Coupled anaerobic methane oxidation and metal reduction in soil under elevated CO2.

Continued current emissions of carbon dioxide (CO2 ) and methane (CH4 ) by human activities will increase global atmospheric CO2 and CH4 concentrations and surface temperature significantly. Fields of paddy rice, the most important form of anthropogenic wetlands, account for about 9% of anthropogenic sources of CH4 . Elevated atmospheric CO2 may enhance CH4 production in rice paddies, potentially reinforcing the increase in atmospheric CH4 . However, what is not known is whether and how elevated CO2 influences CH4 consumption under anoxic soil conditions in rice paddies, as the net emission of CH4 is a balance of methanogenesis and methanotrophy. In this study, we used a long-term free-air CO2 enrichment experiment to examine the impact of elevated CO2 on the transformation of CH4 in a paddy rice agroecosystem. We demonstrate that elevated CO2 substantially increased anaerobic oxidation of methane (AOM) coupled to manganese and/or iron oxides reduction in the calcareous paddy soil. We further show that elevated CO2 may stimulate the growth and metabolism of Candidatus Methanoperedens nitroreducens, which is actively involved in catalyzing AOM when coupled to metal reduction, mainly through enhancing the availability of soil CH4 . These findings suggest that a thorough evaluation of climate-carbon cycle feedbacks may need to consider the coupling of methane and metal cycles in natural and agricultural wetlands under future climate change scenarios.

Seasonal Dynamics of Abundance, Structure, and Diversity of Methanogens and Methanotrophs in Lake Sediments.

Understanding temporal and spatial microbial community abundance and diversity variations is necessary to assess the functional roles played by microbial actors in the environment. In this study, we investigated spatial variability and temporal dynamics of two functional microbial sediment communities, methanogenic Archaea and methanotrophic bacteria, in Lake Bourget, France. Microbial communities were studied from 3 sites sampled 4 times over a year, with one core sampled at each site and date, and 5 sediment layers per core were considered. Microbial abundance in the sediment were determined using flow cytometry. Methanogens and methanotrophs community structures, diversity, and abundance were assessed using T-RFLP, sequencing, and real-time PCR targeting mcrA and pmoA genes, respectively. Changes both in structure and abundance were detected mainly at the water-sediment interface in relation to the lake seasonal oxygenation dynamics. Methanogen diversity was dominated by Methanomicrobiales (mainly Methanoregula) members, followed by Methanosarcinales and Methanobacteriales. For methanotrophs, diversity was dominated by Methylobacter in the deeper area and by Methylococcus in the shallow area. Organic matter appeared to be the main environmental parameter controlling methanogens, while oxygen availability influenced both the structure and abundance of the methanotrophic community.

Large-scale biogeography and environmental regulation of methanotrophic bacteria across boreal inland waters.

Aerobic methanotrophic bacteria (methanotrophs) use methane as a source of carbon and energy, thereby mitigating net methane emissions from natural sources. Methanotrophs represent a widespread and phylogenetically complex guild, yet the biogeography of this functional group and the factors that explain the taxonomic structure of the methanotrophic assemblage are still poorly understood. Here, we used high-throughput sequencing of the 16S rRNA gene of the bacterial community to study the methanotrophic community composition and the environmental factors that influence their distribution and relative abundance in a wide range of freshwater habitats, including lakes, streams and rivers across the boreal landscape. Within one region, soil and soil water samples were additionally taken from the surrounding watersheds in order to cover the full terrestrial-aquatic continuum. The composition of methanotrophic communities across the boreal landscape showed only a modest degree of regional differentiation but a strong structuring along the hydrologic continuum from soil to lake communities, regardless of regions. This pattern along the hydrologic continuum was mostly explained by a clear niche differentiation between type I and type II methanotrophs along environmental gradients in pH, and methane concentrations. Our results suggest very different roles of type I and type II methanotrophs within inland waters, the latter likely having a terrestrial source and reflecting passive transport and dilution along the aquatic networks, but this is an unresolved issue that requires further investigation.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Here, evidence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable isotope experiments confirmed the occurrence of n-damo in the examined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and particulate methane monooxygenase genes demonstrated that M. oxyfera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10(7) gene copies per gram of dry soil. It is estimated that ∼0.51 g of CH4 m(-2) per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents previously unidentified confirmation that the n-damo process is a previously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution.

Variable impact of geochemical gradients on the functional potential of bacteria, archaea, and phages from the permanently stratified Lac Pavin.

