Methane seeps are widespread seafloor ecosystems shaped by the emission of gas from seabed reservoirs. The microorganisms inhabiting methane seeps transform the chemical energy in methane to products that sustain rich benthic communities around the gas leaks. Despite the biogeochemical relevance of microbial methane removal at seeps, the global diversity and dispersion of seep microbiota remain unknown. Here we determined the microbial diversity and community structure of 23 globally distributed methane seeps and compared these to the microbial communities of 54 other seafloor ecosystems, including sulfate–methane transition zones, hydrothermal vents, coastal sediments, and deep-sea surface and subsurface sediments. We found that methane seep communities show moderate levels of microbial richness compared with other seafloor ecosystems and harbor distinct bacterial and archaeal taxa with cosmopolitan distribution and key biogeochemical functions. The high relative sequence abundance of ANME (anaerobic methanotrophic archaea), as well as aerobic Methylococcales, sulfate-reducing Desulfobacterales, and sulfide-oxidizing Thiotrichales, matches the most favorable microbial metabolisms at methane seeps in terms of substrate supply and distinguishes the seep microbiome from other seafloor microbiomes. The key functional taxa varied in relative sequence abundance between different seeps due to the environmental factors, sediment depth and seafloor temperature. The degree of endemism of the methane seep microbiome suggests a high local diversification in these heterogeneous but long-lived ecosystems. Our results indicate that the seep microbiome is structured according to metacommunity processes and that few cosmopolitan microbial taxa mediate the bulk of methane oxidation, with global relevance to methane emission in the ocean. Significance Methane seeps are natural gas leaks at the seafloor that emit methane to the hydrosphere. The emission rates are controlled by methane-oxidizing microorganisms, which shape the ecosystem by supplying energy sources to other microorganisms and animal symbioses. We provide evidence that methane seeps are island-like habitats, harboring distinct microbial communities that share few organisms with other seafloor ecosystems. The seep communities comprise bacteria and archaea that occur worldwide but are locally selected by the environment. These microorganisms show high relative sequence abundances, suggesting high population densities and global relevance for the control of methane emission from the seafloor. At individual seeps, the cosmopolitan microorganisms are associated with a substantial diversity of rare relatives, turning seeps into hotspots of microbial biodiversity.

Around 50% of humankind relies on groundwater as a source of drinking water. Here we investigate the age, geochemistry, and microbiology of 138 groundwater samples from 95 monitoring wells (<250 m depth) located in 14 aquifers in Canada. The geochemistry and microbiology show consistent trends suggesting large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Older groundwaters, especially in aquifers with organic carbon-rich strata, contain on average more cells (up to 1.4 × 10⁷ mL⁻¹) than younger groundwaters, challenging current estimates of subsurface cell abundances. We observe substantial concentrations of dissolved oxygen (0.52 ± 0.12 mg L⁻¹ [mean ± SE]; n = 57) in older groundwaters that seem to support aerobic metabolisms in subsurface ecosystems at an unprecedented scale. Metagenomics, oxygen isotope analyses and mixing models indicate that dark oxygen is produced in situ via microbial dismutation. We show that ancient groundwaters sustain productive communities and highlight an overlooked oxygen source in present and past subsurface ecosystems of Earth.

The methane-emitting cold seeps of Hikurangi margin (New Zealand) are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga. Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets.

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 to 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 to 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.

Emission of the greenhouse gas methane from the seabed is globally controlled by marine aerobic and anaerobic methanotrophs gaining energy via methane oxidation. However, the processes involved in the assembly and dynamics of methanotrophic populations in complex natural microbial communities remain unclear. Here we investigated the development of a methanotrophic microbiome following subsurface mud eruptions at Håkon Mosby mud volcano (1250 m water depth). Freshly erupted muds hosted deep-subsurface communities that were dominated by Bathyarchaeota, Atribacteria and Chloroflexi. Methanotrophy was initially limited to a thin surface layer of Methylococcales populations consuming methane aerobically. With increasing distance to the eruptive center, anaerobic methanotrophic archaea, sulfate-reducing Desulfobacterales and thiotrophic Beggiatoaceae developed, and their respective metabolic capabilities dominated the biogeochemical functions of the community. Microbial richness, evenness, and cell numbers of the entire microbial community increased up to tenfold within a few years downstream of the mud flow from the eruptive center. The increasing diversity was accompanied by an up to fourfold increase in sequence abundance of relevant metabolic genes of the anaerobic methanotrophic and thiotrophic guilds. The communities fundamentally changed in their structure and functions as reflected in the metagenome turnover with distance from the eruptive center, and this was reflected in the biogeochemical zonation across the mud volcano caldera. The observed functional succession provides a framework for the response time and recovery of complex methanotrophic communities after disturbances of the deep-sea bed.