Olga V. Shubenkova

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal.

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.

Faunal communities at sites of gas- and oil-bearing fluids in Lake Baikal

Macro- and meiofaunal communities were examined at four geomorphologically distinct sites with different gas- and oil-bearing fluid characteristics in the northern, central and southern basins of Lake Baikal. All sites had elevated concentrations of bicarbonate, nitrate, sulphate and chloride ions in pore fluids, with highest values at the Frolikha vent. Elevated levels of iron ions were found in pore waters of the St. Petersburg methane seep and the Gorevoy Utes oil seep. The chemical composition of pore waters at the Malenky mud volcano was similar to that reported in earlier work. Consistent with published data, the Frolikha vent (northern basin) and the St. Petersburg methane seep (central basin) were characterised by methane of mixed genesis (thermogenic + biogenic), whereas the methane source was mainly thermogenic at the Gorevoy Utes oil seep (central basin) and biogenic at the Malenky mud volcano (southern basin). In contrast to marine seep ecosystems, the macrofauna was dominated only by amphipods, giant planarians and oligochaetes, whereas bivalves were absent; the meiofauna was similar to its marine counterpart, being dominated by nematodes, cyclops, harpacticoids and ostracods. A statistically significant positive relationship was revealed between faunal abundance and the availability of bacterial mats on seep sediments. Moreover, ANOVA tests showed significant increases in both meiozoobenthic and macrozoobenthic densities at “hot spot” vent/seep sites relative to discharge-free reference sites. The isotopic composition of carbon and nitrogen at various trophic levels of these benthic vent/seep communities was found to differ markedly from that reported by earlier studies for the pelagic and other benthic food webs in Lake Baikal. As in marine seeps, the macrofauna had variable isotopic signatures. Light δ13C and δ15N values suggest the utilization of chemosynthetically fixed and/or methane-derived organic matter. By contrast, the heavy δ13C signatures of some mobile amphipods likely reflect consumption of photosynthetically derived carbon. These findings would at least partly explain why Lake Baikal is a notable outlier in global temperature–biodiversity patterns, exhibiting the highest biodiversity of any lake worldwide but at an extremely cold average temperature.

The First Results of an Investigation into the Phylogenetic Diversity of Microorganisms in Southern Baikal Sediments in the Region of Subsurface Discharge of Methane Hydrates

Phylogenetic analysis of the bacterial communities in Lake Baikal bottom sediments in the region of subsurface methane hydrate discharge has been carried out using data on 16S rRNA sequences. The composition of these microbial communities is shown to be different in different horizons. Methanotrophic bacteria are found in the surface layer (0–5 cm), and uncultured bacteria constitute a great portion of this population. In deeper sediment layers (92–96 cm), a change in the microbial community occurs; specifically, a decreased homology with the known sequences is observed. The new sequences form separate clusters on a phylogenetic tree, indicating the possibly endemic nature of the bacteria revealed. Organisms related to the genus Pseudomonas constitute the main portion of the population. An archaea-related sequence was found in a horizon containing gas hydrate crystals (100–128 cm). Uncultured bacteria remain predominant.

Microbial community of reduced pockmark sediments (Gdansk Deep, Baltic Sea)

The microbial community of reduced pockmark sediments in the Russian sector of the Gdansk Deep, Baltic Sea, was investigated by molecular biological techniques. Fluorescent in situ hybridization was used to determine the numbers of eubacteria, archaea, and sulfate-reducing bacteria. Eubacteria were found to predominate in the upper 10 cm of the sediment (up to 5.3 × 109 cells/g wet sediment), while the number of archaea increased in the 10- to 30-cm layers (up to 2.8 × 109 cells/g wet sediment, which is higher than the number of eubacteria in the same horizons). Analysis of 16S rRNA gene fragments revealed members of the following phyla: Proteobacteria, Chloroflexi, Firmicutes, Planctomycetales, and high-G + C gram-positive bacteria. Sulfate-reducing bacteria (SRBs) of the families Syntrophaceae, Desulfuromonadaceae, and Actinobacteria of the genera Kocuria and Rothia were the predominant groups. Molecular probes were used to determine predominance of Desulfovibrionales in the SRB enrichment cultures obtained from different horizons of pockmark sediments. Three archaeal phylotypes were revealed, belonging to Euryarchaeota. One of these fell into the group of uncultured methanotrophic archaea (ANME-1a), while the other two were most closely related to uncultured methanogens.

