Anaerobic Oxidation Of Methane Activity Research Articles

Humic substances as electron acceptors for anaerobic oxidation of methane driven by ANME-2d

Humic substances (humics) are ubiquitous in terrestrial and aquatic environments where they can serve as electron acceptors for anaerobic oxidation of organic compounds. Methane is a powerful greenhouse gas, as well as the least reactive organic molecule. Anaerobic oxidation of methane (AOM) coupled to microbial reduction of various electron acceptors plays a crucial role in mitigating methane emissions. Here, we reported that humics could serve as terminal electron acceptors for AOM using enriched nitrate-reducing AOM microorganisms. AOM coupled to the reduction of humics was demonstrated based on the production of 13C-labelled carbon dioxide, and AOM activity was evaluated with different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid were electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction, although the partner bacteria NC10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. The potential electron transfer models from ANME-2d to humics were proposed. These results enable a better understanding of available electron acceptors for AOM in natural environments and broaden our insight into the significant role of ANME-2d.

Evaluating radioisotope‐based approaches to measure anaerobic methane oxidation rates in lacustrine sediments

AbstractThe microbial anaerobic oxidation of methane (AOM) is the dominant sink for methane in anoxic sediments. AOM rate measurements are essential for assessing the efficacy of the benthic methane filter to mitigate the evasion of this potent greenhouse gas to the atmosphere. Incubation techniques with trace amounts of radiolabeled substrate (typically 14CH4) represent the most sensitive approach for methane oxidation rate measurements. Yet, radiotracer application can be performed in different ways, rendering the comparability of AOM rate measurements in field and laboratory investigations problematic. We compared four different 14CH4‐based short‐term incubation approaches to quantify methane turnover rates in lake sediments. Three of the applied methods yielded similar and reliable downcore rate profiles. They provided clear evidence for AOM with maximum rates of 15 nmol cm−3 d−1 at ~ 17 cm sediment depth. Using the short‐term slurry incubation (SL) method, however, we were unable to detect the AOM activity maximum that we observed with the other approaches. We hypothesize that changes in the microbial structure and disruption of physical interactions due to mixing of sediments negatively affected the activity of AOM communities and longer incubation times are necessary to enhance the sensitivity of this approach. Minor variabilities in rate measurement that we found in the non‐SL incubations may be related to small‐scale sediment heterogeneity, differential partial methane loss during sample handling, and/or an uneven application of the radiotracer. Whole‐core incubations interfere the least with the in situ conditions, but the ultimate choice of the AOM rate measurement method will depend on the individual sampling requirements.

Active pathways of anaerobic methane oxidation across contrasting riverbeds

Anaerobic oxidation of methane (AOM) reduces methane emissions from marine ecosystems but we know little about AOM in rivers, whose role in the global carbon cycle is increasingly recognized. We measured AOM potentials driven by different electron acceptors, including nitrite, nitrate, sulfate, and ferric iron, and identified microorganisms involved across contrasting riverbeds. AOM activity was confined to the more reduced, sandy riverbeds, whereas no activity was measured in the less reduced, gravel riverbeds where there were few anaerobic methanotrophs. Nitrite-dependent and nitrate-dependent AOM occurred in all sandy riverbeds, with the maximum rates of 61.0 and 20.0 nmol CO2 g−1 (dry sediment) d−1, respectively, while sulfate-dependent and ferric iron-dependent AOM occurred only where methane concentration was highest and the diversity of AOM pathways greatest. Diverse Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea were detected in the sandy riverbeds (16S rRNA gene abundance of 9.3 × 105 to 1.5 × 107 and 2.1 × 104 to 2.5 × 105 copies g−1 dry sediment, respectively) but no other known anaerobic methanotrophs. Further, we found M. oxyfera-like bacteria and M. nitroreducens-like archaea to be actively involved in nitrite- and nitrate/ferric iron-dependent AOM, respectively. Hence, we demonstrate multiple pathways of AOM in relation to methane, though the activities of M. oxyfera-like bacteria and M. nitroreducens-like archaea are dominant.

