Methermicoccus shengliensis gen. nov., sp. nov., a thermophilic, methylotrophic methanogen isolated from oil-production water, and proposal of Methermicoccaceae fam. nov.

bThe zonation of anaerobic methane-cycling Archaea in hydrothermal sediment of Guaymas Basin was studied by general primer pairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (mcrA) and by new groupspecific mcrA and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general mcrA primer pairs in detection sensitivity and phylogenetic coverage. Methanotrophic ANME-1 Archaea were the only group detected with groupspecific primers only. The detection of 14 mcrA lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five mcrA phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of Methanopyraceae and Methermicoccaceae. Downcore diversity decreases from all groups detected in the upper 6 cm (ϳ2 to 40°C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40°C). Despite the presence of hyperthermophilic genera (Methanopyrus, Methanocaldococcus) in cooler surface strata, no genes were detected below 10 cm (>60°C). While mcrAbased and 16S rRNA gene-based community compositions are generally congruent, the deeply branching mcrA cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature.H ydrothermal surface sediments in Guaymas Basin support exceptionally high microbial activity and microbial diversity (1-4). The highly productive overlying water column, combined with terrestrial runoff, leads to sedimentation rates exceeding 1 mm yr Ϫ1 (5) and organic carbon contents of 2 to 4% by weight (6). In addition, the upward flow of hydrothermal fluids supplies hydrocarbons (methane, petroleum) and volatile fatty acids produced by thermal degradation of buried organic matter in deeper sediment layers to surface sediments and their microbial communities (1, 7-9).Methanogenesis and sulfate-dependent methane oxidation in Guaymas Basin are carried out by diverse microbial lineages. 16S rRNA gene-based surveys have detected known methane-cycling groups (Methanococcoides, Methanocorpusculum, Methanoculleus, Methermicoccus, ANME-1, ANME-2) (4, 10Ϫ12), a new lineage of ANME-1, ANME-1Guaymas (12), and unknown deeply branching euryarchaeotal groups within the phylogenetic vicinity of known methane-cycling Archaea (4, 10). Phylogenetic analyses of mcrA, a gene diagnostic of methanogenic and anaerobic methanotrophic Archaea (13,14), indicate an even higher diversity of methane-cycling Archaea than those detected by 16S rRNA gene surveys (Methanoco...

Section: Resultsmentioning

confidence: 95%

Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and Group-Specific PCR Primers

Lever

Teske

2015

“…Genomic DNA was isolated by SDS treatment after grinding under liquid N 2 as previously described (19), and its GϩC content was determined by the thermal denaturation (T m ) method (6,32) and the high-performance liquid chromatography (HPLC) method (35). The genomic DNA of Escherichia coli K-12 was used as a reference.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of Methanothermobacter crinale sp. nov., a Novel Hydrogenotrophic Methanogen from the Shengli Oil Field

Cheng

Dai

et al. 2011

Self Cite

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenic pathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and thermophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generally related to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains, Tm2T and HMD, from the oil sands and oil production water in the Shengli oil field in the People's Republic of China. The type strain, Tm2T , was nonmotile and stained Gram positive. The cells were straight to slightly curved rods (0.3 m in width and 2.2 to 5.9 m in length), but some of them possessed a coccal shape connecting with the rods at the ends. Strain Tm2 T grew with H 2 -CO 2 , but acetate is required. Optimum growth of strain Tm2T occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G؉C content of the genomic DNA was 40.1 mol% ؎ 1.3 mol% (by the thermal denaturation method) or 41.1 mol% (by high-performance liquid chromatography). Analysis of the 16S rRNA gene sequence indicated that Tm2 Methanogenesis is the terminal process of organic compound degradation and plays a major role in the global carbon cycle, occurring in a variety of natural and artificial environments, such as the gastrointestinal tracts of animals, rice paddy soils, deep subsurface marine or freshwater sediments, and anaerobic bioreactors (14, 28). The most important precursors for methane production during anaerobic digestion of organic matter are acetate and H 2 -CO 2 , which are converted into methane by aceticlastic and hydrogenotrophic methanogens (28), respectively. However, an alternative pathway for acetate is syntrophic acetate oxidation followed by hydrogenotrophic methanogenesis (65). Several bacteria responsible for acetate oxidation have been isolated and characterized (17,25,48,60), and a series of reports revealed that this process is present in high-temperature oil reservoirs (15,33,40), thermophilic or mesophilic biogas reactors (16,21,23,43,49), lake sediments (41), and rice paddy soils (27). Interestingly, it is proposed that syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis is the main methanogenic pathway in hightemperature petroleum reservoirs (33) and probably involved in the thermophilic methanogenic degradation of hydrocarbon (15). Cloning of rRNA genes from these environments indicates that the dominant thermophilic hydrogenotrophic methanogens were found to be affiliated with the genus Methanothermobacter. However, to our knowledge, pure isolates have not been described. Here, we report the isolation and characterization of two strains, Tm2T and HMD, belonging to a novel phylotype of Methanothermobacter. MATERIALS AND METHODSSamples and media. The production water of oil reservoirs was collected from the Shengli oil field in 2007 and maintained in our lab at a temperature of 4°C. The oil reservoir is located 1,680 to 1,800 m below the sea...

“…Investigations of microbial communities have used 16S rRNA gene sequence analysis to characterize archaea in the deep subsurface such as in terrestrial rocks (45,52), petroleum reservoirs (8,9,18,39,40,42,58), faults (37,49,57), coal seams (11,48), gas fields (36), and subseafloor gas hydrates (17,33,35). These studies have demonstrated the abundance and diversity of methanoarchaea.…”

mentioning

confidence: 99%

Methanogenic Archaea Isolated from Taiwan's Chelungpu Fault

Lai

2011

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, St545MbT 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 St545MbT were different from those of M. psychrophilus strain R15, and wholegenome 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.