Stratified Community Responses to Methane and Sulfate Supplies in Mud Volcano Deposits: Insights from an In Vitro Experiment

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world''s oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30–55 cm below the sea floor (bsf); the second is at 165–205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry.     

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.     

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.     

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 δ-¹³C values of these lipids (δ-¹³C = −89 to −58‰) 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.     

Microbial methane cycling in sediments of Arctic thermokarst lagoons

Thermokarst lagoons represent the transition state from a freshwater lacustrine to a marine environment, and receive little attention regarding their role for greenhouse gas production and release in Arctic permafrost landscapes. We studied the fate of methane (CH₄) in sediments of a thermokarst lagoon in comparison to two thermokarst lakes on the Bykovsky Peninsula in northeastern Siberia through the analysis of sediment CH₄ concentrations and isotopic signature, methane-cycling microbial taxa, sediment geochemistry, lipid biomarkers, and network analysis. We assessed how differences in geochemistry between thermokarst lakes and thermokarst lagoons, caused by the infiltration of sulfate-rich marine water, altered the microbial methane-cycling community. Anaerobic sulfate-reducing ANME-2a/2b methanotrophs dominated the sulfate-rich sediments of the lagoon despite its known seasonal alternation between brackish and freshwater inflow and low sulfate concentrations compared to the usual marine ANME habitat. Non-competitive methylotrophic methanogens dominated the methanogenic community of the lakes and the lagoon, independent of differences in porewater chemistry and depth. This potentially contributed to the high CH₄ concentrations observed in all sulfate-poor sediments. CH₄ concentrations in the freshwater-influenced sediments averaged 1.34 ± 0.98 μmol g⁻¹, with highly depleted δ¹³C-CH₄ values ranging from −89‰ to −70‰. In contrast, the sulfate-affected upper 300 cm of the lagoon exhibited low average CH₄ concentrations of 0.011 ± 0.005 μmol g⁻¹ with comparatively enriched δ¹³C-CH₄ values of −54‰ to −37‰ pointing to substantial methane oxidation. Our study shows that lagoon formation specifically supports methane oxidizers and methane oxidation through changes in pore water chemistry, especially sulfate, while methanogens are similar to lake conditions.     

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.     

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (¹³C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.     

Use of 16S rRNA Gene Based Clone Libraries to Assess Microbial Communities Potentially Involved in Anaerobic Methane Oxidation in a Mediterranean Cold Seep

This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22–34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0–6 cm). Communities in the middle layer (6–22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study.     

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.     

Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby Mud Volcano, Barents Sea

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56%+/-8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94%+/-2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.     

Anaerobic Methane Oxidation in Enrichment Cultures from Deep Sediments of a Mud Volcano Peschanka (South Baikal)

Under laboratory conditions, the microbial communities of bottom sediments of a mud volcano Peschanka (Lake Baikal) were found to carry out anaerobic methane oxidation (AOM). After 16 days of anaerobic cultivation of the enrichment cultures, methane content in the gas phase decreased, and microbial consortia were established. The content of carbon, nitrogen, and oxygen determined by energy dispersive X-ray spectroscopy (EDS) was higher than in the nearby sediment particles. The presence of bacteria of the NC10 phylum and archaea of the ANME-2d cluster was established by fluorescent in situ hybridization (FISH).     

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.     

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.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. I. Field observations

Observations on the seasonal periodicity in bottom deposits of Lake Vechten indicated an ecological relationship between sulfate-reducing and methane-producing bacteria. Sulfate reducers are most abundant at depths of 0 to 2 cm in the mud at pS^2- values of about 11 and redox potential values of-100 to-150 mV. Maximum number of methane producers are situated at depths of 3 to 6 cm in the mud at pS^2- values of about 14, redox potential values of-250 to-300 mV and maximum values of the methane concentration. During summer stratification the numbers of bacteria increased considerably. However the number of methane producers rose much more than that of the sulfate reducers. Sulfate in the interstitial water of the sediments is reduced by the sulfate reducers and the sulfate concentration limited the latter's abundance. Methane producers are found deeper in the mud at lower concentrations of hydrogen sulphide. Therefore the different localities of the two bacterial groups may be due to sensitivity of methane producers to hydrogen sulphide. Differential counting of the mixed population of methane-producing bacteria showed that acetate-and methyl-alcohol-fermenting types are most abundant at a depth of 5, and formate-and CO₂/H₂-fermenting types at a depth of 3 cm in the mud.     

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.     

微生物介导的甲烷厌氧氧化过程及其影响因子研究进展

温室气体甲烷减排是全球变化领域的研究热点,甲烷厌氧氧化(anaerobic methane oxidation,AOM)过程是一个以前被忽视的甲烷汇,在调控全球甲烷收支平衡及减缓温室效应等方面扮演着十分重要的角色。AOM微生物以甲烷为唯一电子供体,与硫酸盐(SO42-)、亚硝酸盐(NO2-)/硝酸盐(NO3-)、金属离子(Fe3+、Mn4+、Cr6+)等结合完成氧化还原过程,该过程是耦合碳、氮、硫循环的关键环节。本文系统整理分析了不同AOM类型、发生机理、相关功能微生物类群(ANME-1、ANME-2、ANME-3、NC10、MBG-D)及影响AOM过程的关键调控因子的最新研究进展。结果发现,目前80%以上研究都集中在对最常见电子受体类型(SO42-/NO3-/NO2-/Fe3+/Mn4+)的AOM相关过程,而忽视了潜在的新型电子受体(AQDS/HAs O42-/Cr6+/ClO4-等)的耦合作用过程和相对应的微生物类型及作用机理。对未来AOM研究方向提出展望,以期为研究甲烷厌氧氧化菌在不同生态系统中的生态分布及减缓全球温室气体排放提供新的思路。     

Immunological Localization of Coenzyme M Reductase in Anaerobic Methane-Oxidizing Archaea of ANME 1 and ANME 2 Type

The Black Sea is a large, euxinic marine basin, in which the anaerobic oxidation of methane (AOM) plays an important role in the carbon cycle. Methane-related carbonate build-ups, found on the NW' Black Sea shelf are part of an unique microbial ecosystem. Two archaeal guilds are mainly responsible for the AOM: ANME-1 (anaerobic-methane-oxidizing communities)/DSS consortia and ANME-2/greigite-bearing DSS-consortia. These microorganisms constitute a significant sink of methane on earth, but despite their relevance for the global carbon cycle little is known about the biology of AOM. Phylogenetic and biochemical analyses suggested that ANME-archaea have supposedly reversed the methanogenic pathway. Here, we were able to localize methyl-coenzyme M reductase (MCR), which catalyzes the final step of the methane formation, in ultrathin sections. The result was obtained by the immunogold labeling technique using a specific antiserum against the MCR. This technique revealed that the MCR is located in both ANME-1- and ANME-2-archaea. The data also show that MCR-like enzymes are not only encoded in the genomes of ANME-1 and ANME-2, but are, in fact, expressed as cellular proteins at high levels.     

Growth and population dynamics of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a continuous-flow bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (mu(ANME-2) = 0.167 . week(-1)), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (mu(ANME-1) = 0.218 . week(-1)). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 . week(-1), and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

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.