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Fossil benthic foraminifera are used to trace past methane release linked to climate change. However, it is still debated whether isotopic signatures of living foraminifera from methane-charged sediments reflect incorporation of methane-derived carbon. A deeper understanding of isotopic signatures of living benthic foraminifera from methane-rich environments will help to improve reconstructions of methane release in the past and better predict the impact of future climate warming on methane seepage. Here, we present isotopic signatures (δ13C and δ18O) of foraminiferal calcite together with biogeochemical data from Arctic seep environments from c. 1200 m water depth, Vestnesa Ridge, 79° N, Fram Strait. Lowest δ13C values were recorded in shells of Melonis barleeanus, - 5.2‰ in live specimens and - 6.5‰ in empty shells, from sediments dominated by aerobic (MOx) and anaerobic oxidation of methane (AOM), respectively. Our data indicate that foraminifera actively incorporate methane-derived carbon when living in sediments with moderate seepage activity, while in sediments with high seepage activity the poisonous sulfidic environment leads to death of the foraminifera and an overgrowth of their empty shells by methane-derived authigenic carbonates. We propose that the incorporation of methane-derived carbon in living foraminifera occurs via feeding on methanotrophic bacteria and/or incorporation of ambient dissolved inorganic carbon.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

In the current era of rapid climate change, accurate characterization of climate-relevant gas dynamics-namely production, consumption, and net emissions-is required for all biomes, especially those ecosystems most susceptible to the impact of change. Marine environments include regions that act as net sources or sinks for numerous climateactive trace gases including methane (CH4) and nitrous oxide (N2O). The temporal and spatial distributions of CH4 and N2O are controlled by the interaction of complex biogeochemical and physical processes. To evaluate and quantify how these mechanisms affect marine CH4 and N2O cycling requires a combination of traditional scientific disciplines including oceanography, microbiology, and numerical modeling. Fundamental to these efforts is ensuring that the datasets produced by independent scientists are comparable and interoperable. Equally critical is transparent communication within the research community about the technical improvements required to increase our collective understanding of marine CH4 and N2O. A workshop sponsored by Ocean Carbon and Biogeochemistry (OCB) was organized to enhance dialogue and collaborations pertaining to marine CH4 and N2O. Here, we summarize the outcomes from the workshop to describe the challenges and opportunities for near-future CH4 and N2O research in the marine environment.

Abstract. The Southern California Bight is adjacent to one of the world'slargest urban areas, Los Angeles. As a consequence, anthropogenic impactscould disrupt local marine ecosystems due to municipal and industrial wastedischarge, pollution, flood control measures, and global warming. SantaMonica Basin (SMB), due to its unique setting in a low-oxygen and high-sedimentation environment, can provide an excellent sedimentary paleorecordof these anthropogenic changes. This study examined 10 sediment cores,collected from different parts of the SMB between spring and summer 2016,and compared them to existing cores in order to document changes insedimentary dynamics during the last 250 years, with an emphasis on the last40 years. The 210Pb-based mass accumulation rates (MARs) for the deepest and lowest oxygen-containing parts of the SMB basin (900–910 m) have been remarkablyconsistent during the past century, averaging 17.1±0.6 mg cm−2 yr−1. At slightly shallower sites (870–900 m), accumulation ratesshowed more variation but yield the same accumulation rate, 17.9±1.9 mg cm−2 yr−1. Excess 210Pb sedimentation rates were consistentwith rates established using bomb test 137Cs profiles. We also examined14C profiles from two cores collected in the deepest part of the SMB,where fine laminations are present up to about 450 yr BP. These dataindicate that the MAR was slower prior to ∼1900 CE (ratesobtained were 9 and 12 mg cm−2 yr−1). The δ13Corg profilesshow a relatively constant value where laminations are present, suggestingthat the change in sediment accumulation rate is not accompanied by a changein organic carbon sources to the basin. The increase in sedimentation ratetowards the Recent occurs at about the time previous studies predicted anincrease in siltation and the demise of a shelly shelf benthic fauna on theSMB shelf. X-radiographs show finely laminated sediments in the deepest part of thebasin only, with centimeter-scale layering of sediments or no layering whatsoever inshallower parts of the SMB basin. The absence of finely laminated sedimentsin cores MUC 10 (893 m) and MUC 3 (777 m) suggests that the rate at whichanoxia is spreading has not increased appreciably since cores were lastanalyzed in the 1980s. Based on core top data collected during the past halfcentury, sedimentary dynamics within SMB have changed minimally during the last40 years. Specifically, mass accumulation rates, laminated sediment fabric,extent of bioturbation and % Corg have not changed. The onlyparameter that appeared to have changed in the last 450 years was the MAR,with an apparent > 50 % increase occurring between ∼1850 CE and the early 1900s. The post-1900 CE constancy ofsedimentation through a period of massive urbanization in Los Angeles issurprising.

