Composition and Metabolic Potential of Fe(III)-Reducing Enrichment Cultures of Methanotrophic ANME-2a Archaea and Associated Bacteria.

The key microbial group involved in anaerobic methane oxidation is anaerobic methanotrophic archaea (ANME). From a terrestrial mud volcano, we enriched a microbial community containing ANME-2a, using methane as an electron donor, Fe(III) oxide (ferrihydrite) as an electron acceptor, and anthraquinone-2,6-disulfonate as an electron shuttle. Ferrihydrite reduction led to the formation of a black, highly magnetic precipitate. A significant relative abundance of ANME-2a in batch cultures was observed over five subsequent transfers. Phylogenetic analysis revealed that, in addition to ANME-2a, two bacterial taxa belonging to uncultured Desulfobulbaceae and Anaerolineaceae were constantly present in all enrichments. Metagenome-assembled genomes (MAGs) of ANME-2a contained a complete set of genes for methanogenesis and numerous genes of multiheme c-type cytochromes (MHC), indicating the capability of methanotrophs to transfer electrons to metal oxides or to a bacterial partner. One of the ANME MAGs encoded respiratory arsenate reductase (Arr), suggesting the potential for a direct coupling of methane oxidation with As(V) reduction in the single microorganism. The same MAG also encoded uptake [NiFe] hydrogenase, which is uncommon for ANME-2. The MAG of uncultured Desulfobulbaceae contained genes of dissimilatory sulfate reduction, a Wood-Ljungdahl pathway for autotrophic CO2 fixation, hydrogenases, and 43 MHC. We hypothesize that uncultured Desulfobulbaceae is a bacterial partner of ANME-2a, which mediates extracellular electron transfer to Fe(III) oxide.

Moorella sulfitireducens sp. nov., a thermophilic anaerobic bacterium isolated from a terrestrial thermal spring.

In hydrothermal ecosystems, the dissolution of sulfur dioxide in water results in the formation of sulfite, which can be used in microbial metabolism. A limited number of thermophiles have been isolated using sulfite as an electron acceptor. From a terrestrial thermal spring, Sakhalin Island, Russia, we isolated a thermophilic anaerobic bacterium (strain SLA38T). Cells of strain SLA38T were spore-forming straight rods. Growth was observed at temperatures 45-65 °C (optimum at 60 °C) and pH 5.5-9.0 (optimum at pH 6.5-7.0). The novel isolate was capable of anaerobic respiration with sulfite, thiosulfate, fumarate and perchlorate or fermentative growth. Strain SLA38T utilized glycerol, lactate, pyruvate and yeast extract. It grew lithoautotrophically on carbon monoxide with thiosulfate as electron acceptor, producing acetate. The genome size of the isolate was 2.9 Mbp and genomic DNA G + C content was 53.6 mol%. Analysis of the 16S rRNA gene sequences revealed that strain SLA38T belongs to the genus Moorella. Based on the physiological features and phylogenetic analysis, we propose to assign strain SLA38T to a new species of the genus Moorella, as Moorella sulfitireducens sp. nov. The type strain is SLA38T (= DSM 111068T = VKM B-3584T).

Thermosulfuriphilus ammonigenes gen. nov., sp. nov., a thermophilic, chemolithoautotrophic bacterium capable of respiratory ammonification of nitrate with elemental sulfur.

An extremely thermophilic, anaerobic, chemolithoautotrophic bacterium (strain ST65T) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Centre in the south-western Pacific Ocean, at a depth of 1870 m. Cells of strain ST65T were non-motile straight or slightly curved short rods, 0.5-0.6 µm in diameter and 0.8-1.5 µm in length. The temperature range for growth was 47-75 °C, with an optimum at 65 °C. The pH range for growth was 5.5-7.5, with an optimum at pH 6.5. Growth of strain ST65T was observed at NaCl concentrations ranging from 1.5 to 4.5 % (w/v), with an optimum at 2.0-2.5 %. Strain ST65T grew anaerobically with inorganic carbon as a carbon source and with elemental sulfur as an electron donor and nitrate as an electron acceptor producing sulfate and ammonium. It was also able to grow by disproportionation of elemental sulfur, thiosulfate and sulfite. Sulfate was not utilized as an electron acceptor. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to a deep lineage in the phylum Thermodesulfobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the isolate represents a novel species of a new genus, Thermosulfuriphilus ammonigenes gen. nov., sp. nov. ST65T (=DSM 102941T=VKM B-2855T) is the type strain of the type species.

Respiratory Ammonification of Nitrate Coupled to Anaerobic Oxidation of Elemental Sulfur in Deep-Sea Autotrophic Thermophilic Bacteria.

