Microbial life inhabiting subseafloor sediments plays an important role in Earth’s carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (10 2 to 10 3 cells cm −3 ) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH3D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH3D procedure and the well-established 14CH4 radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH3D method were directly and consistently proportional to 14C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method. IMPORTANCE Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH3D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH3D procedure with the well-established 14C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH3D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH3D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.

High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.

Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function.

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.

Abstract. Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring the rates of these metabolisms presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for measuring rates of microbial methane metabolism that is non-toxic, rapid, and relatively high throughput, alleviating some of the current methodological challenges. Specifically, we tested the potential for using monodeuterated methane (CH3D) as a metabolic substrate for measuring the rate of methane activation by quantifying the change in the aqueous D / H ratio over time using a water isotope analyzer. This method represents a non-toxic, comparatively rapid and straightforward approach that is complementary to existing radio (14C)- and stable (13C) carbon isotopic methods; by probing hydrogen atom dynamics, it offers an additional dimension through which to examine the rates and pathways of methane metabolism. We provide direct comparisons between the CH3D procedure and the well-established 14CH4 radiotracer approach for several methanotrophic systems, including type I and type II aerobic methanotroph cultures, and methane seep sediment and carbonate rocks under anoxic and oxic incubation conditions. We also employ this method to investigate the role of pressure on methane oxidation rates in anoxic seep sediment, revealing an 80 % increase at the equivalent of ~ 900 m water depth (40 MPa). The monodeuterated methane approach offers a procedurally straightforward, reliable method that advances three specific aims: 1) the direct comparison of methane oxidation rates between different experimental treatments of the same inoculum; 2) the determination of an absolute scaling factor using paired CH3D and 14C-radiocarbon procedures for new systems of interest; and 3) a continued evaluation of C- and H-atom tracking through methanotrophic metabolisms, with specific foci on enzyme reversibility and anabolic/catabolic branch points. The procedural advantages, consistency, and novel research questions enabled by the monodeuterated methane method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.

Anaerobic oxidation of methane (AOM) impacts carbon cycling by acting as a methane sink and by sequestering inorganic carbon via AOM-induced carbonate precipitation. These precipitates commonly take the form of carbonate nodules that form within methane seep sediments. The timing and sequence of nodule formation within methane seep sediments are not well understood. Further, the microbial diversity associated with sediment-hosted nodules has not been well characterized and the degree to which nodules reflect the microbial assemblage in surrounding sediments is unknown. Here, we conducted a comparative study of microbial assemblages in methane-derived authigenic carbonate nodules and their host sediments using molecular, mineralogical, and geochemical methods. Analysis of 16S rRNA gene diversity from paired carbonate nodules and sediments revealed that both sample types contained methanotrophic archaea (ANME-1 and ANME-2) and syntrophic sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae), as well as other microbial community members. The combination of geochemical and molecular data from Eel River Basin and Hydrate Ridge suggested that some nodules formed in situ and captured the local sediment-hosted microbial community, while other nodules may have been translocated or may represent a record of conditions prior to the contemporary environment. Taken together, this comparative analysis offers clues to the formation regimes and mechanisms of sediment-hosted carbonate nodules.

ABSTRACT Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages. IMPORTANCE Since their discovery in 1984, the global distribution and importance of marine methane seeps have become increasingly clear. Much of our understanding of methane seep microorganisms—from metabolisms to community ecology—has stemmed from detailed studies of seep sediments. However, it has become apparent that carbonates represent a volumetrically significant habitat substrate at methane seeps. Through combined in situ characterization and incubation experiments, this study demonstrates that carbonates host microbial assemblages distinct from and more diverse than those of other seep habitats. This emphasizes the importance of seep carbonates as biodiversity locales. Furthermore, we demonstrate that carbonate-associated microbial assemblages are well adapted to withstand fluctuations in methane seepage, and we gain novel insight into particular taxa that are responsive (or recalcitrant) to changes in seep conditions.

人為的要因による大気中CO2の増加は地球温暖化の主原因と考えられており,早急なCO2隔離・削減技術の開発が求められている.本研究では,ゲスト分子置換法を利用した,海底に産するメタンハイドレートへのCO2封入及びメタン回収が可能かどうかを検討する予備実験として,新潟県上越沖上越海丘において,CO2排出試験及び表層型塊状メタンハイドレートの分解実験を行った.深海底でCO2圧入を行う為,等圧シリンダーポンプ及びヒーターを内蔵したCO2注入用プローブ(ヒートゾンデ)を開発した.メタンハイドレート胚胎堆積物へのCO2注入に先立ち,深海底でのCO2排出を試みたところ,液体CO2がヒートゾンデ先端から排出される様子が観察された後ポンプが動かなくなり,注入作業を行うことが出来なかった.これは装置内に海水が流入して形成されたCO2ハイドレートが流路を塞いだのが原因と考えられ,CO2ハイドレートの形成を防止する改良を装置に施す必要があることが解った.また,ヒートゾンデの熱により海底面に露出したメタンハイドレートが崩壊する様子が観察された.

The importance of tectonics and fluid flow in controlling cold seep habitats has long been appreciated at convergent margins but remains poorly understood in strike-slip systems. Here we present geophysical, geochemical, and biological data from an active methane seep offshore from Del Mar, California, in the inner California borderlands (ICB). The location of this seep appears controlled by localized transpression associated with a step in the San Diego Trough fault zone and provides an opportunity to examine the interplay between fluid expulsion and restraining step overs along strike-slip fault systems. These segment boundaries may have important controls on seep locations in the ICB and other margins characterized by strike-slip faulting (e.g., Greece, Sea of Marmara, and Caribbean). The strike-slip fault systems offshore southern California appear to have a limited distribution of seep sites compared to a wider distribution at convergent plate boundaries, which may influence seep habitat diversity and connectivity.