Benthic microorganisms transported into the water column potentially influence biogeochemical cycles and the pelagic food web structure. In the present study six gas-releasing vent sites in the Coal Oil Point seep field (California) were investigated, and the dislocation of microorganisms from the sediment into the water column via gas bubbles released from the seabed was documented. It was found that the methanotrophs transport efficiency was dependent on the volumetric gas flow, with the highest transport rate of 22.7 × 103 cells mLgas-1 at a volumetric gas flow of 0.07 mLgas s-1, and the lowest rate of 0.2 × 103 cells mLgas-1 at a gas flow of 2.2 mLgas s-1. A simple budget approach showed that this bubble-mediated transport has the potential to maintain a relevant part of the water-column methanotrophs in the seep field. The bubble-mediated link between the benthic and pelagic environment was further supported by genetic analyses, indicating a transportation of methanotrophs of the family Methylomonaceae and oil degrading bacteria of the genus Cycloclasticus from the sediment into the water column. These findings demonstrate that the bubble-mediated transport of microorganisms influences the pelagic microbial abundance and community composition at gas-releasing seep sites.

The Kryos Basin is a deep-sea hypersaline anoxic basin (DHAB) located in the Eastern Mediterranean Sea (34.98°N 22.04°E). It is filled with brine of re-dissolved Messinian evaporites and is nearly saturated with MgCl2-equivalents, which makes this habitat extremely challenging for life. The strong density difference between the anoxic brine and the overlying oxic Mediterranean seawater impedes mixing, giving rise to a narrow chemocline. Here, we investigate the microbial community structure and activities across the seawater-brine interface using a combined biogeochemical, next-generation sequencing, and lipid biomarker approach. Within the interface, we detected fatty acids that were distinctly 13C-enriched when compared to other fatty acids. These likely originated from sulfide-oxidizing bacteria that fix carbon via the reverse tricarboxylic acid cycle. In the lower part of the interface, we also measured elevated rates of methane oxidation, probably mediated by aerobic methanotrophs under micro-oxic conditions. Sulfate reduction rates increased across the interface and were highest within the brine, providing first evidence that sulfate reducers (likely Desulfovermiculus and Desulfobacula) thrive in the Kryos Basin at a water activity of only ~0.4 Aw. Our results demonstrate that a highly specialized microbial community in the Kryos Basin has adapted to the poly-extreme conditions of a DHAB with nearly saturated MgCl2 brine, extending the known environmental range where microbial life can persist.

