Siderite-based anaerobic iron cycle driven by autotrophic thermophilic microbial consortium.

Using a sample from a terrestrial hot spring (pH 6.8, 60 °C), we enriched a thermophilic microbial consortium performing anaerobic autotrophic oxidation of hydrothermal siderite (FeCO3), with CO2/bicarbonate as the electron acceptor and the only carbon source, producing green rust and acetate. In order to reproduce Proterozoic environmental conditions during the deposition of banded iron formation (BIF), we incubated the microbial consortium in a bioreactor that contained an unmixed anoxic layer of siderite, perfectly mixed N2/CO2-saturated liquid medium and microoxic (2% O2) headspace. Long-term incubation (56 days) led to the formation of magnetite (Fe3O4) instead of green rust as the main product of Fe(II) oxidation, the precipitation of newly formed metabolically induced siderite in the anoxic zone, and the deposition of hematite (Fe2O3) on bioreactor walls over the oxycline boundary. Acetate was the only metabolic product of CO2/bicarbonate reduction. Thus, we have demonstrated the ability of autotrophic thermophilic microbial consortium to perform a short cycle of iron minerals transformation: siderite-magnetite-siderite, accompanied by magnetite and hematite accumulation. This cycle is believed to have driven the evolution of the early biosphere, leading to primary biomass production and deposition of the main iron mineral association of BIF.

Syntrophic growth of alkaliphilic anaerobes controlled by ferric and ferrous minerals transformation coupled to acetogenesis.

Redox-active iron minerals can act as energy sources or electron-transferring mediators in microbial syntrophic associations, being important means of interspecies metabolic cooperation in sedimentary environments. Alkaline conditions alter the thermodynamic stability of iron minerals, influencing their availability for interspecies syntrophic interactions. We have modeled anaerobic alkaliphilic microbial associations in ethanol-oxidizing co-culture of an obligate syntroph Candidatus "Contubernalis alkalaceticum" and a facultative lithotroph Geoalkalibacter ferrihydriticus, which is capable of dissimilatory Fe(III) reduction and homoacetogenic oxidation of Fe(II) with CO2. The co-cultures were cultivated with thermodynamically metastable ferric-containing ferrihydrite, or ferrous-containing siderite, or without minerals. Mössbauer spectral analysis revealed the transformation of both minerals to the stable magnetite. In the presence of ferrihydrite, G. ferrihydriticus firstly reduced Fe(III) with ethanol and then switched to syntrophic homoacetogenesis, providing the growth of obligate syntroph on ethanol. The ability of G. ferrihydriticus to accept hydrogen from its syntrophic partner and produce extra acetate from carbonate during ethanol oxidation was confirmed by co-culture growth without minerals. In the presence of siderite, G. ferrihydriticus performed homoacetogenesis using two electron donors simultaneously- siderite and hydrogen. Pieces of evidence for direct and indirect hydrogen-mediated electron exchange between partner organisms were obtained. Relative abundancies of partner organisms and the rate of acetate production by their co-cultures were strongly determined by thermodynamic benefits, which G. ferrihydriticus got from redox transformations of iron minerals. Even the minor growth of G. ferrihydriticus sustained the growth of the syntroph. Accordingly, microbe-to-mineral interactions could represent underestimated drivers of syntrophic interactions in alkaline sedimentary environments.

Permanent Draft Genome Sequence of Desulfurococcus amylolyticus Strain Z-533T, a Peptide and Starch Degrader Isolated from Thermal Springs in the Kamchatka Peninsula and Kunashir Island, Russia.

Desulfurococcus amylolyticus Z-533T, a hyperthermophilic crenarcheon, ferments peptide and starch, generating acetate, isobutyrate, isovalerate, CO2, and hydrogen. Unlike D. amylolyticus Z-1312, it cannot use cellulose and is inhibited by hydrogen. The reported draft genome sequence of D. amylolyticus Z-533T will help to understand the molecular basis for these differences.

Calorithrix insularis gen. nov., sp. nov., a novel representative of the phylum Calditrichaeota.

