Deep-sea hydrothermal vents play an important role in global biogeochemicalcycles, providing biological oases at the seafloor that are supported by the thermal and chemical flux from the Earth's interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated seawater, minerals precipitate to form porous sulphide-sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem 1 Table 2, Supplementary Fig. S1). The DHVE2 were identified in 36 samples and represented up to 10 and 15% of the archaeal population at EPR and ELSC, respectively (as determined by quantitative PCR; Table 1). Generally, the occurrence of DHVE2 appeared to be associated with the fine-grained white elemental sulphur, S 8 , (identified in three different samples by X-ray diffraction, Supplementary Methods) or iron oxyhydroxide (X-ray amorphous -identified based on orange color and texture, Supplementary Table 3) deposits that often coat the outside of mature actively venting sulphide deposits ( Supplementary Fig. S2). Additionally, due to the high guanine and cytosine content of the 16S rRNA sequence and its occasional co-occurrence with thermophiles such as Thermococcus, it has been suggested that the DHVE2 are thermophiles, perhaps heterotrophs whose growth may be stimulated by sulphur 1,16 . A DHVE2 genome fragment from a metagenome library contained a thermostable DNA polymerase, which was further suggestive of the thermophilic nature of this lineage 17 . However, all attempts to culture this group using standard media and conditions to select for sulphate reducers, methanogens or fermenters at 60, 80 and 90°C and around pH 6.5 have been unsuccessful 5 .Based on the predictions that conditions within many sulphide deposits should be acidic, and due to the presence of novel sequences most closely related to the acidophiles, Thermoplasma, Picrophilus and Ferroplasma (Fig. 1 The cells are pleiomorphic to spherical, about 0.6-1 µm in diameter and are motile with a single flagellum (Fig. 2a, b). Some flagella have their proximal end encased in an unusual complex periodic sheath (Fig. 2c). Like many other Archaea (e.g.Methanococcus and Sulfolobus), they are enveloped by a plasma membrane and a single S-layer (Fig. 2a, e). The S-layer, similar to that of Picrophilus oshimae 23 , is thick (~40nm), with a presumed tetragonal (p4) lattice and resembles a delicate bridal veil bordering the cells (Fig. 2a). Although S-layers are quasi-crystalline this S-layer bends into small highly curved structures (Fig. 2d, vesicles). These vesicles bud from the cell partitioning small quantities of cytoplasm that could then anneal to adjacent cells. This is a process that is common amongst their Gram-negative bacterial counterparts 24 . The ability of DHVE2's S-layer to bend into such high curvature structures as these vesicles,suggests that the bonding forces between S-layer subunits are weak or transient. This is unusual 25 ,...

A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen. nov., sp. nov. The strain was enriched from an anaerobic digestor and isolated. Initial isolation was as a monoxen i c prop i on at e-d eg ra d i ng co-cu It u re con t a i n i ng Methanospirillum hungateii JF-lT as an H, -and formate-using partner. Later, an axenic culture was obtained by using crotonate as the catabolic substrate. The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO, and methane from propionate. However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 165 rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter.

We calculated the potential H2 and formate diffusion between microbes and found that at H2 concentrations commonly found in nature, H2 could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H2 concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H2 concentration at the cell surface of H2-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 ,um from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the Km for H2 or formate.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO,, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61"C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16s rRNA indicated that B. infernus should be classified as a new species of the genus Bacilius. B. infernus is the only strictly anaerobic species in the genus Bacillus.

We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 m) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45°C (no growth at <10°C or >55°C). Cells grew with a maximum specific growth rate of 2.43 day ؊1 at 45°C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na ؉ . The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.Marine methane hydrates are an ice-like material in which methane molecules are trapped within cages of the crystalline lattice of water molecules. Methane hydrates form at temperatures of up to about 15 or 16°C when the partial pressure of methane is very high. This occurs in many deep marine sediments, where temperatures are low and the hydrostatic pressure of the water keeps methane in solution at high partial pressures (29). Gas hydrates are globally distributed along coastal margins, trapping enormous volumes of methane, estimated at about twice the amount of all other known fossil fuel reserves (1). Methane hydrates have been estimated to contain roughly 4,000 times today's atmospheric content of methane (4). With most of the methane trapped in known hydrate formations being of biogenic origin, this represents a significant new source of natural gas from biological methanogenesis.Natural gas hydrates typically occur along coastal margins where organic matter accumulates. Microbial numbers are high in these zones: 1.5 ϫ 10 9 cells g Ϫ1 of hydrate-bearing sediments and 1.0 ϫ 10 6 cells ml Ϫ1 within the hydrates themselves (17). Metabolic studies of these sediments, based on incubations with stable isotopes of substrate and subsequent measurement of the isotope-containing product, have demonstrated methanogenesis, sulfate reduction, and methane oxidation (8). Phylogenetic analysis of DNA extracted from marine sedim...

