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Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

 

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter , Methyloversatilis , and Pseudomonas . Subsequent experimental deployments included a methanogen-dominated stage ( Methanobacteriales and Methanomicrobiales ) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells. 

Deep-sea cold seeps are dynamic sources of methane release and unique habitats supporting ocean biodiversity and productivity. Here, we describe newly discovered animal-bacterial symbioses fueled by methane, between two species of annelid (a serpulid Laminatubus and sabellid Bispira ) and distinct aerobic methane-oxidizing bacteria belonging to the Methylococcales, localized to the host respiratory crown. Worm tissue δ 13 C of −44 to −58‰ are consistent with methane-fueled nutrition for both species, and shipboard stable isotope labeling experiments revealed active assimilation of 13 C-labeled methane into animal biomass, which occurs via the engulfment of methanotrophic bacteria across the crown epidermal surface. These worms represent a new addition to the few animals known to intimately associate with methane-oxidizing bacteria and may further explain their enigmatic mass occurrence at 150–million year–old fossil seeps. High-resolution seafloor surveys document significant coverage by these symbioses, beyond typical obligate seep fauna. These findings uncover novel consumers of methane in the deep sea and, by expanding the known spatial extent of methane seeps, may have important implications for deep-sea conservation. 

Ecotones have been described as “biodiversity hotspots” from myriad environments, yet have not been studied extensively in the deep ocean. While physiologically challenging, deep‐water methane seeps host highly productive communities fueled predominantly by chemosynthetic pathways. We hypothesized that the biological and geochemical influence of methane seeps extends into background habitats, resulting in the formation of a “chemotone” where chemosynthesis‐based and photosynthesis‐based communities overlap. To investigate this, we analyzed the macrofaunal assemblages and geochemical properties of sediments collected from “active,” “transition” (potential chemotone), and “background” habitats surrounding five Costa Rican methane seeps (depth range 377–1908 m). Sediment geochemistry demonstrated a clear distinction between active and transition habitats, but not between transition and background habitats. In contrast, biological variables confirmed the presence of a chemotone, characterized by intermediate biomass, a distinct species composition (including habitat endemics and species from both active and background habitats), and enhanced variability in species composition among samples. However, chemotone assemblages were not distinct from active and/or background assemblages in terms of faunal density, biological trait composition, or diversity. Biomass and faunal stable isotope data suggest that chemotones are driven by a gradient in food delivery, receiving supplements from chemosynthetic production in addition to available photosynthetic‐based resources. Sediment geochemistry suggests that chemosynthetic food supplements are delivered across the chemotone at least in part through the water column, as opposed to reflecting exclusivelyin situchemosynthetic production in sediments. Management efforts should be cognisant of the ecological attributes and spatial extent of the chemotone that surrounds deep‐sea chemosynthetic environments.