Nitrite-dependent Anaerobic Methane Oxidation Research Articles

Nitrite-dependent microbial utilization for simultaneous removal of sulfide and methane in sewers

Chemicals are commonly dosed in sewer systems to reduce the emission of hydrogen sulfide (H2S) and methane (CH4), incurring high costs and environmental concerns. Nitrite dosing is a promising approach as nitrite can be produced from urine wastewater, which is a feasible integrated water management strategy. However, nitrite dosing usually requires strict conditions, e.g., relatively high nitrite concentration (e.g., ∼200 mg N/L) and acidic environment, to inhibit microorganisms. In contrast to “microbial inhibition”, this study proposes “microbial utilization” concept, i.e., utilizing nitrite as a substrate for H2S and CH4 consumption in sewer. In a laboratory-scale sewer reactor, nitrite at a relatively low concentrations of 25–48 mg N/L was continuously dosed. Two nitrite-dependent microbial utilization processes, i.e., nitrite-dependent anaerobic methane oxidation (n-DAMO) and microbial sulfide oxidation, successfully occurred in conjunction with nitrite reduction. The occurrence of both processes achieved a 58 % reduction in dissolved methane and over 90 % sulfide removal in the sewer reactor, with microbial activities measured as 15.6 mg CH4/(L·h) and 29.4 mg S/(L·h), respectively. High copy numbers of n-DAMO bacteria and sulfide-oxidizing bacteria (SOB) were detected in both sewer biofilms and sediments. Mechanism analysis confirmed that the dosed nitrite at a relatively low level did not cause the inhibition of sulfidogenic process due to the downward migration of activity zones in sewer sediments. Therefore, the proposed “microbial utilization” concept offers a new alternative for simultaneous removal of sulfide and methane in sewers.

Evolutionary ecology of denitrifying methanotrophic NC10 bacteria in the deep-sea biosphere.

The NC10 phylum links anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophic pathway. Although numerous amplicon-based studies revealed the distribution of this phylum, comprehensive genomic insights and niche characterization in deep-sea environments were still largely unknown. In this study, we extensively surveyed the NC10 bacteria across diverse deep-sea environments, including waters, sediments, cold seeps, biofilms, rocky substrates, and subseafloor aquifers. We then reconstructed and analysed 38 metagenome-assembled genomes (MAGs), and revealed the extensive distribution of NC10 bacteria and their intense selective pressure in these harsh environments. Isotopic analyses combined with gene expression profiling confirmed that active nitrite-dependent anaerobic methane oxidation (n-DAMO) occurs within deep-sea sediments. In addition, the identification of the Wood-Ljungdahl (WL) and 3-hydroxypropionate/4-hydroxybutyrat (3HB/4HP) pathways in these MAGs suggests their capability for carbon fixation as chemoautotrophs in these deep-sea environments. Indeed, we found that for their survival in the oligotrophic deep-sea biosphere, NC10 bacteria encode two branches of the WL pathway, utilizing acetyl-CoA from the carbonyl branch for citric acid cycle-based energy production and methane from the methyl branch for n-DAMO. The observed low ratios of non-synonymous substitutions to synonymous substitutions (pN/pS) in n-DAMO-related genes across these habitats suggest a pronounced purifying selection that is critical for the survival of NC10 bacteria in oligotrophic deep-sea environments. These findings not only advance our understanding of the evolutionary adaptations of NC10 bacteria but also underscore the intricate coupling between the carbon and nitrogen cycles within deep-sea ecosystems, driven by this bacterial phylum.

