Anaerobic Oxidation Of Methane Activity Research Articles

Role and environmental regulation of iron-driven anaerobic methane oxidation in riverine sediment

Iron is an abundant element in the environment and acts as a thermodynamically favorable electron acceptor driving the anaerobic oxidation of methane (AOM). Presently, the role and environmental regulation of iron-driven AOM in rivers, an important source of methane emission, are nearly unknown. Here, we provided direct evidence for iron-driven AOM activity in sediment of a mountainous river (Wuxijiang River, China) through 13C-labeled isotopic experiment. The potential rate of iron-driven AOM ranged between 0.40 and 1.84 nmol 13CO2 g (sediment) d−1, which contributed 36% on average to total AOM activity when combined the potential nitrate- and nitrite-driven AOM rates measured previously. There were significant variations in iron-driven AOM rates among different reaches (upper, middle, and lower) and between seasons (summer and winter). Sediment temperature, pH, and nitrate content were closely associated with the dynamic of AOM activity. Our results indicate that iron-driven AOM has great potential for reducing methane emissions from riverine ecosystems, and suggest the necessity of taking both spatial and temporal scales into account to evaluate the quantitative role of this AOM process.

Geochemical and mineralogical features as indicators of sulfate-methane transition zones in shallow gas-bearing offshore sediments from Zhoushan

Subsurface shallow gas, primarily composed of biogenic methane (>95%), is highly-enriched in global offshore sediments. The redox conditions of methane-rich pore fluid play a crucial role in shaping the geochemical signatures associated with the anaerobic oxidation of methane (AOM) and vertical displacement of sulfate-methane transition zones (SMTZs) within deep sedimentary records. However, long-period, and continuous records of AOM and SMTZs in shallow gas-bearing offshore sediments are sparse. Here, we employed a comprehensive analyses of iron sulfide, elemental, and stable isotopies on a 63-m drilled sediment core (JC-1) from offshore Zhoushan. Four different layers U1–U4 were identified in 11.5–24 mbsf, 24−42 mbsf, 42−51 mbsf, and 51−63 mbsf, respectively, as revealed by the lithofacies and sedimentary structures. By comparing the pyrite contents, total sulfur to total organic carbon ratios, and δ34S-chromium-reducible sulfur values, the commonly seen organoclastic sulfate reduction occurred in U1 and U2, whereas significant AOM activities were found in U1 and U4. Further, the peaks of total sulfur to total organic carbon ratios, sulfur contents, and multiple positive δ34S-chromium-reducible sulfur values indicated the presence of AOM in SMTZs at 18.3, 52, and 59 mbsf. These intervals were featured by a sub-oxic or anoxic condition with strong methane seepages, evident from the scarcity of metals and the enrichment of Mo/Al and U/Al ratios. The presence of multiple SMTZs was consistent with the pore-water data of the adjacent YS4 and YS7 cores. In light of these findings, it is prudent to exercise caution when utilizing geochemical indicators, such as trace element proxies and the δ34S values of sulfide minerals, to comprehend the progression of fluctuating depositional settings in reaction to sea-level fluctuations and shallow gas reservoirs. This study provides a new perspective to better understand the evolution of unsteady depositional environments in response to sea-level changes and shallow gas reservoirs.

Vertical and temporal variations in activity, abundance, and composition of nitrite-driven anaerobic methanotrophs in a paddy field