A moderately thermophilic, anaerobic bacterium designated as strain KRT was isolated from a shallow-water submarine hydrothermal vent (Kunashir Island, Southern Kurils, Russia). Cells of strain KRT were thin (0.2-0.3 µm), flexible, motile, Gram-stain-negative rods of variable length. Optimal growth conditions were pH 6.6, 55 °C and 1-3 % (w/v) NaCl. Strain KRT was able to ferment a wide range of proteinaceous substrates, pyruvate, and mono-, di- and polysaccharides. The best growth occurred with proteinaceous compounds. Nitrate significantly stimulated the growth on proteinaceous substrates decreasing H2 formation, ammonium being the main product of nitrate reduction. Strain KRT did not need the presence of a reducing agent in the medium and tolerated the presence of oxygen in the gas phase up to 3 % (v/v). In the presence of nitrate, aerotolerance of isolate KRT was enhanced up to 6-8 % O2 (v/v). Strain KRT was able to grow chemolithoheterotrophically, oxidizing H2 and reducing nitrate to ammonium. Yeast extract (0.05 g l-1) was required for growth. The G+C content of the genomic DNA of strain KRT was 47.3 mol%. 16S rRNA gene sequence analysis placed isolate KRT in the phylum Calditrichaeota where it represented a novel species of a new genus, for which the name Calorithrix insularis gen. nov., sp. nov. is proposed. The type strain of Calorithrix insularis is KRT (=DSM 101605T=VKM B-3022T).

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.

Thermogladius calderae gen. nov., sp. nov., an anaerobic, hyperthermophilic crenarchaeote from a Kamchatka hot spring.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain 1633T, was isolated from a terrestrial hot spring of the Uzon Caldera (Kamchatka Peninsula, Russia). Cells were regular cocci, 0.5-0.9 µm in diameter, with one flagellum. The temperature range for growth was 80-95 °C, with an optimum at 84 °C. Strain 1633T grew on yeast extract, beef extract, peptone, cellulose and cellobiose. No growth was detected on other sugars or carbohydrates, organic acids, or under autotrophic conditions. The only detected growth products were CO2, acetate, and H2. The growth rate was stimulated by elemental sulfur, which was reduced to hydrogen sulfide. The in silico-calculated G+C content of the genomic DNA of strain 1633T was 55.64 mol%. 16S rRNA gene sequence analysis placed strain 1633T together with the non-validly published 'Thermogladius shockii' strain WB1 in a separate genus-level cluster within the family Desulfurococcaceae. Average nucleotide identity (ANI) results revealed 75.72 % identity between strain 1633T and 'Thermogladius shockii' WB1. Based on these results we propose a novel genus and species with the name Thermogladius calderae gen. nov., sp. nov. The type strain of the type species is 1633T ( = DSM 22663T = VKM B-2946T).

Reclassification of Desulfurococcus mobilis as a synonym of Desulfurococcus mucosus, Desulfurococcus fermentans and Desulfurococcus kamchatkensis as synonyms of Desulfurococcus amylolyticus, and emendation of the D. mucosus and D. amylolyticus species descriptions.

Representatives of the crenarchaeal genus Desulfurococcus are strictly anaerobic hyperthermophiles with an organotrophic type of metabolism. Since 1982, five Desulfurococcus species names have been validly published: Desulfurococcus mucosus, D. mobilis, D. amylolyticus, D. fermentans and D. kamchatkensis. Recently, the genomic sequences of all five species became available, promoting the refinement of their taxonomic status. Analysis of full-length high-quality 16S rRNA gene sequences shows that the sequences of D. mobilis and D. mucosus are 100 % identical and differ by 2.2 % from those of D. amylolyticus, D. fermentans and D. kamchatkensis. The latter three sequences differ from each other by 0.1-0.3 % (99.9 % similarity in the D amylolyticus-D. kamchatkensis pair and 99.7 % in the pairs involving D. fermentans). In silico prediction of DNA-DNA hybridization (DDH) values by comparison of genomes using ggdc 2.0 blast+ at http://ggdc.dsmz.de/ produced results that correlated with the 16S rRNA gene sequence similarity values. In the D. mucosus-D. mobilis and D. amylolyticus-D. kamchatkensis pairs, the predicted DDH values were 99 and 92 %, respectively, much higher than the recommended 70 % species-delimiting DDH value. Between members of different pairs, these values were no higher than 20 %. For D. fermentans, its predicted DDH values were around 70 % with D. amylolyticus and D. kamchatkensis and no higher than 20 % with D. mobilis and D. mucosus. These results indicated that D. mobilis should be reclassified as a synonym of D. mucosus, whereas D. kamchatkensis and D. fermentans should be reclassified as synonyms of D. amylolyticus.

