Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX 86 paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or ␣-or ␤-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.

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 C-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.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain Z-1312T, was isolated from a freshwater hot spring of the Uzon caldera (Kamchatka Peninsula, Russia). The cells were regular cocci, 1–4 μm in diameter, with one long flagellum. The cell envelope was composed of a globular layer attached to the cytoplasmic membrane. The temperature range for growth was 63–89 °C, with an optimum between 80 and 82 °C. The pH range for growth at 80 °C was 4·8–6·8, with an optimum at pH 6·0. Strain Z-1312T grew by hydrolysis and/or fermentation of a wide range of polymeric and monomeric substrates, including agarose, amygdalin, arabinose, arbutin, casein hydrolysate, cellulose (filter paper, microcrystalline cellulose, carboxymethyl cellulose), dextran, dulcitol, fructose, lactose, laminarin, lichenan, maltose, pectin, peptone, ribose, starch and sucrose. No growth was detected on glucose, xylose, mannitol or sorbitol. Growth products when sucrose or starch were used as the substrate were acetate, H2 and CO2. Elemental sulfur, thiosulfate and nitrate added as potential electron acceptors for anaerobic respiration did not stimulate growth when tested with starch as the substrate. H2 at 100 % in the gas phase did not inhibit growth on starch or peptone. The G+C content of the DNA was 42·5 mol%. 16S rRNA gene sequence analysis placed the isolated strain Z-1312T as a member of the genus Desulfurococcus, where it represented a novel species, for which the name Desulfurococcus fermentans sp. nov. (type strain Z-1312T=DSM 16532 T=VKM V-2316T) is proposed.

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.

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°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.

Two novel thermophilic and slightly acidophilic strains, Kam940T 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 °C, with an optimum at 65–70 °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 107 cells ml−1. Sulfate, sulfite, thiosulfate and nitrate added as potential electron acceptors did not stimulate growth when tested with peptone. H2 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 Kam940T. The G+C content of the genomic DNA of strain Kam940T 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 Kam940T 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 Kam940T (=DSM 19380T =VKM B-2539T). 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.

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.Deep-sea hot vents constitute a unique habitat in which chemolithoautotrophic microorganisms thrive by using the energy of inorganic substrates (8). The hot zones of deep-sea hydrothermal environments are inhabited by diverse thermophilic or hyperthermophilic archaea and bacteria, growing at temperatures ranging from moderate to the highest at which microbial growth has been demonstrated (11,18,19). The hydrothermal chimneys of the East Pacific Rise are colonized by alvinellids, Polychaeta worms, which were reported to have no chemosynthetic symbionts but to be accompanied by uncultured proteobacterial epibionts (2, 5) and which live in tubes built from organomineral material of proteinaceous nature (23). The goal of this work was to characterize the thermophilic processes of organic matter production and destruction in the hydrothermal samples from the East Pacific Rise at 13°N and to reveal the influence of molecular hydrogen, a possible electron donor, on the rate of these processes.Sampling procedure and sample characteristics. Deep-sea hydrothermal samples were collected at the 13°N hydrothermal vent field on the East Pacific Rise (depth, 2,650 m) during the AMISTAD (Advanced Microbiological Studies of Thermophiles: Adaptations and Diversity) cruise in 1999. Our study focused on the vent field lying between 12°48Ј18Љ and 12°50Ј32ЉN and between 103°56Ј39Љ and 103°56Ј80ЉW (see Fig. S1 in the supplemental material). Dense populations of Alvinella pompejana, a thermotolerant polychaetous annelid that makes tubes in direct contact with sulfides (3), colonized active structures at the La Chaînette and Grandbonum sites. Pieces of sulfide structures and parts of Alvinella sheaths were collected by the arm of the Nautile, a deep-sea submersible vessel, and lifted to the surface in a submersible insulated basket. Characteristics of these samples are given in Table S1 in the supplemental material.Radioisotopic assays of microbial activity. The samples were prepared for radioisotopic assays immediately following their delivery aboard ship. Fluid/seawater samples were dispensed into 15-ml bottles in 10-ml portions under a flow of nitrogen. Samples of sulfidic structures and tubes of Alvinella were divided into parts of approximately equal volume (ϳ1 cm 3 ), placed in 15-ml bottles, and ...