Paddy soil is an important source of methane (CH4) emissions, and the nitrite-driven anaerobic oxidation of methane (AOM) mediated by Candidatus Methylomirabilis bacteria is a crucial process for CH4 mitigation. However, the vertical and temporal fluctuations in nitrite-driven AOM activity, as well as the abundance and composition of Methylomirabilis bacteria in paddy soil, remain poorly understood. We investigated the potential rate of nitrite-driven AOM and the composition of Methylomirabilis bacteria at varying depths (0–40 cm, at 10 cm intervals) within a paddy soil during different growth stages of rice, including tillering, jointing, flowering, and milky. The potential rate of nitrite-driven AOM ranged from 0.24 to 20.6 nmol CO2 g−1 (dry soil) d−1, and the abundance of Methylomirabilis bacterial 16S rRNA genes varied from 5.8 × 106 to 4.4 × 107 copies g−1 dry soil. The 10–20 cm soil depth showed higher potential activity and bacterial abundance compared to other depths, and the tillering and jointing stages exhibited greater activity and abundance. The composition of Methylomirabilis bacteria showed significant variation across different soil depths but remained stable throughout the growth stages of rice. Soil organic carbon content was positively correlated with potential nitrite-driven AOM rate, and nitrite content was negatively correlated with the abundance of Methylomirabilis bacteria. Soil organic carbon and ammonium content had a significant impact on the composition of Methylomirabilis bacteria. The results underscore the substantial influence of soil depth and the growth stage of rice on nitrite-driven AOM in paddy ecosystems.

Humic substances as electron acceptor for anaerobic oxidation of methane (AOM) and electron shuttle in Mn (IV)-dependent AOM

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. ‘Methanoperedens manganicus’, an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. ‘M. manganicus’ consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. ‘M. manganicus’, and the total archaea population in the microbial community suggested that Ca. ‘M. manganicus’ can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. ‘M. manganicus’. We tested this hypothesis by adding humic acids to a Ca. ‘M. manganicus’ dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

Equal importance of humic acids and nitrate in driving anaerobic oxidation of methane in paddy soils

Methane (CH4) is both generated and consumed in paddy soils, where anaerobic oxidation of methane (AOM) serves as a crucial process for mitigating CH4 emissions. Although the participation of humic acids (HA) and nitrate in AOM has been recognized, their relative roles and significance in paddy soils remain insufficiently investigated. In this study, we explored the potential activity of AOM driven by HA and nitrate, as well as the composition of archaeal communities in paddy soils across different rice growth periods and fertilization treatments. AOM activity ranged from 0.81 to 1.33 and 1.26 to 2.38 nmol of 13CO2 g−1 (dry soil) day−1 with HA and nitrate, respectively. No significant differences (p < 0.05) were observed between the AOM activity driven by HA and nitrate across the three fertilization treatments. According to AOM activity, the annual consumption of CH4 was estimated at approximately 0.49 ± 0.06 and 0.83 ± 0.19 Tg for AOM processes driven by HA and nitrate in Chinese paddy soils. Nitrate-driven AOM activity exhibited a positive (p < 0.05) correlation with the abundance of the ANME-2d mcrA gene but a negative (p < 0.05) correlation with the content of dissolved organic carbon. Intriguingly, HA-driven AOM activity was only correlated positively with the nitrate-driven AOM activity. Soil water content, soil organic carbon, nitrate and nitrite contents were significantly correlated with the relative abundance of methanogenic and methanotrophic archaea. These results identified the potential importance of HA and nitrate in driving AOM processes within paddy soils, providing a comprehensive understanding of the complex microbial processes regulating greenhouse gas emissions from paddy soils.

Lipidomic diversity and proxy implications of archaea from cold seep sediments of the South China Sea