BACKGROUND: Permanently stratified lakes contain diverse microbial communities that vary with depth and so serve as useful models for studying the relationships between microbial community structure and geochemistry. Recent work has shown that these lakes can also harbor numerous bacteria and archaea from novel lineages, including those from the Candidate Phyla Radiation (CPR). However, the extent to which geochemical stratification differentially impacts carbon metabolism and overall genetic potential in CPR bacteria compared to other organisms is not well defined. RESULTS: Here, we determine the distribution of microbial lineages along an oxygen gradient in Lac Pavin, a deep, stratified lake in central France, and examine the influence of this gradient on their metabolism. Genome-based analyses revealed an enrichment of distinct C1 and CO2 fixation pathways in the oxic lake interface and anoxic zone/sediments, suggesting that oxygen likely plays a role in structuring metabolic strategies in non-CPR bacteria and archaea. Notably, we find that the oxidation of methane and its byproducts is largely spatially separated from methane production, which is mediated by diverse communities of sediment methanogens that vary on the centimeter scale. In contrast, we detected evidence for RuBisCO throughout the water column and sediments, including form II/III and form III-related enzymes encoded by CPR bacteria in the water column and DPANN archaea in the sediments. On the whole, though, CPR bacteria and phages did not show strong signals of gene content differentiation by depth, despite the fact that distinct species groups populate different lake and sediment compartments. CONCLUSIONS: Overall, our analyses suggest that environmental gradients in Lac Pavin select for capacities of CPR bacteria and phages to a lesser extent than for other bacteria and archaea. This may be due to the fact that selection in the former groups is indirect and depends primarily on host characteristics. Video Abstract.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave.

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g-1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g-1 DW, 1-3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g-1 DW·h-1 in sediment versus 11.02 ng CH4·g-1 DW·h-1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Soil microbes under threat in the Amazon Rainforest.

Soil microorganisms are sensitive indicators of land-use and climate change in the Amazon, revealing shifts in important processes such as greenhouse gas (GHG) production, but they have been overlooked in conservation and management initiatives. Integrating soil biodiversity with other disciplines while expanding sampling efforts and targeted microbial groups is crucially needed.

MCycDB: A curated database for comprehensively profiling methane cycling processes of environmental microbiomes.

Methane is a critical greenhouse gas with significant impacts on environmental and global change. However, CH4 cycling processes and coupling mechanisms with the biogeochemical cycling of carbon, nitrogen, sulfur and metals in the environment remain elusive. To fill such knowledge gaps, we constructed a manually curated methane cycling database (MCycDB) for comprehensive and accurate analysis of methane cycling microbial communities. MCycDB contains 298 methane cycling gene families covering 10 methane metabolism pathways with 610,208 representative sequences, and associated reference sequences from the NCBI RefSeq database with 48 phyla and 2,197 genera, and five phyla and 100 genera for bacteria and archaea, respectively. Also, homologous groups from public orthology databases were identified and included in MCycDB to reduce false positive assignments. We applied MCycDB to profile methane cycling gene families and associated taxonomic groups from various environments. Gene families involved in methanogenesis were abundant in hot spring sediment and less abundant in freshwater, whereas the ones involved in aerobic oxidation of methane were abundant in permafrost and peatland. This study demonstrates that MCycDB is a useful tool for studying microbially-driven methane cycling processes with high specificity, coverage and accuracy.

Genome-resolved metagenomics reveals novel archaeal and bacterial genomes from Amazonian forest and pasture soils.

Amazonian soil microbial communities are known to be affected by the forest-to-pasture conversion, but the identity and metabolic potential of most of their organisms remain poorly characterized. To contribute to the understanding of these communities, here we describe metagenome-assembled genomes (MAGs) recovered from 12 forest and pasture soil metagenomes of the Brazilian Eastern Amazon. We obtained 11 forest and 30 pasture MAGs (≥50% of completeness and ≤10 % of contamination), distributed among two archaeal and 11 bacterial phyla. The taxonomic classification results suggest that most MAGs may represent potential novel microbial taxa. MAGs selected for further evaluation included members of Acidobacteriota, Actinobacteriota, Desulfobacterota_B, Desulfobacterota_F, Dormibacterota, Eremiobacterota, Halobacteriota, Proteobacteria, and Thermoproteota, thus revealing their roles in carbohydrate degradation and mercury detoxification as well as in the sulphur, nitrogen, and methane cycles. A methane-producing Archaea of the genus Methanosarcina was almost exclusively recovered from pasture soils, which can be linked to a sink-to-source shift after the forest-to-pasture conversion. The novel MAGs constitute an important resource to help us unravel the yet-unknown microbial diversity in Amazonian soils and its functional potential and, consequently, the responses of these microorganisms to land-use change.