Methane mitigation via the nitrite-DAMO process induced by nitrate dosing in sewers

Nitrate or nitrite-dependent anaerobic methane oxidation (n-DAMO) is a microbial process that links carbon and nitrogen cycles as a methane sink in many natural environments. This study demonstrates, for the first time, that the nitrite-dependent anaerobic methane oxidation (nitrite-DAMO) process can be stimulated in sewer systems under continuous nitrate dosing for sulfide control. In a laboratory sewer system, continuous nitrate dosing not only achieved complete sulfide removal, but also significantly decreased dissolved methane concentration by ∼50 %. Independent batch tests confirmed the coupling of methane oxidation with nitrate and nitrite reduction, revealing similar methane oxidation rates of 3.68 ± 0.5 mg CH4 L−1 h−1 (with nitrate as electron acceptor) and 3.57 ± 0.4 mg CH4 L−1 h−1 (with nitrite as electron acceptor). Comprehensive microbial analysis unveiled the presence of a subgroup of the NC10 phylum, namely Candidatus Methylomirabilis (n-DAMO bacteria that couples nitrite reduction with methane oxidation), growing in sewer biofilms and surface sediments with relative abundances of 1.9 % and 1.6 %, respectively. In contrast, n-DAMO archaea that couple methane oxidation solely to nitrate reduction were not detected. Together these results indicated the successful enrichment of n-DAMO bacteria in sewerage systems, contributing to approx. 64 % of nitrite reduction and around 50 % of dissolved methane removal through the nitrite-DAMO process, as estimated by mass balance analysis. The occurrence of the nitrite-DAMO process in sewer systems opens a new path to sewer methane emissions.

Regulation of nitrite-dependent anaerobic methane oxidation bacteria by available phosphorus and microbial communities in lake sediments of cold and arid regions

As global anthropogenic nitrogen inputs continue to rise, nitrite-dependent anaerobic methane oxidation (N-DAMO) plays an increasingly significant role in CH4 consumption in lake sediments. However, there is a dearth of knowledge regarding the effects of anthropogenic activities on N-DAMO bacteria in lakes in the cold and arid regions. Sediment samples were collected from five sampling areas in Lake Ulansuhai at varying depth ranges (0–20, 20–40, and 40–60 cm). The ecological characterization and niche differentiation of N-DAMO bacteria were investigated using bioinformatics and molecular biology techniques. Quantitative PCR confirmed the presence of N-DAMO bacteria in Lake Ulansuhai sediments, with 16S rRNA gene abundances ranging from 1.72 × 104 to 5.75 × 105 copies·g−1 dry sediment. The highest abundance was observed at the farmland drainage outlet with high available phosphorus (AP). Anthropogenic disturbances led to a significant increase in the abundance of N-DAMO bacteria, though their diversity remained unaffected. The heterogeneous community of N-DAMO bacteria was affected by interactions among various environmental characteristics, with AP and oxidation-reduction potential identified as the key drivers in this study. The Mantel test indicated that the N-DAMO bacterial abundance was more readily influenced by the presence of the denitrification genes (nirS and nirK). Network analysis revealed that the community structure of N-DAMO bacteria generated numerous links (especially positive links) with microbial taxa involved in carbon and nitrogen cycles, such as methanogens and nitrifying bacteria. In summary, N-DAMO bacteria exhibited sensitivity to both environmental and microbial factors under various human disturbances. This study provides valuable insights into the distribution patterns of N-DAMO bacteria and their roles in nitrogen and carbon cycling within lake ecosystems.

Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea.

Anaerobic oxidation of methane (AOM) is critical for controlling methane emissions from deep-sea cold seeps. Microbial groups associated with different AOM processes have been briefly investigated, but the AOM activities associated with the utilization of different electron acceptors in the deep-sea methane seeps remain largely unknown. Here, surface sediments (0-4 cm) from two cold seeps (Site F/Haima) and the Xisha trough in the South China Sea (SCS) were incubated to measure AOM potentials driven by nitrite, nitrate, and sulfate as different electron acceptors with isotopically labeled methane gas (13CH4), and the population shifts of microbial communities along the incubation were investigated by high-throughput sequencing and quantitative polymerase chain reaction based on both the 16S rRNA gene and functional genes. Nitrite and nitrate were preferentially used prior to sulfate during the incubation, and nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) contributed more to the methane flux in both the cold seeps and the trough. The highest rates of DAMO and sulfate-dependent anaerobic methane oxidation (SAMO) were detected at Site F, consistent with the transcript abundance of mcrA and dsrB genes during the incubation, and might be explained by the in situ active functional groups. The former process might be explained by Campylobacteria, which could couple sulfide oxidation and nitrate consumption to further participate in the N-DAMO process, while the latter process might be due to the higher proportions of Desulfobacterota. This study elucidates the diversity and potential activities of major microbial groups in the DAMO and SAMO processes in the deep-sea cold seeps using isotopic tracing incubations and provides direct evidence for the occurrence of Nr-/N-DAMO as a previously overlooked microbial methane sink in the deep-sea hydrate-bearing sediments in the SCS. IMPORTANCE Cold seeps occur in continental margins worldwide and are deep-sea oases. Anaerobic oxidation of methane is an important microbial process in the cold seeps and plays an important role in regulating methane content. This study elucidates the diversity and potential activities of major microbial groups in dependent anaerobic methane oxidation and sulfate-dependent anaerobic methane oxidation processes and provides direct evidence for the occurrence of nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) as a previously overlooked microbial methane sink in the hydrate-bearing sediments of the South China Sea. This study provides direct evidence for occurrence of Nr-/N-DAMO as an important methane sink in the deep-sea cold seeps.