Abstract. Benthic microbial methanogenesis is a known source of methane in marine systems. In most sediments, the majority of methanogenesis is located below the sulfate-reducing zone, as sulfate reducers outcompete methanogens for the major substrates hydrogen and acetate. The coexistence of methanogenesis and sulfate reduction has been shown before and is possible through the usage of noncompetitive substrates by methanogens such as methanol or methylated amines. However, knowledge about the magnitude, seasonality, and environmental controls of this noncompetitive methane production is sparse. In the present study, the presence of methanogenesis within the sulfate reduction zone (SRZ methanogenesis) was investigated in sediments (0–30 cm below seafloor, cm b.s.f.) of the seasonally hypoxic Eckernförde Bay in the southwestern Baltic Sea. Water column parameters such as oxygen, temperature, and salinity together with porewater geochemistry and benthic methanogenesis rates were determined in the sampling area Boknis Eck quarterly from March 2013 to September 2014 to investigate the effect of seasonal environmental changes on the rate and distribution of SRZ methanogenesis, to estimate its potential contribution to benthic methane emissions, and to identify the potential methanogenic groups responsible for SRZ methanogenesis. The metabolic pathway of methanogenesis in the presence or absence of sulfate reducers, which after the addition of a noncompetitive substrate was studied in four experimental setups: (1) unaltered sediment batch incubations (net methanogenesis), (2) 14C-bicarbonate labeling experiments (hydrogenotrophic methanogenesis), (3) manipulated experiments with the addition of either molybdate (sulfate reducer inhibitor), 2-bromoethanesulfonate (methanogen inhibitor), or methanol (noncompetitive substrate, potential methanogenesis), and (4) the addition of 13C-labeled methanol (potential methylotrophic methanogenesis). After incubation with methanol, molecular analyses were conducted to identify key functional methanogenic groups during methylotrophic methanogenesis. To also compare the magnitudes of SRZ methanogenesis with methanogenesis below the sulfate reduction zone (> 30 cm b.s.f.), hydrogenotrophic methanogenesis was determined by 14C-bicarbonate radiotracer incubation in samples collected in September 2013.SRZ methanogenesis changed seasonally in the upper 30 cm b.s.f. with rates increasing from March (0.2 nmol cm−3 d−1) to November (1.3 nmol cm−3 d−1) 2013 and March (0.2 nmol cm−3 d−1) to September (0.4 nmol cm−3 d−1) 2014. Its magnitude and distribution appeared to be controlled by organic matter availability, C / N, temperature, and oxygen in the water column, revealing higher rates in the warm, stratified, hypoxic seasons (September–November) compared to the colder, oxygenated seasons (March–June) of each year. The majority of SRZ methanogenesis was likely driven by the usage of noncompetitive substrates (e.g., methanol and methylated compounds) to avoid competition with sulfate reducers, as was indicated by the 1000–3000-fold increase in potential methanogenesis activity observed after methanol addition. Accordingly, competitive hydrogenotrophic methanogenesis increased in the sediment only below the depth of sulfate penetration (> 30 cm b.s.f.). Members of the family Methanosarcinaceae, which are known for methylotrophic methanogenesis, were detected by PCR using Methanosarcinaceae-specific primers and are likely to be responsible for the observed SRZ methanogenesis.The present study indicates that SRZ methanogenesis is an important component of the benthic methane budget and carbon cycling in Eckernförde Bay. Although its contributions to methane emissions from the sediment into the water column are probably minor, SRZ methanogenesis could directly feed into methane oxidation above the sulfate–methane transition zone.

The microbial community response to petroleum seepage was investigated in a whole round sediment core (16 cm length) collected nearby natural hydrocarbon seepage structures in the Caspian Sea, using a newly developed Sediment-Oil-Flow-Through (SOFT) system. Distinct redox zones established and migrated vertically in the core during the 190 days-long simulated petroleum seepage. Methanogenic petroleum degradation was indicated by an increase in methane concentration from 8 μM in an untreated core compared to 2300 μM in the lower sulfate-free zone of the SOFT core at the end of the experiment, accompanied by a respective decrease in the δ13C signal of methane from -33.7 to -49.5‰. The involvement of methanogens in petroleum degradation was further confirmed by methane production in enrichment cultures from SOFT sediment after the addition of hexadecane, methylnapthalene, toluene, and ethylbenzene. Petroleum degradation coupled to sulfate reduction was indicated by the increase of integrated sulfate reduction rates from 2.8 SO42-m-2 day-1 in untreated cores to 5.7 mmol SO42-m-2 day-1 in the SOFT core at the end of the experiment, accompanied by a respective accumulation of sulfide from 30 to 447 μM. Volatile hydrocarbons (C2-C6 n-alkanes) passed through the methanogenic zone mostly unchanged and were depleted within the sulfate-reducing zone. The amount of heavier n-alkanes (C10-C38) decreased step-wise toward the top of the sediment core and a preferential degradation of shorter (n-alkanes (>C30) was seen during the seepage. This study illustrates, to the best of our knowledge, for the first time the development of methanogenic petroleum degradation and the succession of benthic microbial processes during petroleum passage in a whole round sediment core.

