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Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 µg 13C g-1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 µg 15N g-1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation - not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.

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Aerobic methane oxidation coupled with denitrification (AME-D) has garnered significant attention as a promising technology for nitrogen removal from water. Effective biofilm management on the membrane surface is essential to enhance the efficiency of nitrate removal in AME-D systems. In this study, we introduce a novel and scalable layer-structured membrane (LSM) developed using a meticulously designed polyurethane sponge. The application of the LSM in advanced biofilm management for AME-D resulted in a substantial enhancement of denitrification performance. Our experimental results demonstrated remarkable improvements in nitrate-removal flux (92.8 mmol-N m-2 d-1) and methane-oxidation rate (325.6 mmol m-2 d-1) when using an LSM in a membrane biofilm reactor (L-MBfR) compared with a conventional membrane reactor (C-MBfR). The l-MBfR exhibited 12.4-, 6.8- and 3.4-fold increases in nitrate-removal rate, biomass-retention capacity, and methane-oxidation rate, respectively, relative to the control C-MBfR. Notably, the l-MBfR demonstrated a 3.5-fold higher abundance of denitrifying bacteria, including Xanthomonadaceae, Rhodocyclaceae, and Methylophilaceae. In addition, the denitrification-related enzyme activity was twice as high in the l-MBfR than in the C-MBfR. These findings underscore the LSM's ability to create anoxic/anaerobic microenvironments conducive to biofilm formation and denitrification. Furthermore, the LSM exhibited a unique advantage in shaping microbial community structures and facilitating cross-feeding interactions between denitrifying bacteria and aerobic methanotrophs. The results of this study hold great promise for advancing the application of MBfRs in achieving efficient and reliable nitrate removal through the AME-D pathway, facilitated by effective biofilm management.

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Microbial oxidation of organic compounds can promote arsenic release by reducing soil-associated arsenate to the more mobile form arsenite. While anaerobic oxidation of methane has been demonstrated to reduce arsenate, it remains elusive whether and to what extent aerobic methane oxidation (aeMO) can contribute to reductive arsenic mobilization. To fill this knowledge gap, we performed incubations of both microbial laboratory cultures and soil samples from arsenic-contaminated agricultural fields in China. Incubations with laboratory cultures showed that aeMO could couple to arsenate reduction, wherein the former bioprocess was carried out by aerobic methanotrophs and the latter by a non-methanotrophic bacterium belonging to a novel and uncultivated representative of Burkholderiaceae. Metagenomic analyses combined with metabolite measurements suggested that formate served as the interspecies electron carrier linking aeMO to arsenate reduction. Such coupled bioprocesses also take place in the real world, supported by a similar stoichiometry and gene activity in the incubations with natural paddy soils, and contribute up to 76.2% of soil-arsenic mobilization into pore waters in the top layer of the soils where oxygen was present. Overall, this study reveals a previously overlooked yet significant contribution of aeMO to reductive arsenic mobilization.

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Aerobic methane oxidation (MOx) depends critically on the availability of copper (Cu) as a crucial component of the metal centre of particulate methane monooxygenase, one of the main enzymes involved in MOx. Some methanotrophs have developed Cu acquisition strategies, in which they exude Cu-binding ligands termed chalkophores under conditions of low Cu availability. A well-characterised chalkophore is methanobactin (mb), exuded by the microaerophilic methanotroph Methylosinus trichosporium OB3b. Aerobic methanotrophs generally reside close to environmental oxic-anoxic interfaces, where the formation of Cu sulphide phases can aggravate the limitation of bioavailable Cu due to their low solubility. The reactivity of chalkophores towards such Cu sulphide mineral phases has not yet been investigated. In this study, a combination of dissolution experiments and equilibrium modelling was used to examine the dissolution and solubility of bulk and nanoparticulate Cu sulphide minerals in the presence of mb as influenced by pH, oxygen and natural organic matter. In general, we show that mb is effective at increasing the dissolved Cu concentrations in the presence of a variety of Cu sulphide phases that may potentially limit Cu bioavailability. More Cu was mobilised per mole of mb from Cu sulphide nanoparticles compared with well-crystalline bulk covellite (CuS). In general, the efficacy of mb at mobilising Cu from Cu sulphides is pH-dependent. At lower pH, e.g. pH 5, mb was ineffective at solubilizing Cu. The presence of mb increased dissolved Cu concentrations between pH 7 and 8.5, where the solubility of all Cu sulphides is generally low, both in the presence and absence of oxygen. These results suggest that chalkophore-promoted Cu mobilisation from sulphide phases is an effective extracellular mechanism for increasing dissolved Cu concentrations at oxic-anoxic interfaces, particularly in the neutral to slightly alkaline pH range. This suggests that aerobic methanotrophs may be able to fulfil their Cu requirements via the exudation of mb in natural environments where the bioavailability of Cu is constrained by very stable Cu sulphide phases.