Respiratory ammonification of nitrate is the microbial process that determines the retention of nitrogen in an ecosystem. To date, sulfur-dependent dissimilatory nitrate reduction to ammonium has been demonstrated only with sulfide as an electron donor. We detected a novel pathway that couples the sulfur and nitrogen cycles. Thermophilic anaerobic bacteria Thermosulfurimonas dismutans and Dissulfuribacter thermophilus, isolated from deep-sea hydrothermal vents, grew autotrophically with elemental sulfur as an electron donor and nitrate as an electron acceptor producing sulfate and ammonium. The genomes of both bacteria contain a gene cluster that encodes a putative nitrate ammonification enzyme system. Nitrate reduction occurs via a Nap-type complex. The reduction of produced nitrite to ammonium does not proceed via the canonical Nrf system because nitrite reductase NrfA is absent in the genomes of both microorganisms. The genome of D. thermophilus encodes a complete sulfate reduction pathway, while the Sox sulfur oxidation system is missing, as shown previously for T. dismutans. Thus, in high-temperature environments, nitrate ammonification with elemental sulfur may represent an unrecognized route of primary biomass production. Moreover, the anaerobic oxidation of sulfur compounds coupled to growth has not previously been demonstrated for the members of Thermodesulfobacteria or Deltaproteobacteria, which were considered exclusively as participants of the reductive branch of the sulfur cycle.

Microbial diversity and autotrophic activity in Kamchatka hot springs.

Microbial communities of Kamchatka Peninsula terrestrial hot springs were studied using molecular, radioisotopic and cultural approaches. Analysis of 16S rRNA gene fragments performed by means of high-throughput sequencing revealed that aerobic autotrophic sulfur-oxidizing bacteria of the genus Sulfurihydrogenibium (phylum Aquificae) dominated in a majority of streamers. Another widely distributed and abundant group was that of anaerobic bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). Archaea of the genus Vulcanisaeta were abundant in a high-temperature, slightly acidic hot spring, where they were accompanied by numerous Nanoarchaeota, while the domination of uncultured Thermoplasmataceae A10 was characteristic for moderately thermophilic acidic habitats. The highest rates of inorganic carbon assimilation determined by the in situ incubation of samples in the presence of 14C-labeled bicarbonate were found in oxygen-dependent streamers; in two sediment samples taken from the hottest springs this process, though much weaker, was found to be not dependent on oxygen. The isolation of anaerobic lithoautotrophic prokaryotes from Kamchatka hot springs revealed a wide distribution of the ability for sulfur disproportionation, a new lithoautotrophic process capable to fuel autonomous anaerobic ecosystems.

Dissulfurirhabdus thermomarina gen. nov., sp. nov., a thermophilic, autotrophic, sulfite-reducing and disproportionating deltaproteobacterium isolated from a shallow-sea hydrothermal vent.

A thermophilic, anaerobic, chemolithoautotrophic bacterium, strain SH388T, was isolated from a shallow, submarine hydrothermal vent (Kuril Islands, Russia). Cells of strain SH388T were Gram-stain-negative short rods, 0.2-0.4 µm in diameter and 1.0-2.5 µm in length, and motile with flagella. The temperature range for growth was 25-58 °C (optimum 50 °C), and the pH range for growth was pH 5.0-7.0 (optimum pH 6.0-6.5). Growth of strain SH388T was observed in the presence of NaCl concentrations ranging from 0.5 to 4.0 % (w/v) (optimum 2.0-2.5 %). The strain grew chemolithoautotrophically with molecular hydrogen as electron donor, sodium sulfite as electron acceptor and bicarbonate/CO2 as a carbon source. It was also able to grow by disproportionation of sulfite and elemental sulfur but not thiosulfate. Sulfate, Fe(III) and nitrate were not used as electron acceptors either with H2 or organic electron donors. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the class Deltaproteobacteria and was most closely related to Dissulfuribacter thermophilus and Dissulfurimicrobium hydrothermale (91.6 % and 90.4 % sequence similarity). On the basis of its physiological properties and results of phylogenetic analyses, strain SH388T is considered to represent a novel species of a new genus, for which the name Dissulfurirhabdus thermomarina gen. nov., sp. nov. is proposed. The type strain of the species is SH388T (=DSM 100025T=VKM B-2960T). It is the first thermophilic disproportionator of sulfur compounds isolated from a shallow-sea environment.

Thermostilla marina gen. nov., sp. nov., a thermophilic, facultatively anaerobic planctomycete isolated from a shallow submarine hydrothermal vent.