Microbial Selenate Reduction Driven by a Denitrifying Anaerobic Methane Oxidation Biofilm.

Anaerobic oxidation of methane (AOM) plays a crucial role in controlling the flux of methane from anoxic environments. Sulfate-, nitrite-, nitrate-, and iron-dependent methane oxidation processes have been considered to be responsible for the AOM activities in anoxic niches. We report that nitrate-reducing AOM microorganisms, enriched in a membrane biofilm bioreactor, are able to couple selenate reduction to AOM. According to ion chromatography, X-ray photoelectron spectroscopy, and long-term bioreactor performance, we reveal that soluble selenate was reduced to nanoparticle elemental selenium. High-throughput 16S rRNA gene sequencing indicates that Candidatus Methanoperedens and Candidatus Methylomirabilis remained the only known methane-oxidizing microorganisms after nitrate was switched to selenate, suggesting that these organisms could couple anaerobic methane oxidation to selenate reduction. Our findings suggest a possible link between the biogeochemical selenium and methane cycles.

Carbon Geochemistry of Gas Hydrate‐associated Sediments in the Southwestern Taiwan Basin

AbstractMarine gas hydrates, one of the largest methane reservoirs on Earth, may greatly affect the deep sea sedimentary environment and biogeochemistry; however, the carbon geochemistry in gas hydrate‐bearing sediments is poorly understood. In this study, we investigated the carbon variables in sediment core 973‐3 from the southwestern Taiwan Basin in the South China Sea to understand the effect of environmental factors and archaeal communities on carbon geochemistry. The carbon profiles suggest the methanogenesis with the incerase of dissolved inorganic carbon (DIC) and high total organic carbon (TOC) (mean = 0.46%) originated from terrigenous organic matter (mean δ13CTOC value of −23.6±) driven by the abundant methanogen ‘Methanosaeta and Methanomicrobiales'. The active anaerobic oxidation of methane is characterized by the increase of DIC and inorganic carbon (IC), and the depleted δ13CIC, coupled with the increase of TOC and the decrease of δ13CTOC values owing to the methanotroph ‘Methanosarcinales/ANME’ in 430–840 cm. Environmental factors and archaeal communities in core 973‐3 are significantly correlated to carbon variables owing to methane production and oxidation. Our results indicate that the carbon geochemical characteristics are obviously responding to the formation and decomposition of gas hydrates. Furthermore, pH, Eh and grain size, and Methanosaeta greatly affect the carbon geochemistry in gas hydrate‐associated sediments.

Biomarker assessments of sources and environmental implications of organic matter in sediments from potential cold seep areas of the northeastern South China Sea

Multi-biomarker indexes were analyzed for two piston cores from potential cold seep areas of the South China Sea off southwestern Taiwan. Total organic carbon (TOC) normalized terrestrial (n-alkanes) and marine (brassicasterol, dinosterol, alkenones and iso-GDGTs) biomarker contents and ratios (TMBR, 1/P mar-aq, BIT) were used to evaluate the contributions of terrestrial and marine organic matter (TOM and MOM respectively) to the sedimentary organic matter, indicating that MOM dominated the organic sources in Core MD052911 and the sedimentary organic matter in Core ORI-860-22 was mainly derived from terrestrial inputs, and different morphologies were the likely reason for TOM percentage differences. BIT results suggested that river-transported terrestrial soil organic matter was not a major source of TOM of sedimentary organic matter around these settings. Diagnostic biomarkers for methane-oxidizing archaea (MOA) were only detected in one sample at 172 cm depth of Core ORI-860-22, with abnormally high iso-GDGTs content and Methane Index (MI) value (0.94). These results indicated high anaerobic oxidation of methane (AOM) activities at or around 172 cm in Core ORI-860-22. However in Core MD052911, MOA biomarkers were not detected and MI values were lower (0.19–0.38), indicated insignificant contributions of iso-GDGTs from methanotrophic archaea and the absence of significant AOM activities. Biomarker results thus indicated that the discontinuous upward methane seepage and insufficient methane flux could not induce high AOM activities in our sampling sites. In addition, the different patterns of TEX86 and Uk′ 37 temperature in two cores suggested that AOM activities affected TEX86 temperature estimates with lower values in Core ORI-860-22, but not significantly on TEX86 temperature estimates in Core MD052911.