Jelly-like Microbial Mats over Subsurface Fields of Gas Hydrates at the St. Petersburg Methane Seep (Central Baikal)

Jelly-like microbial mat samples were collected from benthic surfaces at the St. Petersburg methane seep located in Central Baikal. The concentrations of certain ions, specifically chloride, bromide, sulphate, acetate, iron, calcium, and magnesium, were 2–40 times higher in the microbial mats than those in the pore and bottom water. A large number of diatom valves, cyanobacteria, and filamentous, rod-shaped and coccal microorganisms were found in the samples of bacterial mats using light, epifluorescence and scanning microscopy.Comparative analysis of a 16S rRNA gene fragment demonstrated the presence of bacteria and archaea belonging to the following classes and phyla: Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia, Cytophaga-Flavobacteria-Bacteroidetes, Cyanobacteria, and Euryarchaeota. The chemical composition and phylogenetic structure of the microbial community showed that the life activity of the mat occurs due to methane and its derivatives involved. Values of δ13C for the microbial mats varied from −73.6‰ to −65.8‰ and for animals from −68.9‰ to −36.6‰. Functional genes of the sequential methane oxidation (pmoA and mxaF) and different species of methanotrophic bacteria inhabiting cold ecosystems were recorded in the total DNA. Like in other psychroactive communities, the destruction of organic substances forming formed as a result of methanotrophy, terminates at the stage of acetate formation in the microbial mats of Lake Baikal (1,400 m depth). Its further transformation is limited by hydrogen content and carried out in the subsurface layers of sediments.

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).

Transformation of Organic Matter by a Microbial Community in Sediments of Lake Baikal under Experimental Thermobaric Conditions of Protocatagenesis

ABSTRACT Early diagenesis of organic matter in bottom sediments of Lake Baikal is a focus of many geochemical studies, because it is one of the few sites of petroleum formation in a nonmarine environment. Although Baikal is a rift lake and considered one of the prospective fields for deep biosphere investigations, the transformation processes of organic matter by microbial communities from deep bottom sediments and likely entering of the microorganisms from deep sediments into the near-surface sediments were not previously studied in Lake Baikal. The natural microbial community from near-surface sediments of the cold methane seep Goloustnoe (Southern Baikal Basin) was incubated with methane and the diatom Synedra acus at 80°C and 49.5 atm to simulate catagenesis. The 11-month incubation yielded the enrichment culture of viable thermophilic microorganisms. Their presence in low-temperature sediment layers may be indicative of their migration through fault zones together with gas-bearing fluids. After culturing, molecular biological methods allowed for the detection of both widespread microorganisms and unique clones whose phylogenetic status is currently unknown. The sediment after the experiment showed the formation of polycyclic aromatic hydrocarbon, retene. Retene can be either a conifer or algal biomarker, thus, interpretation of paleoclimate data is tenuous.

Isolation of total bacterial DNA for ecological characterization of bottom sediments of Lake Baikal

It is known that the conventional microbiological methods of investigation reveal not more than 1% of the species of microorganisms actually inhabiting one biotope or another. The most complete characterization of the microbial community is achieved by investigating DNA isolated from the samples differing from each other in ecological conditions. The sediments of Lake Baikal are characterized by a high content of humic compounds which hinders molecular biological analyses. Because of this, the procedure of DNA isolation is a step o critical importance for the analysis of bottom sediments.In the present work we compare the methods of isolation of the whole bacterial DNA from the bottom sediments of Lake Baikal. The most efficient method allowing us to obtained DNA preparations for further molecular investigation was chosen.

[Comparative molecular biological analysis of the microbial community of the holocene and pleistocene deposits of Posol'skaya Shoal, Lake Baikal].

The bacterial diversity was studied in sediment layers of Posol’skaya Shoal station (Southern Baikal) belonging to different periods. A set of primers specific to individual bacterial groups was used to analyze the 16S rRNA gene fragments. The bacterial diversity in the Holocene deposits was found to be higher than in the Pleistocene ones. In the upper sediments, a positive PCR reaction with bacterial primers and with specific cyanobacterial and archaebacterial primers was detected. The following phylogenetic groups were revealed in the microbial community of the surface horizon: green nonsulfur bacteria, δ-proteobacteria, β-proteobacteria (Nitrospirae), α-proteobacteria, acidobacteria, crenarchaeota, euryarchaeota, and groups of uncultured bacteria. From the DNA of the Pleistocene deposits, the PCR product was obtained only with bacterial primers. The representatives of the genus Pseudomonas were most closely related to the sequences obtained (95–97% homology).

Diversity of cultured aerobic organisms in the areas of natural oil seepage on Lake Baikal

The diversity of cultured aerobic organisms collected from water samples and bottom sediment from two areas of natural oil seepage on Lake Baikal has been researched. Representatives of Alpha-, Beta-, and Gammaproteobacteria have been found in samples collected near the Bol’shaya Zelenovskaya River mouth, while near Cape Gorevoi Utes Betaproteobacteria were absent. Most cultures are characterized by a sufficiently high homology level (96–100%) with nucleotide sequences from the international database.