Anaerobic microbial hydrocarbon degradation is a major biogeochemical process at marine seeps. Here we studied the response of the microbial community to petroleum seepage simulated for 190 days in a sediment core from the Caspian Sea using a sediment-oil-flow-through (SOFT) system. Untreated (without simulated petroleum seepage) and SOFT sediment microbial communities shared 43% bacterial genus-level 16S rRNA-based operational taxonomic units (OTU0.945) but shared only 23% archaeal OTU0.945. The community differed significantly between sediment layers. The detection of fourfold higher deltaproteobacterial cell numbers in SOFT than in untreated sediment at depths characterized by highest sulfate reduction rates and strongest decrease of gaseous and mid-chain alkane concentrations indicated a specific response of hydrocarbon-degrading Deltaproteobacteria. Based on an increase in specific CARD-FISH cell numbers, we suggest the following groups of sulfate-reducing bacteria to be likely responsible for the observed decrease in aliphatic and aromatic hydrocarbon concentration in SOFT sediments: clade SCA1 for propane and butane degradation, clade LCA2 for mid- to long-chain alkane degradation, clade Cyhx for cycloalkanes, pentane and hexane degradation, and relatives of Desulfobacula for toluene degradation. Highest numbers of archaea of the genus Methanosarcina were found in the methanogenic zone of the SOFT core where we detected preferential degradation of long-chain hydrocarbons. Sequencing of masD, a marker gene for alkane degradation encoding (1-methylalkyl)succinate synthase, revealed a low diversity in SOFT sediment with two abundant species-level MasD OTU0.96.

Despite long-standing interest in the biogeochemistry of the Santa Barbara Basin (SBB), there are no direct rate measurements of different nitrogen transformation processes. We investigated benthic nitrogen cycling using in situ incubations with 15 NO 3- addition and quantified the rates of total nitrate (NO 3-) uptake, denitrification, anaerobic ammonia oxidation (anammox), N 2 O production, and dissimilatory nitrate reduction to ammonia (DNRA). Denitrification was the dominant NO 3- reduction process, while anammox contributed 0 %–27 % to total NO 3- reduction. DNRA accounted for less than half of NO 3- reduction except at the deepest station at the center of the SBB where NO 3- concentration was lowest. NO 3- availability and sediment total organic carbon content appeared to be two key controls on the relative importance of DNRA. The increasing importance of fixed N retention via DNRA relative to fixed N loss as NO 3- deficit intensifies suggests a negative feedback loop that potentially contributes to stabilizing the fixed N budget in the SBB. Nitrous oxide (N 2 O) production as a fraction of total NO 3- reduction ranged from 0.2 % to 1.5 %, which was higher than previous reports from nearby borderland basins. A large fraction of NO 3- uptake was unaccounted for by NO 3- reduction processes, suggesting that intracellular storage may play an important role. Our results indicate that the SBB acts as a strong sink for fixed nitrogen and potentially a net source of N 2 O to the water column. [ABSTRACT FROM AUTHOR]

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~10³ cells mL-1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator. [ABSTRACT FROM AUTHOR]