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Membrane biofilm (MBf) technology is a promising biological water treatment process that combines membrane aeration with biofilms. To expand its application in the treatment of toxic organic wastewater, methane/air gas mixture-MBfs ((CH4 + Air)-MBfs) and air-MBfs were coupled to enhance the treatment of p-nitroaniline (PNA) wastewater. Based on exploration of the membrane permeability of methane and oxygen, a hybrid MBf reactor was constructed, and the degradation characteristics of PNA and the coupling effects of (CH4 + Air)-MBfs and air-MBfs were studied. The permeation flux of methane was found to be 1.114 g/(m2 d) when using a methane/air gas mixture at an aeration pressure of 10 kPa, and this result was better than that when methane was used as the aeration gas alone. Aeration with a methane/air gas mixture provided conditions for realizing aerobic methane oxidation; the aerobic methane oxidation that occurred in the (CH4 + Air)-MBfs promoted the reduction of PNA, and the intermediates of PNA degradation were further degraded by the air-MBfs. At an influent PNA membrane area load of 1.67 g/(m2 d), the PNA removal load reached 187.30 g/(m3 d). The coupling of MBfs took advantage of different matrix-based MBfs and promoted the degradation of PNA by utilizing the synergistic effects of various functional microorganisms.

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Anaerobic technologies are consolidated for sewage treatment and are the core processes for mining marketable products from waste streams. However, anaerobic effluents are supersaturated with methane, which represents a liability regarding greenhouse gas emissions. Meanwhile, anaerobic technologies are not capable of nitrogen removal, which is required to ensure environmental protection. Methane oxidation and denitrification processes can be combined to address both issues concurrently. Aerobic methane oxidizers can release intermediate organic compounds that can be used by conventional denitrifiers as electron donors. Alternatively, anoxic methanotrophic species combine methane oxidation with either nitrate or nitrite reduction in the same metabolism. Engineered systems need to overcome the long doubling times and low NOx consumption rates of anoxic methanotrophic microorganisms. Another commonly reported bottleneck of methanotrophic denitrification relates to gas-liquid mass transfer limitations. Although anaerobic effluents are supersaturated with methane, experimental setups usually rely on methane supply in a gaseous mode. Hence, possibilities for the application of methane-oxidation coupled to denitrification in full scale might be overlooked. Moreover, syntrophic relationships among methane oxidizers, denitrifiers, nitrifiers, and other microorganisms (such as anammox) are not well understood. Integrating mixed populations with various metabolic abilities could allow for more robust methane-driven wastewater denitrification systems. This review presents an overview of the metabolic capabilities of methane oxidation and denitrification and discusses technological aspects that allow for the application of methanotrophic denitrification at larger scales.