Cold seeps on the continental margins are characterized by intense microbial activities that consume a large portion of methane by anaerobic methanotrophic archaea (ANME) through anaerobic oxidation of methane (AOM). Although ANMEs are known to contain unique ether lipids that may have an important function in marine carbon cycling, their full lipidomic profiles and functional distribution in particular cold-seep settings are still poorly characterized. Here, we combined the 16S rRNA gene sequencing and lipidomic approaches to analyze archaeal communities and their lipids in cold seep sediments with distinct methane supplies from the South China Sea. The archaeal community was dominated by ANME-1 in the moderate seepage area with strong methane emission. Low seepage area presented higher archaeal diversity covering Lokiarchaeia, Bathyarchaeia, and Thermoplasmata. A total of 55 core lipids (CLs) and intact polar lipids (IPLs) of archaea were identified, which included glycerol dialkyl glycerol tetraethers (GDGTs), hydroxy-GDGTs (OH-GDGTs), archaeol (AR), hydroxyarchaeol (OH-AR), and dihydroxyarchaeol (2OH-AR). Diverse polar headgroups constituted the archaeal IPLs. High concentrations of dissolved inorganic carbon (DIC) with depleted δ13CDIC and high methane index (MI) values based on both CLs (MICL) and IPLs (MIIPL) indicate that ANMEs were active in the moderate seepage area. The ANME-2 and ANME-3 clades were characterized by enhanced glycosidic and phosphoric diether lipids production, indicating their potential role in coupling carbon and phosphurus cycling in cold seep ecosystems. ANME-1, though representing a smaller proportion of total archaea than ANME-2 and ANME-3 in the low seepage area, showed a positive correlation with MIIPL, indicating a different mechanism contributing to the IPL-GDGT pool. This also suggests that MIIPL could be a sensitive index to trace AOM activities performed by ANME-1. Overall, our study expands the understanding of the archaeal lipid composition in the cold seep and improves the application of MI using intact polar lipids that potentially link to extent ANME activities.

Identifying Putative Subsurface Microbial Drivers of Methane Flux on Earth and Mars

On Earth microorganisms are critical drivers of the methane cycle, both producing and consuming methane (Boetius et al. 2000, Knittel and Boetius 2009, Orphan et al. 2001). Molecular and isotopic-based investigations of archaeal-bacterial consortia catalyzing the anaerobic oxidation of methane (AOM) in marine methane seeps identified the pivotal role of these microorganisms in mitigating the release of methane into the atmosphere (Knittel and Boetius 2009, Orphan et al. 2001). In the marine environment, AOM is predominantly carried out by closely associated consortia of methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) coupling methane oxidation to sulfate reduction in the absence of oxygen. Wolf Spring (WS), Axel Heiberg Island, Nunavut is a hypersaline cold spring methane seep and the only known terrestrial permafrost hosted methane seep known to host ANME-1 archaea associated with AOM (Niederberger et al. 2010, Magnuson et al. 2022). Wolf Spring is an unparalleled analogue for putative subsurface brines and sites of methane release on Mars. Enigmatic observations of methane in the near-surface Martian atmosphere remain a tantalizing potential biosignature. The combination of field site characterization, microbial microcosm experiments, and in situ methane monitoring represents a coordinated interdisciplinary effort to identify methane driven microbial metabolisms not only critical to understanding methane flux in the Arctic, but also as possible drivers to the methane cycle on Mars. Detailed microbial characterization of these springs has identified a chemotrophic community dominated by sulfur cycling (Altshuler et al. 2022, Niederberger et al. 2010). To date, microbial and geochemical characterization has been carried out on sediment samples to a few centimeters depth. This study expands on these initial studies, with the successful collection and analysis of deeper sediment cores at WS focusing on AOM activity to better understand the microorganisms involved and the methane cycling capacity at depth. Two decades of observing methane on Mars (Mumma et al. 2009) have generated data indicative of a dynamic, geochemical system characterized by a profile similar to the release of methane from seeps on Earth (Etiope and Oehler 2019) producing both distinct pulses known as plumes and slow background seepage. These observations suggest as of yet unknown geochemical and potentially geobiological methane sources and sinks. While methane can be produced abiotically (Etiope and Lollar 2013), on Earth most methane is biogenic. Determining the biogenicity of CH4 is non-trivial and requires a correlated approach including determination of carbon isotopes. In terrestrial systems, biogenic CH4 is 13C depleted. To characterize methane sources and sinks on Mars, near surface measurements at a frequency not possible with existing instrumentation are required. We are currently developing off-axis integrated cavity-enhanced output (OA-ICOS) spectrometry as a portable trace gas analyzer capable of obtaining high frequency measurements of methane at the sub-ppb level (Sapers et al. 2021). Optimizing OA-ICOS trace methane measurements at WS will help refine sensitivity and measurement cadence in a Mars-like environment as well as providing new remote methane monitoring capabilities for Arctic methane emissions. We are currently developing in situ 12CH4:13CH4 capabilities using OA-ICOS technology. The importance of δ 13C as a biosignature is summarized in Fig. 1.