A Novel Laboratory-Scale Mesocosm Setup to Study Methane Emission Mitigation by Sphagnum Mosses and Associated Methanotrophs.

Degraded peatlands are often rewetted to prevent oxidation of the peat, which reduces CO2 emission. However, the created anoxic conditions will boost methane (CH4) production and thus emission. Here, we show that submerged Sphagnum peat mosses in rewetted-submerged peatlands can reduce CH4 emission from peatlands with 93%. We were able to mimic the field situation in the laboratory by using a novel mesocosm set-up. By combining these with 16S rRNA gene amplicon sequencing and qPCR analysis of the pmoA and mmoX genes, we showed that submerged Sphagnum mosses act as a niche for CH4 oxidizing bacteria. The tight association between Sphagnum peat mosses and methane oxidizing bacteria (MOB) significantly reduces CH4 emissions by peatlands and can be studied in more detail in the mesocosm setup developed in this study.

Resilience of methane cycle and microbial functional genes to drought and flood in an alkaline wetland: A metagenomic analysis.

Alkaline wetlands distributed in arid or semi-arid areas are hotspots of methane (CH4) emissions. Periods of drought and flood, although regular, are stressful events encountered by methanogenic anaerobes in alkaline wetlands. To investigate the response of the CH4 cycle of alkaline wetlands to such stresses, we take Zhalong wetland as an example, then determined the CH4 flux and soil microbiomes in the wetland during wet, dry, and flooded periods. The in-situ CH4 flux in the wet period was 9.55-17.29 mg‧m-2‧h-1, but sharply degraded to 3.37-6.61 mg‧m-2‧h-1 in the dry period. It resumed to 4.51-20.80 mg‧m-2‧h-1 when the wetland was flooded again, which indicated that methanogenesis is quite resilient to drought. Syntrophic acetogenesis, and subsequently aceticlastic methanogenesis, were the dominant methanogenic pathways and resisted drought. Members belonging to Syntrophobacterales were the dominant syntrophic acetogens. They enter a viable but non-culturable (VBNC) state to resist drought. The dominant Methanosarcinales have the ability to repair reactive oxygen species damage during dry periods. The community of CH4 sink was governed by anaerobic methanotrophs, which entered a VBNC state or used repair systems to survive dry periods. This study revealed the responses of the CH4 cycle and microbial functional genes to drought and flood in alkaline wetlands.

Microbial Communities Involved in Methane, Sulfur, and Nitrogen Cycling in the Sediments of the Barents Sea.

A combination of physicochemical and radiotracer analysis, high-throughput sequencing of the 16S rRNA, and particulate methane monooxygenase subunit A (pmoA) genes was used to link a microbial community profile with methane, sulfur, and nitrogen cycling processes. The objects of study were surface sediments sampled at five stations in the northern part of the Barents Sea. The methane content in the upper layers (0-5 cm) ranged from 0.2 to 2.4 µM and increased with depth (16-19 cm) to 9.5 µM. The rate of methane oxidation in the oxic upper layers varied from 2 to 23 nmol CH4 L-1 day-1 and decreased to 0.3 nmol L-1 day-1 in the anoxic zone at a depth of 16-19 cm. Sulfate reduction rates were much higher, from 0.3 to 2.8 µmol L-1 day-1. In the surface sediments, ammonia-oxidizing Nitrosopumilaceae were abundant; the subsequent oxidation of nitrite to nitrate can be carried out by Nitrospira sp. Aerobic methane oxidation could be performed by uncultured deep-sea cluster 3 of gamma-proteobacterial methanotrophs. Undetectable low levels of methanogenesis were consistent with a near complete absence of methanogens. Anaerobic methane oxidation in the deeper sediments was likely performed by ANME-2a-2b and ANME-2c archaea in consortium with sulfate-reducing Desulfobacterota. Sulfide can be oxidized by nitrate-reducing Sulfurovum sp. Thus, the sulfur cycle was linked with the anaerobic oxidation of methane and the nitrogen cycle, which included the oxidation of ammonium to nitrate in the oxic zone and denitrification coupled to the oxidation of sulfide in the deeper sediments. Methane concentrations and rates of microbial biogeochemical processes in sediments in the northern part of the Barents Sea were noticeably higher than in oligotrophic areas of the Arctic Ocean, indicating that an increase in methane concentration significantly activates microbial processes.

