Diversity and distribution of methanotrophic archaea at cold seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Diversity and Distribution of Methanotrophic Archaea at Cold Seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Identification of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rod-shaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.     

New insight into stratification of anaerobic methanotrophs in cold seep sediments

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions.     

An anaerobic methane-oxidizing community of ANME-1b archaea in hypersaline Gulf of Mexico sediments

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 microM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.     

Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.     

An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 μM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.     

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.     

Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-Oxidizing Archaea

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.     

Endophytic Fusarium verticillioides reduces disease severity caused by Ustilago maydis on maize

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.     

Distribution of anaerobic methane-oxidizing and sulfate-reducing communities in the G11 Nyegga pockmark,Norwegian Sea

Pockmarks are seabed geological structures sustaining methane seepage in cold seeps. Based on RNA-derived sequences the active fraction of the archaeal community was analysed in sediments associated with the G11 pockmark, in the Nyegga region of the Norwegian Sea. The anaerobic methanotrophic Archaea (ANME) and sulfate-reducing bacteria (SRB) communities were studied as well. The vertical distribution of the archaeal community assessed by PCR-DGGE highlighted the presence of ANME-2 in surface sediments, and ANME-1 in deeper sediments. Enrichments of methanogens showed the presence of hydrogenotrophic methanogens of the Methanogenium genus in surface sediment layers as well. The active fraction of the archaeal community was uniquely composed of ANME-2 in the shallow sulfate-rich sediments. Functional methyl coenzyme M reductase gene libraries showed that sequences affiliated with the ANME-1 and ANME-3 groups appeared in the deeper sediments but ANME-2 dominated both surface and deeper layers. Finally, dissimilatory sulfite reductase gene libraries revealed a high SRB diversity (i.e. Desulfobacteraceae, Desulfobulbaceae, Syntrophobacteraceae and Firmicutes) in the shallow sulfate-rich sediments. The SRB diversity was much lower in the deeper section. Overall, these results show that the microbial community in sediments associated with a pockmark harbour classical cold seep ANME and SRB communities.     

Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME‐1 group

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate‐reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME‐1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82–90% of a composite ANME‐1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME‐1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron‐accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]‐hydrogenases are lacking in the dataset, genes for an [FeFe]‐hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c‐type cytochromes were identified, which have not yet been correlated with methanogenesis‐related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME‐1 to the bacterial sulfate‐reducing partner.     

Archaeal and anaerobic methane oxidizer communities in the Sonora Margin cold seeps,Guaymas Basin (Gulf of California)

Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the ‘BIG'' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel ‘ANME-2c Sonora'' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.     

A distinct freshwater-adapted subgroup of ANME-1 dominates active archaeal communities in terrestrial subsurfaces in Japan

Anaerobic methane-oxidizing archaea (ANME) are known to play an important role in methane flux, especially in marine sediments. The 16S rRNA genes of ANME have been detected in terrestrial freshwater subsurfaces. However, it is unclear whether ANME are actively involved in methane oxidation in these environments. To address this issue, Holocene sediments in the subsurface of the Kanto Plain in Japan were collected for biogeochemical and molecular analysis. The potential activity of the anaerobic oxidation of methane (AOM) (0.38-3.54 nmol cm?3 day?1) was detected in sediment slurry incubation experiments with a (13) CH(4) tracer. Higher AOM activity was observed in low-salinity treatment compared with high-salinity condition (20‰), which supports the adaptation of ANME in freshwater habitats. The 16S rRNA sequence analysis clearly revealed the presence of a distinct subgroup of ANME-1, designated ANME-1a-FW. Phylogenetic analysis of the mcrA genes also implied the presence of the distinct subgroup in ANME-1. ANME-1a-FW was found to be the most dominant active group in the archaeal communities on the basis of 16S rRNA analysis (75.0-93.8% of total archaeal 16S rRNA clones). Sulfate-reducing bacteria previously known as the syntrophic bacterial partners of ANME-1 was not detected. Our results showed that ANME-1a-FW is adapted to freshwater habitats and is responsible for AOM in terrestrial freshwater subsurface environments.     

Aerobic and anaerobic methanotrophs in the Black Sea water column

Inputs of CH(4) from sediments, including methane seeps on the continental margin and methane-rich mud volcanoes on the abyssal plain, make the Black Sea the world's largest surface water reservoir of dissolved methane and drive a high rate of aerobic and anaerobic oxidation of methane in the water column. Here we present the first combined organic geochemical and molecular ecology data on a water column profile of the western Black Sea. We show that aerobic methanotrophs type I are responsible for methane oxidation in the oxic water column and ANME-1- and ANME-2-related organisms for anaerobic methane oxidation. The occurrence of methanotrophs type I cells in the anoxic zone suggests that inactive cells settle to deeper waters. Molecular and biomarker results suggest that a clear distinction between the occurrence of ANME-1- and ANME-2-related lineages exists, i.e. ANME-1-related organisms are responsible for anaerobic methane oxidation below 600 m water depth, whereas ANME-2-related organisms are responsible for this process in the anoxic water column above approximately 600 m water depth.     

Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.     

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.