U-Th chronology and formation controls of methane-derived authigenic carbonates from the Hola trough seep area, northern Norway

We investigated methane-derived authigenic carbonate (MDAC) crusts and nodules from a cold seep site on the northern Norwegian continental shelf in ca. 220m water depth to determine the timing and mode of their formation. Gas bubbling observed during remotely operated vehicle (ROV)-assisted sampling of MDAC crusts revealed ongoing seep activity. Authigenic carbonates were present as crusts on the seafloor and as centimetre-size carbonate-cemented nodules at several intervals within an adjacent sediment core. Aragonite-dominated mineralogy of the MDAC crusts suggests formation close to the seafloor at higher rates of sulphate-dependent anaerobic oxidation of methane (AOM). In contrast, dolomite-cemented nodules are consistent with the formation at the sulphate-methane-transition zone deeper within the sediment at lower rates of AOM. The δ13C-carbonate values of bulk rock and of micro-drilled aragonite samples vary between −22.2‰ and −34.6‰ (VPDB). We interpret the carbon in aragonite to be mainly derived from the anaerobic oxidation of thermogenic methane, with a minor contribution from seawater dissolved inorganic carbon (DIC). AOM activity is supported by high concentrations of AOM-related biomarkers of archaea (archaeol and 2-sn-hydroxyarchaeol) and sulphate-reducing bacteria (iso and anteiso-C15:0 fatty acids) in the crusts. The dolomite nodules exhibit higher δ13C-carbonate values (−12‰ VPDB) suggesting a smaller amount of methane-derived carbon, presumably due to the contribution of DIC migrating from depth, and lower AOM rates. The latter is supported by orders of magnitude lower concentrations of archaeol and sn-2-hydroxyarchaeol in the sediment interval containing the largest dolomite nodules. δ18O values of pure aragonite samples and dolomite nodules indicate the precipitation of carbonate close to isotopic equilibrium with seawater and no influence of gas hydrate-derived water. U-Th dating of two MDAC crusts shows that they formed between 1.61±0.02 and 4.39±1.63ka BP and between 2.65±0.02 and 4.32±0.08ka BP. We infer both a spatial and temporal change in methane flux and related MDAC formation at this seep site. These changes might be caused by regional seismic events that can affect pore pressure or re-activation of migration pathways thus facilitating fluid flow from deep sources towards the seabed.

Anaerobic Methane Oxidation Driven by Microbial Reduction of Natural Organic Matter in a Tropical Wetland.