A novel thermophilic planctomycete (strain SVX8T) was isolated from a shallow submarine hydrothermal vent, Vulcano Island, Italy. The temperature range for growth was 30-68 °C, with an optimum at 55 °C. The pH range for growth was 5.0-9.0, with an optimum at pH 7.0-8.0. Growth was observed at NaCl concentrations ranging from 0.8 to 4.5 % (w/v) with an optimum at 2.5-3.5 % (w/v). The isolate grew anaerobically using a number of mono-, di- and polysaccharides as electron donors and nitrate or elemental sulfur as electron acceptors or by fermentation. Nitrate was reduced to nitrite; sulfur was reduced to sulfide. Strain SVX8T did not grow at atmospheric concentration of oxygen but grew microaerobically (up to 2 % oxygen in the gas phase). The G+C content of the DNA of strain SVX8T was 58.5 mol%. Based on phylogenetic position and phenotypic features, the new isolate is considered to represent a novel species belonging to a new genus in the order Planctomycetales, for which the name Thermostilla marina gen. nov., sp. nov. is proposed. The type strain of Thermostilla marina is SVX8T ( = JCM 19992T = VKM B-2881T). Strain SVX8T is the first thermophilic planctomycete isolated from a marine environment.

Inmirania thermothiophila gen. nov., sp. nov., a thermophilic, facultatively autotrophic, sulfur-oxidizing gammaproteobacterium isolated from a shallow-sea hydrothermal vent.

A novel thermophilic, facultatively autotrophic bacterium, strain S2479T, was isolated from a thermal spring located in a tidal zone of a geothermally heated beach (Kuril Islands, Russia). Cells of strain S2479T were rod-shaped and motile with a Gram-negative cell-wall type. The temperature range for growth was 35-68 °C (optimum 65 °C), and the pH range for growth was pH 5.5-8.8 (optimum pH 6.5). Growth of strain S2479T was observed in the presence of NaCl concentrations ranging from 0.5 to 3.5 % (w/v) (optimum 1.5-2.0 %). The strain oxidized sulfur and thiosulfate as sole energy sources for autotrophic growth under anaerobic conditions with nitrate as electron acceptor. Strain S2479T was also capable of heterotrophic growth by reduction of nitrate with oxidation of low-chain fatty acids and a limited number of other carboxylic acids or with complex proteinaceous compounds. Nitrate was reduced to N2. Sulfur compounds were oxidized to sulfate. Strain S2479T did not grow aerobically during incubation at atmospheric concentration of oxygen but was able to grow microaerobically (1 % of oxygen in gas phase). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was a member of the family Ectothiorhodospiraceae, order Chromatiales, class Gammaproteobacteria. On the basis of phylogenetic and phenotypic properties, strain S2479T represents a novel species of a new genus, for which the name Inmirania thermothiophila gen. nov., sp. nov. is proposed. The type strain of the type species is S2479T ( = DSM 100275T = VKM B-2962T).

Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov., thermophilic anaerobic representatives of the phylum Planctomycetes.

Two novel strains of thermophilic planctomycetes were recovered from terrestrial and subterranean habitats. Strain R1(T) was isolated from a hot spring (Kunashir Island, Russia) and strain SBP2(T) was isolated from a deep gold mine (South Africa). Both isolates grew in the temperature range 30-60 °C and pH range 5.0-8.0. Strain R1(T) grew optimally at 60 °C and pH 6.0-6.5; for SBP2(T) optimal conditions were at 52 °C and pH 7.5-8.0. Both strains were capable of anaerobic respiration with nitrate and nitrite as electron acceptors as well as of microaerobic growth. They also could grow by fermentation of mono-, di- and polysaccharides. Based on their phylogenetic position and phenotypic features we suggest that the new isolates represent two novel species belonging to a new genus in the order Planctomycetales, for which the names Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov. are proposed. The type strain of Thermogutta terrifontis, the type species of the genus, is R1(T) ( = DSM 26237(T) = VKM B-2805(T)), and the type strain of Thermogutta hypogea is SBP2(T) ( = JCM 19991(T) = VKM B-2782(T)).

Complete genome sequence of strain 1860, a crenarchaeon of the genus Pyrobaculum able to grow with various electron acceptors.

Strain 1860, a novel member of the genus Pyrobaculum, is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.

Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia).

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87 degrees C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or alpha- or beta-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellulosiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.

PCR-based identification of hyperthermophilic archaea of the family Thermococcaceae.

A method for rapid detection and identification of hyperthermophilic archaea of the family Thermococcaceae based on PCR amplification of 16S rRNA gene fragments with primers TcPc 173F (5'-TCCCCCATAGGYCTGRGGTACTGGAAGGTC-3') and TcPc 589R (5'-GCCGTGRGATTTCGCCAGGGACTTACGGGC-3') was developed and used for identification of new isolates.