Abstract. The potential coupling of nitrogen (N2) fixation and sulfate reduction (SR) was explored in sediments of the Peruvian oxygen minimum zone (OMZ). Sediment samples were retrieved by a multiple corer at six stations along a depth transect (70–1025 m water depth) at 12° S, covering anoxic and hypoxic bottom water conditions. Benthic N2 fixation, determined by the acetylene reduction assay, was detected at all sites, with highest rates between 70 and 253 m and lower rates at greater depth. SR rates decreased with increasing water depth. N2 fixation and SR overlapped in sediments, suggesting a potential coupling of both processes. However, a weak positive correlation of their activity distribution was detected by principle component analysis. A potential link between N2 fixation and sulfate-reducing bacteria was indicated by the molecular analysis of nifH genes. Detected nifH sequences clustered with the sulfate-reducing bacteria Desulfonema limicola at the 253 m station. However, nifH sequences of other stations clustered with uncultured organisms, Gammaproteobacteria, and Firmicutes (Clostridia) rather than with known sulfate reducers. The principle component analysis revealed that benthic N2 fixation in the Peruvian OMZ is controlled by organic matter (positive) and free sulfide (negative). No correlation was found between N2 fixation and ammonium concentrations (even at levels > 2022 µM). N2 fixation rates in the Peruvian OMZ sediments were in the same range as those measured in other organic-rich sediments.

Microbial transformation of hydrocarbons to methane is an environmentally relevant process taking place in a wide variety of electron acceptor-depleted habitats, from oil reservoirs and coal deposits to contaminated groundwater and deep sediments. Methanogenic hydrocarbon degradation is considered to be a major process in reservoir degradation and one of the main processes responsible for the formation of heavy oil deposits and oil sands. In the absence of external electron acceptors such as oxygen, nitrate, sulfate or Fe(III), fermentation and methanogenesis become the dominant microbial metabolisms. The major end product under these conditions is methane, and the only electron acceptor necessary to sustain the intermediate steps in this process is CO2, which is itself a net product of the overall reaction. We are summarizing the state of the art and recent advances in methanogenic hydrocarbon degradation research. Both the key microbial groups involved as well as metabolic pathways are described, and we discuss the novel insights into methanogenic hydrocarbon-degrading populations studied in laboratory as well as environmental systems enabled by novel cultivation-based and molecular approaches. Their possible implications on energy resources, bioremediation of contaminated sites, deep-biosphere research, and consequences for atmospheric composition and ultimately climate change are also addressed.

Large amounts of the greenhouse gas methane are released from the seabed to the water column, where it may be consumed by aerobic methanotrophic bacteria. The size and activity of methanotrophic communities, which determine the amount of methane consumed in the water column, are thought to be mainly controlled by nutrient and redox dynamics. Here, we report repeated measurements of methanotrophic activity and community size at methane seeps west of Svalbard, and relate them to physical water mass properties and modelled ocean currents. We show that cold bottom water, which contained a large number of aerobic methanotrophs, was displaced by warmer water with a considerably smaller methanotrophic community within days. Ocean current simulations using a global ocean/sea-ice model suggest that this water mass exchange is consistent with short-term variations in the meandering West Spitsbergen Current. We conclude that the shift from an offshore to a nearshore position of the current can rapidly and severely reduce methanotrophic activity in the water column. Strong fluctuating currents are common at many methane seep systems globally, and we suggest that they affect methane oxidation in the water column at other sites, too.