Wetlands constitute the main natural source of methane on Earth due to their high content of natural organic matter (NOM), but key drivers, such as electron acceptors, supporting methanotrophic activities in these habitats are poorly understood. We performed anoxic incubations using freshly collected sediment, along with water samples harvested from a tropical wetland, amended with 13C-methane (0.67 atm) to test the capacity of its microbial community to perform anaerobic oxidation of methane (AOM) linked to the reduction of the humic fraction of its NOM. Collected evidence demonstrates that electron-accepting functional groups (e.g., quinones) present in NOM fueled AOM by serving as a terminal electron acceptor. Indeed, while sulfate reduction was the predominant process, accounting for up to 42.5% of the AOM activities, the microbial reduction of NOM concomitantly occurred. Furthermore, enrichment of wetland sediment with external NOM provided a complementary electron-accepting capacity, of which reduction accounted for ∼100 nmol 13CH4 oxidized · cm-3 · day-1 Spectroscopic evidence showed that quinone moieties were heterogeneously distributed in the wetland sediment, and their reduction occurred during the course of AOM. Moreover, an enrichment derived from wetland sediments performing AOM linked to NOM reduction stoichiometrically oxidized methane coupled to the reduction of the humic analogue anthraquinone-2,6-disulfonate. Microbial populations potentially involved in AOM coupled to microbial reduction of NOM were dominated by divergent biota from putative AOM-associated archaea. We estimate that this microbial process potentially contributes to the suppression of up to 114 teragrams (Tg) of CH4 · year-1 in coastal wetlands and more than 1,300 Tg · year-1, considering the global wetland area.IMPORTANCE The identification of key processes governing methane emissions from natural systems is of major importance considering the global warming effects triggered by this greenhouse gas. Anaerobic oxidation of methane (AOM) coupled to the microbial reduction of distinct electron acceptors plays a pivotal role in mitigating methane emissions from ecosystems. Given their high organic content, wetlands constitute the largest natural source of atmospheric methane. Nevertheless, processes controlling methane emissions in these environments are poorly understood. Here, we provide tracer analysis with 13CH4 and spectroscopic evidence revealing that AOM linked to the microbial reduction of redox functional groups in natural organic matter (NOM) prevails in a tropical wetland. We suggest that microbial reduction of NOM may largely contribute to the suppression of methane emissions from tropical wetlands. This is a novel avenue within the carbon cycle in which slowly decaying NOM (e.g., humic fraction) in organotrophic environments fuels AOM by serving as a terminal electron acceptor.

Anaerobic Methanotrophic Archaea of the ANME-2d Cluster Are Active in a Low-sulfate, Iron-rich Freshwater Sediment.

ANaerobic MEthanotrophic (ANME) archaea remove the greenhouse gas methane from anoxic environments and diminish its flux to the atmosphere. High methane removal efficiencies are well documented in marine environments, whereas anaerobic oxidation of methane (AOM) was only recently indicated as an important methane sink in freshwater systems. Freshwater AOM-mediating microorganisms lack taxonomic identification and only little is known about metabolic adaptions to prevailing biogeochemical conditions. One of the first study sites providing information about AOM activity in freshwater sediment is Lake Ørn, a low-sulfate, iron-rich Danish lake. With the aim to identify freshwater AOM-mediating archaea, we incubated AOM-active anoxic, nitrate-free freshwater sediment from Lake Ørn with 13C-labeled methane (13CCH4) and 13C-labeled bicarbonate (13CDIC) and followed the assimilation of 13C into RNA by stable isotope probing. While AOM was active, 13CCH4 and probably also 13CDIC were incorporated into uncultured archaea of the Methanosarcinales-related cluster ANME-2d, whereas other known ANME lineages were not detected. This finding strongly suggests that ANME-2d archaea perform AOM coupled to sulfate and/or iron reduction and may have the capability of mixed assimilation of CH4 and DIC. ANME-2d archaea may thus play an important role in controlling methane emissions from nitrate-depleted and low-sulfate freshwater systems.

High Sulfur Isotope Fractionation Associated with Anaerobic Oxidation of Methane in a Low-Sulfate, Iron-Rich Environment

Sulfur isotope signatures provide key information for the study of microbial activity in modern systems and the evolution of the Earth surface redox system. Microbial sulfate reducers shift sulfur isotope distributions by discriminating against heavier isotopes. This discrimination is strain-specific and often suppressed at sulfate concentrations in the lower micromolar range that are typical to freshwater systems and inferred for ancient oceans. Anaerobic oxidation of methane (AOM) is a sulfate-reducing microbial process with a strong impact on global sulfur cycling in modern habitats and potentially in the geological past, but its impact on sulfur isotope signatures is poorly understood, especially in low sulfate environments. We investigated sulfur cycling and 34S fractionation in a low-sulfate freshwater sediment with biogeochemical conditions analogous to Early Earth environments. The zone of highest AOM activity was associated in situ with a zone of strong 34S depletions in the pool of reduced sulfur species, indicating a coupling of sulfate reduction and AOM at sulfate concentrations < 50 µmol L-1. In slurry incubations of AOM-active sediment, the addition of methane stimulated sulfate reduction and induced a bulk sulfur isotope effect of ~29 ‰. Our results imply that sulfur isotope signatures may be strongly impacted by AOM even at sulfate concentrations two orders of magnitude lower than at present oceanic levels. Therefore, we suggest that sulfur isotope fractionation during AOM must be considered when interpreting 34S signatures in modern and ancient environment.