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AIMS: Aerobic methane oxidation coupled to denitrification (AME-D) is a promising process for removing nitrate from groundwater and yet its microbial mechanism and ecological implications are not fully understood. This study used RNA stable isotope probing (RNA-SIP) and high-throughput sequencing to identify the micro-organisms that are actively involved in aerobic methane oxidation within a denitrifying biofilm. METHODS AND RESULTS: Two RNA-SIP experiments were conducted to investigate labelling of RNA and methane monooxygenase (pmoA) transcripts when exposed to 13 C-labelled methane over a 96-hour time period and to determine active bacteria involved in methane oxidation in a denitrifying biofilm. A third experiment was performed to ascertain the extent of 13 C labelling of RNA using isotope ratio mass spectrometry (IRMS). All experiments used biofilm from an established packed bed reactor. IRMS confirmed 13 C enrichment of the RNA. The RNA-SIP experiments confirmed selective enrichment by the shift of pmoA transcripts into heavier fractions over time. Finally, high-throughput sequencing identified the active micro-organisms enriched with 13 C. CONCLUSIONS: Methanotrophs (Methylovulum spp. and Methylocystis spp.), methylotrophs (Methylotenera spp.) and denitrifiers (Hyphomicrobium spp.) were actively involved in AME-D. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use RNA-SIP and high-throughput sequencing to determine the bacteria active within an AME-D community.

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Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

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Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu+. Other metal ions, however, are bound but not reduced, e.g., K+. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K+ or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.

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Being an energetic fuel, methane is able to support microbial growth and drive the reduction of various electron acceptors. These acceptors include a broad range of oxidized contaminants (e.g., nitrate, nitrite, perchlorate, bromate, selenate, chromate, antimonate and vanadate) that are ubiquitously detected in water environments and pose threats to human and ecological health. Using methane as electron donor to biologically reduce these contaminants into nontoxic forms is a promising solution to remediate polluted water, considering that methane is a widely available and inexpensive electron donor. The understanding of methane-based biological reduction processes and the responsible microorganisms has grown in the past decade. This review summarizes the fundamentals of metabolic pathways and microorganisms mediating microbial methane oxidation. Experimental demonstrations of methane as an electron donor to remove oxidized contaminants are summarized, compared, and evaluated. Finally, the review identifies opportunities and unsolved questions that deserve future explorations for broadening understanding of methane oxidation and promoting its practical applications.

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Endevours on the enhancement of nitrate removal efficiency during methane oxidation coupled with denitrification (AME-D) has always overlooked the role of membrane employed. It would be highly beneficial to enrich the biomass content and to manage biofilm on the membrane, in the utilization of methane and denitrification. In this study, an innovative and scalable double-layer membrane (DLM) was designed and prepared for a membrane biofilm reactor (MBfR), to simultaneously enhance nitrate removal flux and methane utilization efficiency during aerobic methane oxidation coupled with the denitrification (AME-D) process. The DLM allowed quick bacterial attachment and biomass accumulation for biofilm growth, which would be then self-regulated for well distribution of functional microbes on/within the DLM. Upon a high biofilm density of over 70 g-VSS m-2 achieved on the DLM, the methane utilization efficiency of the MBfR was enhanced significantly to over 1.3 times than the control MBfR with conventional polypropylene membrane. The MBfR employed DLM also demonstrated the maximum nitrate removal flux of 740 mg-NO3--N m-2 d-1 that was approximately 1.64 times of that in control MBfR at continuous-mode operation. This DLM indeed favored the enrichment of Type II aerobic methanotrophs of Methylocystaceae, and methanol-utilization denitrifiers of Rhodocyclaceae that preferentially utilize methanol as the cross-feeding intermediates to promote the methane utilization efficiency, and thus to enhance the nitrate removal flux. These results raised from new designed DLM confirmed the importance of membrane surface properties on the effectiveness of MBfR, and offered great potential to address challenging problems of MBfRs during engineering application.