Horizontal and vertical distribution of nitrate-driven anaerobic methane oxidation process in coastal wetlands under different plant species cover across southeast China

Coastal wetlands are an important methane emission source. Nitrate-driven anaerobic oxidation of methane (AOM), catalyzing via a novel clade of anaerobic methanotrophic archaea (ANME-2d), is a potentially important process for methane emission mitigation in coastal wetlands. However, the influence of regional and local environmental conditions, as well as plant species cover on this AOM process, remains unclear. Here, we investigated the horizontal and vertical distribution of nitrate-driven AOM activity and ANME-2d archaeal community in four coastal wetlands under both native and invasive plant species across southeast China. It was estimated that this AOM process could potentially reduce 5.7–9.5% methane emissions from the studied wetlands. Generalized linear mixed-effects model showed that sampling locations and sediment layers had greater impact on nitrate-driven AOM activity than plant species cover, and all these factors significantly affected the abundance of ANME-2d archaea. Nitrate-driven AOM activity was higher in low-latitude coastal wetlands than in high-latitude areas, and the activity positively correlated with mean annual temperature. Furthermore, both nitrate-driven AOM activity and ANME-2d archaeal abundance showed significant vertical variations along sediment profiles, with higher values in the slightly alkaline surface sediment. The plant species cover was found to impact the ANME-2d archaeal abundance and diversity by regulating the availability of substrates for the archaea. Overall, our study provides valuable insights into effects of regional and local environmental parameters and plant species cover on the horizontal and vertical distribution of nitrate-driven AOM activity and ANME-2d archaeal community in coastal wetlands.

Enhanced anaerobic oxidation of methane with the coexistence of iron oxides and sulfate fertilizer in paddy soil

Iron oxides and sulfate are usually abundant in paddy soil, but their role in reducing methane emissions is little known. In this work, paddy soil was anaerobically cultivated with ferrihydrite and sulfate for 380 days. An activity assay, inhibition experiment, and microbial analysis were conducted to evaluate the microbial activity, possible pathways, and community structure, respectively. The results showed that anaerobic oxidation of methane (AOM) was active in the paddy soil. The AOM activity was much higher with ferrihydrite than sulfate, and an extra 10% of AOM activity was stimulated when ferrihydrite and sulfate coexisted. The microbial community was highly similar to the duplicates but totally different with different electron acceptors. The microbial abundance and diversity decreased due to the oligotrophic condition, but mcrA-carrying archaea increased 2–3 times after 380 days. Both the microbial community and the inhibition experiment implied that there was an intersection between iron and sulfur cycles. A “cryptic sulfur cycle” might link the two cycles, in which sulfate was quickly regenerated by iron oxides, and it might contribute 33% of AOM in the tested paddy soil. Complex links between methane, iron, and sulfur geochemical cycles occur in paddy soil, which may be significant in reducing methane emissions from rice fields.

Spatio-temporal variations of activity of nitrate-driven anaerobic oxidation of methane and community structure of Candidatus Methanoperedens-like archaea in sediment of Wuxijiang river

