Methanoperedens Nitroreducens Research Articles

Characterization of the redox-active extracellular polymeric substances in an anaerobic methanotrophic consortium

Anaerobic oxidation of methane (AOM) is a microbial process of importance in the global carbon cycle. AOM is predominantly mediated by anaerobic methanotrophic archaea (ANME), the physiology of which is still poorly understood. Here we present a new addition to the current physiological understanding of ANME by examining, for the first time, the biochemical and redox-active properties of the extracellular polymeric substances (EPS) of an ANME enrichment culture. Using a ‘Candidatus Methanoperedens nitroreducens’-dominated methanotrophic consortium as the representative, we found it can produce an EPS matrix featuring a high protein-to-polysaccharide ratio of ∼8. Characterization of EPS using FTIR revealed the dominance of protein-associated amide I and amide II bands in the EPS. XPS characterization revealed the functional group of C-(O/N) from proteins accounted for 63.7% of total carbon. Heme-reactive staining and spectroscopic characterization confirmed the distribution of c-type cytochromes in this protein-dominated EPS, which potentially enabled its electroactive characteristic. Redox-active c-type cytochromes in EPS mediated the EET of ‘Ca. M. nitroreducens’ for the reduction of Ag+ to metallic Ag, which was confirmed by both ex-situ experiments with extracted soluble EPS and in-situ experiments with pristine EPS matrix surrounding cells. The formation of nanoparticles in the EPS matrix during in-situ extracellular Ag + reduction resulted in a relatively lower intracellular Ag distribution fraction, beneficial for alleviating the Ag toxicity to cells. The results of this study provide the first biochemical information on EPS of anaerobic methanotrophic consortia and a new insight into its physiological role in AOM process.

Nitrate-dependent anaerobic methane oxidation coupled to Fe(III) reduction as a source of ammonium and nitrous oxide

‘Candidatus Methanoperedens nitroreducens’ is an archaeal methanotroph with global importance that links carbon and nitrogen cycles and great potential for sustainable operation of wastewater treatment. It has been reported to mediate the anaerobic oxidation of methane through a reverse methanogenesis pathway while reducing nitrate to nitrite. Here, we demonstrate that ‘Ca. M. nitroreducens’ reduces ferric iron forming ammonium (23.1 %) and nitrous oxide (N2O, 46.5 %) from nitrate. These results are supported with the upregulation of genes coding for proteins responsible for dissimilatory nitrate reduction to ammonium (nrfA), N2O formation (norV, cyt P460), and multiple multiheme c-type cytochromes for ferric iron reduction. Concomitantly, an increase in the N2O-reducing SJA-28 lineage and a decrease in the nitrite-reducing ‘Candidatus Methylomirabilis oxyfera’ are consistent with the changes in ‘Ca. M. nitroreducens’ end products. These findings demonstrate the highly flexible physiology of ‘Ca. M. nitroreducens’ in anaerobic ecosystems with diverse electron acceptor conditions, and further reveals its roles in linking methane oxidation to global biogeochemical cycles. ‘Ca. M. nitroreducens’ could significantly affect the bioavailability of nitrogen sources as well as the emission of greenhouse gas in natural ecosystems and wastewater treatment plants.

Probing Denitrifying Anaerobic Methane Oxidation via Antimicrobial Intervention: Implications for Innovative Wastewater Management.

Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO) has the potential for application in wastewater treatment plants. However, our understanding of the N-DAMO process is primarily based on studies conducted on environmental samples or enrichment cultures using metagenomic approaches. To gain deeper insights into N-DAMO, we used antimicrobial compounds to study the function and physiology of 'Candidatus Methanoperedens nitroreducens' and 'Candidatus Methylomirabilis oxyfera' in N-DAMO enrichment cultures. We explored the effects of inhibitors and antibiotics and investigated the potential application of N-DAMO in wastewater contaminated with ammonium and heavy metals. Our results showed that 'Ca. M. nitroreducens' was susceptible to puromycin and 2-bromoethanesulfonate, while the novel methanogen inhibitor 3-nitrooxypropanol had no effect on N-DAMO. Furthermore, 'Ca. M. oxyfera' was shown to be susceptible to the particulate methane monooxygenase inhibitor 1,7-octadiyne and a bacteria-suppressing antibiotic cocktail. The N-DAMO activity was not affected by ammonium concentrations below 10 mM. Finally, the N-DAMO community appeared to be remarkably resistant to lead (Pb) but susceptible to nickel (Ni) and cadmium (Cd). This study provides insights into microbial functions in N-DAMO communities, facilitating further investigation of their application in methanogenic, nitrogen-polluted water systems.

Multi-heme cytochrome-mediated extracellular electron transfer by the anaerobic methanotroph ‘Candidatus Methanoperedens nitroreducens’

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by ‘Candidatus Methanoperedens nitroreducens’, an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that ‘Ca. M. nitroreducens’ uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.

Long-term fertilization enhances the activity of anaerobic oxidation of methane coupled to nitrate reduction and associated microbial abundance in paddy soils

Anaerobic oxidation of methane coupled to nitrate reduction (nitrate-coupled AOM) is performed by Candidatus Methanoperedens nitroreducens (M. nitroreducens)-related archaea, and is recently recognized as a crucial component of the global carbon cycle. The input of fertilizers is an essential agricultural practice that greatly impacts methane (CH4) production and emission. However, the significance of nitrate-coupled AOM in CH4 cycling and its response to fertilization in rice fields remain unclear. In this study, the potential nitrate-coupled AOM rates and the communities of M. nitroreducens-related archaea in rice fields were examined at different soil layers (0–10, 10–20, and 30–40 cm) at tillering, elongation, flowering, and ripening stages under three long-term fertilization treatments (CK-without fertilizer, CF-chemical fertilization, or CFS-chemical fertilization with straw incorporation). The results indicated that both CF (1.07 nmol 13CO2 g−1 d−1) and CFS (1.21 nmol 13CO2 g−1 d−1) treatments significantly promoted the potential nitrate-coupled AOM rates compared to CK (0.53 nmol 13CO2 g−1 d−1). A greater response of potential activity to fertilization was observed at plough layer (upper 20 cm) and during elongation stage. The abundance of M. nitroreducens-related archaea under CF (1.12 × 107 copies g−1) and CFS (1.62 × 107 copies g−1) treatments was significantly greater than that under CK (6.93 × 106 copies g−1). Conversely, the growth response of these archaeal to fertilization was stronger at deeper layer (30–40 cm). Moreover, no significant change was observed in the community composition of M. nitroreducens-related archaea among treatments. Correlation analysis suggested that the variations of soil organic carbon, NH4+, and NO3− contents caused by fertilization were key factors influencing the potential nitrate-coupled AOM rates and M. nitroreducens-related archaeal abundance. Our findings provide the first evidence for positive response of nitrate-coupled AOM to long-term fertilization, demonstrating its potential to act as an important process for mitigating CH4 emission in rice fields.

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.

Salinity effect on an anaerobic methane- and ammonium-oxidising consortium: Shifts in activity, morphology, osmoregulation and syntrophic relationship