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Mangrove forests are one of the important ecosystems in tropical coasts because of their high primary production, which they sustain by sequestering a substantial amount of CO2 into plant biomass. These forests often experience various levels of inundation and play an important role in CH4 emissions, but the taxonomy of methanotrophs in these systems remains poorly understood. In this study, DNA-based stable isotope probing showed significant niche differentiation in active aerobic methanotrophs in response to niche differentiation in upstream and downstream mangrove soils of the Tamsui estuary in northwestern Taiwan, in which salinity levels differ between winter and summer. Methylobacter and Methylomicrobium-like Type I methanotrophs dominated methane-oxidizing communities in the field conditions and were significantly 13C-labeled in both upstream and downstream sites, while Methylobacter were well adapted to high salinity and low temperature. The Type II methanotroph Methylocystis comprised only 10-15% of all the methane oxidizers in the upstream site but less than 5% at the downstream site under field conditions. 13C-DNA levels in Methylocystis were significantly lower than those in Type I methanotrophs, while phylogenetic analysis further revealed the presence of novel methane oxidizers that are phylogenetically distantly related to Type Ia in fresh and incubated soils at a downstream site. These results suggest that Type I methanotrophs display niche differentiation associated with environmental differences between upstream and downstream mangrove soils.

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Nitrate removal efficiency of aerobic methane oxidation coupled with denitrification (AME-D) process was elevated by enhancing the methanol-linked synergy in a membrane biofilm reactor (MBfR) under a low O2:CH4 ratio. After 140 days' enrichment, the nitrate removal rate increased significantly from 3 to 4 mg-N L-1 d-1 to 22.09 ± 1.21 mg-N L-1 d-1 and the indicator, mol CH4 consumed/mol reduced NO3--N (C/N ratio), decreased to 1.79 which was very close to the theoretical minimum value (1.27-1.39). The increased nitrate removal efficiency was largely related to the enhanced relationship between aerobic methanotrophs and methanol-utilizing denitrifiers. Type I methanotrophs and some denitrifiers, especially those potential methanol-utilizing denitrifiers from Methylobacillus, Methylotenera, Methylophilus and Methyloversatilis, were abundant in the MBfR sludge. Aerobic methanotrophs and potential methanol-utilizing denitrifiers were closely associated in many globular aggregates (5-10 µm diameter) in the MBfR sludge, which may have promoted the denitrifiers to capture methanol released by methanotrophs efficiently. If we assume methanol is the only cross-feeding intermediate in the MBfR, about 38-60% of the CH4 supplied would be converted to methanol and secreted rather than continuing to be oxidized. At least 63% of this secreted methanol should be utilized for denitrification instead of being oxidized by oxygen in the MBfR. These findings suggest that the nitrate removal efficiency of the AME-D process could be significantly improved.

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Recent investigations demonstrate that some coastal wetlands are atmospheric methane sinks, but the regulatory mechanisms are not clear. Here, the main pathway and operator of methane oxidation in the Yellow River Delta (YRD) wetland, a methane source in the wet season but a methane sink in the dry season, were investigated. The anaerobic oxidation of methane (AOM) and aerobic methane oxidation (AMO) abilities of wetland soil were measured, and the microbial community structure was analyzed. The experimental results showed that AMO was active throughout the year. In contrast, AOM was weak and even undetected. The microbial community analysis indicated that Methylomicrobium and Methylobacter potentially scavenged methane in oxic environments. A representative strain of Methylobacter, which was isolated from the soil, presented a strong AMO ability at high concentrations of methane and air. Overall, this study showed that active AMO performing by Methylobacter may account for methane sink in the YRD wetland during the dry season. Our research not only has determined the way in which methane sinks are formed but also identified the potential functional microbes. In particular, we confirmed the function of potential methanotroph by pure culture. Our research provides biological evidence for why some wetlands have methane sink characteristics, which may help to understand the global methane change mechanism.

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Chromate contamination can pose a high risk to both the environment and public health. Previous studies have shown that CH4-based membrane biofilm reactor (MBfR) is a promising method for chromate removal. In this study, we developed a multispecies biofilm model to study chromate reduction and its interaction with nitrate reduction in a CH4-based MBfR. The model-simulated results were consistent with the experimental data reported in the literature. The model showed that the presence of nitrate in the influent promoted the growth of heterotrophs, while suppressing methanotrophs and chromate reducers. Moreover, it indicated that a biofilm thickness of 150 µm and an influent dissolved oxygen concentration of 0.5 mg O2/L could improve the reactor performance by increasing the chromate removal efficiency under the simulated conditions.

