Carbon-dependent growth, community structure and methane oxidation performance of a soil-derived methanotrophic mixed culture.

Soil-borne methane-oxidizing microorganisms act as a terrestrial methane (CH4) sink and are potentially useful in decreasing global CH4 emissions. Understanding the ecophysiology of methanotrophs is crucial for a thorough description of global carbon cycling. Here, we report the in situ balance of soils from abandoned landfills, meadows and wetlands, their capacities to produce and oxidize CH4 at laboratory-scale and the isolation of a soil-borne methanotrophic-heterotrophic mixed culture that was used for carbon (C1 and C2) feeding experiments. We showed that even with similar soil properties, the in situ CH4 balance depends on land-use. Different soils had different potentials to adapt to increased CH4 availability, leading to the highest CH4 oxidation capacities for landfill and wetland soils. The most efficient mixed culture isolated from the landfill was dominated by the methanotrophs Methylobacter sp. and Methylosinus sp., which were accompanied by Variovorax sp. and Pseudomonas sp. and remained active in oxidizing CH4 when supplied with additional C-sources. The ratios between type I and type II methanotrophs and between methanotrophic and heterotrophic bacteria changed when C-sources were altered. A significant effect of the application of the mixed culture on the CH4 oxidation of soils was established but the extent varied depending on soil type.

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane.

Hydrogenotrophic methanogens can use gaseous substrates, such as H2 and CO2, in CH4 production. H2 gas is used to reduce CO2. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH4 production from CO2 and H2. CO2 and H2 were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5-5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH4 production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH4 production process. The results show that acidic operation of a CH4 production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.

Methanobacterium aggregans sp. nov., a hydrogenotrophic methanogenic archaeon isolated from an anaerobic digester.

A novel, strictly anaerobic, hydrogenotrophic methanogen, strain E09F.3T, was isolated from a commercial biogas plant in Germany. Cells of E09F.3T were Gram-stain-negative, non-motile, slightly curved rods, long chains of which formed large aggregates consisting of intertwined bundles of chains. Cells utilized H2+CO2 and, to a lesser extent, formate as substrates for growth and methanogenesis. The optimal growth temperature was around 40 °C; maximum growth rate was obtained at pH around 7.0 with approximately 6.8 mM NaCl. The DNA G+C content of strain E09F.3T was 39.1 mol%. Phylogenetic analyses based on 16S rRNA and mcrA gene sequences placed strain E09F.3T within the genus Methanobacterium. On the basis of 16S rRNA gene sequence similarity, strain E09F.3T was closely related to Methanobacterium congolense CT but morphological, physiological and genomic characteristics indicated that strain E09F.3T represents a novel species. The name Methanobacterium aggregans sp. nov. is proposed for this novel species, with strain E09F.3T ( = DSM 29428T = JCM 30569T) as the type strain.

Methanobacterium paludis sp. nov. and a novel strain of Methanobacterium lacus isolated from northern peatlands.

Two mesophilic, hydrogenotrophic methanogens, designated strains SWAN1T and AL-21, were isolated from two contrasting peatlands: a near circumneutral temperate minerotrophic fen in New York State, USA, and an acidic boreal poor fen site in Alaska, USA, respectively. Cells of the two strains were rod-shaped, non-motile, stained Gram-negative and resisted lysis with 0.1% SDS. Cell size was 0.6×1.5-2.8 µm for strain SWAN1T and 0.45-0.85×1.5-35 µm for strain AL-21. The strains used H2/CO2 but not formate or other substrates for methanogenesis, grew optimally around 32-37 °C, and their growth spanned through a slightly low to neutral pH range (4.7-7.1). Strain AL-21 grew optimally closer to neutrality at pH 6.2, whereas strain SWAN1T showed a lower optimal pH at 5.4-5.7. The two strains were sensitive to NaCl with a maximal tolerance at 160 mM for strain SWAN1T and 50 mM for strain AL-21. Na2S was toxic at very low concentrations (0.01-0.8 mM), resulting in growth inhibition above these values. The DNA G+C content of the genomes was 35.7 mol% for strain SWAN1T and 35.8 mol% for strain AL-21. Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains are members of the genus Methanobacterium. Strain SWAN1T shared 94-97% similarity with the type strains of recognized species of the genus Methanobacterium, whereas strain AL-21 shared 99% similarity with Methanobacterium lacus 17A1T. On the basis of phenotypic, genomic and phylogenetic characteristics, strain SWAN1T (=DSM 25820T=JCM 18151T) is proposed as the type strain of a novel species, Methanobacterium paludis sp. nov., while strain AL-21 is proposed as a second strain of Methanobacterium lacus.

