Nitrate-dependent Anaerobic Methane Oxidation Research Articles

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.

How to Provide Nitrite Robustly for Anaerobic Ammonium Oxidation in Mainstream Nitrogen Removal.

Innovation in decarbonizing wastewater treatment is urgent in response to global climate change. The practical implementation of anaerobic ammonium oxidation (anammox) treating domestic wastewater is the key to reconciling carbon-neutral management of wastewater treatment with sustainable development. Nitrite availability is the prerequisite of the anammox reaction, but how to achieve robust nitrite supply and accumulation for mainstream systems remains elusive. This work presents a state-of-the-art review on the recent advances in nitrite supply for mainstream anammox, paying special attention to available pathways (forward-going (from ammonium to nitrite) and backward-going (from nitrate to nitrite)), key controlling strategies, and physiological and ecological characteristics of functional microorganisms involved in nitrite supply. First, we comprehensively assessed the mainstream nitrite-oxidizing bacteria control methods, outlining that these technologies are transitioning to technologies possessing multiple selective pressures (such as intermittent aeration and membrane-aerated biological reactor), integrating side stream treatment (such as free ammonia/free nitrous acid suppression in recirculated sludge treatment), and maintaining high activity of ammonia-oxidizing bacteria and anammox bacteria for competing oxygen and nitrite with nitrite-oxidizing bacteria. We then highlight emerging strategies of nitrite supply, including the nitrite production driven by novel ammonia-oxidizing microbes (ammonia-oxidizing archaea and complete ammonia oxidation bacteria) and nitrate reduction pathways (partial denitrification and nitrate-dependent anaerobic methane oxidation). The resources requirement of different mainstream nitrite supply pathways is analyzed, and a hybrid nitrite supply pathway by combining partial nitrification and nitrate reduction is encouraged. Moreover, data-driven modeling of a mainstream nitrite supply process as well as proactive microbiome management is proposed in the hope of achieving mainstream nitrite supply in practical application. Finally, the existing challenges and further perspectives are highlighted, i.e., investigation of nitrite-supplying bacteria, the scaling-up of hybrid nitrite supply technologies from laboratory to practical implementation under real conditions, and the data-driven management for the stable performance of mainstream nitrite supply. The fundamental insights in this review aim to inspire and advance our understanding about how to provide nitrite robustly for mainstream anammox and shed light on important obstacles warranting further settlement.

Effects of magnetite on nitrate-dependent anaerobic oxidation of methane in an anaerobic membrane biofilm reactor: Metatranscriptomic analysis and mechanism prediction

Magnetite has been widely reported to accelerate methane production in anaerobic digestion. However, the effects of magnetite on the process of methane consumption, specifically nitrate-dependent anaerobic oxidation of methane (AOM), and its underlying mechanisms remain poorly understood. In this study, the addition of magnetite significantly improved the removal of nitrate in an anaerobic membrane biofilm reactor (AnMBfR) (25.2 ± 2.5 mg NO3−-N/L/d) compared to that in an AnMBfR without magnetite (8.7 ± 4.2 mg NO3−-N/L/d). Metatranscriptomic analysis showed that genes encoding type IV pilus assembly proteins were highly expressed in nitrate/nitrite-reducing bacteria in the biofilms of the two AnMBfRs. Specifically, the transcript abundance of these genes was observed to be higher in the presence of magnetite (gene expression levels, log2FPKM = 13.08) compared to the absence of magnetite (12.04). Biofilms with magnetite exhibited a linear increase in electron transfer resistance with increasing temperature, and the decrease in pH significantly increased their conductivity, indicating a metallic-like conductivity. Contrarily, an increase in temperature or decrease in pH did not influence the resistance or conductivity of biofilms without magnetite. Some of these cytochrome c (CYC)-like proteins were electrically active in the electrochemical Fourier transform infrared spectra and were closely associated with the outer-membrane multi-heme c-type cytochromes (OMCs). The intensities of the OMC-like bands in the surface-enhanced resonance Raman spectra with biofilms in the presence of magnetite were higher than those without magnetite. This study suggests that magnetite promotes the nitrate-dependent AOM process because of the formation of methanotrophic consortia based on direct interspecies electron transfer (DIET).

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.

Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings

The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea.

Nitrate leaching and its implication for Fe and As mobility in a Southeast Asian aquifer.