Nitrate-driven anaerobic oxidation of methane (AOM), catalyzing by Candidatus Methanoperedens-like archaea, is a new addition in the global CH4 cycle. This AOM process acts as a novel pathway for CH4 emission reduction in freshwater aquatic ecosystems; however, its quantitative importance and regulatory factors in riverine ecosystems are nearly unknown. Here, we examined the spatio-temporal changes of the communities of Methanoperedens-like archaea and nitrate-driven AOM activity in sediment of Wuxijiang River, a mountainous river in China. These archaeal community composition varied significantly among reaches (upper, middle, and lower reaches) and between seasons (winter and summer), but their mcrA gene diversity showed no significant spatial or temporal variations. The copy numbers of Methanoperedens-like archaeal mcrA genes were 1.32 × 105–2.47 × 107 copies g−1 (dry weight), and the activity of nitrate-driven AOM was 0.25–1.73 nmol CH4 g−1 (dry weight) d−1, which could potentially reduce 10.3% of CH4 emissions from rivers. Significant spatio-temporal variations of mcrA gene abundance and nitrate-driven AOM activity were found. Both the gene abundance and activity increased significantly from upper to lower reaches in both seasons, and were significantly higher in sediment collected in summer than in winter. In addition, the variations of Methanoperedens-like archaeal communities and nitrate-driven AOM activity were largely impacted by the sediment temperature, NH4+ and organic carbon contents. Taken together, both time and space scales need to be considered for better evaluating the quantitative importance of nitrate-driven AOM in reducing CH4 emissions from riverine ecosystems.

Anaerobic methane-oxidizing activity in a deep underground borehole dominantly colonized by Ca. Methanoperedenaceae.

The family Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high-pressure laboratory incubation system, we investigated 214- and 249-m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome-resolved metagenomics, respectively. The groundwater samples amended with 13 C-labelled methane and amorphous Fe(III) were incubated at a pressure of 1.6MPa. After 3-7 days of incubation, the AOM rate was 45.8 ± 19.8nM/day in 214-m groundwater. However, almost no activity was detected from 249-m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214-m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249-m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214-m deep subsurface borehole interval. Although the presence of Fe-bearing phyllosilicates was demonstrated in the 214-m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe-bearing phyllosilicates as in situ electron acceptors by high-pressure incubation amended with the Fe-bearing phyllosilicates.

Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance (≤1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface.

Sulfate differentially stimulates but is not respired by diverse anaerobic methanotrophic archaea.

Sulfate-coupled anaerobic oxidation of methane (AOM) is a major methane sink in marine sediments. Multiple lineages of anaerobic methanotrophic archaea (ANME) often coexist in sediments and catalyze this process syntrophically with sulfate-reducing bacteria (SRB), but the potential differences in ANME ecophysiology and mechanisms of syntrophy remain unresolved. A humic acid analog, anthraquinone 2,6-disulfonate (AQDS), could decouple archaeal methanotrophy from bacterial sulfate reduction and serve as the terminal electron acceptor for AOM (AQDS-coupled AOM). Here in sediment microcosm experiments, we examined variations in physiological response between two co-occurring ANME-2 families (ANME-2a and ANME-2c) and tested the hypothesis of sulfate respiration by ANME-2. Sulfate concentrations as low as 100 µM increased AQDS-coupled AOM nearly 2-fold matching the rates of sulfate-coupled AOM. However, the SRB partners remained inactive in microcosms with sulfate and AQDS and neither ANME-2 families respired sulfate, as shown by their cellular sulfur contents and anabolic activities measured using nanoscale secondary ion mass spectrometry. ANME-2a anabolic activity was significantly higher than ANME-2c, suggesting that ANME-2a was primarily responsible for the observed sulfate stimulation of AQDS-coupled AOM. Comparative transcriptomics showed significant upregulation of ANME-2a transcripts linked to multiple ABC transporters and downregulation of central carbon metabolism during AQDS-coupled AOM compared to sulfate-coupled AOM. Surprisingly, genes involved in sulfur anabolism were not differentially expressed during AQDS-coupled AOM with and without sulfate amendment. Collectively, this data indicates that ANME-2 archaea are incapable of respiring sulfate, but sulfate availability differentially stimulates the growth and AOM activity of different ANME lineages.