Methanobacterium movilense sp. nov., a hydrogenotrophic, secondary-alcohol-utilizing methanogen from the anoxic sediment of a subsurface lake.

A novel strain of methanogenic archaea, designated MC-20(T), was isolated from the anoxic sediment of a subsurface lake in Movile Cave, Mangalia, Romania. Cells were non-motile, Gram-stain-negative rods 3.5-4.0 µm in length and 0.6-0.7 µm in width, and occurred either singly or in short chains. Strain MC-20(T) was able to utilize H2/CO2, formate, 2-propanol and 2-butanol as substrate, but not acetate, methanol, ethanol, dimethyl sulfide, monomethylamine, dimethylamine or trimethylamine. Neither trypticase peptone nor yeast extract was required for growth. The major membrane lipids of strain MC-20(T) were archaeol phosphatidylethanolamine and diglycosyl archaeol, while archaeol phosphatidylinositol and glycosyl archaeol were present only in minor amounts. Optimal growth was observed at 33 °C, pH 7.4 and 0.08 M NaCl. Based on phylogenetic analysis of 16S rRNA gene sequences, strain MC-20(T) was closely affiliated with Methanobacterium oryzae FPi(T) (similarity 97.1%) and Methanobacterium lacus 17A1(T) (97.0%). The G+C content of the genomic DNA was 33.0 mol%. Based on phenotypic and genotypic differences, strain MC-20(T) was assigned to a novel species of the genus Methanobacterium for which the name Methanobacterium movilense sp. nov. is proposed. The type strain is MC-20(T) ( = DSM 26032(T) = JCM 18470(T)).

Methanobacterium lacus sp. nov., isolated from the profundal sediment of a freshwater meromictic lake.

An autotrophic, hydrogenotrophic methanogen, designated strain 17A1(T), was isolated from the profundal sediment of the meromictic Lake Pavin, France. The cells of the novel strain, which were non-motile, Gram-staining-negative rods that measured 2-15 µm in length and 0.2-0.4 µm in width, grew as filaments. Strain 17A1(T) grew in a mineral medium and its growth was stimulated by the addition of yeast extract, vitamins, acetate or rumen fluid. Penicillin, vancomycin and kanamycin reduced growth but did not completely inhibit it. Growth occurred at 14-41 °C (optimum 30 °C), at pH 5.0-8.5 (optimum pH 6.5) and with 0-0.4 M NaCl (optimum 0.1 M). The novel strain utilized H(2)/CO(2) and methanol/H(2) as substrates but not formate, acetate, methylamine/H(2), isobutanol or 2-propanol. Its genomic DNA G+C content was 37.0 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, strain 17A1(T) appeared to be a member of the genus Methanobacterium, with Methanobacterium beijingense 8-2(T) (96.3% sequence similarity) identified as the most closely related established species. Based on phenotypic and phylogenetic data, strain 17A1(T) represents a novel species of methanogen within the genus Methanobacterium, for which the name Methanobacterium lacus sp. nov. is proposed. The type strain is 17A1(T) (=DSM 24406(T)=JCM 17760(T)).

Diversity and quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy subjects.

AIM: To investigate the diversity, levels and proportions of Archaea in the subgingival biofilm of generalized aggressive periodontitis (GAgP; n=30) and periodontally healthy (PH; n=30) subjects. MATERIALS AND METHODS: Diversity was determined by sequencing archaeal 16S rRNA gene libraries from 20 samples (10/group). The levels and proportions of Archaea were analysed by quantitative PCR (qPCR) in four and two samples/subject in GAgP and PH groups, respectively. RESULTS: Archaea were detected in 27/28 subjects and 68% of the sites of the GAgP group, and in 26/30 subjects and 58.3% sites of the PH group. Methanobrevibacter oralis was found in all 20 samples studied, Methanobacterium curvum/congolense in three GAgP and six PH samples, and Methanosarcina mazeii in four samples from each group. The levels and proportions of Archaea were higher in GAgP than in PH, whereas no differences were observed between the two probing depth category sites from the GAgP group. CONCLUSION: Archaea were frequently found in subjects with periodontal health and GAgP, especially M. oralis. However, the higher levels and proportions (Archaea/total prokaryotes) of this domain observed in GAgP in comparison with PH subjects indicate a possible role of some of these microorganisms as an environmental modifier in GAgP.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis.

BACKGROUND AND OBJECTIVE: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. MATERIAL AND METHODS: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. RESULTS: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. CONCLUSION: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Methanobacterium movens sp. nov. and Methanobacterium flexile sp. nov., isolated from lake sediment.