The drinking water quality in Southeast Asia is at risk due to arsenic (As) groundwater contamination. Intensive use of fertilizers may lead to nitrate (NO3-) leaching into aquifers, yet very little is known about its effect on iron (Fe) and As mobility in water. We ran a set of microcosm experiments using aquifer sediment from Vietnam supplemented with 15NO3- and 13CH4. To assess the effect of nitrate-dependent anaerobic methane oxidation (N-DAMO) we also inoculated the sediment with two different N-DAMO enrichment cultures. We found that native microorganisms and both N-DAMO enrichments could efficiently consume all NO3- in 5 days. However, CH4 oxidation was observed only in the inoculated microcosms, suggesting that the native microbial community did not perform N-DAMO. In uninoculated microcosms, NO3- was preferentially used over Fe(III) as an electron acceptor and consequently inhibited Fe(III) reduction and As mobilization. The addition of N-DAMO enrichment cultures led to Fe(III) reduction and stimulated As and Mn release into the water. The archaeal community in all treatments was dominated by Ca. Methanoperedens while the bacterial community consisted of various denitrifiers. Our results suggest that input of N fertilizers to the aquifer decreases As mobility and that CH4 cannot serve as an electron donor for NO3- reduction.

Nitrate-dependent anaerobic methane oxidation (N-DAMO) as a bioremediation strategy for waters affected by agricultural runoff.

Agricultural drainage ditches are subjected to high anthropogenic nitrogen input leading to eutrophication and greenhouse gas emissions. Nitrate-dependent anaerobic methane oxidation (N-DAMO) could be a promising remediation strategy to remove methane (CH4) and nitrate (NO3-) simultaneously. Therefore, we aimed to evaluate the potential of N-DAMO to remove excess NO3- and decrease CH4 release from agricultural drainage ditches. Microcosm experiments were conducted using sediment and surface water collected from three different sites: a sandy-clay ditch (SCD), a freshwater-fed peatland ditch (FPD), and a brackish peatland ditch (BPD). The microcosms were inoculated with an N-DAMO enrichment culture dominated by Candidatus Methanoperedens and Candidatus Methylomirabilis and supplemented with 13CH4 and 15NO3-. A significant decrease in CH4 and NO3- concentration was only observed in the BPD sediment. In freshwater sediments (FPD and SCD) the effect of N-DAMO inoculation on CH4 and NO3- removal was negligible, likely because N-DAMO microorganisms were outcompeted by heterotrophic denitrifiers consuming NO3- much faster. Overall, our results suggest that bioaugmentation with N-DAMO might be a potential strategy for decreasing NO3- concentrations and CH4 emission in brackish ecosystems with increasing agricultural activities where the native microbial community is incapable of efficient denitrification.

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.

Response of denitrifying anaerobic methane oxidation enrichment to salinity stress: Process and microbiology

Denitrifying anaerobic methane oxidation (DAMO) is a novel biological process which could decrease nitrogen pollution and methane emission simultaneously in wastewater treatment. Salinity as a key environmental factor has important effects on microbial community and activity, however, it remains unclear for DAMO microorganisms. In this study, response of the enrichment of DAMO archaea and bacteria to different salinity was investigated from the aspect of process and microbiology. The results showed that the increasing salinity from 0.14% to 25% evidently deteriorated DAMO process, with the average removal rate of nitrate and methane decreased from 1.91 mg N/(L·d) to 0.07 mg N/(L·d) and 3.22 μmol/d to 0.59 μmol/d, respectively. The observed IC50 value of salinity on the DAMO culture was 1.73%. Further microbial analyses at the gene level suggested that the relative abundance of DAMO archaea in the enrichment decreased to 46%, 39%, 38% and 33% of the initial value. However, DAMO bacteria suffered less impact with the relative abundance maintaining over 75% of the initial value (except 1% salinity). In functional genes of DAMO bacteria, pmoA, decreased gradually from 100% to 86%, 43%, 15% and 2%, while mcrA (DAMO archaea) maintained at 67%–97%. This difference probably indicated DAMO bacteria appeared functional inhibition prior to community inhibition, which was opposite for the DAMO archaea. Results above-mentioned concluded that, though the process of nitrate-dependent anaerobic methane oxidation was driven by the couple of DAMO archaea and bacteria, they individually featured different response to high salinity stress. These findings could be helpful for the application of DAMO-based process in high salinity wastewater treatment, and also the understanding to DAMO microorganisms.

Nitrate-dependent anaerobic methane oxidation and chemolithotrophic denitrification in a temperate eutrophic lake.

