Yulia A. Frank

Sulfate Reduction Potential in Sediments in the Norilsk Mining Area, Northern Siberia

Abstract The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a 35SO4 2− tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm−3 day−1. Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 107 cells ml−1. The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28°C vs. 4°C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4°C, but they were also found in mesophilic enrichments.

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.

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs, Western Siberia, Russia

ABSTRACT The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.

Stable and Variable Parts of Microbial Community in Siberian Deep Subsurface Thermal Aquifer System Revealed in a Long-Term Monitoring Study

The goal of this work was to study the diversity of microorganisms inhabiting a deep subsurface aquifer system in order to understand their functional roles and interspecies relations formed in the course of buried organic matter degradation. A microbial community of a deep subsurface thermal aquifer in the Tomsk Region, Western Siberia was monitored over the course of 5 years via a 2.7 km deep borehole 3P, drilled down to a Palaeozoic basement. The borehole water discharges with a temperature of ca. 50°C. Its chemical composition varies, but it steadily contains acetate, propionate, and traces of hydrocarbons and gives rise to microbial mats along the surface flow. Community analysis by PCR-DGGE 16S rRNA genes profiling, repeatedly performed within 5 years, revealed several dominating phylotypes consistently found in the borehole water, and highly variable diversity of prokaryotes, brought to the surface with the borehole outflow. The major planktonic components of the microbial community were Desulfovirgula thermocuniculi and Methanothermobacter spp. The composition of the minor part of the community was unstable, and molecular analysis did not reveal any regularity in its variations, except some predominance of uncultured Firmicutes. Batch cultures with complex organic substrates inoculated with water samples were set in order to enrich prokaryotes from the variable part of the community. PCR-DGGE analysis of these enrichments yielded uncultured Firmicutes, Chloroflexi, and Ignavibacteriae. A continuous-flow microaerophilic enrichment culture with a water sample amended with acetate contained Hydrogenophilus thermoluteolus, which was previously detected in the microbial mat developing at the outflow of the borehole. Cultivation results allowed us to assume that variable components of the 3P well community are hydrolytic organotrophs, degrading buried biopolymers, while the constant planktonic components of the community degrade dissolved fermentation products to methane and CO2, possibly via interspecies hydrogen transfer. Occasional washout of minor community components capable of oxygen respiration leads to the development of microbial mats at the outflow of the borehole where residual dissolved fermentation products are aerobically oxidized. Long-term community analysis with the combination of molecular and cultivation techniques allowed us to characterize stable and variable parts of the community and propose their environmental roles.

Characterization and Genome Analysis of the First Facultatively Alkaliphilic Thermodesulfovibrio Isolated from the Deep Terrestrial Subsurface

Members of the genus Thermodesulfovibrio belong to the Nitrospirae phylum and all isolates characterized to date are neutrophiles. They have been isolated from terrestrial hot springs and thermophilic methanogenic anaerobic sludges. Their molecular signatures have, however, also been detected in deep subsurface. The purpose of this study was to characterize and analyze the genome of a newly isolated, facultatively alkaliphilic Thermodesulfovibrio from a 2 km deep aquifer system in Western Siberia, Russia. The new isolate, designated N1, grows optimally at pH 8.5 and at 65°C. It is able to reduce sulfate, thiosulfate or sulfite with a limited range of electron donors, such as formate, pyruvate, and lactate. Analysis of the 1.93 Mb draft genome of strain N1 revealed that it contains a set of genes for dissimilatory sulfate reduction, including sulfate adenyltransferase, adenosine-5′-phosphosulfate reductase AprAB, membrane-bound electron transfer complex QmoABC, dissimilatory sulfite reductase DsrABC, and sulfite reductase-associated electron transfer complex DsrMKJOP. Hydrogen turnover is enabled by soluble cytoplasmic, membrane-linked, and soluble periplasmic hydrogenases. The use of thiosulfate as an electron acceptor is enabled by a membrane-linked molybdopterin oxidoreductase. The N1 requirement for organic carbon sources corresponds to the lack of the autotrophic C1-fixation pathways. Comparative analysis of the genomes of Thermodesulfovibrio (T. yellowstonii, T. islandicus, T. àggregans, T. thiophilus, and strain N1) revealed a low overall genetic diversity and several adaptive traits. Consistent with an alkaliphilic lifestyle, a multisubunit Na+/H+ antiporter of the Mnh family is encoded in the Thermodesulfovibrio strain N1 genome. Nitrogenase genes were found in T. yellowstonii, T. aggregans, and T. islandicus, nitrate reductase in T. islandicus, and cellulose synthetase in T. aggregans and strain N1. Overall, our results provide genomic insights into metabolism of the Thermodesulfovibrio lineage in microbial communities of the deep subsurface biosphere.