Analysis of the complete genome of Fervidococcus fontis confirms the distinct phylogenetic position of the order Fervidicoccales and suggests its environmental function.

The complete genome of the obligately anaerobic crenarchaeote Fervidicoccus fontis Kam940(T), a terrestrial hot spring inhabitant with a growth optimum of 65-70 °C, has been sequenced and analyzed. The small 1.3-Mb genome encodes several extracellular proteases and no other extracellular hydrolases. No complete pathways of carbohydrate catabolism were found. Genes coding for enzymes necessary for amino acid transamination and further oxidative decarboxylation are present. The genome encodes no mechanisms of acyl-CoA and acetyl-CoA oxidation. Two [NiFe]-hydrogenases are encoded: a membrane-bound energy-converting hydrogenase and a cytoplasmic one. The ATP-synthase is H(+)-dependent as inferred from the amino acid sequence of the membrane rotor subunit. On the whole, genome analysis shows F. fontis to be a peptidolytic heterotroph with a restricted biosynthetic potential, which is in accordance with its phenotypic properties. The analysis of phylogenetic markers and of the distribution of best blastp hits of F. fontis proteins in the available genomes of Crenarchaeota supports distinct phylogenetic position of the order Fervidicoccales as a separate lineage adjoining the heterogeneous order Desulfurococcales. In addition, certain F. fontis genomic features correlate with its adaptation to temperatures of 60-80 °C, which are lower than temperatures preferred by Desulfurococcales.

Brockia lithotrophica gen. nov., sp. nov., an anaerobic thermophilic bacterium from a terrestrial hot spring.

A novel thermophilic bacterium, strain Kam1851(T), was isolated from a terrestrial hot spring of the Uzon Caldera, Kamchatka Peninsula, Russia. Cells of strain Kam1851(T) were spore-forming rods with a gram-positive type of cell wall. Growth was observed between 46 and 78 °C, and pH 5.5-8.5. The optimal growth (doubling time, 6.0 h) was at 60-65 °C and pH 6.5. The isolate was an obligate anaerobe growing in pre-reduced medium only. It grew on mineral medium with molecular hydrogen or formate as electron donors, and elemental sulfur, thiosulfate or polysulfide as electron acceptors. The main cellular fatty acids were C(16 : 0) (34.2 %), iso-C(16 : 0) (18 %), C(18 : 0) (12.8 %) and iso-C(17 : 0) (11.1 %). The G+C content of the genomic DNA of strain Kam1851(T) was 63 mol%. 16S rRNA gene sequence analysis showed that strain Kam1851(T) belonged to the order Thermoanaerobacterales, but it was not closely related to representatives of any genera with validly published names. The most closely related strains, which had no more than 89.2 % sequence similarity, were members of the genera Ammonifex and Caldanaerobacter. On the basis of its phylogenetic position and novel phenotypic features, isolate Kam1851(T) is proposed to represent a novel species in a new genus, Brockia lithotrophica gen. nov., sp. nov.; the type strain of Brockia lithotrophica is Kam1851(T) ( = DSM 22653(T) = VKM B-2685(T)).

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

Uncultured archaea dominate in the thermal groundwater of Uzon Caldera, Kamchatka.

The thermoacidophilic microbial community inhabiting the groundwater with pH 4.0 and temperature 50°C at the East Thermal Field of Uzon Caldera, Kamchatka, was examined using pyrosequencing of the V3 region of the 16S rRNA gene. Bacteria comprise about 30% of microorganisms and are represented primarily by aerobic lithoautotrophs using the energy sources of volcanic origin--thermoacidophilic methanotrophs of the phylum Verrucomicrobia and Acidithiobacillus spp. oxidising metals and reduced sulfur compounds. More than 70% of microbial population in this habitat were represented by archaea, in majority affiliated with "uncultured" lineages. The most numerous group (39% of all archaea) represented a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales. Another abundant group (33% of all archaea) was related to MCG1 lineage of the phylum Crenarchaeota, originally detected in the Yellowstone hot spring as the environmental clone pJP89. The organisms belonging to these two groups are widely spread in hydrothermal environments worldwide. These data indicate an important environmental role of these two archaeal groups and should stimulate the investigation of their metabolism by cultivation or metagenomic approaches.