Methane Seep in Shallow-Water Permeable Sediment Harbors High Diversity of Anaerobic Methanotrophic Communities, Elba, Italy.

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.

Characteristics of authigenic pyrite and its sulfur isotopes influenced by methane seep at Core A, Site 79 of the middle Okinawa Trough

The anaerobic oxidation of methane (AOM) has strongly developed at Core A, Site 79 of the middle Okinawa Trough, East China Sea, and a large amount of authigenic pyrite is preserved in the surface sediment. In this study, we analyze the characteristics of the authigenic pyrite and its sulfur isotopic values. The authigenic pyrite is stripy and tubular, and there were foraminifera compartments filled with pyrite. The pyrite is extracted using chromium reduction, and the values of δ 34S are found to lie between -41.20‰ and 8.92‰ V-CDT. The bulk pyrite tends to be more enriched in 34S with increasing depth. Particularly, the δ 34S value of the pyrite lies between -32.73‰ and -41.20‰ V-CDT above 278 cmbsf, but it quickly increases below this depth (-21.49‰–8.92‰ V-CDT). At the same time, the total sulfur content of the pyrite shows an abrupt increase above 100 cmbsf but is otherwise stable between 1.04% and 0.55% below 100 cmbsf. The stable and negative values of δ 34S and the decreasing values of total sulfur above 278 cmbsf indicate reduced AOM activities in 17.18–5.3 ka. In addition, the increasing δ 34S and pyrite content indicate strong AOM development and methane seep below 278 cmbsf in 18.8–17.18 ka. In particular, the highest positive value of δ 34S occurring in 18.78 ka indicates the most intense AOM activity. The shallow sulfate-methane interface (SMI) and high methane flux below marine sediments also strongly support this activity.

Response of anaerobic methanotrophs and benthic foraminifera to 20 years of methane emission from a gas blowout in the North Sea

Methane emissions from marine sediments are partly controlled by microbial anaerobic oxidation of methane (AOM). AOM provides a long-term sink for carbon through precipitation of methane-derived authigenic carbonates (MDAC). Estimates on the adaptation time of this benthic methane filter as well as on the establishment of related processes and communities after an onset of methane seepage are rare. In the North Sea, considerable amounts of methane have been released since 20 years from a man-made gas blowout offering an ideal natural laboratory to study the effects of methane seepage on initially “pristine” sediment. Sediment cores were taken from the blowout crater and a reference site (50 m distance) in 2011 and 2012, respectively, to investigate porewater chemistry, the AOM community and activity, the presence of authigenic carbonates, and benthic foraminiferal assemblages. Potential AOM activity (up to 3060 nmol cm−3 sediment d−1 or 375 mmol m−2 d−1) was detected only in the blowout crater up to the maximum sampling depth of 18 cm. CARD-FISH analyzes suggest that monospecific ANME-2 aggregates were the only type of AOM organisms present, showing densities (up to 2.2*107 aggregates cm−3) similar to established methane seeps. No evidence for recent MDAC formation was found using stable isotope analyzes (δ13C and δ18O). In contrast, the carbon isotopic signature of methane was recorded by the epibenthic foraminifer Cibicides lobatulus (δ13C −0.66‰). Surprisingly, the foraminiferal assemblage in the blowout crater was dominated by Cibicides and other species commonly found in the Norwegian Channel and fjords, indicating that these organisms have responded sensitively to the specific environmental conditions at the blowout. The high activity and abundance of AOM organisms only at the blowout site suggests adaptation to a strong increase in methane flux in the order of at most two decades. High gas discharge dynamics in permeable surface sediments facilitate fast sulfate replenishing and stimulation of AOM. The accompanied prevention of total alkalinity build-up in the porewater thereby appears to inhibit the formation of substantial methane-derived authigenic carbonate at least within the given time window.

