Archaeal diversity in naturally occurring and impacted environments from a tropical region

AIMS: To evaluate archaeal diversity in natural and impacted habitats from Rio de Janeiro state, Brazil, a tropical region of South America. METHODS AND RESULTS: 16S rRNA gene was amplified directly by polymerase chain reaction (PCR) from genomic DNA, extracted from Guanabara Bay (GB) water, halomarine sediment (HS), municipal landfill leachate, agricultural soil and wastewater treatment (WT) system. Five archaeal 16S rDNA clone libraries were constructed. A total of 123 clones, within the five libraries analysed, were clustered into 29 operational taxonomic units, related to cultivated (24%) and uncultivated (76%) organisms. Rarefaction analysis showed that the libraries contained different levels of diversity. PCR-denaturing gradient gel electrophoresis (DGGE) of 16S-23S intergenic spacer regions confirmed the presence of a dominant phylotype, revealed by the WT system clone library. CONCLUSIONS: Archaeal communities of impacted environments seem to be confined to specific ecosystems with similar physicochemical properties, while communities from natural environments appear to be widely distributed. The presence of a high number of phylotypes related to uncultivated organisms suggests new archaeal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, the analysis of archaeal diversity in tropical environments from Brazil, and adds sequences from this region to the developing database of 16S rRNA clone libraries from environmental samples.     

A novel haloarchaeal-related lineage is widely distributed in deep oceanic regions

During our study of the 16S rRNA gene sequence-based archaeal diversity of a deep-sea site located at a 3000 m depth at the Antarctic Polar Front, we detected several phylotypes ascribed to already known Group II and III Euryarchaeota, and a cluster of distinct sequences that branched off at the base of haloarchaea. The position of this lineage (marine Group IV) was very robust using distance (neighbour-joining) and maximum-likelihood methods. Subsequently, we designed specific primers for the detection of this archaeal group in other marine environments using polymerase chain reaction amplification and sequence comparison. Group IV archaea were found in the Antarctic area (across a gradient from the Southern ocean to the South Atlantic), and also in North Atlantic and Mediterranean waters. In all oceanic locations, Group IV archaea were never detected in surface waters, but were vertically distributed in the deepest part of the water column.     

Relationships between bacterial diversity and environmental variables in a tropical marine environment, Rio de Janeiro

This study is the first to apply a comparative analysis of environmental chemistry, microbiological parameters and bacterioplankton 16S rRNA clone libraries from different areas of a 50 km transect along a trophic gradient in the tropical Guanabara Bay ecosystem. Higher bacterial diversity was found in the coastal area, whereas lower richness was observed in the more polluted inner bay water. The significance of differences between clone libraries was examined with libshuff statistics. Paired reciprocal comparisons indicated that each of the libraries differs significantly from the others, and this is in agreement with direct interpretation of the phylogenetic tree. Furthermore, correspondence analyses showed that some taxa are related to specific abiotic, trophic and microbiological parameters in Guanabara Bay estuarine system.     

A nested PCR approach for improved recovery of archaeal 16S rRNA gene fragments from freshwater samples

In a survey on the presence of archaea in a number of European lakes, it was found that known archaeal primer sets for PCR were not suited for use in freshwater environment, as some lack selectivity, while others were too selective. A nested PCR was developed for denaturing gradient gel electrophoresis (DGGE) with primer sets 21F–958R and Parch519f–Arch915r, respectively. After sequencing of the DGGE bands obtained by this nested method, 93% of the sequences were of archaeal origin. More diverse archaeal DGGE patterns were found as compared with other PCR methods. The nested PCR-DGGE method presented here is therefore a reliable tool to analyze the archaeal diversity in freshwater habitats, revealing even more widespread diversity of the archaea.     

Genetic diversity of archaea in deep-sea hydrothermal vent environments.

Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.     

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.     

Environmental shaping of sponge associated archaeal communities

Background

Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood.

Methodology/Principal Findings

We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum.

Conclusion/Significance

The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.     

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.     

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFA-accumulation/degradation cycles.     