Deciphering cryptic methane cycling: Coupling of methylotrophic methanogenesis and anaerobic oxidation of methane in hypersaline coastal wetland sediment

Methanogenesis has recently been shown to fuel anaerobic oxidation of methane (AOM) within the sulfate-reducing zone of marine sediments, coining the term “cryptic methane cycle”. Here we present research on the relationship between methanogenesis and AOM in a shallow hypersaline pool (∼130 PSU) within a southern California coastal wetland. Sediment (top 20 cm) was subjected to geochemical analyses, in-vitro slurry experiments, and radiotracer incubations using 35S-SO42−, 14C-mono-methylamine, and 14C-CH4, to study sulfate reduction, methylotrophic methanogenesis, and AOM. An adapted radioisotope method was used to follow cryptic methane cycling in 14C-mono-methylamine labeling incubations with increasing incubation times (1 hour to three weeks). Results showed peaks in AOM (max 13 nmol cm−3 d−1) and sulfate reduction activity (max 728 nmol cm−3 d−1) within the top 6 cm. Below 6 cm, AOM activity continued (max 15 nmol cm−3 d−1), while sulfate reduction was absent despite 67 mM sulfate, suggesting AOM was coupled to the reduction of iron. Methane concentrations were low (<50 nM) throughout the sediment. Batch sediment slurry incubations with methylated substrates (mono-methylamine and methanol) stimulated methanogenesis, pointing to the presence of methylotrophic methanogens. Incubations with 14C-mono-methylamine revealed the simultaneous activity of methanogenesis and coupled AOM through the stepwise transfer of 14C from mono-methylamine to CO2 via methane. Our results suggest that AOM is a crucial process in coastal wetland sediments to prevent the buildup of methane in the sulfate-reducing zone. We propose that cryptic methane cycling has been largely overlooked in coastal wetlands resulting in incomplete understanding of carbon cycling in this environment.

Activity, abundance and community composition of nitrite-dependent methanotrophs in response to fertilization in paddy soils

Nitrite-dependent anaerobic oxidation of methane (AOM), which is mediated by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria, has the great potential to reduce methane emissions from paddy ecosystems. Fertilization, a necessary practice for increasing crop yields, could greatly affect nitrite-dependent AOM process. Here, we investigated the abundance, community composition and activity of M. oxyfera-like bacteria at different layers (0–10, 20–30 and 40–50 cm) of paddy soils under three fertilization treatments, including unfertilized control (CK), chemical fertilization (CF) and organic fertilization (OF). The 16S rRNA gene abundance of M. oxyfera-like bacteria in soils under CF (6.1 × 106–2.2 × 107 copies g−1 dry soil) and OF (8.9 × 106–1.8 × 107 copies g−1 dry soil) treatments was higher than that under CK treatment (5.0 × 106–1.5 × 107 copies g−1 dry soil). An obvious separation of M. oxyfera-like bacterial communities was observed between different fertilization treatments, and a higher diversity of these bacteria was detected in CF and OF soils. The nitrite-dependent AOM activity was increased in soils under CF (3.6–11.5 nmol CO2 g−1 (dry soil) d−1) and OF (3.4–7.2 nmol CO2 g−1 (dry soil) d−1) treatments as compared to CK (1.6–4.4 nmol CO2 g−1 (dry soil) d−1) treatment, particularly at the layer of 20–30 cm. Overall, our results demonstrated that the communities and activity of M. oxyfera-like bacteria responded positively to both chemical and organic fertilization, which expanded our knowledge of the role of nitrite-dependent AOM in regulating methane emissions from paddy ecosystems.

Biomarker and Isotopic Composition of Seep Carbonates Record Environmental Conditions in Two Arctic Methane Seeps