High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions

The role of anaerobic oxidation of methane (AOM) in wetlands, the largest natural source of atmospheric methane, is poorly constrained. Here we report rates of microbially mediated AOM (average rate=20 nmol cm(-3) per day) in three freshwater wetlands that span multiple biogeographical provinces. The observed AOM rates rival those in marine environments. Most AOM activity may have been coupled to sulphate reduction, but other electron acceptors remain feasible. Lipid biomarkers typically associated with anaerobic methane-oxidizing archaea were more enriched in (13)C than those characteristic of marine systems, potentially due to distinct microbial metabolic pathways or dilution with heterotrophic isotope signals. On the basis of this extensive data set, AOM in freshwater wetlands may consume 200 Tg methane per year, reducing their potential methane emissions by over 50%. These findings challenge precepts surrounding wetland carbon cycling and demonstrate the environmental relevance of an anaerobic methane sink in ecosystems traditionally considered strong methane sources.

Geochemical constraints on the methane seep activity in western slope of the middle Okinawa Trough, the East China Sea

Anaerobic oxidation of methane and sulfate reduction was studied in the pore waters of four cores at two stations of the middle Okinawa Trough. Pore water vertical distributions of sulfate, methane, sulfide, total alkalinity, ammonium, and phosphate were determined in this study. Our results show strong linear sulfate concentration gradients of 6.83 mmol m−1 in Core A and 5.96 mmol m−1 in Core C, which were collected from two stations. Concurrent variations of methane, total alkalinity and hydrogen sulfide all exhibit steep increases with depth at both cores, which indicate active methane seep activities around two stations. Pore water ammonium and phosphate concentrations reveal minor influences of organic matter degradation on sulfate reduction at two stations. Sulfate methane interface (SMI) was extrapolated from linear sulfate profiles in methane seep cores. Shallower SMI depths (A: 4.9 mbsf; C: 5.4 mbsf) indicate strong methane fluxes and active anaerobic oxidation of methane in the underlying sediments.

Stratified community responses to methane and sulfate supplies in mud volcano deposits: insights from an in vitro experiment.

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world's oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30–55 cm below the sea floor (bsf); the second is at 165–205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry.

Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling

Two ∼6m long sediment cores were collected along the ∼300m isobath on the Alaskan Beaufort Sea continental margin. Both cores showed distinct sulfate-methane transition zones (SMTZ) at 105 and 120cm below seafloor (cmbsf). Sulfate was not completely depleted below the SMTZ but remained between 30 and 500μM. Sulfate reduction and anaerobic oxidation of methane (AOM) determined by radiotracer incubations were active throughout the methanogenic zone. Although a mass balance could not explain the source of sulfate below the SMTZ, geochemical profiles and correlation network analyses of biotic and abiotic data suggest a cryptic sulfur cycle involving iron, manganese and barite. Inhibition experiments with molybdate and 2-bromoethanesulfonate (BES) indicated decoupling of sulfate reduction and AOM and competition between sulfate reducers and methanogens for substrates. While correlation network analyses predicted coupling of AOM to iron reduction, the addition of manganese or iron did not stimulate AOM. Since none of the classical archaeal anaerobic methanotrophs (ANME) were abundant, the involvement of unknown or unconventional phylotypes in AOM is conceivable. The resistance of AOM activity to inhibitors implies deviation from conventional enzymatic pathways. This work suggests that the classical redox cascade of electron acceptor utilization based on Gibbs energy yields does not always hold in diffusion-dominated systems, and instead biotic processes may be more strongly coupled to mineralogy.

A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor.

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.