Methane seeps are among the most productive habitats along continental margins, as anaerobic methane-oxidizing euryarchaeaota and sulfur-metabolizing deltaproteobacteria form the biological base of a dynamic deep-sea ecosystem. The degree of methane seepage therefore represents one important variable in ecosystem dynamics, and the recent discovery of carbonate-hosted endolithic methanotrophy exposes another potentially discriminating factor: physical substrate type. Methanotrophic microbial communities have been detected within diverse seep-associated habitats, including unlithified sediments, protolithic carbonate nodules, and lithified carbonate slabs and chemoherms of distinct mineralogies. However, a systematic assessment of the diversity and community structure associated with these different habitats has been lacking. In this study, microbial aggregate analysis, microbial abundance quantification, mineralogical identification, and archaeal and bacterial 16S rRNA gene clone libraries were used to deconvolve the relationships between seepage activity, substrate type, and microbial community structure. We report prevalent methane-oxidizing archaeal lineages in both active and low-activity seep settings, and a strong community dependence on both seepage activity and substrate type. Statistical treatments of relative taxa abundances indicate that archaeal community structure is more dependent on the degree of methane seepage than physical substrate type; bacterial assemblages appear to be more strongly influenced by the type of colonization substrate than seepage activity. These findings provide a window into the determinants of community structure and function, improving our understanding of potential elemental cycling at seep sites.

The Sr/Ca ratio of coral aragonite is used to reconstruct past sea surface temperature (SST). Twenty‐one laboratories took part in an interlaboratory study of coral Sr/Ca measurements. Results show interlaboratory bias can be significant, and in the extreme case could result in a range in SST estimates of 7°C. However, most of the data fall within a narrower range and the Porites coral reference material JCp‐1 is now characterized well enough to have a certified Sr/Ca value of 8.838 mmol/mol with an expanded uncertainty of 0.089 mmol/mol following International Association of Geoanalysts (IAG) guidelines. This uncertainty, at the 95% confidence level, equates to 1.5°C for SST estimates using Porites, so is approaching fitness for purpose. The comparable median within laboratory error is <0.5°C. This difference in uncertainties illustrates the interlaboratory bias component that should be reduced through the use of reference materials like the JCp‐1. There are many potential sources contributing to biases in comparative methods but traces of Sr in Ca standards and uncertainties in reference solution composition can account for half of the combined uncertainty. Consensus values that fulfil the requirements to be certified values were also obtained for Mg/Ca in JCp‐1 and for Sr/Ca and Mg/Ca ratios in the JCt‐1 giant clam reference material. Reference values with variable fitness for purpose have also been obtained for Li/Ca, B/Ca, Ba/Ca, and U/Ca in both reference materials. In future, studies reporting coral element/Ca data should also report the average value obtained for a reference material such as the JCp‐1.

Precipitation of carbonate minerals can play important roles in geological carbon sequestration and engineered processes for mineral carbonation. Influences of temperature, solution composition, and the presence of a solid substrate on the nucleation and precipitation of magnesium carbonate minerals were examined in a set of batch experiments. Conditions studied are relevant to full-scale geological carbon sequestration systems. Aqueous phase analysis by inductively coupled plasma mass spectrometry quantified the extent of precipitation. X-ray diffraction analysis was conducted to identify solids. Temperature significantly affected the identity of the solid obtained. At 25 degrees C and 60 degrees C the solids were magnesium carbonate minerals, and at 100 degrees C the solid phase was identified as brucite [Mg(OH)(2)]. Although magnesite (MgCO3) was predicted to be the most thermodynamically stable magnesium carbonate phase, no magnesite precipitated and instead metastable magnesium carbonate phases formed. Evolution of dissolved concentrations was consistent with precipitation of these metastable phases. Presence of the magnesium silicate forsterite had no measurable effect on the rate or extent of precipitation. Mineralization in geological systems is likely to also be controlled by ionic strength, pressure, and mineralogy of the host formation.

Deep-sea scleractinian corals precipitate aragonite skeletons that provide valuable archives of past ocean conditions. During calcification biological mediation causes variability in trace metal incorporation and isotopic ratios of the aragonite such that signals caused by environmental controls can be overwhelmed. This complicates the interpretation of geochemical proxies used for paleo-reconstructions. In this study we examine the environmental controls on the Mg/Li ratio of 34 individuals from seven genera of deep-sea scleractinian corals: Desmophyllum, Balanophyllia, Caryophyllia, Enallopsammia, Flabellum, Trochocyanthus, and Lophelia. In addition we examine the microscale distributions of Mg and Li in Desmophyllum and Balanophyllia using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Both Mg/Ca and Li/Ca ratios increased by more than a factor of two in the center of calcification regions compared to the outer, fibrous regions of the coral skeleton. As a result, replicate ~10mg subsamples of coral show less variability in the Mg/Li ratio than Mg/Ca. Microscale Mg and Li results are consistent with Rayleigh-type incorporation of trace metals with additional processes dominating the composition within centers of calcification. Comparison of Mg/Li to seawater properties near the site of collection shows that the ratio is not controlled by either carbonate ion or salinity. It appears that temperature is the major control on the Mg/Li ratio. For all 34 samples the temperature correlation (R2=0.62) is significantly better than for Mg/Ca (R2=0.06). For corals of the family Caryophyllidae the R2 value increases to 0.82 with the exclusion of one sample that was observed to have an altered, chalky texture. Despite this excellent correlation the scatter in the data suggests that the Mg/Li ratio of deep-sea corals cannot be used to reconstruct temperature to better than approximately ±1.6°C without better temperature control and additional calibration points on modern coral samples.