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.     

Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review

Evidence supporting a key role for anaerobic methane oxidation in the global methane cycle is reviewed. Emphasis is on recent microbiological advances. The driving force for research on this process continues to be the fact that microbial communities intercept and consume methane from anoxic environments, methane that would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments. Geochemical evidence for this process has been observed in numerous marine sediments along the continental margins, in methane seeps and vents, around methane hydrate deposits, and in anoxic waters. The anaerobic oxidation of methane is performed by at least two phylogenetically distinct groups of archaea, the ANME-1 and ANME-2. These archaea are frequently observed as consortia with sulfate-reducing bacteria, and the metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. The archaeal member of a consortium apparently oxidizes methane and shuttles reduced compounds to the sulfate-reducing bacteria. Despite recent advances in understanding anaerobic methane oxidation, uncertainties still remain regarding the nature and necessity of the syntrophic association, the biochemical pathway of methane oxidation, and the interaction of the process with the local chemical and physical environment. This review will consider the microbial ecology and biogeochemistry of anaerobic methane oxidation with a special emphasis on the interactions between the responsible organisms and their environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant (13)C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. (13)C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of delta-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong (13)C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant (13)C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

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.     

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.     

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.     

Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant ¹³C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. ¹³C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of δ-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong ¹³C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant ¹³C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

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 (μ_ANME-2 = 0.167 · week⁻¹), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (μ_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.     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

Patterns of 15N assimilation and growth of methanotrophic ANME-2 archaea and sulfate-reducing bacteria within structured syntrophic consortia revealed by FISH-SIMS

Methane release from the oceans is controlled in large part by syntrophic interactions between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (DSS), frequently found as organized consortia. An understanding of the specifics of this symbiotic relationship and the metabolic heterogeneity existing between and within individual methane-oxidizing aggregates is currently lacking. Here, we use the microanalytical method FISH-SIMS (fluorescence in situ hybridization-secondary ion mass spectrometry) to describe the physiological traits and anabolic activity of individual methanotrophic consortia, specifically tracking ¹⁵N-labelled protein synthesis to examine the effects of organization and size on the metabolic activity of the syntrophic partners. Patterns of ¹⁵N distribution within individual aggregates showed enhanced ¹⁵N assimilation in ANME-2 cells relative to the co-associated DSS revealing a decoupling in anabolic activity between the partners. Protein synthesis in ANME-2 cells was sustained throughout the core of individual ANME-2/DSS consortia ranging in size range from 4 to 20 μm. This indicates that metabolic activity of the methane-oxidizing archaea is not limited to, or noticeably enhanced at the ANME−2/DSS boundary. Overall, the metabolic activity of both syntrophic partners within consortia was greater than activity measured in representatives of the ANME-2 and DSS observed alone, with smaller ANME-2/DSS aggregates displaying a tendency for greater ¹⁵N uptake and doubling times ranging from 3 to 5 months. The combination of ¹⁵N-labelling and FISH-SIMS provides an important perspective on the extent of heterogeneity within methanotrophic aggregates and may aid in constraining predictive models of activity and growth by these syntrophic consortia.     

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.     

Insights into the genomes of archaea mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.     

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.     

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.     

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.     

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.     

A Microbial Mat of a Large Sulfur Bacterium Preserved in a Miocene Methane-Seep Limestone

A Miocene methane-seep limestone from the Romagna Apennine (Pietralunga, Italy) was found to contain an extraordinarily well-preserved microbial mat consisting of filamentous fossils. Individual filaments of the lithified Pietralunga mat are 50 to 80 μ m in diameter and resemble the sulfide-oxidizing bacterium Beggiatoa. Mats of sulfur bacteria are common around modern methane-seeps, but have not yet been reported from ancient seep limestones. This is thought to be related to the conditions prevailing in metabolically active mats of sulfur bacteria that do not favor carbonate formation. The preservation of the Pietralunga mat was most likely caused by a sudden change from oxidizing to anoxic conditions, leading to the rapid carbonate precipitation induced by anaerobic oxidation of methane. Lipid biomarkers specific for archaea and sulfate-reducing bacteria linked with the anaerobic oxidation of methane co-occur with compounds derived from methanotrophic bacteria and ciliates. These findings confirm a close proximity of oxic and anoxic conditions, as required for the growth of sulfide-oxidizing bacteria in the methane-based ecosystem. The lack of earlier reports on fossilized thiotrophic mats in seep limestones is most likely related to the rarity of environmental changes rapid enough to preserve the filaments rather than to a lower frequency of thiotrophic mats around methane-seeps in the geological past.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.