Present-day activity of cold seeps in the ocean is evident from direct observations of methane emanating from the seafloor, the presence of chemosynthetic organisms, or the quantification of high gas concentrations in sediment pore waters and the water column. Verifying past cold seep activity and biogeochemical characteristics is more challenging but may be reconstructed from proxy records of authigenic seep carbonates. Here, we investigated the lipid-biomarker inventory, carbonate mineralogy, and stable carbon and oxygen isotope compositions of seep-associated carbonates from two active Arctic methane seeps, located to the northwest (Vestnesa Ridge; ∼1,200 m water depth) and south (Storfjordrenna; ∼380 m water depth) offshore Svalbard. The aragonite-dominated mineralogy of all but one carbonate sample indicate precipitation close to the seafloor in an environment characterized by high rates of sulfate-dependent anaerobic oxidation of methane (AOM). In contrast, Mg-calcite rich nodules sampled in sediments of Storfjordrenna appear to have formed at the sulfate-methane-transition zone deeper within the sediment at lower rates of AOM. AOM activity at the time of carbonate precipitation is indicated by the 13C-depleted isotope signature of the carbonates [−20 to −30‰ Vienna Pee Dee Belemnite (VPDB)], as well as high concentrations of 13C-depleted lipid biomarkers diagnostic for anaerobic methanotrophic archaea (archaeol and sn2-hydroxyarchaeol) and sulfate-reducing bacteria (iso and anteiso-C15:0 fatty acids) in the carbonates. We also found 13C-depleted lipid biomarkers (diploptene and a 4α-methyl sterol) that are diagnostic for bacteria mediating aerobic oxidation of methane (MOx). This suggests that the spatial separation between AOM and MOx zones was relatively narrow at the time of carbonate formation, as is typical for high methane-flux regimes. The seep-associated carbonates also displayed relatively high δ18O values (4.5–5‰ VPDB), indicating the presence of 18O-enriched fluids during precipitation, possibly derived from destabilized methane gas hydrates. Based on the combined isotopic evidence, we suggest that all the seep carbonates resulted from the anaerobic oxidation of methane during intense methane seepage. The seepage likely was associated to gas hydrates destabilization, which led to the methane ebullition from the seafloor into the water column.

Genomic and enzymatic evidence of acetogenesis by anaerobic methanotrophic archaea

Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem.

Different responses of nitrite- and nitrate-dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir

Nitrite (NO2−)- and nitrate (NO3−)-dependent anaerobic oxidation of methane (AOM) are two new additions in microbial methane cycle, which potentially act as important methane sinks in freshwater aquatic systems. Here, we investigated spatial variations of community composition, abundance and potential activity of NO2−- and NO3−-dependent anaerobic methanotrophs in the sediment of Jiulonghu Reservoir (Zhejiang Province, China), a freshwater reservoir having a gradient of increasing nitrogen loading from upstream to downstream regions. High-throughput sequencing of total bacterial and archaeal 16S rRNA genes showed the cooccurrence of Candidatus Methylomirabilis oxyfera (M. oxyfera)-like and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like anaerobic methanotrophs in the examined reservoir sediments. The community structures of these methanotrophs differed substantially between the sediments of upstream and downstream regions. Quantitative PCR suggested higher M. oxyfera-like bacterial abundance in the downstream (8.6 × 107 to 2.8 × 108 copies g−1 dry sediment) than upstream sediments (2.4 × 107 to 3.5 × 107 copies g−1 dry sediment), but there was no obvious difference in M. nitroreducens-like archaeal abundance between these sediments (3.7 × 105 to 4.8 × 105 copies g−1 dry sediment). The 13CH4 tracer experiments suggested the occurrence of NO2−- and NO3−-dependent AOM activities, and their rates were 4.7–14.1 and 0.8–2.6 nmol CO2 g−1 (dry sediment) d−1, respectively. Further, the rates of NO2−-dependent AOM in downstream sediment were significantly higher than those in upstream sediment. The NO3− concentration was the key factor affecting the spatial variations of abundance and activity of NO2−-dependent anaerobic methanotrophs. Overall, our results showed different responses of NO2−- and NO3−-dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir.

