microbial Communities in Sediments from Southern Baikal Containing Fe/Mn Concretions

Massive parallel sequencing of the 16S rRNA gene fragments was used to investigate the composition and diversity of microbial communities in sediments from Southern Baikal to a depth of 9 cm with 1-cm step. In the layers from the sediment surface to the lower border of oxygen penetration (2 cm), organotrophic bacteria with high similarity to the heterotrophic species Luteolibacter luojiensis constituted the largest fraction of the community. In the formation zone of Fe/Mn crusts (3–5 cm), Proteobacteria and Actinobacteria predominated in the community, while the share of Cyanobacteria was considerable. The lower reduced layers showed an increased contribution of the Bacteroidetes, while the shares of the taxa predominant in the higher layers remained significant. Analysis of archaeal 16S rRNA gene amplicons revealed predominance of the soil and aquatic Thaumarchaeota (Marine Group I lineage), which are involved in anaerobic ammonium oxidation, practically in all sediment layers. The buried oxidized layer (6–7 cm), where members of the uncultured Marine Benthic Group D lineage of the order Thermoplasmatales (Euryarchaeota) predominated, was an exception in this regard. Small numbers of archaea of the Baikal-1 lineages (below 1%) were observed in the communities from the 6–7 and 7–8 cm layers, while the archaea involved in anaerobic methane oxidation (including the ANME-2d group) were not detected.

The Ability of Microbial Community of Lake Baikal Bottom Sediments Associated with Gas Discharge to Carry Out the Transformation of Organic Matter under Thermobaric Conditions.

The ability to compare the composition and metabolic potential of microbial communities inhabiting the subsurface sediment in geographically distinct locations is one of the keys to understanding the evolution and function of the subsurface biosphere. Prospective areas for study of the subsurface biosphere are the sites of hydrocarbon discharges on the bottom of the Lake Baikal rift, where ascending fluxes of gas-saturated fluids and oil from deep layers of bottom sediments seep into near-surface sediment. The samples of surface sediments collected in the area of the Posolskaya Bank methane seep were cultured for 17 months under thermobaric conditions (80°C, 5 MPa) with the addition of complementary organic substrate, and a different composition for the gas phase. After incubation, the presence of intact cells of microorganisms, organic matter transformation and the formation of oil biomarkers was confirmed in the samples, with the addition of Baikal diatom alga Synedra acus detritus, and gas mixture CH4:H2:CO2. Taxonomic assignment of the 16S rRNA sequence data indicates that the predominant sequences in the enrichment were Sphingomonas (55.3%), Solirubrobacter (27.5%) and Arthrobacter (16.6%). At the same time, in heat-killed sediment and in sediment without any additional substrates, which were cultivated in a CH4 atmosphere, no geochemical changes were detected, nor the presence of intact cells and 16S rRNA sequences of Bacteria and Archaea. This data may suggest that the decomposition of organic matter under culturing conditions could be performed by microorganisms from low-temperature sediment layers. One possible explanation of this phenomenon is migration of the representatives of the deep thermophilic community through fault zones in the near surface sediment layers, together with gas-bearing fluids.