Screening and Isolation of PHB-Producing Bacteria in a Polluted Marine Microbial Mat

The characteristics of microbial mats within the waste stream from a seafood cannery were compared to a microbial community at a pristine site near a sandy beach at Puerto San Carlos, Baja California Sur, Mexico. Isolation of poly-beta-hydroxybutyrate (PHB)-producing bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stains with Sudan Black and Nile Red, and chemical extraction of PHB were used as a culture-dependent strategy for the detection of PHB-producing bacteria. The culture-independent approach included denaturing gradient gel electrophoresis of phylotypes of 16S rRNA of microbial communities from environmental samples. Significant differences in community structure were found among the polluted and pristine sites. These differences were correlated with the physicochemical characteristics of the seawater column. At the polluted site, the seawater was rich in nutrients (ammonia, phosphates, and organic matter), compared to the pristine location. Partial sequencing of 16S rDNA of cultures of bacteria producing PHB included Bacillus and Staphylococcus at both sites; Paracoccus and Micrococcus were found only at the polluted site and Rhodococcus and Methylobacterium were found only at the pristine site. Bands of the sequences of 16S rDNA from both field samples in the denaturing gradient gel electrophoresis (DGGE) analyses affiliated closely only with bacterial sequences of cultures of Bacillus and Staphylococcus. High concentrations of organic and inorganic nutrients at the polluted site had a clear effect on the composition and diversity of the microbial community compared to the unpolluted site.     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

Archaeal diversity and community development in deep-sea hydrothermal vents

Over the past 35 years, researchers have explored deep-sea hydrothermal vent environments around the globe and studied a number of archaea, their unique metabolic and physiological properties, and their vast phylogenetic diversity. Although the pace of discovery of new archaeal taxa, phylotypes and phenotypes in deep-sea hydrothermal vents has slowed recently, bioinformatics and interdisciplinary geochemistry-microbiology approaches are providing new information on the diversity and community composition of archaea living in deep-sea vents. Recent investigations have revealed that archaea could have originated and dispersed from ancestral communities endemic to hydrothermal vents into other biomes on Earth, and the community structure and productivity of chemolithotrophic archaea are controlled primarily by variations in the geochemical composition of hydrothermal fluids.     

Archaeal habitats--from the extreme to the ordinary

The domain Archaea represents a third line of evolutionary descent, separate from Bacteria and Eucarya. Initial studies seemed to limit archaea to various extreme environments. These included habitats at the extreme limits that allow life on earth, in terms of temperature, pH, salinity, and anaerobiosis, which were the homes to hyper thermo philes, extreme (thermo)acidophiles, extreme halophiles, and methanogens. Typical environments from which pure cultures of archaeal species have been isolated include hot springs, hydrothermal vents, solfataras, salt lakes, soda lakes, sewage digesters, and the rumen. Within the past two decades, the use of molecular techniques, including PCR-based amplification of 16S rRNA genes, has allowed a culture-independent assessment of microbial diversity. Remarkably, such techniques have indicated a wide distribution of mostly uncultured archaea in normal habitats, such as ocean waters, lake waters, and soil. This review discusses organisms from the domain Archaea in the context of the environments where they have been isolated or detected. For organizational purposes, the domain has been separated into the traditional groups of methanogens, extreme halophiles, thermoacidophiles, and hyperthermophiles, as well as the uncultured archaea detected by molecular means. Where possible, we have correlated known energy-yielding reactions and carbon sources of the archaeal types with available data on potential carbon sources and electron donors and acceptors present in the environments. From the broad distribution, metabolic diversity, and sheer numbers of archaea in environments from the extreme to the ordinary, the roles that the Archaea play in the ecosystems have been grossly underestimated and are worthy of much greater scrutiny.     