Two mesophilic methanogenic strains, designated TS-2(T) and GH(T), were isolated from sediments of Tuosu lake and Gahai lake, respectively, in the Qaidam basin, Qinghai province, China. Cells of both isolates were rods (about 0.3-0.5×2-5 µm) with blunt rounded ends and Gram-staining-positive. Strain TS-2(T) was motile with one or two polar flagella and used only H(2)/CO(2) for growth and methanogenesis. Strain GH(T) was non-motile, used both H(2)/CO(2) and formate and displayed a variable cell arrangement depending on the substrate: long chains when growing in formate (50 mM) or under high pressure H(2) and single cells under low pressure H(2). Phylogenetic analysis based on 16S rRNA gene sequences placed the two isolates in the genus Methanobacterium. Strain TS-2(T) was most closely related to Methanobacterium alcaliphilum NBRC 105226(T) (96% 16S rRNA gene sequence similarity). Phylogenetic analysis based on the alpha subunit of methyl-coenzyme M reductase also supported the affiliation of the two isolates with the genus Methanobacterium. DNA-DNA relatedness between the isolates and M. alcaliphilum DSM 3387(T) was 39-53%. Hence we propose two novel species, Methanobacterium movens sp. nov. (type strain TS-2(T)=AS 1.5093(T)=JCM 15415(T)) and Methanobacterium flexile sp. nov. (type strain GH(T)=AS 1.5092(T)=JCM 15416(T)).

Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., mesophilic methanogens isolated from salty environments.

Two methane-producing archaea, designated Mic5c12(T) and Mic6c05(T), were isolated from sludge deposited in a crude oil storage tank and a tubercle on the interior of a pipe transporting natural gas-containing brine, respectively. The isolates were Gram-staining-variable, non-motile rods and grew only on H(2)/CO(2). Strain Mic6c05(T) produced methane from some alcohols without showing any growth; strain Mic5c12(T) did not utilize alcohols. The optimum growth conditions for strain Mic5c12(T) were 35 °C, pH 6.5 and 0-0.68 M NaCl and for strain Mic6c05(T) were 40 °C, pH 6.0-7.5 and 0.34 M NaCl. Strain Mic5c12(T) was halotolerant and strain Mic6c05(T) was halophilic. Comparative 16S rRNA gene sequence analysis revealed that strains Mic5c12(T) and Mic6c05(T) belonged to the genus Methanobacterium and their closest relative was Methanobacterium subterraneum A8p(T) (97.3 and 97.9 % 16S rRNA gene sequence similarity, respectively). The findings from the 16S rRNA gene sequence analyses were supported by analysis of McrA, the alpha subunit of methyl-coenzyme M reductase. On the basis of phylogenetic analyses and phenotypic characteristics, two novel species are proposed, Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., with type strains Mic5c12(T) (=NBRC 105198(T) =DSM 22353(T)) and Mic6c05(T) (=NBRC 105197(T) =DSM 21974(T)), respectively.

Methanobacterium arcticum sp. nov., a methanogenic archaeon from Holocene Arctic permafrost.

A mesophilic, non-motile, hydrogenotrophic, rod-shaped methanogen, designated M2(T), was isolated from Holocene permafrost sediments of the Kolyma lowland in the Russian Arctic. Cells were 3-6 µm long and 0.45-0.5 µm wide. Strain M2(T) grew on H(2)/CO(2) and formate. Optimum conditions for growth were 37°C, pH 6.8-7.2 and 0.1 M NaCl. The DNA G+C content was 38.1 mol%. On the basis of 16S rRNA gene sequence comparison with known methanogens, strain M2(T) was affiliated with the genus Methanobacterium and was most closely related to Methanobacterium veterum MK4(T) and Methanobacterium bryantii DSM 863(T) (both 99 % 16S rRNA gene sequence similarity). However, no significant DNA-DNA relatedness was observed between strain M2(T) and these type strains. We propose that strain M2(T) represents a novel species, with the name Methanobacterium arcticum sp. nov., with type strain M2(T) (=DSM 19844(T) =VKM B-2371(T)).

Methanogenic archaea isolated from Taiwan's Chelungpu fault.

Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification, and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, St545Mb(T) of newly proposed species Methanolobus chelungpuianus and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 m below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod-shaped organism capable of using H(2)-CO(2) and formate as substrates for methanogenesis. The morphological, biochemical, and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545Mb(T), isolated from a sandstone sample at 545 mbls, is a nonmotile, irregular, coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545Mb(T) was 99.0% similar to that of Methanolobus psychrophilus strain R15 and was 96 to 97.5% similar to the those of other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545Mb(T) were different from those of M. psychrophilus strain R15, and whole-genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545Mb(T) (DSM 19953(T); BCRC AR10030; JCM 15159) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.