While the emissions of methane (CH4) by natural systems have been widely investigated, CH4 aquatic sinks are still poorly constrained. Here, we investigated the CH4 cycle and its interactions with nitrogen (N), iron (Fe) and manganese (Mn) cycles in the oxic-anoxic interface and deep anoxic waters of a small, meromictic and eutrophic lake, during two summertime sampling campaigns. Anaerobic CH4 oxidation (AOM) was measured from the temporal decrease of CH4 concentrations, with the addition of three potential electron acceptors (NO3-, iron oxides (Fe(OH)3) and manganese oxides (MnO2)). Experiments with the addition of either 15N-labeled nitrate (15N-NO3-) or 15N-NO3- combined with sulfide (H2S), to measure denitrification, chemolithotrophic denitrification and anaerobic ammonium oxidation (anammox) rates, were also performed. Measurements showed AOM rates up to 3.8µmol CH4 L-1 d-1 that strongly increased with the addition of NO3- and moderately increased with the addition of Fe(OH)3. No stimulation was observed with MnO2 added. Potential denitrification and anammox rates up to 63 and 0.27µmol N2 L-1 d-1, respectively, were measured when only 15N-NO3- was added. When H2S was added, both denitrification and anammox rates increased. Altogether, these results suggest that prokaryote communities in the redoxcline are able to efficiently use the most available substrates.

The anaerobic oxidation of methane in paddy soil by ferric iron and nitrate, and the microbial communities involved

The anaerobic oxidation of methane (AOM) mediated by microorganisms is a key process in the reduction of methane emissions, and AOM-coupled electron acceptors have been shown to regulate methane emissions into the atmosphere in marine systems. Paddy fields are a significant source of methane and account for 20% of global methane emissions, but the effect of electron acceptors on the methane emission process in flooded paddy fields has been poorly characterized. This study aimed to determine whether the electron acceptors ferric iron and nitrate, and biochar, acting as an electron shuttle, can regulate the AOM process in paddy soil, with or without interaction between biochar and these two electron acceptors. We also aimed to characterize which microorganisms are actively involved. Here, we added 13C-labeled CH4 (13CH4) into anaerobic microcosms to evaluate the role of electron acceptors by measuring the methane oxidation rate and the enrichment of 13C-labeled CO2 (13CO2). We then combined DNA-stable isotope probing with amplicon sequencing to study the active microorganisms. We found for the first time that, in addition to nitrate, ferric iron can also effectively promote AOM in paddy soil. However, there was no significant effect of biochar. Ferric iron-dependent AOM was mainly carried out by iron-reducing bacteria (Geobacter, Ammoniphilus and Clostridium), and nitrate-dependent AOM was mainly by nitrate-reducing bacteria (Rhodanobacter, Paenibacillus and Planococcus). Our results demonstrate that the AOM process, regulated by the electron acceptors ferric iron and nitrate, can alleviate methane emission from paddy soil. The potentially active microorganisms related to electron acceptor reduction may be crucial for this methane sink and deserve further research.

Simultaneous nitrate and sulfate dependent anaerobic oxidation of methane linking carbon, nitrogen and sulfur cycles

ANaerobic MEthanotrophic (ANME) archaea are critical microorganisms mitigating methane emission from anoxic zones. In previous studies, sulfate-dependent anaerobic oxidation of methane (AOM) and nitrate-dependent AOM, performed by different clades of ANME archaea, were detected in marine sediments and freshwater environments, respectively. This study shows that simultaneous sulfate- and nitrate-dependent AOM can be mediated by a clade of ANME archaea, which may occur in estuaries and coastal zones, at the interface of marine and freshwater environments enriched with sulfate and nitrate. Long-term (~1,200 days) performance data of a bioreactor, metagenomic analysis and batch experiments demonstrated that ANME-2d not only conducted AOM coupled to reduction of nitrate to nitrite, but also coupled to the conversion of sulfate to sulfide, in collaboration with sulfate-reducing bacteria (SRB). Sulfide was oxidized back to sulfate by sulfide-oxidizing autotrophic denitrifiers with nitrate or nitrite as electron acceptors, in turn alleviating sulfide accumulation. In addition, dissimilatory nitrate reduction to ammonium performed by ANME-2d was detected, providing substrates to Anammox. Metatranscriptomic analysis revealed significant upregulation of flaB in ANME-2d and pilA in Desulfococcus, which likely resulted in the formation of unique nanonets connecting cells and expanding within the biofilm, and putatively providing structural links between ANME-2d and SRB for electron transfer. Simultaneous nitrate- and sulfate-dependent AOM as observed in this study could be an important link between the carbon, nitrogen and sulfur cycles in natural environments, such as nearshore environments.