Nitrate-dependent anaerobic methane oxidation (AOM) is a microbial process of both ecological significance for global methane mitigation and application potential for wastewater treatment. It is mediated by organisms belonging to the archaeal family ‘Candidatus Methanoperedenaceae’, which have so far mainly been found in freshwater environments. Their potential distribution in saline environments and their physiological responses to salinity variation were still poorly understood. In this study, the responses of the freshwater 'Candidatus Methanoperedens nitroreducens’-dominated consortium to different salinities were investigated using short- and long-term setups. Short-term exposure to salt stress significantly affected nitrate reduction and methane oxidation activities over the tested concentration range of 15–200‰ NaCl, and ‘Ca. M. nitroreducens’ showed the higher tolerance to high salinity stress than its partner of anammox bacteria. At high salinity concentration, near marine conditions of 37‰, the target organism ‘Ca. M. nitroreducens’ showed stabilized nitrate reduction activity of 208.5 µmol day−1 gCDW−1 in long-term bioreactors over 300 days, in comparison to 362.9 and 334.3 µmol day−1 gCDW−1 under low-salinity conditions (1.7‰ NaCl) and control conditions (∼15‰ NaCl). Different partners of ‘Ca. M. nitroreducens’ evolved in the consortia with three different salinity conditions, suggesting the different syntrophic mechanisms shaped by changes in salinity. A new syntrophic relationship between ‘Ca. M. nitroreducens’ and Fimicutes and/or Chloroflexi denitrifying populations was identified under the marine salinity condition. Metaproteomic analysis shows that the salinity changes lead to higher expression of response regulators and selective ion (Na+/H+) channeling proteins that can regulate the osmotic pressure between the cell and its environment. The reverse methanogenesis pathway was, however, not impacted. The finding of this study has important implications for the ecological distribution of the nitrate-dependent AOM process in marine environments and the potential of this biotechnological process for the treatment of high-salinity industrial wastewater.

Formate as an Alternative Electron Donor for the Anaerobic Methanotrophic Archaeon Candidatus ‘Methanoperedens nitroreducens’

A metagenomic survey predicted that some anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae can use formate as an alternative electron donor, although this process has yet to be experimentally verified. In this study, formate was fed to an enrichment culture dominated by a member of Methanoperedenaceae, Candidatus ‘Methanoperedens nitroreducens’. The culture indeed oxidized formate and reduced nitrate simultaneously in the absence or presence of methane. The fdhAB genes for formate metabolism were identified in the genome of Ca. ‘M. nitroreducens’, and RT-qPCR results showed their expression levels increased 2–6-fold when formate was consumed. This observation was in line with the results of metaproteomic analysis, which showed that the expression levels of formate dehydrogenase (FdhAB) was increased in the presence of formate. Together, the findings strongly suggested that formate can be an alternative electron donor for Ca. ‘M. nitroreducens’, revealing its versatile metabolic potential. As formate is a product or intermediate compound of many microbial processes, the capability to utilize formate could aid ANME in coping with the dynamic availability of methane in natural or anthropogenic environments. Our findings demonstrate the need for additional research on the feasibility of Ca. ‘M. nitroreducens’ and other ANME utilizing alternative electron donors other than methane.

Anaerobic methane oxidation: High-rate performance of a continuous bioreactor using nitrate and nitrite as electron acceptors

The anaerobic oxidation of methane coupled to denitrification (N-AOM) is an innovative biotechnology able to simultaneously remove dissolved methane and NO3–/NO2–, minimizing the environmental impact of anaerobic effluents discharge. Most N-AOM studies have been implemented in batch reactors. However, studies in continuous systems are required to assess the potential of this biotechnology for full-scale applications. In this work, a continuous N-AOM reactor was implemented with a hydraulic retention time as short as 17 h. Process performance was evaluated using NO3– alone and in a mixture with NO2–. Methane elimination capacity (EC) values from 20 to 39 g m-3h−1 were reached when NO3– was supplied as the only electron acceptor. When the mixture of NO3– and NO2– was fed, EC values from 48 to 61 g m-3h−1 were recorded, which constitute the best N-AOM performance so far reported in the literature. The amount of nitrogen directed to N2O formation was always below 0.55% of the total nitrogen removed. Data from the reactor and further batch tests evidenced that the pH value was a critical factor influencing methane oxidation, denitrification, and N2O emission. Microbial characterization using 16S rRNA sequencing and metagenomics revealed that the archaeon Methanoperedens nitroreducens and the bacterium Methylomirabilis oxyfera were not present in the reactor. Instead, bacteria belonging to Proteobacteria and Bacteroidetes phyla, and archaea from the Euryarchaeota and Halobacterota phyla were the most abundant microorganisms. Based on the metagenomic analysis, the potential metabolic mechanisms involved in the N-AOM process were proposed.

Anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens' has a pleomorphic life cycle.

'Candidatus Methanoperedens' are anaerobic methanotrophic (ANME) archaea with global importance to methane cycling. Here meta-omics and fluorescence in situ hybridization (FISH) were applied to characterize a bioreactor dominated by 'Candidatus Methanoperedens nitroreducens' performing anaerobic methane oxidation coupled to nitrate reduction. Unexpectedly, FISH revealed the stable co-existence of two 'Ca. M. nitroreducens' morphotypes: the archetypal coccobacilli microcolonies and previously unreported planktonic rods. Metagenomic analysis showed that the 'Ca. M. nitroreducens' morphotypes were genomically identical but had distinct gene expression profiles for proteins associated with carbon metabolism, motility and cell division. In addition, a third distinct phenotype was observed, with some coccobacilli 'Ca. M. nitroreducens' storing carbon as polyhydroxyalkanoates. The phenotypic variation of 'Ca. M. nitroreducens' probably aids their survival and dispersal in the face of sub-optimal environmental conditions. These findings further demonstrate the remarkable ability of members of the 'Ca. Methanoperedens' to adapt to their environment.

Effect of gradual increase of atmospheric CO2 concentration on nitrate-dependent anaerobic methane oxidation in paddy soils

Nitrate-dependent anaerobic oxidation of methane (AOM) is a new pathway to reduce methane emissions from paddy ecosystems. The elevated atmospheric CO2 concentration can affect methane emissions from paddy ecosystems, but its impact on the process of nitrate-dependent AOM is poorly known. Based on the automatic CO2 control platform with open top chambers and the 13CH4 stable isotope experiments, the responses of the activity of nitrate-dependent AOM, abundance and community composition of Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea to the gradual increase of CO2 concentration were investigated in paddy fields. We set up two CO2 concentration treatments, including an ambient CO2 and a gradual increase of CO2(increase of 40 μL·L-1 per year above ambient CO2 concentration until 160 μL·L-1). The results showed the nitrate-dependent AOM rate of 0.7-11.3 nmol CO2·g-1·d-1 in the studied paddy fields, and quantitative PCR showed the abundance of M. nitroreducens-like archaeal mcrA genes of 2.2×106-8.5×106 copies·g-1. Compared to the ambient CO2 treatment, the slow elevated CO2 treatment enhanced the nitrate-dependent AOM rate and stimulated the abundance of M. nitroreducens-like archaea, particularly in 5-10 cm soil layer. The gradual increased CO2 concentration treatment did not change the community composition of M. nitroreducens-like archaea, but significantly decreased their diversity. The soil organic carbon content was an important factor influencing the nitrate-dependent AOM process. Overall, our results showed that the gradual increase of CO2 concentration could promote the nitrate-dependent AOM, suggesting its positive role in mitigating methane emissions from paddy ecosystems under future climate change.

Polyhydroxyalkanoate-driven current generation via acetate by an anaerobic methanotrophic consortium

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH4) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by ‘Candidatus Methanoperedens nitroreducens’ in a CH4-fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH4 directly. In the absence of CH4, current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of ‘Ca. M. nitroreducens’ to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems.

Fast development of microbial cultures for the anaerobic oxidation of CH4 coupled to denitrification employing widely available inocula

This work assessed the potential of two widely available inocula for the implementation of anaerobic oxidation of methane coupled with NO3- reduction (N-AOM). Methane consumption via N-AOM was observed within the first 40–67 days of culture using secondary activated sludge (AS) and a mixture of AS and anaerobic granular sludge (AS+AGS) as inoculum. Maximum methane removal rates of 0.13 ± 0.02 and 0.17 ± 0.01 gCH4 m−3 h−1 were recorded for AS and AS+AGS cultures, respectively. 16 S rRNA high throughput sequencing applied to biomass samples throughout incubation revealed the absence of the archaeon Methanoperedens nitroreducens and the bacterium Methylomirabilis oxyfera in AS and AS+AGS cultures. Instead, members from the Bacteroidetes, Chloroflexi, Proteobacteria, and Euryarchaeota lineages were predominant in the cultures. Nitrous oxide, an undesirable intermediate of nitrate reduction, was not detected in measurements performed at the end of the incubations. The methane consumption rates recorded are comparable with N-AOM systems requiring acclimation periods of up to 600 days. Therefore, this work confirmed that N-AOM processes might be implemented with inocula available in bulk amounts in short periods, which facilitates the application of this process for the treatment of residual methane in wastewater treatment plants and other waste management facilities.