Origin of lipid biomarkers in mud volcanoes from the Alboran Sea, western Mediterranean

Abstract. Mud volcanoes (MVs) are the most prominent indicators of active methane/hydrocarbon venting at the seafloor on both passive and active continental margins. Their occurrence in the western Mediterranean is patent at the West Alboran Basin, where numerous MVs develop overlaying a major sedimentary depocentre containing overpressured shales. Although some of these MVs have been studied, the detailed biogeochemistry of expelled mud so far has not been examined in detail. This work provides the first results on the composition and origin of organic matter, anaerobic oxidation of methane (AOM) processes and general characteristics on MV dynamics using lipid biomarkers as the main tool. Lipid biomarker analysis was performed on MV expelled material (mud breccias) and interbedded hemipelagic sediments from Perejil, Kalinin and Schneider's Heart MVs located in the northwest margin of the Alboran Sea. The n alkane distributions and n alkane-derived indices (CPI and ACL), in combination with the epimerization degree of hopanes (22S/(22S+22R)) indicate that all studied mud breccia have a similar biomarker composition consisting of mainly thermally immature organic matter with an admixture of petroleum-derived compounds. This concordant composition indicates that common source strata must feed all three studied MVs. The past or present AOM activity was established using lipid biomarkers specific for anaerobic methanotrophic archaea (irregular isoprenoids and dialkyl glycerol diethers) and the depleted carbon isotope composition (δ13C) of crocetane/phytane. The presence of these lipid biomarkers, together with the low amounts of detected glycerol dialkyl glycerol tetraethers, is consistent with the dominance of anaerobic methanotrophs of the ANME-2 over ANME-1, at least in mud breccia from Perejil MVs. In contrast, the scarce presence or lack of these AOM-related lipid biomarkers in sediments from Kalinin and Schneider's Heart MVs, suggests that no recent active methane seepage has occurred at these sites. Moreover, the observed methane concentrations support the current activity of Perejil MV, and the very low methane seepage activity in Kalinin and Schneider's Heart MVs.

Spatial variation in shallow sediment methane sources and cycling on the Alaskan Beaufort Sea Shelf/Slope

The MITAS (Methane in the Arctic Shelf/Slope) expedition was conducted during September, 2009 onboard the U.S. Coast Guard Cutter (USCGC) Polar Sea (WAGB-11), on the Alaskan Shelf/Slope of the Beaufort Sea. Expedition goals were to investigate spatial variations in methane source(s), vertical methane flux in shallow sediments (<10 mbsf), and methane contributions to shallow sediment carbon cycling. Three nearshore to offshore transects were conducted across the slope at locations approximately 200 km apart in water column depths from 20 to 2100 m. Shallow sediments were collected by piston cores and vibracores and samples were analyzed for sediment headspace methane (CH4), porewater sulfate (SO42−), chloride (Cl−), and dissolved inorganic carbon (DIC) concentrations, and CH4 and DIC stable carbon isotope ratios (δ13C). Downward SO42− diffusion rates estimated from sediment porewater SO42− profiles were between −15.4 and −154.8 mmol m−2 a−1 and imply a large spatial variation in vertical CH4 flux between transects in the study region. Lowest inferred CH4 fluxes were estimated along the easternmost transect. Higher inferred CH4 flux rates were observed in the western transects. Sediment headspace δ13CCH4 values ranged from −138 to −48‰, suggesting strong differences in shallow sediment CH4 cycling within and among sample locations. Measured porewater DIC concentrations ranged from 2.53 mM to 79.39 mM with δ13CDIC values ranging from −36.4‰ to 5.1‰. Higher down-core DIC concentrations were observed to occur with lower δ13C where an increase in δ13CCH4 was measured, indicating locations with active anaerobic oxidation of methane. Shallow core CH4 production was inferred at the two western most transects (i.e. Thetis Island and Halkett) through observations of low δ13CCH4 coupled with elevated DIC concentrations. At the easternmost Hammerhead transect and offshore locations, δ13CCH4 and DIC concentrations were not coupled suggesting less rapid methane cycling. Results from the MITAS expedition represent one of the most comprehensive studies of methane source(s) and vertical methane flux in shallow sediments of the U.S. Alaskan Beaufort Shelf to date and show geospatially variable sediment methane flux that is highly influenced by the local geophysical environment.