V. A. Korneeva

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.

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.

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.

Anaerobic Microbial Community in the Aerobic Water and at the Oxic/Anoxic Interface in the Black Sea

Fluorescent in situ hybridization (FISH) was used to analyze the abundance and phylogenetic composition of physiologically active anaerobic microbial communities [sulfate-reducing bacteria (SRB) and methanogenic archaea] in the aerobic waters and in the oxic/anoxic transitional zone (chemocline) of the Black Sea. Biogenic sulfate reduction and methane formation were detected at these horizons by radioisotope techniques. Numerous SRB phylogenetically related to Desulfotomaculum (30.5% of detected bacterial cells), Desulfovibrio (29.6%), and Desulfobacter (6.7%) were detected in the aerobic zone at a depth of 30 m, whereas Desulfomicrobium-related bacteria (33.5%) were prevalent in chemocline at a depth of 150 m. In the oxic subsurface water layer, Methanomicrobiales-related archaea and subgroup 1 methanogens constituted up to 62 and 35.3% of archaeal cell, respectively. The active cells of sulfate-reducing and methanogenic microorganisms were much more abundant in the samples collected in summer than in winter from the deep-sea zone. The presence of physiologically active anaerobic microorganisms in oxic and chemocline waters of the Black Sea correlates with the hydrochemical data on the presence of sulfide and methane at corresponding depths.

Molecular detection of methanogenic archaea in the Black Sea oxidized waters

525 Methanogenic archaea are traditionally considered strictly anaerobic microorganisms. However, some methanogens, which possess the enzymatic mechaa nisms of antioxidative defense, may survive at low oxyy gen concentrations [1] and retain their activity in the anoxic microniches formed within oxic habitats (e.g., diatom shells, copepod fecal pellets, and other detrital microparticles) [2, 3]. Earlier, radioisotope analysis revealed methanogenesis not only in anoxic waters, but also in the oxidized surface waters of the Black Sea [4]. The presence of viable archaea in the Black Sea surface waters and in the chemocline zone was origii nally demonstrated using fluorescence in situ hybridd ization (FISH) [5]. Subsequent FISH investigation of the Black Sea oxic waters revealed the presence of physiologically active methanogenic archaea of subb groups I (genera Methanobacterium, Methanobrevii bacter, and Methanosphaera) and II (order Methaa nomicrobiales) [6]. The goal of the present work was to detect methaa nogenic archaea in the oxic water column and in the chemocline zone of the Black Sea using PCR analysis. Water samples from the depths from 30 to 200 m were collected with a submerged pump in July, 2010, on board the Ashamba research vessel (Southern Branch of Shirshov Institute of Oceanology, Russian Academy of Sciences) at a 10000m deep station (44°29.85Ј N, 37°55.24Ј E, 9 miles from the Blue Bay town of the Gelendzhik region). Since methanogenic archaea are known to exist in the water column not only as freeeliving cells, but may also be associated with microparticles [2, 3], the water samples for PCR detection (5 L each) were filtered sequentially through GF/C coarseepore glass fiber fill ters and through 0.222µm bacterial filters (Millipore, United States). The filters were stored in the TE buffer (pH 8.0) : ethanol mixture (1 : 1) at 4°С. After the fill ters were ground in liquid nitrogen, DNA was extracted using the Genomic DNA Purification Kit (Fermentas, Lithuania). The oligonucleotide primers [7, 8] (Syntol, Russia) used for PCR are listed in the table. PCR amplification was performed in the final reaction volume of 25 µL, which contained ~25 ng DNA template, 400 µM dNTP (Fermentas), 500 nM each primer (Syntol), and 2.5 U Taq DNA polyy merase, in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, United States) under the recommended cycling parameters [7, 8]. The 16S rRNA gene fragments were separated by denaturing gradient gel electrophoresis (DGGE) in 6.5% polyacrylamide gel with a 40 to …

A psychrophilic sulfate-reducing bacterium from the Black Sea aerobic water

752 Although traditionally sulfateereducing bacteria (SRB) have been considered strict anaerobes, they are capable of survival in the biotopes affected by oxygen, such as activated sludge, cyanobacterial mats, water column, bottom sediments, etc. [1, 2]. The Black Sea is a meromictic water body. Its anaerobic, sulfideerich deep waters do not mix with the upper, oxic water layer. Fluorescent in situ hybridd ization revealed viable SRB cells (Desulfotomaculum, Desulfovibrio, and Desulfobacter) in both anaerobic and aerobic zones of the Black Sea [3]. We therefore attempted to obtain SRB enrichments and pure cull tures from the aerobic water column of the Black Sea. To obtain enrichment cultures, 900mL water samm ples from the depth of 30 m were concentrated on nitrocellulose filters. The filters were then placed into Hungate tubes with Widdel medium for marine sulfate reducers supplemented with vitamins and yeast extract [4]. Trace element solution was prepared according to Pfennig [5]. The medium was supplemented with MnSO 4 (400 mg/L). The pure culture was obtained using the same medium from the colonies formed in sequential dilutions on solid medium. The colonies were transferred into liquid medium. Sulfide formaa tion was used to assess bacterial growth. Sulfide was determined colorimetrically [6]. The taxonomic position of the SRB isolates was determined by analysis of the 16S rRNA gene sequence. Chromosome DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega, United States) according to the manufacturers recc ommendations. The universal primer system [7] was used for a polymerase chain reaction and sequencing of the amplified 16S rRNA gene fragments. The PCR mixture (25 µL contained the following: 1 µM of each primer, 200 µM dNTP, 1 U BioTaq DNA polymerase (Dialat, Russia), and 50 ng DNA template. PCR products were analyzed by electrophoresis in 2% agaa rose gel in TAE buffer at 6 V/cm and staining with ethidium bromide. Gel images were obtained using a Gel Doc XR documentation system (BiooRad, United States). PCR products were isolated and purified using the Wizard PCR Preps DNA Purification System (Promega, United States) according to the manufacc turers recommendations. The sequencing was carried out in the service laboratory (Syntol, Russia) on an ABI Prism 3100 automatic sequencer (Applied Bioo systems, United States) using the BigDye Terminator v3.0 reagent kit and the universal bacterial primers 11F and 1492R. Analysis of the sequences was carried out using the BLAST software package The ultrastructure of the cells …