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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.

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.

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.

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).

A strain of methanogenic bacterium Px1 was isolated from an anaerobic digester in the department of environmental sciences, TsingHua University, which treated the wastewater from Beijing Beer Factory. The isolate is curved rod, light-yellow colony, uses only H2 + CO2 as the substrate for CH4 formation. On the basis of physiology, morphology, structural characteristics and phylogenetic analysis, it is turned out that strain Px1 is a new species of the genus Methanobacterium, the name Methanobacterium curvum sp. nov. is proposed.