Molecular indicators of methane metabolisms at cold seeps along the United States Atlantic Margin

Anaerobic oxidation of methane (AOM) and the environmental conditions supporting AOM on continental margins is an essential component to global methane budgets. Diagnostic lipid biomarkers and their compound specific isotope analysis preserved in authigenic carbonates at cold seeps can serve as “fingerprints” to archaeal−bacterial consortia involved in AOM. However, despite the discovery of several hundreds of seeps along the United States Atlantic Margin (USAM), there are relatively few biomarker investigations of cold seep carbonates along this passive margin. A lipid biomarker, carbon isotope, and DNA marker gene study was therefore undertaken to determine the microbial origins of authigenic carbonates from two USAM seeps, Norfolk and the Baltimore Canyon seep fields. Results from this study capture a distinct archaeal lipid signature from putative methanotrophic archaea, including archaeol (I), sn-2-hydroxyarchaeol, 2,6,10,15,19-pentamethylicosane (PMI), and crocetane. The 13C-depleted AOM-related archaeal lipid samples (i.e., archaeol: −91.6‰, sn-2-hydroxyarchaeol: −129.2‰, PMI −92.8‰, and crocetane: −70.9‰) confirm the dominance of methane assimilation and isotope fractionation during AOM. These results are consistent with the detection of archaeal anaerobic methanotrophs (ANMEs) based on 16S rRNA gene sequencing. The Norfolk authigenic carbonate contained ANME-1a, -1b, 2a-2b, and 2c whereas only the ANME-2 clade was detected at Baltimore and present as the subclusters 2a-2b and -2b. The ANME-2d clade may also be present, particularly at the Baltimore seep site, given the high abundance of Candidatus Methanoperedens nitroreducens detected in the mcrA gene sequencing. The presence of terminally branched fatty acids, antesio- and iso-C15:0 components, as well as C16:1ω7 with δ13C values as low as −107.6‰, are indicative of sulfate-reducing bacteria (SRB) at the Norfolk seep site and supports syntrophy of SRB with methane-oxidizing archaea. In contrast, nitrate-driven AOM in syntrophy M. nitroreducens at the Baltimore seep site is consistent with elevated fatty acid δ13C values and lack of Deltaproteobacteria at the Baltimore seep site. Taken together, the range in lipid composition, distribution, and carbon isotopic composition observed at the Norfolk and Baltimore seep sites suggests AOM is performed by multiple archaea instead of a single species and may be paired with either or both nitrate- and sulfate-reduction. Given the heterogeneous nature of cold seep ecosystems, this study fills a critical spatial gap in our knowledge of AOM activity at two seep sites along a passive margin.

Formation of authigenic carbonates at a methane seep site in the middle Okinawa Trough, East China Sea

The formation of authigenic carbonates at cold seep is closely related to underlying methane seepage and anaerobic oxidation of methane (AOM), and these processes can be reflected in the geochemical characteristics of pore water in the sediments. In terms of petrological, mineralogical and geochemical analyses of research materials, including authigenic carbonates and pore water samples from the middle Okinawa Trough, East China Sea,these results show that authigenic carbonate minerals are mainly composed of high-magnesium calcite, low-magnesium calcite, dolomite, and iron dolomite and exhibit extremely negative carbon isotopic compositions (δ13C = −52.30‰ to −31.10‰ V-PDB) and positive oxygen isotopic compositions (δ18O = 3.36‰ to 7.27‰ V-PDB). Geochemical analyses of pore water suggest linear sulfate gradients and steep increase of methane and total alkalinity concentrations with depth, which indicate sulfate-induced AOM activities. Based on the sulfate concentration reduction, carbon isotope characteristics of dissolved inorganic carbon (DIC) and increased methane concentration, the sulfate-methane interface (SMI) is considered to be shallow. These data, combined with the variations in ion concentrations in pore water, suggest that the leakage of biogenic methane and intense AOM resulted in the formation of abundant authigenic carbonates, which inherited light carbon (12C)-enriched isotopic signature of methane in the shallow sediments. Combined geochemical characteristics of pore water and authigenic carbonates, intense methane seep activities and associated AOM can be inferred both in present-day and ancient geological records on the western slope of the middle Okinawa Trough.