Regulation of coastal methane sinks by a structured gradient of microbial methane oxidizers.

Coastal wetlands are widely recognized as atmospheric methane sources. However, recent field studies suggest that some coastal wetlands could also act as methane sinks, but the mechanism is not yet clear. Here, we investigated methane oxidation with different electron acceptors (i.e., oxygen, nitrate/nitrite, sulfate, Fe(III) and Mn(IV)) in four coastal wetlands in China using a combination of molecular biology methods and isotopic tracing technologies. The geochemical profiles and in situ Gibbs free energies suggest that there was significant nitrite-dependent anaerobic oxidation of methane (nitrite-AOM) in the sub-surface sediments; this was subsequently experimentally verified by both the microbial abundance and activity. Remarkably, the methanotrophic communities seemed to exist in the sediments as layered structures, and the surface aerobic methane-oxidizing bacteria were able to take up atmospheric methane at a rate of 0.10-0.18 nmol CH4 day-1 cm-2, while most, if not all, sedimentary methane was being completely consumed by anaerobic methanotrophs (23-58% by methane oxidizers in phylum NC10). These results suggest that coastal methane sinks might be governed by diverse microbial communities where NC10 methane oxidizers contributed significantly. This finding helps to better understand and predict the coastal methane cycle and reduce uncertainties in the estimations of the global methane flux.

[Methane Cycle Anaerobic Membrane Bioreactor with Desulfurization for Treating High Sulfate Organic Wastewater at Normal Temperature].

The operational performance of a methane cycle anaerobic membrane bioreactor (MCAnMBR) with desulfurization for the treatment of high sulfate organic wastewater was investigated. Within 63 days, the MCAnMBR with desulfurization was started successfully at a normal temperature of 26-34℃. The two main problems at the beginning of debugging, sludge loss and increased pH, were effectively solved by adjusting the gas path system and adding methanol to the inflow for acclimation. The results show that the inflow concentration of SO42- from 650 mg·L-1to 5800 mg·L-1can be treated well with the conditions of hydraulic retention time (HRT)=120 h, a volume loading of 3.61-4.36 kg·(m3·d)-1, pH of 7.18-7.61, and temperature of 23.3-25.4℃. The effluent chemical oxygen demand (COD) concentration could be lowered to 23 mg·L-1. The total removal rate of COD could remain stable at 96.23%-99.77%, and the SO42- conversion rate was about 83.83%-95.51%. These results indicated that the MCAnMBR reactor with desulfurization could effectively solve the problem of secondary sulfide inhibition. A gradient experiment revealed that the high Na2SO4 inflow could be treated when the influent COD ranged from 18000 mg·L-1to 21000 mg·L-1, the concentration of SO42- ranged between 9082 mg·L-1 and 9600 mg·L-1, and the COD/SO42-=2.

Metagenomic analysis of nitrogen and methane cycling in the Arabian Sea oxygen minimum zone.

Oxygen minimum zones (OMZ) are areas in the global ocean where oxygen concentrations drop to below one percent. Low oxygen concentrations allow alternative respiration with nitrate and nitrite as electron acceptor to become prevalent in these areas, making them main contributors to oceanic nitrogen loss. The contribution of anammox and denitrification to nitrogen loss seems to vary in different OMZs. In the Arabian Sea, both processes were reported. Here, we performed a metagenomics study of the upper and core zone of the Arabian Sea OMZ, to provide a comprehensive overview of the genetic potential for nitrogen and methane cycling. We propose that aerobic ammonium oxidation is carried out by a diverse community of Thaumarchaeota in the upper zone of the OMZ, whereas a low diversity of Scalindua-like anammox bacteria contribute significantly to nitrogen loss in the core zone. Aerobic nitrite oxidation in the OMZ seems to be performed by Nitrospina spp. and a novel lineage of nitrite oxidizing organisms that is present in roughly equal abundance as Nitrospina. Dissimilatory nitrate reduction to ammonia (DNRA) can be carried out by yet unknown microorganisms harbouring a divergent nrfA gene. The metagenomes do not provide conclusive evidence for active methane cycling; however, a low abundance of novel alkane monooxygenase diversity was detected. Taken together, our approach confirmed the genomic potential for an active nitrogen cycle in the Arabian Sea and allowed detection of hitherto overlooked lineages of carbon and nitrogen cycle bacteria.