Genomic insights into a new acidophilic, copper-resistant Desulfosporosinus isolate from the oxidized tailings area of an abandoned gold mine

Microbial sulfate reduction in acid mine drainage is still considered to be confined to anoxic conditions, although several reports have shown that sulfate-reducing bacteria occur under microaerophilic or aerobic conditions. We have measured sulfate reduction rates of up to 60 nmol S cm(-3) day(-1) in oxidized layers of gold mine tailings in Kuzbass (SW Siberia). A novel, acidophilic, copper-tolerant Desulfosporosinus sp. I2 was isolated from the same sample and its genome was sequenced. The genomic analysis and physiological data indicate the involvement of transporters and additional mechanisms to tolerate metals, such as sequestration by polyphosphates. Desulfosporinus sp. I2 encodes systems for a metabolically versatile life style. The genome possessed a complete Embden-Meyerhof pathway for glycolysis and gluconeogenesis. Complete oxidation of organic substrates could be enabled by the complete TCA cycle. Genomic analysis found all major components of the electron transfer chain necessary for energy generation via oxidative phosphorylation. Autotrophic CO2 fixation could be performed through the Wood-Ljungdahl pathway. Multiple oxygen detoxification systems were identified in the genome. Taking into account the metabolic activity and genomic analysis, the traits of the novel isolate broaden our understanding of active sulfate reduction and associated metabolism beyond strictly anaerobic niches.

An Acidophilic Desulfosporosinus Isolated from the Oxidized Mining Wastes in the Transbaikal Area

Dissimilatory sulfate reduction plays an important role in removal of dissolved metals from acidic mine waters. Although this process was convincingly shown to occur in acidic waste of metal recovery, few isolates of acidophilic/acid-tolerant sulfate reducers are known. We isolated a new acidophilic sulfidogen, strain BG, from the oxidized acidic waste of the Bom-Gorkhon tungsten deposit, Transbaikalia, Russia. Phylogenetic analysis of its 16S rRNA gene sequence made it possible to identify it as a member of the genus Desulfosporosinus. Unlike other known acidophilic sulfate reducers of this genus, strain BG was tolerant to high copper concentrations (up to 5 g/L), could grow on organic acids at low ambient pH, and formed crystalline copper sulfides (covellite and chalcopyrite). Molecular analysis of the phylotypes predominating in oxidized waste and in enrichment cultures confirmed the presence of various Desulfosporosinus strains.

A metagenomic window into the 2-km-deep terrestrial subsurface aquifer revealed multiple pathways of organic matter decomposition

We have sequenced metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia. Our goal was the recovery of near-complete genomes of the community members to enable accurate reconstruction of metabolism and ecological roles of the microbial majority, including previously unstudied lineages. The water, obtained via a 2.6 km deep borehole 1-R, was anoxic, with a slightly alkaline pH, and a temperature around 45°C. Microbial community, as revealed by 16S rRNA gene profiling over 2 years, mostly consisted of sulfate-reducing Firmicutes and Deltaproteobacteria, and uncultured lineages of the phyla Chlorofexi, Ignavibacteriae and Aminicenantes (OP8). 25 composite genomes with more than 90% completeness were recovered from metagenome and used for metabolic reconstruction. Members of uncultured lineages of Chlorofexi and Ignavibacteriae are likely involved in degradation of carbohydrates by fermentation, and are also capable of aerobic and anaerobic respiration. The Chlorofexi bacterium has the Wood-Ljungdahl pathway of CO2 fixation. The recently identified candidate phylum Riflebacteria accounted for 5%-10% of microbial community. Metabolic reconstruction of a member of Riflebacteria predicted that it is an anaerobe capable to grow on carbohydrates by fermentation or dissimilatory Fe(III) reduction.

Phylogeny and physiology of candidate phylum BRC1 inferred from the first complete metagenome-assembled genome obtained from deep subsurface aquifer

Candidate bacterial phylum BRC1 has been identified in a broad range of mostly organic-rich oxic and anoxic environments through molecular analysis of microbial communities. None of the members of BRC1 have been cultivated and only a few draft genome sequences have been obtained from metagenomes or as a result of single-cell sequencing. We have reconstructed complete genome of BRC1 bacterium, BY40, from metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia, and used it for metabolic reconstruction and comparison with existing genomic data. Analysis of 3.3Mb genome of BY40 bacterium revealed numerous glycoside hydrolases that could enable utilization of carbohydrates, including enzymes of chitin-degradation pathway. The bacterium lacks flagellar machinery but the twitching motility is encoded. The reconstructed central metabolism revealed pathways enabling the fermentation of organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Phylogenetic analysis using BY40 genome supported the phylum level classification of BRC1 lineage. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Sumerlaea chitinivorans, within a candidate phylum Sumerlaeota.

Selection for novel, acid-tolerant Desulfovibrio spp. from a closed Transbaikal mine site in a temporal pH- gradient bioreactor

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.