Methanobacterium kanagiense sp. nov., a hydrogenotrophic methanogen, isolated from rice-field soil.

A pure culture of an obligately anaerobic, hydrogenotrophic, methanogenic archaeon, designated strain 169(T), which grows with hydrogen and carbon dioxide as the sole energy and carbon sources, was isolated from an anaerobic propionate-oxidizing enrichment culture originally obtained as an inoculant from rice-field soil in Japan. Cells of strain 169(T) were non-motile, Gram-reaction-variable and rod-shaped or slightly curved rods with rounded ends (1.6-5.0 × 0.35-0.5 µm). Strain 169(T) had fimbriae at both ends of the cell (up to ~10 per cell) but did not possess flagella. Ultrathin sections showed a single-layered, electron-dense cell wall about 6 nm thick, which is typical of Gram-positive bacteria. Growth was observed at 15 °C-45 °C (optimum 40 °C), at pH  6.5-9.6 (optimum pH 7.5-8.5) and in 0-70 g NaCl l(-1) (0-1.2 M) (optimum 5 g NaCl l(-1); 0.086 M). Strain 169(T) utilized only hydrogen and carbon dioxide as energy and carbon sources. The DNA G+C content was 39.3 mol%. The results of 16S rRNA gene sequence analysis indicated that strain 169(T) was most closely related to Methanobacterium subterraneum DSM 11074(T) (96.8 % sequence similarity) and Methanobacterium formicicum DSM 1535(T) (96.4 %). On the basis of its morphological, physiological and phylogenetic characteristics, strain 169(T) ( = DSM 22026(T) = JCM 15797(T)) represents a novel species of the genus Methanobacterium, for which the name Methanobacterium kanagiense sp. nov. is proposed.

Methanobrevibacter phylotypes are the dominant methanogens in sheep from Venezuela.

Rumen methanogens in sheep from Venezuela were examined using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE) profiles prepared from pooled and individual PCR products from the rumen contents from 10 animals. A total of 104 clones were examined, revealing 14 different 16S rRNA gene sequences or phylotypes. Of the 14 phylotypes, 13 (99 of 104 clones) belonged to the genus Methanobrevibacter, indicating that the genus Methanobrevibacter is the most dominant component of methanogen populations in sheep in Venezuela. The largest group of clones (41 clones) was 97.9-98.5% similar to Methanobrevibacter gottschalkii. Two sequences were identified as possible new species, one belonging to the genus Methanobrevibacter and the other belonging to the genus Methanobacterium. DGGE analysis of the rumen contents from individual animals also revealed 14 different bands with a range of 4-9 bands per animal.

Effects of temperature on the diversity and community structure of known methanogenic groups and other archaea in high Arctic peat.

Archaeal populations are abundant in cold and temperate environments, but little is known about their potential response to climate change-induced temperature changes. The effects of temperature on archaeal communities in unamended slurries of weakly acidic peat from Spitsbergen were studied using a combination of fluorescent in situ hybridization (FISH), 16S rRNA gene clone libraries and denaturing gradient gel electrophoresis (DGGE). A high relative abundance of active archaeal cells (11-12% of total count) was seen at low temperatures (1 and 5 degrees C), and this community was dominated by Group 1.3b Crenarchaeota and the euryarchaeal clusters rice cluster V (RC-V), and Lake Dagow sediment (LDS). Increasing temperature reduced the diversity and relative abundance of these clusters. The methanogenic community in the slurries was diverse and included representatives of Methanomicrobiales, Methanobacterium, Methanosarcina and Methanosaeta. The overall relative abundance and diversity of the methanogenic archaea increased with increasing temperature, in accordance with a strong stimulation of methane production rates. However, DGGE profiling showed that the structure of this community changed with temperature and time. While the relative abundance of some populations was affected directly by temperature, the relative abundance of other populations was controlled by indirect effects or did not respond to temperature.

Thermophilic methanogenic Archaea in compost material: occurrence, persistence and possible mechanisms for their distribution to other environments.

