Methanogen Diversity Evidenced by Molecular Characterization of Methyl Coenzyme M Reductase A (mcrA) Genes in Hydrothermal Sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H₂/CO₂- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H₂/CO₂, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

Investigation of the methanogen population structure and activity in a brackish lake sediment

The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Phylogenetic characterization of methanogenic assemblages in eutrophic and oligotrophic areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.     

Molecular diversity of methanogens in feedlot cattle from Ontario and Prince Edward Island, Canada

The molecular diversity of rumen methanogens in feedlot cattle and the composition of the methanogen populations in these animals from two geographic locations were investigated using 16S rRNA gene libraries prepared from pooled PCR products from 10 animals in Ontario (127 clones) and 10 animals from Prince Edward Island (114 clones). A total of 241 clones were examined, with Methanobrevibacter ruminantium accounting for more than one-third (85 clones) of the clones identified. From these 241 clones, 23 different 16S rRNA phylotypes were identified. Feedlot cattle from Ontario, which were fed a corn-based diet, revealed 11 phylotypes (38 clones) not found in feedlot cattle from Prince Edward Island, whereas the Prince Edward Island cattle, which were fed potato by-products as a finishing diet, had 7 phylotypes (42 clones) not found in cattle from Ontario. Five sequences, representing the remaining 161 clones (67% of the clones), were common in both herds. Of the 23 different sequences, 10 sequences (136 clones) were 89.8 to 100% similar to those from cultivated methanogens belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales, and the remaining 13 sequences (105 clones) were 74.1 to 75.8% similar to those from Thermoplasma volcanium and Thermoplasma acidophilum. Overall, nine possible new species were identified from the two clone libraries, including two new species belonging to the order Methanobacteriales and a new genus/species within the order Methanosarcinales. From the present survey, it is difficult to conclude whether the geographical isolation between these two herds or differences between the two finishing diets directly influenced community structure in the rumen. Further studies are warranted to properly assess the differences between these two finishing diets.     

Phylogenetic Diversity of the Archaeal Community in a Continental High-Temperature,Water-Flooded Petroleum Reservoir

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

The impact of aridification and vegetation type on changes in the community structure of methane-cycling microorganisms in Japanese wetland soils

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.     

Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.     

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1)

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake.     

Phylogenetic relationships of symbiotic methanogens in diverse termites

Termites harbor symbiotic microorganisms in their gut which emit methane. The phylogeny of the termite methanogens was inferred without cultivation based on nucleotide sequences of PCR-amplified 16S ribosomal RNA genes. Seven methanogen sequences from four termite species were newly isolated, and together with those previously published, these sequences were phylogenetically compared. The termite methanogen sequences were divided into three clusters. Two clusters of sequences, derived from the gut DNA of so-called higher termites, were related to methanogens in the orders Methanosarcinales or Methanomicrobiales. All of the sequences in the case of lower termites were closely related to the genus Methanobrevibacter. However, most of the termite symbionts were found to be distinct from known methanogens. They are not dispersed among diverse methanogen species, but rather formed unique lineages in the phylogenetic trees.     

Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16S rRNAs

DNAs from 16 species of archaebacteria including 6 novel isolates were hybridized with 16S rRNAs from 7 species representing different orders or groups of the urkingdom of archaebacteria. The yields, normalized for the number of genes per microgram of DNA, and the temperature stabilities of all hybrids were determined and related to each other. A taxonomic tree constructed from such fractional stability data reveals the same major divisions as that derived from comparative cataloging of 16S rRNA sequences. The extreme halophiles appear however as a distinct order besides the three known divisions of methanogens. The methanogens, the halophiles and Thermoplasma form one of two clearly recognizable branches of the archaebacterial urkingdom. The order represented by Sulfolobus and the related novel order Thermoproteales form the other branch. Three novel genera, Thermoproteus, Desulfurococcus and the "stiff filaments" represent three families of this order. The extremely thermophilic methanogen Methanothermus fervidus belongs to the Methanobacteriales. SN1, a methanogen from Italy, appears as another species of the genus Methanococcus. Another novel methanogen, M3, represents a genus or family of the order Methanomicrobiales.     

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.     

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33°56'N, 102°52'E) region and under the annual temperature around 1°C. Previous studies indicated that Zoige wetland was one of the CH₄ emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales , could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10⁷ cells per gram of soil, accounting for about 30% of the total Archeae . By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO₂, methanol or trimethylamine was determined to increase to 10⁸ cells ml⁻¹, but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30°C and 15°C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae , and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.     

Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The delta-(13)C values of these lipids (delta-(13)C = -89 to -58 per thousand) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.     

Prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin

The community compositions of Bacteria and Archaea were investigated in deep, sub-seafloor sediments from the highly productive Peru Margin (ODP Leg 201, sites 1228 and 1229, c. 25 km apart) down to nearly 200 m below the seafloor using taxonomic (16S rRNA) and functional (mcrA and dsrA) gene markers. Bacterial and archaeal groups identified from clone libraries of 16S rRNA gene sequences at site 1229 agreed well with sequences amplified from bands excised from denaturing gradient gel electrophoresis (DGGE) depth profiles, with the exception of the Miscellaneous Crenarchaeotic Group (MCG). This suggested that the prokaryotic community at site 1228, obtained from DGGE profiling alone, was reliable. Sites were dominated by Bacteria in the Gammaproteobacteria, Chloroflexi (green non-sulphur bacteria) and Archaea in the MCG and South African Gold Mine Euryarchaeotic Group, although community composition changed with depth. The candidate division JS1 was present throughout both sites but was not dominant. The populations identified in the Peru Margin sediments consisted mainly of prokaryotes found in other deep subsurface sediments, and were more similar to communities from the Sea of Okhotsk (pelagic clays) than to those from the low organic carbon Nankai Trough sediments. Despite broad similarities in the prokaryotic community at the two sites, there were some differences, as well as differences in activity and geochemistry. Methanogens (mcrA) within the Methanosarcinales and Methanobacteriales were only found at site 1229 (4 depths analysed), whereas sulphate-reducing prokaryotes (dsrA) were only found at site 1228 (one depth), and these terminal-oxidizing prokaryotes may represent an active community component present at low abundance. This study clearly demonstrates that the deep subsurface sediments of the Peru Margin have a large diverse and metabolically active prokaryotic population.