How efficiently does a metabolically enhanced system with denitrifying anaerobic methane oxidizing microorganisms remove antibiotics?

In this work, the novel N-damo (Nitrite dependent anaerobic methane oxidation) process was investigated at high biomass activities for its potential to remove simultaneously nitrite and methane, as well as selected antibiotics commonly found in sewage in trace amounts. For this purpose, two MBRs were operated at three high nitrite loading rates (NLRs), namely 76 ± 9.9, 161.5 ± 11.4 and 215.2 ± 24.2 mg N–NO⁻2 L−1 d−1, at long-term operation. The MBRs performance achieved a significantly high nitrite removal activity for an N-damo process (specific denitrifying activity of up to 540 mg N–NO⁻2 g−1 VSS d−1), even comparable to heterotrophic denitrification values. In this study, we have implemented a novel operational strategy that sets our work apart from previous studies with similar bioreactors. Specifically, we have introduced Cerium as a trace element in the feeding medium, which serves as a key differentiating factor. It allowed maintaining a stable reactor operation at high NLRs. Microbial community composition evidenced that both MBRs were dominated with N-damo bacteria (67–87% relative abundance in period III and I, respectively). However, a decrease in functional N-damo bacteria (Candidatus Methylomirabilis) abundance was observed during the increase in biomass activity and concentration, concomitantly with an increase of the other minor families (Hypomicrobiaceae and Xanthobacteraceae). Most of the selected antibiotics showed high biotransformation such as sulfamethoxazole, trimethoprim, cefalexin and azithromycin, whereas others such as roxithromycin and clarithromycin were only partially degraded (20–35%). On the contrary, ciprofloxacin showed almost no removal. Despite the metabolic enhancement, no apparent increase on the antibiotic removal was observed throughout the operation, suggesting that microbiological composition was of greater influence than its primary metabolic activity on the removal of antibiotics.

Microbial Niche Differentiation during Nitrite-Dependent Anaerobic Methane Oxidation.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) has been demonstrated to play important roles in the global methane and nitrogen cycle. However, despite diverse n-DAMO bacteria widely detected in environments, little is known about their physiology for microbial niche differentiation. Here, we show the microbial niche differentiation of n-DAMO bacteria through long-term reactor operations combining genome-centered omics and kinetic analysis. With the same inoculum dominated by both species "Candidatus Methylomirabilis oxyfera" and "Candidatus Methylomirabilis sinica", n-DAMO bacterial population was shifted to "Ca. M. oxyfera" in a reactor fed with low-strength nitrite, but shifted to "Ca. M. sinica" with high-strength nitrite. Metatranscriptomic analysis showed that "Ca. M. oxyfera" harbored more complete function in cell chemotaxis, flagellar assembly, and two-component system for better uptake of nitrite, while "Ca. M. sinica" had a more active ion transport and stress response system, and more redundant function in nitrite reduction to mitigate nitrite inhibition. Importantly, the half-saturation constant of nitrite (0.057 mM vs 0.334 mM NO2-) and inhibition thresholds (0.932 mM vs 2.450 mM NO2-) for "Ca. M. oxyfera" vs "Ca. M. sinica", respectively, were highly consistent with genomic results. Integrating these findings demonstrated biochemical characteristics, especially the kinetics of nitrite affinity and inhibition determine niche differentiation of n-DAMO bacteria.