The Santa Barbara Basin naturally experiences transient deoxygenation due to its unique geological setting in the southern California Borderland and seasonal changes in ocean currents. Long-term measurements of the basin showed that anoxic events and subsequent nitrate exhaustion in the bottom waters have been occurring more frequently and lasting longer over the past decade. One characteristic of the Santa Barbara Basin is the seasonal development of extensive mats of benthic nitrate-reducing sulfur-oxidizing bacteria, which are found at the sediment–water interface when the basin's bottom waters reach anoxia but still provide some nitrate. To assess the mat's impact on the benthic and pelagic redox environment, we collected biogeochemical sediment and benthic flux data in November 2019, after anoxia developed in the deepest waters of the basin and dissolved nitrate was depleted (down to 9.9 µ M). We found that the development of mats was associated with a shift from denitrification to dissimilatory nitrate reduction to ammonium. The zone of sulfate reduction appeared near the sediment–water interface in sediment hosting these ephemeral white mats. We found that an exhaustion of iron oxides in the surface sediment was an additional prerequisite for mat proliferation. Our research further suggests that cycles of deoxygenation and reoxygenation of the benthic environment result in extremely high benthic fluxes of dissolved iron from the basin's sediment. This work expands our understanding of nitrate-reducing sulfur-oxidizing mats and their role in sustaining and potentially expanding marine anoxia. [ABSTRACT FROM AUTHOR]

The trace metal iron (Fe) is an essential micronutrient that controls phytoplankton productivity, which subsequently affects organic matter cycling with feedback on the cycling of macronutrients. Along the continental margin of the US West Coast, high benthic Fe release has been documented, in particular from deep anoxic basins in the Southern California Borderland. However, the influence of this Fe release on surface primary production remains poorly understood. In the present study from the Santa Barbara Basin, in situ benthic Fe fluxes were determined along a transect from shallow to deep sites in the basin. Fluxes ranged between 0.23 and 4.9 mmol m -2 d -1 , representing some of the highest benthic Fe fluxes reported to date. To investigate the influence of benthic Fe release from the oxygen-deficient deep basin on surface phytoplankton production, we combined benthic flux measurements with numerical simulations using the Regional Ocean Modeling System coupled to the Biogeochemical Elemental Cycling (ROMS-BEC) model. For this purpose, we updated the model Fe flux parameterization to include the new benthic flux measurements from the Santa Barbara Basin. Our simulations suggest that benthic Fe fluxes enhance surface primary production, supporting a positive feedback on benthic Fe release by decreasing oxygen in bottom waters. However, a reduction in phytoplankton Fe limitation by enhanced benthic fluxes near the coast may be partially compensated for by increased nitrogen limitation further offshore, limiting the efficacy of this positive feedback. [ABSTRACT FROM AUTHOR]

Methane in oil reservoirs originates mostly from thermogenic sources, yet secondary microbial methane production from petroleum biodegradation is known to be pervasive. The conventional approach for identifying this secondary microbial methane commonly relies on geochemical characteristics of other gas molecules such as the carbon isotopic composition of carbon dioxide and propane. This information is sometimes obscured by isotopic variations in source material and may not be available in certain geological reservoirs. To better constrain the presence of secondary microbial methane, we studied the clumped isotopologue compositions of methane in terrestrial Azerbaijanian mud volcanoes, which support the occurrence of secondary microbial gas. Here, a deficit in Δ12CH2D2 of thermogenic methane occurs due to different δD of hydrogen sources that contribute to the formation of methane molecules (i.e., combinatorial effect). The Δ12CH2D2 is expected to move toward equilibrium as thermal maturity increases. More importantly, both Δ13CH3D and Δ12CH2D2 values of methane approach low-temperature thermodynamic equilibrium in most gases, suggesting that the original thermogenic methane has been altered by newly formed microbial methane in addition to isotope exchange among methane molecules catalyzed by the methyl-coenzyme M reductase enzyme. We conclude that methane clumped isotopes provide a unique proxy for identifying secondary microbial methane and understanding the exact evolution stages for natural gases. [ABSTRACT FROM AUTHOR]