Response of the Anaerobic Methanotrophic Archaeon Candidatus "Methanoperedens nitroreducens" to the Long-Term Ferrihydrite Amendment.

Anaerobic methanotrophic (ANME) archaea can drive anaerobic oxidation of methane (AOM) using solid iron or manganese oxides as the electron acceptors, hypothetically via direct extracellular electron transfer (EET). This study investigated the response of Candidatus “Methanoperedens nitroreducens TS” (type strain), an ANME archaeon previously characterized to perform nitrate-dependent AOM, to an Fe(III)-amended condition over a prolonged period. Simultaneous consumption of methane and production of dissolved Fe(II) were observed for more than 500 days in the presence of Ca. “M. nitroreducens TS,” indicating that this archaeon can carry out Fe(III)-dependent AOM for a long period. Ca. “M. nitroreducens TS” possesses multiple multiheme c-type cytochromes (MHCs), suggesting that it may have the capability to reduce Fe(III) via EET. Intriguingly, most of these MHCs are orthologous to those identified in Candidatus “Methanoperedens ferrireducens,” an Fe(III)-reducing ANME archaeon. In contrast, the population of Ca. “M. nitroreducens TS” declined and was eventually replaced by Ca. “M. ferrireducens,” implying niche differentiation between these two ANME archaea in the environment.

A Genome-Scale Metabolic Model of Methanoperedens nitroreducens: Assessing Bioenergetics and Thermodynamic Feasibility.

Methane is an abundant low-carbon fuel that provides a valuable energy resource, but it is also a potent greenhouse gas. Therefore, anaerobic oxidation of methane (AOM) is an essential process with central features in controlling the carbon cycle. Candidatus ‘Methanoperedens nitroreducens’ (M. nitroreducens) is a recently discovered methanotrophic archaeon capable of performing AOM via a reverse methanogenesis pathway utilizing nitrate as the terminal electron acceptor. Recently, reverse methanogenic pathways and energy metabolism among anaerobic methane-oxidizing archaea (ANME) have gained significant interest. However, the energetics and the mechanism for electron transport in nitrate-dependent AOM performed by M. nitroreducens is unclear. This paper presents a genome-scale metabolic model of M. nitroreducens, iMN22HE, which contains 813 reactions and 684 metabolites. The model describes its cellular metabolism and can quantitatively predict its growth phenotypes. The essentiality of the cytoplasmic heterodisulfide reductase HdrABC in the reverse methanogenesis pathway is examined by modeling the electron transfer direction and the specific energy-coupling mechanism. Furthermore, based on better understanding electron transport by modeling, a new energy transfer mechanism is suggested. The new mechanism involves reactions capable of driving the endergonic reactions in nitrate-dependent AOM, including the step reactions in reverse canonical methanogenesis and the novel electron-confurcating reaction HdrABC. The genome metabolic model not only provides an in silico tool for understanding the fundamental metabolism of ANME but also helps to better understand the reverse methanogenesis energetics and its thermodynamic feasibility.