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Microbial aerobic methane oxidation (MAMO) has been considered as an environmental-friendly method for mitigating methane emission from municipal landfill sites. Soil column has in a landfill cover under one-dimensional (1-D) condition. However, most of the published soil column tests failed to simulate 1-D heat transfer due to the use of thermal conductive boundary at the sidewall. In the present study, a heavily instrumented soil column was developed to quantify the effects of thermal boundary condition on the methane oxidation efficiency under different ambient temperatures in landfill cover soil. The sidewall of the soil column was thermally insulated to ensure 1-D heat transport as would have been typically expected in the field condition. Two soil column tests with and without thermal insulation were conducted at a range of controlled ambient temperatures from 15 to 30°C, for studying how soil moisture, matric suction, gas pressure, soil temperature and gas concentration evolve with MAMO. The test results reveal that ignoring thermal insulation in a soil column test would result in a greater loss of soil heat generation by MAMO and hence oxidation efficiency by up to 100% for the range of temperature considered. When the ambient temperature increased to 30°C (but less than the optimum temperature for MAMO), the MAMO efficiency increased abruptly at first but then decreased substantially with time, and this is likely due to the accumulation of biomass generated by MAMO.

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Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named "Methylotetracoccus oryzae" C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems.IMPORTANCE Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions.

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Methane oxidation coupled to selenate reduction has been suggested as a promising technology to bio-remediate selenium contaminated environments. However, the effect of dissolved oxygen (DO) on this process remained unclear. Here, we investigate the feasibility of selenate removal at two distinct DO concentrations. A membrane biofilm reactor (MBfR) was initially fed with ∼5 mg Se/L and then lowered to ∼1 mg Se/L of selenate, under anoxic condition containing ∼0.2 mg/L of influent DO. Selenate removal reached approximately 90% without selenite accumulation after one-month operation. Then 6-7 mg/L of DO was introduced and showed no apparent effect on selenate reduction in the subsequent operation. Electron microscopy suggested elevated oxygen exposure did not affect microbial shapes. 16S rDNA sequencing showed the aerobic methanotroph Methylocystis increased, while possible selenate reducers, Ignavibacterium and Bradyrhizobium, maintained stable after oxygen boost. Gene analysis indicated that nitrate/nitrite reductases positively correlated with selenate removal flux and were not remarkably affected by oxygen addition. Reversely, enzymes related with aerobic methane oxidation were obviously improved. This study provides a potential technology for selenate removal from oxygenated environments in a methane-based MBfR.

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The discovery of denitrifying anaerobic methane oxidation (DAMO) has not only improved our understanding of global methane and nitrogen cycles, but also provided new technology options for removal of nitrate from nitrate-contaminated water. Previous studies have demonstrated DAMO organisms could remove nitrate and nitrite from wastewater under strictly anaerobically conditions. In the study, we investigate the feasibility of nitrate removal from groundwater, which contains dissolved oxygen in addition to nitrate. A membrane biofilm reactor (MBfR), inoculated with DAMO co-culture, was capable of treating synthetic groundwater containing highly contaminated nitrate (50  mg N/L) and oxygen (7-9 mg O2/L), with a maximum volumetric nitrate removal rate of 45 mg N/L-d. Accumulations of acetate and propionate were observed in some transient periods, indicating the possible involvement of acetate and propionate as intermediates in methane oxidation. The 16 S rRNA gene amplicon sequencing revealed that Candidatus Methylomirabilis, a known bacterial DAMO organism able to couple nitrite reduction with anaerobic oxidation of methane (AOM), was the dominant population. No archaeal DAMO organisms that are capable of coupling nitrate to AOM were observed, however, considerable amount of denitrifiers were developed in this system. Based on known metabolisms of these microorganisms and a series of batch studies, it was assumed that methane was oxidized into volatile fatty acids (VFAs) under oxygen-limiting conditions, then the generated VFAs served as carbon sources for these heterotrophic denitrifiers to remove nitrate. This study offers a potential technology for nitrate removal from groundwater by DAMO process in MBfR.

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The O2:CH4 ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O2:CH4 ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O2:CH4 ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems.