Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane.
ABSTRACT: Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.
Project description:The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments into the hydrosphere. AOM is largely mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), and has mainly been investigated in deep-sea sediments. Here we studied methane seepage at four spots located at 12 m water depth in coastal, organic carbon depleted permeable sands off the Island of Elba (Italy). We combined biogeochemical measurements, sequencing-based community analyses and in situ hybridization to investigate the microbial communities of this environment. Increased alkalinity, formation of free sulfide and nearly stoichiometric methane oxidation and sulfate reduction rates up to 200 nmol g(-1) day(-1) indicated the predominance of sulfate-coupled AOM. With up to 40 cm thickness the zones of AOM activity were unusually large and occurred in deeper sediment horizons (20-50 cm below seafloor) as compared to diffusion-dominated deep-sea seeps, which is likely caused by advective flow of pore water due to the shallow water depth and permeability of the sands. Hydrodynamic forces also may be responsible for the substantial phylogenetic and unprecedented morphological diversity of AOM consortia inhabiting these sands, including the clades ANME-1a/b, ANME-2a/b/c, ANME-3, and their partner bacteria SEEP-SRB1a and SEEP-SRB2. High microbial dispersal, the availability of diverse energy sources and high habitat heterogeneity might explain that the emission spots shared few microbial taxa, despite their physical proximity. Although the biogeochemistry of this shallow methane seep was very different to that of deep-sea seeps, their key functional taxa were very closely related, which supports the global dispersal of key taxa and underlines strong selection by methane as the predominant energy source. Mesophilic, methane-fueled ecosystems in shallow-water permeable sediments may comprise distinct microbial habitats due to their unique biogeochemical and physical characteristics. To link AOM phylotypes with seep habitats and to enable future meta-analyses we thus propose that seep environment ontology needs to be further specified.
Project description:The flux of methane, a potent greenhouse gas, from the seabed is largely controlled by anaerobic oxidation of methane (AOM) coupled to sulfate reduction (S-AOM) in the sulfate methane transition (SMT). S-AOM is estimated to oxidize 90% of the methane produced in marine sediments and is mediated by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria. An additional methane sink, i.e., iron oxide coupled AOM (Fe-AOM), has been suggested to be active in the methanic zone of marine sediments. Geochemical signatures below the SMT such as high dissolved iron, low to undetectable sulfate and high methane concentrations, together with the presence of iron oxides are taken as prerequisites for this process. So far, Fe-AOM has neither been proven in marine sediments nor have the governing key microorganisms been identified. Here, using a multidisciplinary approach, we show that Fe-AOM occurs in iron oxide-rich methanic sediments of the Helgoland Mud Area (North Sea). When sulfate reduction was inhibited, different iron oxides facilitated AOM in long-term sediment slurry incubations but manganese oxide did not. Especially magnetite triggered substantial Fe-AOM activity and caused an enrichment of ANME-2a archaea. Methane oxidation rates of 0.095 ± 0.03 nmol cm-3 d-1 attributable to Fe-AOM were obtained in short-term radiotracer experiments. The decoupling of AOM from sulfate reduction in the methanic zone further corroborated that AOM was iron oxide-driven below the SMT. Thus, our findings prove that Fe-AOM occurs in methanic marine sediments containing mineral-bound ferric iron and is a previously overlooked but likely important component in the global methane budget. This process has the potential to sustain microbial life in the deep biosphere.
Project description:Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.
Project description:In marine sediments the anaerobic oxidation of methane with sulfate as electron acceptor (AOM) is responsible for the removal of a major part of the greenhouse gas methane. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME) and their specific partner bacteria. The physiology of these organisms is poorly understood, which is due to their slow growth with doubling times in the order of months and the phylogenetic diversity in natural and in vitro AOM enrichments. Here we study sediment-free long-term AOM enrichments that were cultivated from seep sediments sampled off the Italian Island Elba (20°C; hereon called E20) and from hot vents of the Guaymas Basin, Gulf of California, cultivated at 37°C (G37) or at 50°C (G50). These enrichments were dominated by consortia of ANME-2 archaea and Seep-SRB2 partner bacteria (E20) or by ANME-1, forming consortia with Seep-SRB2 bacteria (G37) or with bacteria of the HotSeep-1 cluster (G50). We investigate lipid membrane compositions as possible factors for the different temperature affinities of the different ANME clades and show autotrophy as characteristic feature for both ANME clades and their partner bacteria. Although in the absence of additional substrates methane formation was not observed, methanogenesis from methylated substrates (methanol and methylamine) could be quickly stimulated in the E20 and the G37 enrichment. Responsible for methanogenesis are archaea from the genus Methanohalophilus and Methanococcoides, which are minor community members during AOM (1-7‰ of archaeal 16S rRNA gene amplicons). In the same two cultures also sulfur disproportionation could be quickly stimulated by addition of zero-valent colloidal sulfur. The isolated partner bacteria are likewise minor community members (1-9‰ of bacterial 16S rRNA gene amplicons), whereas the dominant partner bacteria (Seep-SRB1a, Seep-SRB2, or HotSeep-1) did not grow on elemental sulfur. Our results support a functioning of AOM as syntrophic interaction of obligate methanotrophic archaea that transfer non-molecular reducing equivalents (i.e., via direct interspecies electron transfer) to obligate sulfate-reducing partner bacteria. Additional katabolic processes in these enrichments but also in sulfate methane interfaces are likely performed by minor community members.
Project description:Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem.
Project description:Methane seeps are widespread seafloor ecosystems shaped by complex physicochemical-biological interactions over geological timescales, and seep microbiomes play a vital role in global biogeochemical cycling of key elements on Earth. However, the mechanisms underlying the coexistence of methane-cycling microbial communities remain largely elusive. Here, high-resolution sediment incubation experiments revealed a cryptic methane cycle in the South China Sea (SCS) methane seep ecosystem, showing the coexistence of sulfate (SO4 2-)- or iron (Fe)-dependent anaerobic oxidation of methane (AOM) and methylotrophic methanogenesis. This previously unrecognized methane cycling is not discernible from geochemical profiles due to high net methane consumption. High-throughput sequencing and Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) results suggested that anaerobic methane-oxidizing archaea (ANME)-2 and -3 coupled to sulfate-reducing bacteria (SRB) carried out SO4 2--AOM, and alternative ANME-2 and -3 solely or coupled to iron-reducing bacteria (IRB) might participate in Fe-AOM in sulfate-depleted environments. This finding suggested that ANME could alter AOM metabolic pathways according to geochemical changes. Furthermore, the majority of methylotrophic methanogens belonged to Methanimicrococcus, and hydrogenotrophic and acetoclastic methanogens were likely inhibited by sulfate or iron respiration. Fe-AOM and methylotrophic methanogenesis are overlooked potential sources and sinks of methane in methane seep ecosystems, thus influencing methane budgets and even the global carbon budget in the ocean.
Project description:Sedimentary biofilms comprising microbial communities mediating the anaerobic oxidation of methane are rare. Here, we describe two biofilm communities discovered in sediment cores recovered from Arctic cold seep sites (gas hydrate pingos) in the north-western Barents Sea, characterized by steady methane fluxes. We found macroscopically visible biofilms in pockets in the sediment matrix at the depth of the sulphate-methane-transition zone. 16S rRNA gene surveys revealed that the microbial community in one of the two biofilms comprised exclusively of putative anaerobic methanotrophic archaea of which ANME-1 was the sole archaeal taxon. The bacterial community consisted of relatives of sulphate-reducing bacteria (SRB) belonging to uncultured Desulfobacteraceae clustering into SEEP-SRB1 (i.e. the typical SRB associated to ANME-1), and members of the atribacterial JS1 clade. Confocal laser scanning microscopy demonstrates that this biofilm is composed of multicellular strands and patches of ANME-1 that are loosely associated with SRB cells, but not tightly connected in aggregates. Our discovery of methanotrophic biofilms in sediment pockets closely associated with methane seeps constitutes a hitherto overlooked and potentially widespread sink for methane and sulphate in marine sediments.
Project description:The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed "Mat Mound") were characterized by porewater geochemistry of methane, C2-C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.
Project description:The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with (13)C-labeled CH4 ((13)CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, (13)C-labeled CO2 ((13)CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with (13)CO2 production and no methanogenesis occurred, excluding TMO as a possible source for (13)CO2 production from (13)CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.
Project description:Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to (15)[Formula: see text] and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were (15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis-including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)-were all observed to be actively synthesized ((15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of (15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.