Anaerobic methanotrophic community of a 5346‐m‐deep vesicomyid clam colony in the Japan Trench

Vesicomyidae clams harbor sulfide‐oxidizing endosymbionts and are typical members of cold seep communities where active venting of fluids and gases takes place. We investigated the central biogeochemical processes that supported a vesicomyid clam colony as part of a locally restricted seep community in the Japan Trench at 5346 m water depth, one of the deepest seep settings studied to date. An integrated approach of biogeochemical and molecular ecological techniques was used combining in situ and ex situ measurements. In sediment of the clam colony, low sulfate reduction rates (maximum 128 nmol mL^?1 day^?1) were coupled to the anaerobic oxidation of methane. They were observed over a depth range of 15 cm, caused by active transport of sulfate due to bioturbation of the vesicomyid clams. A distinct separation between the seep and the surrounding seafloor was shown by steep horizontal geochemical gradients and pronounced microbial community shifts. The sediment below the clam colony was dominated by anaerobic methanotrophic archaea (ANME‐2c) and sulfate‐reducing Desulfobulbaceae (SEEP‐SRB‐3, SEEP‐SRB‐4). Aerobic methanotrophic bacteria were not detected in the sediment, and the oxidation of sulfide seemed to be carried out chemolithoautotrophically by Sulfurovum species. Thus, major redox processes were mediated by distinct subgroups of seep‐related microorganisms that might have been selected by this specific abyssal seep environment. Fluid flow and microbial activity were low but sufficient to support the clam community over decades and to build up high biomasses. Hence, the clams and their microbial communities adapted successfully to a low‐energy regime and may represent widespread chemosynthetic communities in the Japan Trench. In this regard, they contributed to the restricted deep‐sea trench biodiversity as well as to the organic carbon availability, also for non‐seep organisms, in such oligotrophic benthic environment of the dark deep ocean.     

Activity,Distribution, and Diversity of Sulfate Reducers and Other Bacteria in Sediments above Gas Hydrate (Cascadia Margin,Oregon)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. Recent investigations showed that another characteristic feature of cold seeps is the occurrence of methanotrophic archaea, which can be identified by specific biomarker lipids and 16S rDNA analysis. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH), bacterial production, enzyme activity, and sulfate reduction rates. Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6 2 10 10 cells cm m 3 ) and sulfate reduction rates (up to 8 w mol cm m 3 d m 1 ) in hydrate-bearing sediments, as well as a high bacterial diversity, especially in the group of i -proteobacteria including members of the branches Desulfosarcina/Desulfococcus , Desulforhopalus , Desulfobulbus , and Desulfocapsa . Most of the diversity of sulfate-reducing bacteria in hydrate-bearing sediments comprises seep-endemic clades, which share only low similarities with previously cultured bacteria.     

Quantification of mcrA by quantitative fluorescent PCR in sediments from methane seep of the Nankai Trough

A quantitative fluorogenic PCR method for group-specific methyl coenzyme M reductase subunit A genes (mcrA) from methanotrophic archaea was established and applied to the characterization of microbial communities in anoxic methane seep sediments at the accretionary prism of the Nankai Trough. All of the previously identified subgroups of anaerobic methanotroph (ANME) mcrA genes were detected in the cores up to 25 cm below the seafloor, but distributional patterns of mcrA genes were found to differ according to depth. These findings suggest a distinct distribution of phylogenetically and physiologically diverse methanotrophic archaea that mediate methane oxidation in the anoxic sediments. This quantification method will contribute to future investigations of methanotrophic microbial ecosystems in anoxic marine sediments.     

Microbial communities from Arctic marine sediments respond slowly to methane addition during ex situ enrichments

Anaerobic methanotrophic archaea (ANME) consume methane in marine sediments, limiting its release to the water column, but their responses to changes in methane and sulfate supplies remain poorly constrained. To address how methane exposure may affect microbial communities and methane- and sulfur-cycling gene abundances in Arctic marine sediments, we collected sediments from offshore Svalbard that represent geochemical horizons where anaerobic methanotrophy is expected to be active, previously active, and long-inactive based on reaction-transport biogeochemical modelling of porewater sulfate profiles. Sediment slurries were incubated at in situ temperature and pressure with different added methane concentrations. Sediments from an active area of seepage began to reduce sulfate in a methane-dependent manner within months, preceding increased relative abundances of anaerobic methanotrophs ANME-1 within communities. In previously active and long-inactive sediments, sulfur-cycling Deltaproteobacteria became more dominant after 30 days, though these communities showed no evidence of methanotrophy after nearly 8 months of enrichment. Overall, enrichment conditions, but not methane, broadly altered microbial community structure across different enrichment times and sediment types. These results suggest that active ANME populations may require years to develop, and consequently microbial community composition may affect methanotrophic responses to potential large-scale seafloor methane releases in ways that provide insight for future modelling studies.     

Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.     