Since compost is widely used as soil amendment and the fact that during the processing of compost material high amounts of microorganisms are released into the air, we investigated whether compost may act as a carrier for thermophilic methanogens to temperate soils. All eight investigated compost materials showed a clear methane production potential between 0.01 and 0.98 micromol CH(4) g dw(-1)h(-1) at 50 degrees C. Single strand conformation polymorphism (SSCP) and cloning analysis indicated the presence of Methanosarcina thermophila, Methanoculleus thermophilus, and Methanobacterium formicicum. Bioaerosols collected during the turning of a compost pile showed both a highly similar SSCP profile compared to the corresponding compost material and clear methane production during anoxic incubation in selective medium at 50 degrees C. Both observations indicated a considerable release of thermophilic methanogens into the air. To analyse the persistence of compost-borne thermophilic methanogens in temperate oxic soils, we therefore studied their potential activity in compost and compost/soil mixtures, which was brought to a meadow soil, as well as in an agricultural soil fertilised with compost. After 24h anoxic incubation at 50 degrees C, all samples containing compost showed a clear methanogenic activity, even 1 year after application. In combination with the in vitro observed resilience of the compost-borne methanogens against desiccation and UV radiation we assume that compost material acts as an effective carrier for the distribution of thermophilic methanogens by fertilisation and wind.

Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester.

After immobilization of anaerobes on polyurethane foam in a thermophilic, fixed-bed, anaerobic digester supplied with acetate, the results of real-time PCR analysis indicated that the major immobilized methanogenic archaea were Methanosarcina spp., and that the major free-living methanogenic archaea were Methanosarcina and Methanobacterium spp. 16S rRNA gene densities of Methanosarcina spp. and Methanobacterium spp. immobilized on the polyurethane foam were 7.6x10(9) and 2.6x10(8) copies/cm3, respectively. Immobilized methanogenic archaea could be concentrated 1000 times relative to those in the original anaerobically digested sludge from a completely mixed thermophilic digester supplied with cattle waste. On the other hand, immobilized bacteria could be concentrated only 10 times. The cell densities of the immobilized methanogenic archaea and bacteria were higher than those of the free-living methanogenic archaea and bacteria in the reactor. The results of clone analysis indicate that the major methanogenic archaea of the original thermophilic sludge are members of the order Methanomicrobiales, and that the major methanogenic archaea immobilized on the polyurethane foam are Methanosarcina spp., and those of the liquid phase are Methanobacterium spp. The results of the real time PCR analysis approximately agree with those of the clone analysis. These results indicate that real-time PCR analysis is useful for quantitatively describing methanogenic communities.

[Study on microbial community in methanogenic granular sludge by FISH and DGGE].

Four methanogenic granules taken from an anaerobic reactor in different periods were investigated by FISH and DGGE, the eubacterial and archaeal community in these granules was researched and the phylogenetic analysis of dominant archaea was also studied. The FISH results indicated that the quantity of eubacteria was much more than archaea in the methanogenic granule and most eubacteria were located in the out layer of granule, while most archaea were located in the inner layer. The DGGE fingerprints indicated that as the organic loading rate of the reactor increased and the operating time elapsed, the eubacterial community was kept stable relatively, while the archaeal community was changed significantly, which resulted in the gradual decrease of the archaeal varieties. As seven typical bands were cut and sequenced, the results indicated that the dominant species of archaea in granule of the last period were Methanocor pusculum, Methanobacterium, Methanosaeta, and etc.

Desulfotomaculum and Methanobacterium spp. dominate a 4- to 5-kilometer-deep fault.

Alkaline, sulfidic, 54 to 60 degrees C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO4(2-) in the presence of high pH, high concentrations of H2 and CH4, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO4(2-) reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.

Methanobacterium beijingense sp. nov., a novel methanogen isolated from anaerobic digesters.

Two methanogenic strains, 8-2T and 4-1, with rod-shaped (0.4-0.5 x 3-5 microm), non-motile cells, sometimes observed in chains, were isolated from two anaerobic digesters in Beijing, China. The two strains used H2/CO2 and formate for growth and produced methane. The temperature range for growth was 25-50 degrees C, with fastest growth at 37 degrees C. The pH ranges for growth and methane production were 6.5-8.0 for strain 8-2T and 6.8-8.6 for strain 4-1, with the fastest growth at pH 7.2 for strain 8-2T and pH 7.5-7.7 for strain 4-1. The G+C content of genomic DNA for strain 8-2T was 38.9 mol%. The similarity levels of the 16S rRNA sequence of strain 8-2T with other species of the genus Methanobacterium ranged from 93.8 to 96.0 %. Based on the phylogenetic analysis and phenotypic characteristics, the novel species Methanobacterium beijingense sp. nov. is proposed, with the type strain 8-2T (=DSM 15999T=CGMCC 1.5011T).