Diversity of geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses

The abundance, distribution, and phylogenetic diversity of members of the Fe(III)-reducing family Geobacteraceae were studied along a gradient of metal contaminants in Lake Coeur d’Alene, Idaho. Partial 16S rRNA gene fragments were amplified by PCR using primers directed toward conserved regions of the gene within the family Geobacteraceae. Analysis of amplicons separated by denaturing gradient gel electrophoresis (DGGE) suggested within-site variation was as great as between-site variation. Amplicons were cloned and grouped by RFLP type and DGGE migration distance and representatives were sequenced. Grouping clones with 3% or less sequence dissimilarity, 15 distinct phylotypes were identified compared to 16 distinct DGGE bands. Only 1 phylotype was recovered from all sites. This clone, B14, is most closely related to Geobacter metallireducens and constituted a greater portion of the pristine community than of the contaminated communities. A second phylotype, Q2, predominated in the contaminated communities and was notably absent from the pristine libraries. Clone Q2 presents a high degree of sequence similarity to two Geobacter spp. previously isolated from this region of Lake Coeur d’Alene. Six phylotypes were unique to the contaminated sediments, whereas two were found only in the pristine sediments. Indices of diversity (Shannon and Simpson) were consistently higher when calculated with DGGE data than when clone library data were used. Most-probable-number PCR and real-time PCR suggested that the Geobacteraceae phylotypes were spread relatively evenly across all three sites along the gradient. Our data indicate that the Geobacteraceae are diverse and abundant in Lake Coeur d’Alene sediments, regardless of metals content. These results provide insight into the ability of dissimilatory Fe(III)-reducing bacteria to colonize habitats with elevated metal concentrations, and they have important implications for the management and remediation of metal-contaminated sites.     

Phylogenetic Analyses of Archaeal Ribosomal DNA Sequences from Salt Pan Sediment of Mumbai,India

The archaeal diversity in salt pan sediment from Mumbai, India, was investigated by 16S rDNA-dependent molecular phylogeny. Small-subunit rRNA (16S rDNA) from salt pan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. Thirty-two unique phylotypes were obtained by PCR-based RFLP of 16S rRNA genes using endonucleases Hae111 and Msp1, which were most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain Archaea including both Crenarchaeota and Euryarcheaota. However, none of the retrieved Crenarchaeota sequences could be grouped with previously cultured Crenarchaeota. Of all the Euryarcheaota sequences, three sequences were related to Haloarchaea, two were related to cultured Methanosarcina sp., and the remaining sequences were affiliated with uncultured Methanosarcina sp., Methanogenium sp., and Methanolobus sp. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of a highly diverse archaeal population and may represent novel sequences and organisms specially adapted to the salt pan sediment of Mumbai. These findings are of fundamental value for understanding the complexity of salt pan ecosystems.     

Is it possible to calibrate the pollution level of the region of Algiers (Mediterranean Sea) by exploiting marine macrophytes?

In the absence of water-quality data, a biological indicator is the only way to estimate the pollution level. Samples of macrophytes from exposed shallow rocky substrata of the region of Algiers (Algeria, Mediterranean Sea) were collected in supposedly (in the absence of available pollution data) polluted and pristine waters. These samples were compared to a set of samples spanning a known pollution gradient found near Marseilles (France) and to some samples from a variety of other Mediterranean localities. All samples were collected in similar conditions. The diversity point (i.e., the number of species per sample) was not greater at Cherchell (control site) than at the three sites in the Bay of Algiers. Analysis of the dataset was successful in ranking the Algerian sites, but failed to calibrate the pollution level of the Algerian sites by inserting Algerian samples within the pollution gradient of Marseilles. In contrast, regional characteristics of the macrophyte communities appear to be largely prevalent. This means that water-quality biological indicators and indices based upon marine macrophytes, at least for the open waters and exposed shallow Mediterranean habitats studied here, could be reliable within a given region, but may require validation and/or adjustment, perhaps considerable, for other regions.     

Phylogenetic Characterization of Archaea in Saltpan Sediments

A study was undertaken to investigate the presence of archaeal diversity in saltpan sediments of Goa, India by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from saltpan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. 10 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonuclease Msp 1, which was most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain archaea including both crenarchaeota and euryarcheaota. None of the retrieved crenarchaeota sequences could be grouped with previously cultured crenarchaeota however; two sequences were related with haloarchaea. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of low archaeal population but showed yet unclassified species, may specially adapted to the salt pan sediment of Goa.     

A hotspot for cold crenarchaeota in the neuston of high mountain lakes

We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters ( n  = 10 lakes; R ² > 0.80; P  < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes.     

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.     

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.