A novel methane-producing archaeon, strain SMSP T , was isolated from an anaerobic, propionatedegrading enrichment culture that was originally obtained from granular sludge in a mesophilic upflow anaerobic sludge blanket (UASB) reactor used to treat a beer brewery effluent. Cells were non-motile, blunt-ended, straight rods, 1.0-2.6 mm long by 0.5 mm wide; cells were sometimes up to 7 mm long. Asymmetrical cell division was observed in rod-shaped cells. Coccoid cells (0.5-1.0 mm in diameter) were also observed in mid-to late-exponential phase cultures. Growth was observed between 10 and 40 6C (optimum, 30-33 6C) and pH 7.0 and 7.6 (optimum, pH 7.4). The G+C content of the genomic DNA was 56.2 mol%. The strain utilized formate and hydrogen for growth and methane production. Based on comparative sequence analyses of the 16S rRNA and mcrA (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane-producing pathway) genes, strain SMSP T was affiliated with group E1/E2 within the order Methanomicrobiales. The closest relative based on both 16S rRNA and mcrA gene sequences was Methanoregula boonei 6A8 T (96.3 % 16S rRNA gene sequence similarity, 85.4 % deduced McrA amino acid sequence similarity). The percentage of 16S rRNA gene sequence similarity indicates that strain SMSP T and Methanoregula boonei 6A8 T represent different species within the same genus. This is supported by our findings of shared phenotypic properties, including cell morphology and growth temperature range, and phenotypic differences in substrate usage and pH range. Based on these genetic and phenotypic properties, we propose that strain SMSP T represents a novel species of the genus Methanoregula, for which we propose the name Methanoregula formicica sp. nov., with the type strain SMSP T (5NBRC 105244The group E1/E2, originally called the R10 group or fen cluster, is a family-level clade within the order Methanomicrobiales, which comprises H 2 /CO 2 -using methanogenic archaea (Hales et al., 1996; Bräuer et al., 2006b;Galand et al., 2002;Cadillo-Quiroz et al., 2006). This clade has long been recognized as an uncultured group; however, to date, three methanogenic strains have been isolated and characterized: Methanoregula boonei 6A8 T (Bräuer et al., 2006a(Bräuer et al., , 2011, Methanosphaerula palustris E1-9cT (Cadillo-Quiroz et al., 2008) and Methanolinea tarda NOBI-1 T (Imachi et al., 2008). Methanoregula boonei and Methanosphaerula palustris were isolated from peatlands, while Methanolinea tarda was isolated from a methanogenic digester. In addition to the these isolates, many 16S rRNA gene clones belonging to group E1/E2 have been retrieved from a wide variety of anoxic environments, such as bogs and fens (Bräuer et al., 2006a, b;Cadillo-Quiroz et al., 2006Chan et al., 2002;Galand et al., 2002), methanogenic sludges (Imachi et al., 2008; Chen et al., 2004Chen et al., , 2009Narihiro et al., 2009) Supplementary Fig. S1, available in IJSEM Online), indicating the widespread distribution of E1/E2 methanogens.Recen...

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.

Methanolinea mesophila sp. nov., a hydrogenotrophic methanogen isolated from rice field soil, and proposal of the archaeal family Methanoregulaceae fam. nov. within the order Methanomicrobiales