A. L. Bryukhanov

Investigation of the sulfate-reducing bacterial community in the aerobic water and chemocline zone of the Black Sea by the fish technique

Fluorescent in situ hybridization (FISH) was used to analyze the abundance and phylogenetic composition of sulfate-reducing bacteria in the aerobic waters and in the oxic/anoxic transitional zone (chemocline) of the Black Sea, where biogenic formation of reduced sulfur compounds was detected by radioisotope techniques. Numerous sulfate-reducing bacteria of the genera Desulfotomaculum (30.5% of detected bacterial cells), Desulfovibrio (29.6%), and Desulfobacter (6.7%) were revealed in the aerobic zone at a depth of 30 m, while Desulfomicrobium-related bacteria (33.5%) were prevalent in the upper chemocline waters at 150-m depth. Active cells of sulfate-reducing bacteria were much more abundant in the samples collected in summer than in the winter samples from the deep-sea zone. The presence of physiologically active sulfate reducers in oxic and chemocline waters of the Black Sea correlates with the hydrochemical data on the presence of reduced sulfur compounds in the aerobic water column.

Activity and structure of the sulfate-reducing bacterial community in the sediments of the southern part of Lake Baikal

The rates of sulfate reduction (SR) and the diversity of sulfate-reducing bacteria (SRB) were studied in the sediments of the Posol’skaya Banka elevation in the southern part of Lake Baikal. SR rates varied from 1.2 to 1641 nmol/(dm3 day), with high rates (>600 nmol/(dm3 day)) observed at both deep-water stations and in subsurface silts. Integral SR rates calculated for the uppermost 50 cm of the sediments were higher for gas-saturated and gas hydrate-bearing sediments than in those with low methane content. Enrichment cultures were obtained in Widdel medium for freshwater SRB. Analysis of the 16S rRNA gene fragments from clone libraries obtained from the enrichments revealed the presence of SRB belonged to the genus Desulfosporosinus, with D. lacus as the most closely related member (capable of sulfate, sulfite, and thiosulfate reduction), as well as members of the order Clostridiales.The rates of sulfate reduction (SR) and the diversity of sulfate-reducing bacteria (SRB) were studied in the sediments of the Posolskaya banka elevation in the southern part of Lake Baikal. SR rates varied from 1.2 to 1641 nmol/(dm3 day), with high rates (> 600 nmol/(dm3 day)) observed at both deep-water stations and in subsurface silts. Integral SR rates calculated for the uppermost 50 cm of the sediments were higher for gas-saturated and gas hydrate-bearing sediments than in those with low methane content. Enrichment SRB cultures were obtained in Widdel medium for freshwater SRB. Analysis of the 16S rRNA gene fragments from clone libraries obtained from the enrichments revealed the presence of SRB belonged to Desulfosporosinus genus, with D. lacus as the most closely related member (capable of sulfate, sulfite, and thiosulfate reduction), as well as members of the order Clostridiales.

Extracellular Protein Metabolite of Luteococcus japonicus subsp. casei Reactivates Cells Subjected to Oxidative Stress

A protein exometabolite isolated from the culture liquid of Luteococcus japonicus subsp. casei reactivates the cells of this microorganism following H2O2- or paraquat-induced oxidative stress. The resistance of L. casei cells to these oxidizers is accounted for by the high activity of superoxide dismutase and catalase. The effect of the protein exometabolite is universal, in that it reactivates the cells after UV irradiation, heating, or oxidative stress. However, cells subjected to oxidative stress are significantly less susceptible to the reactivating effect as compared to their UV-irradiated or heated counterparts. Possible causes of these differences are discussed.

Structure of the Archaeal Community in the Black Sea Photic Zone

Qualitative and quantitative analysis of the structure of the archaeal community of the photic zone of the Black Sea water column was carried out. Real-time PCR revealed 2 × 104 archaeal cells/mL (4.2% of the total cell number) at a 15-m depth. The structure of archaeal communities in the subsurface water column was investigated using the sequencing by synthesis technology (Illumina/Solexa) of the 16S rRNA genes. The Marine Group II phylogenetic cluster belonging to the phylum Euryarchaeota was the most numerous archaeal group (1.2–1.7 × 104 cells/mL). The Marine Group I phylogenetic cluster (phylum Thaumarchaeota) was the second most numerous group (40% of the free-living archaea or 7.7 × 103 cells/mL). Sequences of the ‘Nitrosopumilus’ cluster were revealed among Marine Group I sequences due to high homology (over 90%). A group of archaea belonging to the Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG-6) (phylum Euryarchaeota) was also detected. The 16S rRNA gene sequences belonging to this cluster were revealed only in the suspension fraction. High homology level (over 90%) suggested classification of most DHVEG-6 sequences within the ‘Parvarchaeum’ cluster. In spite of a noticeable methane peak detected at 15-m depth, no sequences of methanogens were found.

The sulfate-reducing bacterial community of sulfide-rich water of the Ust’-Kachka resort spring, Perm Krai, Russia

Microbiological investigation of the highly mineralized, sulfide-rich cold spring of the Ust’-Kachka resort was carried out. The total number and biomass of microbial cells were 50 × 103 cell/mL and 15 μg/L, respectively. While the total microbial number was low, the sulfate reduction rate determined by the radioisotope method was relatively high (0.575 mg S L−1 day−1). An enrichment culture was obtained on Widdel medium. According to the results of cloning the 16S rRNA gene fragments with subsequent restriction analysis, the dominant organisms were group 6 sulfate-reducing bacteria (Desulfovibrio-Desulfomicrobium) and the microorganism exhibiting 99% similarity to the anaerobic haloalkaliphilic bacterium Halanaerobium hydrogeniformans.