Water column dynamics control nitrite-dependent anaerobic methane oxidation by Candidatus “Methylomirabilis” in stratified lake basins

We investigated microbial methane oxidation in the water column of two connected but hydrodynamically contrasting basins of Lake Lugano, Switzerland. Both basins accumulate large amounts of methane in the water column below their chemoclines, but methane oxidation efficiently prevents methane from reaching surface waters. Here we show that in the meromictic North Basin water column, a substantial fraction of methane was eliminated through anaerobic methane oxidation (AOM) coupled to nitrite reduction by Candidatus Methylomirabilis. Incubations with 14CH4 and concentrated biomass from this basin showed enhanced AOM rates with nitrate (+62%) and nitrite (+43%). In the more dynamic South Basin, however, aerobic methanotrophs prevailed, Ca. Methylomirabilis was absent in the anoxic water column, and no evidence was found for nitrite-dependent AOM. Here, the duration of seasonal stratification and anoxia seems to be too short, relative to the slow growth rate of Ca. Methylomirabilis, to allow for the establishment of anaerobic methanotrophs, in spite of favorable hydrochemical conditions. Using 16 S rRNA gene sequence data covering nearly ten years of community dynamics, we show that Ca. Methylomirabilis was a permanent element of the pelagic methane filter in the North Basin, which proliferated during periods of stable water column conditions and became the dominant methanotroph in the system. Conversely, more dynamic water column conditions led to a decline of Ca. Methylomirabilis and induced blooms of the faster-growing aerobic methanotrophs Methylobacter and Crenothrix. Our data highlight that physical (mixing) processes and ecosystem stability are key drivers controlling the community composition of aerobic and anaerobic methanotrophs.

Positive response of nitrite-dependent anaerobic methane oxidation to both gradual and abrupt increases of atmospheric CO2 concentration in paddy soils

The global ongoing increase in the atmospheric CO2 concentration is a gradual rather than an abrupt process. Although the abrupt increase in atmospheric CO2 concentration has been reported to greatly affect nitrite-dependent anaerobic methane oxidation (n-damo), the effect of a gradual increase in atmospheric CO2 concentration on n-damo remains unclear. To better evaluate the role of n-damo in paddy soils, this study investigated the responses of n-damo to different CO2 treatments: the ambient CO2 concentration (CK), a gradual increase (GI), and an abrupt increase (AI) in the CO2 concentration across different growth periods and soil depths. The 13CH4 stable isotope and quantitative PCR results revealed that compared to the CK, GI and AI significantly increased the activity of n-damo by 91.6 % and 90.0 % (p < 0.05), respectively, and also significantly increased the abundance of Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria by 67.7 % and 98.6 % (p < 0.05), respectively. Although there was no significant variation between GI and AI. In addition, the activity and abundance differed significantly between growth period and soil depth. The high-throughput sequencing results showed that GI and AI had little effect on bacterial community, while the soil depth had a significant impact on bacterial community. The changes in several soil physiochemical factors (e.g., the contents of soil water, organic carbon, and inorganic nitrogen) had important impacts on n-damo activity and the bacterial community structure. Overall, the results indicate a positive and similar response of n-damo to both gradual and abrupt increases in the atmospheric CO2 concentration in paddy soils.

Occurrence of nitrite-dependent anaerobic methane oxidation bacteria in the continental shelf sediments

Nitrite-dependent anaerobic methane oxidation (N-damo) is a key bioprocess coupling global carbon and nitrogen cycles and is mediated by NC10 bacteria. So far, the distribution of N-damo bacteria in marine sediments has rarely been reported. In this study, the sediments from the Bohai Sea, Yellow Sea and East China Sea were taken as the research objects, and the ecological distribution of N-damo bacteria was investigated by quantitative PCR and amplicon sequencing. Quantitative PCR results demonstrated that the highest average copy number of N-damo bacterial 16S rRNA gene was in the Bohai Sea, followed by the East China Sea, while the lowest was observed in the Yellow Sea. Based on the OTU numbers, the N-damo bacterial diversity was highest in East China Sea, followed by the Bohai Sea, while lowest in the Yellow Sea. The N-damo bacterial community structure exhibited an obvious spatial distribution among the three seas. Sediment nitrite nitrogen content is the key environmental factor affecting the abundance and diversity of N-damo bacteria, and sediment ammonia nitrogen content is the key environmental factor affecting the community structure of N-damo bacteria.