Methanogeniumfrigidum sp. nov. was isolated from the perennially cold, anoxic hypolimnion of Ace Lake in the Vesfold Hills of Antarctica. The cells were psychrophilic, exhibiting most rapid growth at 15°C and no growth at temperatures above 18 to 20°C. The cells were irregular, nonmotile caccoids (diameter, 1.2 to 2.5 pm) that occurred singly and grew by CO, reduction by using H, as a reductant. Formate could replace H,, but growth was slower. Acetate, methanol, and trimethylamine were not catabolized. Cells grew with acetate as the only organic compound in the culture medium, but growth was much faster in medium also supplemented with peptones and yeast extract. The cells were slightly halophilic; good growth occurred in medium supplemented with 350 to 600 mM Na+, but no growth occurred with 100 or 850 mM Na+. The pH range for growth was 6.5 to 7.9; no growth occurred at pH 6.0 or 8.5. Growth was slow (maximum specific growth rate, 0.24 day-'; doubling time, 2.9 days). This is the first report of a psychrophilic methanogen growing by CO, reduction.

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53°N, 167°W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO 2 -reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.More than 85% of the ocean's organic carbon is deposited in shallow, anoxic marine sediments (8). Methanogens play an important role in the degradation of this organic matter by converting low-molecular-weight compounds into methane. In marine sediments, methane is present in dissolved form in the pore water, as free gas, or trapped within methane hydrates. Molecular and isotopic data suggest that most of the methane produced in marine sediments is of biogenic origin (31); however, methanogens that inhabit permanently cold marine sediments have been poorly characterized.Several studies have investigated the microbial diversity in cold marine sediments (12,43,46); however, the role that these microbes play in mediating geochemical processes is still not completely understood. In addition to methanogens, uncultivated lineages of other Archaea have been identified in marine sediments (22,29,32,37,41,67). These include phylotypes belonging to the marine benthic groups B (MBG-B), C, and D and the anaerobic methane-oxidizing (ANME) archaeal groups ANME-1 and ANME-2.Skan Bay has previously served as a model system for studying low-temperature microbial processes in marine sediments (2,3,47,51,66). The sediments contain clear environmental gradients (66), and the complete redox sequence from oxygen to methane occurs within 0.5 m of the surface, allowing for good depth resolution of microbial activities (20). Previous analyses of Skan Bay sediments have shown that microbes mediate important geochemical processes, including sulfate reduction, methane production, and the anaerobic oxidation of methane (AOM) (66). The specific aim of this study was to identify and cultivate Archaea involved in methane cycling at Skan Bay. Cultivation-independent and -dependent techniques were employed to characterize the archaeal populations at various depths and therefore along...

Three novel strains of methylotrophic methanogens were isolated from Skan Bay, Alaska, by using anaerobic cultivation techniques. The water was 65 m deep at the sampling site. Strains AK-4 (=OCM 774), AK-5 T (=OCM 775 T =DSM 17273 T ) and AK-9 (=OCM 793) were isolated from the sulfate-reducing zone of the sediments. Each of the strains was a non-motile coccus and occurred singly. Cells grew with trimethylamine as a catabolic substrate and strain AK-4 could also catabolize methanol. Yeast extract and trypticase peptones were not required for growth, but their addition to the culture medium slightly stimulated growth. Each of the strains grew at temperatures of 5-28 6C; they were slight halophiles and grew fastest in the neutral pH range. Analysis of the 16S rRNA gene sequences indicated that strain AK-4 was most closely related to Methanosarcina baltica. DNA-DNA hybridization studies showed 88 % relatedness, suggesting that strain AK-4 represents a novel strain within this species. Strains AK-5 T and AK-9 had identical 16S rRNA gene sequences that were most closely related to the sequence of Methanococcoides burtonii (99?8 % sequence similarity). DNA-DNA hybridization studies showed that strains AK-5 T and AK-9 are members of the same species (88 % relatedness value), but strain AK-5 T had a DNA-DNA relatedness value of only 55 % to Methanococcoides burtonii. This indicates that strains AK-5 T and AK-9 should be considered as members of a novel species in the genus Methanococcoides. We propose the name Methanococcoides alaskense sp. nov., with strain AK-5 T (=OCM 775 T =DSM 17273 T ) as the type strain.In the marine environment, sulfate reduction is the dominant microbial process in the upper sediment layers. Generally, when sulfate is present, sulfate reduction is the major catabolic process and methanogenesis is limited. Because of their higher affinity for hydrogen and acetate, sulfate-reducing bacteria out-compete methanogens for these important substrates (King et al., 1983;King, 1984;Oremland & Taylor, 1978). As a result, methanogenesis becomes a dominant process only in deeper sediments in which the sulfate ions have been exhausted. The limited methane production that occurs together with sulfate reduction is due to the activity of methylotrophic methanogens (King, 1984;Oremland & Taylor, 1978;Oremland & Polcin, 1982).Methanogens that belong to the family Methanosarcinaceae are characterized as having the broadest substrate range of methanogens; many can grow by reducing CO 2 with H 2 or by the splitting of acetate and all can grow by dismutating methyl compounds (Kendall & Boone, 2004). The family Methanosarcinaceae includes eight genera: Methanosarcina, Methanolobus, Methanococcoides, Methanohalobium, Methanohalophilus, Methanosalsum, Methanomethylovorans and Methanimicrococcus. The only described species of Methanimicrococcus, Methanimicrococcus blatticola, is unique among the Methanosarcinaceae because it reduces methylated compounds only in the presence of H 2 (Sprenger et al., 2000). The GenBank/EMB...