[Long-term storage of obligate anaerobic microorganisms in glycerol].

We evaluated the possibility of storing the cultures of obligate anaerobic microorganisms (clostridia, acetogenic and sulfate-reducing bacteria, and methanogenic archaea) in 25% glycerol at −70°C for a long time (up to 3 years). This method of storage is adequate for preserving cell viability in the majority of obligate anaerobes.

Susceptibility of Archaea to the Antibiotic Effect of the Parasporal Inclusion Proteins from Different Bacillus thuringiensis subspecies

The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri. The minimal inhibitory concentrations of these proteins were 20 to 50 μg/ml. Lysozyme exhibited similar bactericidal effect on archaea. The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.

Components of antioxidant systems in the cells of aerotolerant sulfate-reducing bacteria of the genus Desulfovibrio (strains A2 and TomC) isolated from metal mining waste

Two strains of sulfate-reducing bacteria of the genus Desulfovibrio (A2 and TomC) isolated from metal mining waste were able to grow on agar Postgate C nutrient medium under microaerobic conditions. Since their growth in liquid nutrient medium was just slightly affected by 1% O2 (initial concentration in the gas phase) and 0.05–0.1 mM H2O2, these strains were relatively oxygen-tolerant. Only the presence of oxidants in high concentrations (5–10% О2 or 0.3–1.0 mM H2O2) resulted in practically complete inhibition of their growth. Strain A2 was more resistant to oxidative stresses than strain TomC. Activities of the key enzymes of antioxidant defense—superoxide dismutase (SOD), catalase, and peroxidase—were revealed in the cell-free extracts of strain A2 grown under strict anaerobic conditions. While strain TomC was found to possess no peroxidase activity, its catalase activity was much higher than that of strain A2 (36 and 2 U/mg protein, respectively). SOD activity of both strains was almost the same (5 U/mg protein). Sublethal H2O2 doses (concentration of 0.05–0.15 mM and exposure for 45–240 min) resulted in a drastic increase of catalase activity, especially in strain A2. Sublethal О2 doses (1–2% in the gas phase) had no significant effect on activities of the antioxidant enzymes of both strains. The cytochrome composition determined from the absolute absorption spectra of the whole cells of strains TomC and A2 revealed the presence of the c heme (438 and 831 pmol/mg protein) and the d heme (336 and 303 pmol/mg protein, respectively). The presence of the d heme indicated the presence of the bd heme–heme quinol oxidase, which together with the c heme may provide for the functioning of the electron transport segment of the antioxidant defensive system, which is responsible for aerotolerance of sulfate-reducing bacteria.

Detection of anaerobic sulfate-reducing bacteria in oxygen-containing upper water layers of the Black and Baltic seas

Fluorescent in situ hybridization (FISH) and PCR were used for analysis of phylogenetic structure of anaerobic sulfate-reducing bacterial communities in oxygen-containing upper water layers of meromictic basins: the Black Sea and the Gdansk Deep of the Baltic Sea. In the Black Sea (continental slope at depths 30–70 m), cells of sulfate-reducing bacteria (SRB) hybridizing with 16S rRNA-specific FISH-probes for Desulfotomaculum, Desulfobacter, and Desulfovibrio genera were revealed, whereas Desulfomicrobium-related bacteria were prevalent in the chemocline zone at a 150-m depth. Besides Desulfotomaculum (SRB subgroup 1), Desulfobacter (SRB subgroup 4), and Desulfovibrio-Desulfomicrobium (SRB subgroup 6), nested PCR with the use of 16S rRNA gene-specific primers detected the presence of Desulfococcus–Desulfonema–Desulfosarcina (SRB subgroup 5) in the oxygen-containing water column of the Black and Baltic seas. Active enrichment SRB culture that contained bacterium Desulfosporosinus sp. as a major component was obtained from the Black Sea water sample collected at a 70-m depth.

Sulfate-reducing bacterial communities in the water column of the Gdansk Deep (Baltic Sea)

Biodiversity of sulfate-reducing bacterial communities in the water column of the Gdansk Deep, Baltic Sea, where H2S had been detected in near-bottom layers, was analyzed by PCR with primers for the 16S rRNA genes of six major phylogenetic subgroups of sulfate-reducing bacteria (SRB). Using denaturing gradient gel electrophoresis followed by sequencing, the nucleotide sequences of reamplified dsrB gene fragments from investigated water samples were determined. For the first time the presence of nucleotide sequences of the dsrB gene was detected by PCR in the water samples from all hydrochemical layers, including subsurface oxic waters. The presence of the 16S rRNA genes of representatives of Desulfotomaculum, Desulfococcus-Desulfonema-Desulfosarcina, and Desulfovibrio-Desulfomicrobium SRB subgroups was also revealed throughout the water column of the Gdansk Deep. Analysis of translated amino acid sequences encoded by the dsrB gene demonstrated the highest homology with the relevant sequences of uncultured SRB from various marine habitats.