Distribution pattern of N-damo bacteria along an anthropogenic nitrogen input gradient from the coastal mangrove wetland to the South China sea sediments

Microbial nitrite-dependent anaerobic methane oxidation (n-damo) process is important for mitigating methane emission and anthropogenic nitrogen inputs in the marine environment. However, the distribution pattern of n-damo bacteria along an anthropogenic N-input gradient from the coastal wetland to the pristine South China Sea is poorly understood. This study investigated the diversity and abundance of n-damo bacteria in samples collected along a N-input gradient from Mai Po (MP) mangrove wetland sediments of the Pearl River Estuary (PRE) to the deep ocean sediments of the South China Sea (SCS). Retrieved 16S rDNA sequences showed a shift of n-damo community composition of complex structures with both freshwater and marine n-damo lineages in MP intertidal sediments to marine dominated characteristic in SCS sediments. The observed variation of Shannon and Chao1 indexes of n-damo bacteria shared a similar trend of a decrease at first followed by an increase along the targeting gradient with previously investigated methanogens, anaerobic methanotrophic archaea, ammonia-oxidizing archaea and ammonia-oxidizing bacteria, but had a reverse pattern with anammox bacteria. The community structure of pmoA gene sequences contained freshwater lineages only in SCS continental shelf sediments closer to the PRE, and turned to group with other marine samples in deeper and pristine sediments. Results suggested that n-damo bacteria might be a major contributor to anaerobic denitrification in the SCS sediments because their abundances were much higher than previously studied anammox bacteria in the same sample set. The distribution pattern of n-damo bacterial diversity, richness and abundance along the anthropogenic N-input gradient implies that they could be used as a bio-indicator for monitoring the anthropogenic/terrestrial inputs in marine environments.

Spatial variations of activity and community structure of nitrite-dependent anaerobic methanotrophs in river sediment

Rivers are an important site for methane emissions and reactive nitrogen removal. The process of nitrite-dependent anaerobic methane oxidation (n-damo) links the global carbon cycle and the nitrogen cycle, but its role in methane mitigation and nitrogen removal in rivers is poorly known. In the present study, we investigated the activity, abundance, and community composition of n-damo bacteria in sediment of the upper, middle, and lower reaches of Wuxijiang River (Zhejiang Province, China). The 13CH4 stable isotope experiments showed that the methane oxidation activity of n-damo was 0.11–1.88 nmol CO2 g−1 (dry sediment) d−1, and the activity measured from the middle reaches was significantly higher than that from the remaining regions. It was estimated that 3.27 g CH4 m−2 year−1 and 8.72 g N m−2 year−1 could be consumed via n-damo. Quantitative PCR confirmed the presence of n-damo bacteria, and their 16S rRNA gene abundance varied between 5.45 × 105 and 5.86 × 106 copies g−1 dry sediment. Similarly, the abundance of n-damo bacteria was significantly higher in the middle reaches. High-throughput sequencing showed a high n-damo bacterial diversity, with totally 152 operational taxonomic units being detected at 97 % sequence similarity cut-off. In addition, the n-damo bacterial community composition also varied spatially. The inorganic nitrogen (NH4+, NO2−, NO3−) level was found to be the key environmental factor controlling the n-damo activity and bacterial community composition. Overall, our results showed the spatial variations and environmental regulation of the activity and community structure of n-damo bacteria in river sediment, which expanded our understanding of the quantitative importance of n-damo in both methane oxidation and reactive nitrogen removal in riverine systems.

