D. A. Ivasenko

Copper resistance in Desulfovibrio strain R2.

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria.

Precipitation of Cu-sulfides by copper-tolerant Desulfovibrio isolates

The purpose of this work was to isolate Cu-tolerant sulfate-reducers that could be used to produce copper sulfides under pure culture conditions. Three sulfate-reducing bacteria were isolated from wastewater effluents of a zinc-smelter in the Urals and their tolerance to copper varied between 325 and 2600 mg Cu l −1 . Analysis of 16S rRNA gene sequences placed the isolates in the genus Desulfovibrio. The isolates showed pronounced saccharolytic growth. Growing cultures precipitated Cu 2+ as covellite (CuS) within less than a week. Extended incubation for 1 month lead to the formation of chalcocite (Cu2S) and chalcopyrite (CuFeS2).

Effect of metal concentration on the microbial community in acid mine drainage of a polysulfide ore deposit

The composition of microbial communities of acid mine drainage (AMD) in two wells drilled in the terrace of the Sherlovaya Gora open-cast polymetallic ores mine (Eastern Siberia) was studied. While drainage water filling two wells, ShG14-1 and ShG14-8, had similar values of pH (2.6), Eh (447–494 mV), and temperature (6.5°C), the water in the first well contained more metals and sulfate. The water in ShG14-1 and ShG14-8 contained, respectively, 1898 and 434 mg/L of iron, 734 and 49 mg/L of manganese, 81 and 7 mg/L of copper, 3597 and 787 mg/L of zinc, and 15990 and 3632 mg/L of sulfate. Molecular analysis of the microbial communities was performed using pyrosequencing of the 16S rRNA gene fragments. The ShG14-8 microbial community included such bacterial taxa typically found in AMD sites as Gallionella (38.8% of total 16S rRNA gene sequences), Ferrovum (4.4%), Acidiphilium (9.1%), Acidisphaera (8.2%), Acidithiobacillus (7.2%), and Leptospirillum (4.6%). In the ShG14-1 sample with higher content of metals, strict acidophiles Acidithiobacillus (16.0%) and Leptospirillum (25.4%) were more abundant, while Gallionella, Ferrovum, Acidiphilium and Acidisphaera were almost absent. Ferrimicrobium (16.8%) and Sulfobacillus (1.4%) were detected in ShG14-1 but not in ShG14-8. Thus, the increase in concentration of metals in the acid mine drainage water under the same value of total acidity substantially altered the composition of the microbial community, preventing the development of “moderate” alpha- and beta-proteobacterial acidophiles, so that the community was dominated by the bacteria characteristic of the extremely acidic drainage waters.

A novel uncultured bacterium of the family Gallionellaceae: Description and genome reconstruction based on metagenomic analysis of microbial community in acid mine drainage

Drainage waters at the metal mining areas often have low pH and high content of dissolved metals due to oxidation of sulfide minerals. Extreme conditions limit microbial diversity in such habitats. A microbial community of cold acid mine drainage (6.5°C, pH 2.65) at the Sherlovaya Gora polymetallic open-cast mine (Transbaikal region, Eastern Siberia, Russia) was studied using metagenomic techniques. Most of microorganisms belonged to a single uncultured lineage representing a new species of the Betaproteobacteria genus Gallionella. Bacteria of the genera Thiobacillus, Acidobacterium, Acidisphaera, and Acidithiobacillus were the minor components of the community. Almost complete (3.4 Mb) composite genome of the new bacterial lineage designated Candidatus Gallionella acididurans ShG14-8 was reconstructed using metagenomic data. Genome analysis revealed that Fe(II) oxidation probably involved the cytochromes localized on the outer cell membrane. The electron transport chain included NADH dehydrogenase, a cytochrome bc1 complex, an alternative complex III, and bd-, cbb3-, and bo3-types cytochrome oxidases. Oxidation of reduced sulfur compounds probably involved the Sox system, sulfide–quinone oxidoreductase, adenyl sulfate reductase, and sulfate adenyltransferase. The genes involved in autotrophic carbon assimilation via the Calvin cycle were present, while no pathway for nitrogen fixation was revealed. High numbers of RND metal transporters and P type ATPases were probably responsible for resistance to heavy metals. The new microorganism was an aerobic chemolithoautotroph that belonged to the group of psychrotolerant iron- and sulfur-oxidizing acidophiles of the family Gallionellaceae, which are widely distributed in acid mine drainage.