Bio-reduced graphene oxide on hollow fibers as gas-diffusible anodes for enhancing bioelectrochemical methane oxidation

Bioelectrochemical methane oxidation provides opportunities for conversion of methane into electricity, fuels with higher energy intensity and value-added chemicals. Previous studies indicate that methane bioavailability due to low solubility of methane and sluggish extracellular electron transfer (EET) of methane-oxidizing consortium are two of main kinetic limitations for the performance of methane-fuelled bioelectrochemical systems. In this study, tubular gas-diffusible electrodes were synthesized through in situ bio-reduction of graphene oxidation (GO) on hollow fibers (HFs). A special methanotrophic consortium dominated by ‘Candidatus ‘Methanoperedens nitroreducens’ was found to be capable of reducing GO to reduced graphene oxide (rGO) with c-type cytochrome playing an essential role in its EET. We used this new-found feature for self-assembly of highly conductive rGO on HFs, thereby yielding methanotrophic biofilm/rGO matrix wrapped HFs as gas diffusible electrodes. The rGO-deposited HFs boosted the current output associated with the bioelectrochemical methane oxidation by 6 times, compared to an unamended control. This improvement can be ascribed to the development of an rGO network on the HFs, which converted commercial HFs to conductive electrodes and increased the contact area between microbes and electrodes by incorporating biomass in the three-dimensional microporous rGO scaffold. The strategy developed in this study can be extended to other bioelectrochemical systems suffering from the issue of low aqueous solubility.

Unraveling Nitrogen, Sulfur, and Carbon Metabolic Pathways and Microbial Community Transcriptional Responses to Substrate Deprivation and Toxicity Stresses in a Bioreactor Mimicking Anoxic Brackish Coastal Sediment Conditions

Microbial communities are key drivers of carbon, sulfur, and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation, and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Ca. Nitrobium versatile, identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The ANaerobic Methanotroph (ANME) Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). During the ammonium removal experiment, increased DNRA was unable to sustain anaerobic ammonium oxidation (anammox) activity. After ammonium was reintroduced, a novel anaerobic bacterial methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila, while the anammox Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra. At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductases. Our results indicate that some community members could be more resilient to the tested experimental conditions than others, and that some community functions such as methane and sulfur oxidation coupled to denitrification can remain stable despite large shifts in microbial community structure. Further studies on complex bioreactor enrichments are required to elucidate coastal ecosystem responses to future disturbances.

Microbe-mediated reduction of methane emission in rice-frog crop ecosystem

Rice–frog crop ecosystem (RFCE) can significantly reduce CH4 emission from paddy soils to mitigate global warming. However, information on the correlation of soil microbial communities with paddy soil CH4 emission in RFCEs remains limited. This study evaluated the influence of frogs on CH4 flux, paddy soil/water properties, and abundance/community composition of methanogens and methanotrophs in an RFCE between 2018 and 2019. The study parameters were compared between “with frogs” (RFCE; 15,000 frogs ha−1) and “without frogs” (control check [CK]) conditions. Frogs in paddies significantly decreased the CH4 flux during the rice-growing seasons (p < 0.05) but increased the paddy water/soil dissolved oxygen (DO) concentration, redox potential (Eh), and soil organic carbon (SOC) content. Compared with the CK, the RFCE significantly decreased the relative abundance of the soil methanogen species Candidatus Methanoperedens nitroreducens and Methanocella arvoryzae, genera Candidatus Methanoperedens and Methanocella, and families Methanoperedenaceae and Methanocellaceae (p < 0.05); however, it significantly increased the abundance of the soil methanotroph species Methylosinus sp. LW2, Methylocystis sp. KS3, and Methylocystis sp. KS30; genera Methylosinus and Methylocystis; and family Methylocystaceae (p < 0.05). Furthermore, variation partitioning analysis revealed that oxygen (DO and Eh) explained 41.2% of the variation in the abundance of soil methanogens, which was greater than the effects of C/N (38.2%) and SOC content (38.1%). For soil methanotrophs, oxygen (DO and Eh) explained 45.6% of the variation, which was more than the effects of C/N or pH. Oxygen concentration in the paddy soil/water was the driving factor for the change in the composition of soil methanogenic and methanotrophic microbial communities. Bioturbation due to the frogs increased oxygen availability in the paddy soil, decreased CH4 production by soil methanogens, and increased CH4 oxidation by type II methanotrophs, all of which reduced CH4 emission. Thus, this study provides strong evidence for the microbial mechanisms underlying the reduction of CH4 emission from RFCE.

Spatial and temporal variations of the community structure and abundance of Candidatus Methanoperedens nitroreducens-like archaea in paddy soils

Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea, catalyzing anaerobic methane oxidation coupled with nitrate reduction, could play an important role in reducing methane emissions from paddy ecosystems. Currently, the spatial and temporal variations of the communities of M. nitroreducens-like archaea and the factors regulating their distribution in paddy soils are poorly known. Here, we examined the diversity, community composition and abundance of these anaerobic methanotrophs at four representative depths (0–10, 10–20, 20–30 and 30–40 cm) of paddy soils across four rice growth stages (tillering, jointing, flowering and milky stages). High-throughput sequencing of M. nitroreducens-like archaeal mcrA genes showed that their community structure varied among different depths. In contrast, the community structure of these archaea remained relatively stable across rice growth stages. Further, both soil depth and rice growth stage had a significant impact on the abundance of mcrA genes. The gene abundance varied from 7.6 × 105 to 5.0 × 106 copies g−1 dry soil with higher values in 20–30 cm soils, and the abundance was found to be decreased from tillering to milky stages. The soil organic carbon content and ammonium content were the key factors affecting the community structure of these archaea. Our results indicated that both soil depth and crop growth stage greatly affected the abundance of M. nitroreducens-like archaea, while their community structure seemed to be more responsive to soil depth. Taken together, both space and time scales should be considered for better understanding the role of M. nitroreducens-like archaea reducing methane emissions from paddy ecosystems.

Microbial abundance and activity of nitrite/nitrate-dependent anaerobic methane oxidizers in estuarine and intertidal wetlands: Heterogeneity and driving factors

Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is a crucial link between carbon and nitrogen cycles in estuarine and coastal ecosystems. However, the factors that affect the heterogeneous variability in n-DAMO microbial abundance and activity across estuarine and intertidal wetlands remain unclear. This study examined the spatiotemporal variations in n-DAMO microbial abundance and associated activity in different estuarine and intertidal habitats via quantitative PCR and 13C stable isotope experiments. The results showed that Candidatus 'Methylomirabilis oxyfera' (M. oxyfera)-like DAMO bacteria and Candidatus 'Methanoperedens nitroreducens' (M. nitroreducens)-like DAMO archaea cooccurred in estuarine and intertidal wetlands, with a relatively higher abundance of the M. oxyfera-like bacterial pmoA gene (4.0 × 106-7.6 × 107 copies g−1 dry sediment) than the M. nitroreducens-like archaeal mcrA gene (4.5 × 105-9.4 × 107 copies g−1 dry sediment). The abundance of the M. oxyfera-like bacterial pmoA gene was closely associated with sediment pH and ammonium (P<0.05), while no significant relationship was detected between M. nitroreducens-like archaeal mcrA gene abundance and the measured environmental parameters (P>0.05). High n-DAMO microbial activity was observed, which varied between 0.2 and 84.3 nmol 13CO2 g−1 dry sediment day−1 for nitrite-DAMO bacteria and between 0.4 and 32.6 nmol 13CO2 g−1 dry sediment day−1 for nitrate-DAMO archaea. The total n-DAMO potential tended to be higher in the warm season and in the upstream freshwater and low-salinity estuarine habitats and was significantly related to sediment pH, total organic carbon, Fe(II), and Fe(III) contents (P<0.05). In addition to acting as an important methane (CH4) sink, n-DAMO microbes had the potential to consume a substantial amount of reactive N in estuarine and intertidal environments, with estimated nitrogen elimination rates of 0.5-224.7 nmol N g−1 dry sediment day−1. Overall, our investigation reveals the distribution pattern and controlling factors of n-DAMO bioprocesses in estuarine and intertidal marshes and gains a better understanding of the coupling mechanisms between carbon and nitrogen cycles.