Nitrogen cycling and microbial cooperation in the terrestrial subsurface

The nitrogen cycle plays a major role in aquatic nitrogen transformations, including in the terrestrial subsurface. However, the variety of transformations remains understudied. To determine how nitrogen cycling microorganisms respond to different aquifer chemistries, we sampled groundwater with varying nutrient and oxygen contents. Genes and transcripts involved in major nitrogen-cycling pathways were quantified from 55 and 26 sites, respectively, and metagenomes and metatranscriptomes were analyzed from a subset of oxic and dysoxic sites (0.3-1.1 mg/L bulk dissolved oxygen). Nitrogen-cycling mechanisms (e.g. ammonia oxidation, denitrification, dissimilatory nitrate reduction to ammonium) were prevalent and highly redundant, regardless of site-specific physicochemistry or nitrate availability, and present in 40% of reconstructed genomes, suggesting that nitrogen cycling is a core function of aquifer communities. Transcriptional activity for nitrification, denitrification, nitrite-dependent anaerobic methane oxidation and anaerobic ammonia oxidation (anammox) occurred simultaneously in oxic and dysoxic groundwater, indicating the availability of oxic-anoxic interfaces. Concurrent activity by these microorganisms indicates potential synergisms through metabolite exchange across these interfaces (e.g. nitrite and oxygen). Fragmented denitrification pathway encoding and transcription was widespread among groundwater bacteria, although a considerable proportion of associated transcriptional activity was driven by complete denitrifiers, especially under dysoxic conditions. Despite large differences in transcription, the capacity for the final steps of denitrification was largely invariant to aquifer conditions, and most genes and transcripts encoding N2O reductases were the atypical Sec-dependant type, suggesting energy-efficiency prioritization. Results provide insights into the capacity for cooperative relationships in groundwater communities, and the richness and complexity of metabolic mechanisms leading to the loss of fixed nitrogen.

Response of potential activity, abundance and community composition of nitrite-dependent anaerobic methanotrophs to long-term fertilization in paddy soils.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) catalysed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a novel pathway in regulating methane (CH4 ) emissions from paddy fields. Nitrogen fertilization is essential to improve rice yields and soil fertility; however, its effect on the n-damo process is largely unknown. Here, the potential n-damo activity, abundance and community composition of M. oxyfera-like bacteria were investigated in paddy fields under three long-term (32 years) fertilization treatments, i.e. unfertilized control (CK), chemical fertilization (NPK) and straw incorporation with chemical fertilization (SNPK). Relative to the CK, both NPK and SNPK treatments significantly (p < 0.05) increased the potential n-damo activity (88%-110%) and the abundance (52%-105%) of M. oxyfera-like bacteria. The variation of soil organic carbon (OrgC) content and inorganic nitrogen content caused by the input of chemical fertilizers and straw returning were identified as the key factors affecting the potential n-damo activity and the abundance of M. oxyfera-like bacteria. However, the community composition and diversity of M. oxyfera-like bacteria did not change significantly by the input of fertilizers. Overall, our results provide the first evidence that long-term fertilization greatly stimulates the n-damo process, indicating its active role in controlling CH4 emissions from paddy fields.

Potential role of nitrite-dependent anaerobic methane oxidation in methane consumption and nitrogen removal in Chinese paddy fields

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by bacteria closely related to Candidatus Methylomirabilis oxyfera, links the global carbon and nitrogen cycles. Currently, the contribution of n-damo in controlling methane emissions and nitrogen removal, and the key regulatory factors of this process in Chinese paddy fields are poorly known. Here, soil samples from 20 paddy fields located in different climate zones across China were collected to examine the n-damo activity and bacterial communities. The n-damo activity and bacterial abundance varied from 1.05 to 5.97 nmol CH4 g−1 (dry soil) d−1 and 2.59 × 105 to 2.50 × 107 copies g−1 dry soil, respectively. Based on the n-damo activity, it was estimated that approximately 0.91 Tg CH4 and 2.17 Tg N could be consumed annually via n-damo in Chinese paddy soils. The spatial variations in n-damo activity and community structure of n-damo bacteria were significantly (p < 0.05) affected by the soil ammonium content, labile organic carbon content and pH. Furthermore, significant differences in n-damo activity, bacterial abundance and community composition were observed among different climate zones. The n-damo activity was found to be positively correlated with the mean annual air temperature. Taken together, our results demonstrated the potential importance of n-damo in both methane consumption and nitrogen removal in Chinese paddy soils, and this process was regulated by local soil and climatic factors.