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 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.     

Activities and distribution of methanogenic and methane‐oxidizing microbes in marine sediments from the Cascadia Margin

We investigated methane production and oxidation and the depth distribution and phylogenetic affiliation of a functional gene for methanogenesis, methyl coenzyme M reductase subunit A (mcrA), at two sites of the Integrated Ocean Drilling Program Expedition 311. These sites, U1327 and U1329, are respectively inside and outside the area of gas hydrate distribution on the Cascadia Margin. Radiotracer experiments using ¹⁴C‐labelled substrates indicated high potential methane production rates in hydrate‐bearing sediments [128–223 m below seafloor (mbsf)] at U1327 and in sediments between 70 and 140 mbsf at U1329. Tracer‐free experiments indicated high cumulative methane production in sediments within and below the gas hydrate layer at U1327 and in sediments below 70 mbsf at U1329. Stable tracer experiments using ¹³C‐labelled methane showed high potential methane oxidation rates in near‐surface sediments and in sediments deeper than 100 mbsf at both sites. Results of polymerase chain reaction amplification of mcrA in DNA were mostly consistent with methane production: relatively strong mcrA amplification was detected in the gas hydrate‐bearing sediments at U1327, whereas at U1329, it was mainly detected in sediments from around the bottom‐simulating reflector (126 mbsf). Phylogenetic analysis of mcrA separated it into four phylotype clusters: two clusters of methanogens, Methanosarcinales and Methanobacteriales, and two clusters of anaerobic methanotrophic archaea, ANME‐I and ANME‐II groups, supporting the activity measurement results. These results reveal that in situ methanogenesis in deep sediments probably contributes to gas hydrate formation and are inconsistent with the geochemical model that microbial methane currently being generated in shallow sediments migrates downward and contributes to the hydrate formation. At Site U1327, gas hydrates occurred in turbidite sediments, which were absent at Site U1329, suggesting that a geological setting suitable for a gas hydrate reservoir is more important for the accumulation of gas hydrate than microbiological properties.     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.     

Prokaryotic communities of the north-eastern Mongolian soda lakes

We have studied the activity and composition of several geochemically significant physiological groups of bacteria in more than twenty alkaline salt lakes of the north-east Mongolia steppe with water salinity from 3 to 390 g l^?1 and pH values ranging from 9.0 to 10.6. Active and diverse microbial communities have been found in most of the lakes. The methanotrophic bacteria were represented by the Type I members. Among the culturable forms of sulfur-oxidizing bacteria obligately chemolithoautotrophic and haloalkaliphilic representatives of the genera Thioalkalimicrobium and Thioalkalivibrio were detected in the sediments at high numbers (up to 10⁶ cells ml^?1). The largest population of anaerobic phototrophic bacteria was represented by purple sulfur bacteria of the Ectothiorhodospiraceae family. Salinity was the key factor in determining the activity and the composition of the microbial communities. The most diverse and active prokaryotic populations, including aerobic and anaerobic phototrophic, methanogenic, methanotrophic, sulfur-oxidizing, sulfate-reducing and nitrifying bacteria, were found in lakes with salinity less than 60 g l^?1. In hypersaline lakes with a salinity >100 g l^?1, the sulfur cycle remained active due to the activity of extremely halotolerant and alkaliphilic sulfur bacteria, while other important functional groups responsible for nitrification and methane oxidation processes were not detected. Overall, the prokaryotic communities of the Mongolian alkaline salt lakes represent an interesting new example of a diverse community of haloalkaliphilic bacteria well adopted to a broad salinity range.     

Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.     

Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.     

Sulfate reduction,methanogenesis, and methane oxidation in the Holocene sediments of the Vyborg Bay,Baltic Sea

Methane content and the rates of microbial processes of the carbon and sulfur cycles were determined for the sediments of the Vyborg Bay, Baltic Sea. Formation of the gas-bearing surface sediments in the Vyborg Bay was found to depend on the activity of the modern microbial processes of the transformation of organic matter, resulting in production of significant amounts of reduced gases (methane and hydrogen sulfide). Rapid consumption of sulfate in the course of sulfate reduction coupled to organic matter decomposition both suppressed anaerobic oxidation of methane and promoted microbial methanogenesis. The gasbearing sediments of this area therefore become a source of methane, and methane concentration in the near-bottom water increases significantly.     

Microbial rRNA gene expression and co‐occurrence profiles associate with biokinetics and elemental composition in full‐scale anaerobic digesters

This study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full‐scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH₃‐N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse‐transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH₃‐N, Fe, S, Mo and Ni. A co‐occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy‐sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Enrichment of ANME‐2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with ¹³C labeled methane, ¹³C labeled inorganic carbon was produced and the AOM rate based on ¹³C‐inorganic carbon was 1.2 μmol/(g_dw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME‐2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM.     

Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities

The anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing-bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethanesulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.     

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.     

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.