Activity and distribution of thermophilic prokaryotes in hydrothermal fluid, sulfidic structures, and sheaths of alvinellids (East Pacific Rise, 13°N).

Processes of inorganic carbon assimilation, methanogenesis, sulfate reduction, and acetate oxidation to CO(2) occurring in samples from the East Pacific Rise at 13°N were traced, using radioisotopically labeled substrates, at temperatures ranging from 65 to 100°C. Molecular hydrogen stimulated lithotrophic methanogenesis and sulfate reduction but inhibited inorganic carbon assimilation. Active mineralization of acetate was observed in an organic-rich Alvinella-associated system at 80°C. Members of the Thermococcales were the most numerous hyperthermophilic archaea in these samples, their density achieving 10(8) cells per cm(3), while the numbers of cultured hydrogen-utilizing thermophilic lithotrophs were several orders of magnitude lower.

Fervidicoccus fontis gen. nov., sp. nov., an anaerobic, thermophilic crenarchaeote from terrestrial hot springs, and proposal of Fervidicoccaceae fam. nov. and Fervidicoccales ord. nov.

Two novel thermophilic and slightly acidophilic strains, Kam940(T) and Kam1507b, which shared 99 % 16S rRNA gene sequence identity, were isolated from terrestrial hot springs of the Uzon caldera on the Kamchatka peninsula. Cells of both strains were non-motile, regular cocci. Growth was observed between 55 and 85 degrees C, with an optimum at 65-70 degrees C (doubling time, 6.1 h), and at pH 4.5-7.5, with optimum growth at pH 5.5-6.0. The isolates were strictly anaerobic organotrophs and grew on a narrow spectrum of energy-rich substrates, such as beef extract, gelatin, peptone, pyruvate, sucrose and yeast extract, with yields above 10(7) cells ml(-1). Sulfate, sulfite, thiosulfate and nitrate added as potential electron acceptors did not stimulate growth when tested with peptone. H(2) at 100 % in the gas phase inhibited growth on peptone. Glycerol dibiphytanyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl rings were present in the lipid fraction of isolate Kam940(T). The G+C content of the genomic DNA of strain Kam940(T) was 37 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were archaea of the phylum Crenarchaeota, only distantly related to the cultured members of the class Thermoprotei (no more than 89 % identity), and formed an independent lineage adjacent to the orders Desulfurococcales and Acidilobales and clustering only with uncultured clones from hot springs of Yellowstone National Park and Iceland as the closest relatives. On the basis of their phylogenetic position and novel phenotypic features, isolates Kam940(T) and Kam1507b are proposed to be assigned to a new genus and species, Fervidicoccus fontis gen. nov., sp. nov. The type strain of Fervidicoccus fontis is strain Kam940(T) (=DSM 19380(T) =VKM B-2539(T)). The phylogenetic data as well as phenotypic properties suggest that the novel crenarchaeotes form the basis of a new family, Fervidicoccaceae fam. nov., and order, Fervidicoccales ord. nov., within the class Thermoprotei.

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.

Distribution of Crenarchaeota representatives in terrestrial hot springs of Russia and Iceland.

Culture-independent (PCR with Crenarchaeota-specific primers and subsequent denaturing gradient gel electrophoresis) and culture-dependent approaches were used to study the diversity of Crenarchaeota in terrestrial hot springs of the Kamchatka Peninsula and the Lake Baikal region (Russia) and of Iceland. Among the phylotypes detected there were relatives of both cultured (mainly hyperthermophilic) and uncultured Crenarchaeota. It was found that there is a large and diverse group of uncultured Crenarchaeota that inhabit terrestrial hot springs with moderate temperatures (55 to 70 degrees C). Two of the lineages of this group were given phenotypic characterization, one as a result of cultivation in an enrichment culture and another one after isolation of a pure culture, "Fervidococcus fontis," which proved to be a moderately thermophilic, neutrophilic (optimum pH of 6.0 to 7.5), anaerobic organotroph.