Nitrite-dependent anaerobic oxidation decreases methane emissions from peatlands

Peatlands are C-rich but N-poor ecosystems that function as an important C sink but also as significant CH4 sources and thus contribute to global warming. To confirm that N fertilization strongly reduces peatland CH4 emissions by stimulating nitrite-dependent anaerobic methane oxidation (n-damo), we investigated CH4 efflux and concentration in the soil profiles of a peatland subjected to long-term N fertilization. Contrary to previously reported increase of CH4 emissions from paddy, forest and grassland soils, N fertilization of peatland reduced CH4 efflux by 87%, especially during the hottest summer months (from 3.8 mg m−2 h−1 in the unfertilized control to 0.5 mg m−2 h−1 in the N-fertilized site). The CH4 efflux was strongly reduced because the high organic matter content of the peat indirectly stimulated n-damo by providing CH4 (methanogens use organic matter to produce methane) and nitrite (denitrifiers use organic matter as an energy source). N fertilization reduced the CH4 concentration by 50–95% (from 7800–9500 ppm in the unfertilized control to 370–4800 ppm in the N-fertilized site) in the top 30-cm soil, whereas the abundance of Methylomirabilis bacteria increased for 170% and its activity raised for 220% compared to the unfertilized control soil. Consequently, the topsoil is a hotspot of Methylomirabilis activity that use NO2− to oxidize CH4 and play an important role in reducing CH4 emissions in N-fertilized peatlands.

Nitrite-dependent anaerobic methane oxidation bacteria and potential in permafrost region of Daxing'an Mountains.

Nitrite-dependent anaerobic methane oxidation (n-damo) acts as a crucial link between biogeochemical carbon and nitrogen cycles. Nevertheless, very few studies have characterized n-damo microorganisms in high-latitude permafrost regions. Therefore, this study investigated the vertical distribution and diversity of n-damo bacterial communities in soil from three forest types in the permafrost regions of the Daxing'an Mountains. A total of 11 and 8 operational taxonomic units (OTUs) of n-damo 16S rRNA and pmoA genes were observed, respectively. Remarkable spatial variations in n-damo bacteria community richness, diversity, and structure were observed at different soil depths. Moreover, the abundances of n-damo bacteria (16S rRNA and pmoA genes) varied between 1.55 × 104 to 1.47 × 105 and 1.31 × 103 to 3.11 × 104 copies g-1 dry soil (ds), as demonstrated by quantitative PCR analyses. 13CH4 stable isotope tracer assays indicated that the potential n-damo rates varied from 0 to 1.26nmolg-1day-1, with the middle layers (20-40cm and 40-60cm) exhibiting significantly higher values than the upper (0-20cm) and deeper layers (80-100cm) in all three forest types. Redundancy analyses (RDA) indicated that total organic carbon (TOC), nitrate (NO3--N), and nitrite (NO2--N) were key modulators of the distribution of n-damo bacterial communities. This study thus demonstrated the widespread occurrence of n-damo bacteria in cold and high-latitude regions of forest ecosystems and provided important insights into the global distribution of these bacteria. KEY POINTS: • This study detected n-damo bacteria in soil samples obtained from the permafrost region of three forest types in the Daxing'an Mountains. • The community composition of n-damo bacteria was mainly affected by soil depth and not forest type. • The abundances of n-damo bacteria first increased and then decreased at higher soil depths.