Uncultured bacteria and methanogenic archaea predominate in the microbial community of Western Siberian deep subsurface aquifer

V. V. Kadnikova, b, *, Yu. A. Frankc, A. V. Mardanovb, A. V. Beletskiib, D. A. Ivasenkoc, N. V. Pimenovd, O. V. Karnachukc, and N. V. Ravina, b aFaculty of Biology, Lomonosov Moscow State University, Moscow, Russia bInstitute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia cTomsk State University, Russia dWinogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia *e-mail: vkadnikov@bk.ru Received October 28, 2016

Variability of the composition of the microbial community of the deep subsurface thermal aquifer in Western Siberia

The deep subsurface biosphere is one of the least studied ecosystems on Earth, containing communities of extremophilic microorganisms. The present work was aimed at molecular genetic characterization of microbial communities of underground thermal waters in Western Siberia, lying at depths of 2–3 km. Water samples were collected from the 5P oil-exploration well, drilled to a depth of 2.8 km near the village Chazhemto (Tomsk region). The water had a temperature of about 20°C, a neutral pH and a low redox potential (–304 mV). Underground aquifers have a complex structure and may contain both planktonic microorganisms and those immobilized on the surface of rocks in the form of biofilms, which may be washed out and detected in the water flowing out of the well. Community composition was analyzed by amplification and pyrosequencing of the 16S rRNA gene fragments in seven water samples taken at different times during 26 hours. Bacteria, which constituted about half of the community, were represented mainly by uncultured lineages of the phyla Firmicutes, Ignavibacteria, Chloroflexi, Bacteroidetes, and Proteobacteria. Archaea belonged mainly to known methanogens of the genera Methanothermobacter, Methanosaeta, and Methanomassiliicoccus. Analysis of the samples taken at different times revealed large variations in the content of most groups of bacteria, with a decrease in Firmicutes abundance accompanied by an increase in the shares of Ignavibacteria and Chloroflexi. The share of archaea of the genus Methanothermobacter varied slightly during the day, while significant variations were observed for the phylotypes assigned to Methanosaeta and Methanomassiliicoccus. Hydrogenotrophic archaea of the genus Methanothermobacter are probably a permanent component of the microbial community occurring in the planktonic state, while most of the identified groups of bacteria are present in biofilms or spatially localized parts of the underground water reservoir, the material of which accidentally enters the well.

Lignite coal burning seam in the remote Altai Mountains harbors a hydrogen-driven thermophilic microbial community

Thermal ecosystems associated with underground coal combustion sites are rare and less studied than geothermal features. Here we analysed microbial communities of near-surface ground layer and bituminous substance in an open quarry heated by subsurface coal fire by metagenomic DNA sequencing. Taxonomic classification revealed dominance of only a few groups of Firmicutes. Near-complete genomes of three most abundant species, ‘Candidatus Carbobacillus altaicus’ AL32, Brockia lithotrophica AL31, and Hydrogenibacillus schlegelii AL33, were assembled. According to the genomic data, Ca. Carbobacillus altaicus AL32 is an aerobic heterotroph, while B. lithotrophica AL31 is a chemolithotrophic anaerobe assimilating CO2 via the Calvin cycle. H. schlegelii AL33 is an aerobe capable of both growth on organic compounds and carrying out CO2 fixation via the Calvin cycle. Phylogenetic analysis of the large subunit of RuBisCO of B. lithotrophica AL31 and H. schlegelii AL33 showed that it belongs to the type 1-E. All three Firmicutes species can gain energy from aerobic or anaerobic oxidation of molecular hydrogen, produced as a result of underground coal combustion along with other coal gases. We propose that thermophilic Firmicutes, whose spores can spread from their original geothermal habitats over long distances, are the first colonizers of this recently formed thermal ecosystem.

Sulfate-reducing bacteria in the microbial community of acidic drainage from a gold deposit tailing storage

A. V. Mardanova, *, A. V. Beletskiia, D. A. Ivasenkob, N. V. Pimenovc, O. V. Karnachukb, and N. V. Ravina aInstitute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia bTomsk State University, Tomsk, Russia cWinogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia *e-mail: mardanov@biengi.ac.ru Received September 29, 2016