The recently discovered cryptic methane cycle in the sulfate-reducing zone of marine and wetland sediment couples methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). Here we present evidence of cryptic methane cycling activity within the upper regions of the sulfate-reducing zone, along a depth transect within the Santa Barbara Basin, off the coast of California, USA. The top 0–20 cm of sediment from each station was subjected to geochemical analyses and radiotracer incubations using 35 S–SO 42- , 14 C–mono-methylamine, and 14 C–CH 4 to find evidence of cryptic methane cycling. Methane concentrations were consistently low (3 to 16 µ M) across the depth transect, despite AOM rates increasing with decreasing water depth (from max 0.05 nmol cm -3 d -1 at the deepest station to max 1.8 nmol cm -3 d -1 at the shallowest station). Porewater sulfate concentrations remained high (23 to 29 mM), despite the detection of sulfate reduction activity from 35 S–SO 42- incubations with rates up to 134 nmol cm -3 d -1. Metabolomic analysis showed that substrates for methanogenesis (i.e., acetate, methanol and methylamines) were mostly below the detection limit in the porewater, but some samples from the 1–2 cm depth section showed non-quantifiable evidence of these substrates, indicating their rapid turnover. Estimated methanogenesis from mono-methylamine ranged from 0.2 to 0.5 nmol cm -3 d -1. Discrepancies between the rate constants (k) of methanogenesis (from 14 C–mono-methylamine) and AOM (from either 14 C–mono-methylamine-derived 14 C–CH 4 or from directly injected 14 C–CH 4) suggest the activity of a separate, concurrent metabolic process directly metabolizing mono-methylamine to inorganic carbon. We conclude that the results presented in this work show strong evidence of cryptic methane cycling occurring within the top 20 cm of sediment in the Santa Barbara Basin. The rapid cycling of carbon between methanogenesis and methanotropy likely prevents major build-up of methane in the sulfate-reducing zone. Furthermore, our data suggest that methylamine is utilized by both methanogenic archaea capable of methylotrophic methanogenesis and non-methanogenic microbial groups. We hypothesize that sulfate reduction is responsible for the additional methylamine turnover, but further investigation is needed to elucidate this metabolic activity. [ABSTRACT FROM AUTHOR]

The recently discovered cryptic methane cycle in the sulfate-reducing zone of marine and wetland sediments couples methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). Here we present evidence of cryptic methane cycling activity within the upper regions of the sulfate-reducing zone, along a depth transect within the Santa Barbara Basin, off the coast of California, USA. The top 0–20 cm of sediment from each station was subjected to geochemical analyses and radiotracer incubations using 35S-SO42-, 14C-mono-methylamine, and 14C- CH4 to find evidence of cryptic methane cycling. Methane concentrations were consistently low (~3 to ~16 µM) across the depth transect, despite AOM rates increasing with decreasing water depth (from max 0.05 nmol cm-3 d-1 at the deepest station to max 1.8 nmol cm-3 d-1 at the shallowest station). Porewater sulfate concentrations remained high (~23 mM to ~29 mM), despite the detection of sulfate reduction activity from 35S-SO42- incubations with rates up to 134 nmol cm-3 d-1. Metabolomic analysis showed that substrates for methanogenesis (i.e., acetate, methanol and methylamines) were mostly below the detection limit in the porewater, but some samples from the 1–2 cm depth section showed non-quantifiable evidence of these substrates, indicating their rapid turnover. Estimated methanogenesis from mono-methylamine ranged from 0.2 nmol to 0.5 nmol cm-3 d-1. Discrepancies between the rate constants (K1) of methanogenesis (from 14C- mono-methylamine) and AOM (from either 14C- mono-methylamine-derived 14C-CH4 or from directly injected 14C-CH4) suggest the activity of a separate, concurrent metabolic process directly metabolizing mono-methylamine to inorganic carbon. We conclude that the results presented in this work show strong evidence of cryptic methane cycling occurring within the top 20 cm of sediment in the Santa Barbara Basin. The rapid cycling of carbon between methanogenesis and methanotropy likely prevents major build-up of methane in the sulfate-reducing zone. Furthermore, our data suggest that methylamine is utilized by both methanogenic archaea capable of methylotrophic methanogenesis and non-methanogenic microbial groups. We hypothesize that sulfate reduction is responsible for the additional methylamine turnover but further investigation is needed to elucidate this metabolic activity. [ABSTRACT FROM AUTHOR]