Spatial and temporal characteristics of community structure of nitrite-dependent anaerobic methane-oxidizing bacteria in paddy soil

Paddy fields are one of the most important methane sources, which have great impacts on climate change. The nitrite-dependent anaerobic methane oxidation, by NC10 phylum bacteria-Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria, is a new process regulating methane emission from paddy fields. However, little is known about the spatial and temporal variations of M. oxyfera-like bacterial communities and the regulating factors in paddy soils. We investigated the community composition, diversity, and abundance of M. oxyfera-like bacteria in 0-40 cm depth of paddy soils at key growth stages of rice, including tillering, jointing, flowering, and milky stages. Results of high-throughput sequencing showed that community composition of M. oxyfera-like bacteria differed significantly among different soil layers, while no significant variation was observed among different rice growth stages. The diversity of M. oxyfera-like bacteria increased with soil depth. Real-time quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria ranged from 5.73×106 to 2.56×107 copies·g-1 (dry weight), with the highest gene abundance in the 10-20 cm layer. Further, the abundance of these bacteria showed a decreasing trend with rice growth. Soil organic carbon content and soil pH were correlated with the M. oxyfera-like bacterial community structures and abundance. In all, our results suggested a certain degree of heterogeneity of spatial and temporal distribution of M. oxyfera-like bacterial communities in paddy soils, which was largely influenced by soil organic carbon and soil pH.

NO3- is an important driver of nitrite-dependent anaerobic methane oxidation bacteria and CH4 fluxes in the reservoir riparian zone.

Nitrite-dependent anaerobic methane oxidation (N-DAMO) is an important biological process that combines microbial nitrogen and carbon cycling and is mainly carried out by nitrite-dependent anaerobic methane-oxidizing bacteria. The discovery of this microbial process has changed the conventional view of methane oxidation and nitrogen loss. In this study, the abundance, diversity, and community structure of N-DAMO bacteria were investigated based on high-throughput sequencing and fluorescence quantitative PCR measurements. We examined environmental factors driving the variations of CH4 fluxes and N-DAMO bacterial using correlation analysis and redundancy analysis. We found low CH4 fluxes and abundant N-DAMO bacteria in the riparian zone. After decomposing the effects of single variables and exploring them, NO3- was the only significant factor that significantly correlated with the abundance and richness of the N-DAMO community and gas fluxes (p < 0.05). Under the influence of three different land use types, the increase in NO3- (grassland vs. woodland and sparse woods, + 132.81% and + 106.25%) caused structural changes in the composition of the N-DAMO bacterial community, increasing its abundance (- 9.58% and + 21.19%), thus promoting the oxidation of CH4 and reduced CH4 emissions (+ 4.78% and + 35.63%) from the riparian zone. Appropriate NO3- input helps maintain the existing low methane emission fluxes in the riparian zone of the reservoir.

Response of nitrite-dependent anaerobic methanotrophs to elevated atmospheric CO2 concentration in paddy fields

Nitrite-dependent anaerobic methane oxidation (n-damo) catalyzed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a new pathway for the regulation of methane emissions from paddy fields. Elevated atmospheric CO2 concentrations (e[CO2]) can indirectly affect the structure and function of microbial communities. However, the response of M. oxyfera-like bacteria to e[CO2] is currently unknown. Here, we investigated the effect of e[CO2] (ambient CO2 + 200 ppm) on community composition, abundance, and activity of M. oxyfera-like bacteria at different depths (0–5, 5–10, and 10–20 cm) in paddy fields across multiple rice growth stages (tillering, jointing, and flowering). High-throughput sequencing showed that e[CO2] had no significant effect on the community composition of M. oxyfera-like bacteria. However, quantitative PCR suggested that the 16S rRNA gene abundance of M. oxyfera-like bacteria increased significantly in soil under e[CO2], particularly at the tillering stage. Furthermore, 13CH4 tracer experiments showed potential n-damo activity of 0.31–8.91 nmol CO2 g−1 (dry soil) d−1. E[CO2] significantly stimulated n-damo activity, especially at the jointing and flowering stages. The n-damo activity and abundance of M. oxyfera-like bacteria increased by an average of 90.9% and 50.0%, respectively, under e[CO2]. Correlation analysis showed that the increase in soil dissolved organic carbon content caused by e[CO2] had significant effects on the activity and abundance of M. oxyfera-like bacteria. Overall, this study provides the first evidence for a positive response of M. oxyfera-like bacteria to e[CO2], which may help reduce methane emissions from paddy fields under future climate change conditions.