A novel identified Pseudomonas aeruginosa, which exhibited nitrate- and nitrite-dependent methane oxidation abilities, could alleviate the disadvantages caused by nitrate supplementation in rumen fluid fermentation.

After the occurrence of nitrate-dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO3 or NaNO2 as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO2 enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further invitro rumen fermentation showed that supplementation with the isolated P.aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.

Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes

Abstract. Nitrate pollution of freshwaters and methane emissions into the atmosphere are crucial factors in deteriorating the quality of drinking water and in contributing to global climate change. The n-damo (nitrite-dependent anaerobic methane oxidation), nitrate-dependent anaerobic methane oxidation and the anaerobic oxidation of ammonium (anammox) represent two microbially mediated processes that can reduce nitrogen loading of aquatic ecosystems and associated methane emissions to the atmosphere. Here, we report vertical concentration and stable-isotope profiles of CH4, NO3-, NO2-, and NH4+ in the water column of Fohnsee (lake in southern Bavaria, Germany) that may indicate linkages between denitrification, anaerobic oxidation of methane (AOM), and anammox. At a water depth from 12 to 20 m, a methane–nitrate transition zone (NMTZ) was observed, where δ13C values of methane and δ15N and δ18O of dissolved nitrate markedly increased in concert with decreasing concentrations of methane and nitrate. These data patterns, together with the results of a simple 1-D diffusion model linked with a degradation term, show that the nonlinear methane concentration profile cannot be explained by diffusion and that microbial oxidation of methane coupled with denitrification under anaerobic conditions is the most parsimonious explanation for these data trends. In the methane zone at the bottom of the NMTZ (20 to 22 m) δ15N of ammonium increased by 4 ‰, while ammonium concentrations decreased. In addition, a strong 15N enrichment of dissolved nitrate was observed at a water depth of 20 m, suggesting that anammox is occurring together with denitrification. The conversion of nitrite to N2 and nitrate during anammox is associated with an inverse N isotope fractionation and may explain the observed increasing offset (Δδ15N) of 26 ‰ between δ15N values of dissolved nitrate and nitrite at a water depth of 20 m compared to the Δδ15Nnitrate-nitrite of 11 ‰ obtained in the NMTZ at a water depth between 16 and 18 m. The associated methane concentration and stable-isotope profiles indicate that some of the denitrification may be coupled to AOM, an observation supported by an increased concentration of bacteria known to be involved in n-damo/denitrification with AOM (NC10 and Crenothrix) and anammox (“Candidatus Anammoximicrobium”) whose concentrations were highest in the methane and ammonium oxidation zones, respectively. This study shows the potential for a coupling of microbially mediated nitrate-dependent methane oxidation with anammox in stratified freshwater ecosystems, which may be important for affecting both methane emissions and nitrogen concentrations in lakes.

Biogas-driven complete nitrogen removal from wastewater generated in side-stream partial nitritation

Anaerobic digestion is an attractive process in wastewater treatment plants (WWTPs) to achieve simultaneous sludge reduction and energy recovery. While converting the majority of organic carbon to biogas (mainly consisting 60%CH4 + 40%CO2), the high-strength anaerobic digestion liquor consists of a high level of nitrogen concentration. The feasibility of utilizing biogas produced in-situ to achieve satisfactory nitrogen removal performance from partially nitrified anaerobic digestion liquor was examined in this study. To this end, a membrane biofilm reactor (MBfR) was used to couple nitrite- or nitrate-dependent anaerobic methane oxidation (n-DAMO) and anammox microorganisms, which was supplied with synthetic biogas and partially nitrified anaerobic digestion liquor (470 mg NH4+-N/L + 560 mg NO2−-N/L). The MBfR achieved not only nearly complete nitrogen removal (~99%), but also a practically useful nitrogen removal rate above 1 kg N/m3/d. Due to the acidification caused by excessive CO2 supply from biogas, pH dropping was observed. Two corresponding strategies, i.e., intermittent alkali dosing and intermittent nitrogen gas flushing, were developed to control the pH at neutral. Mass balance based on batch tests and microbial community analysis by 16S rRNA gene amplicon sequencing both showed the joint contribution of anammox bacteria and anaerobic methane oxidizers to the nitrogen removal. This study proved the potential and capacity of MBfR to access complete nitrogen removal from high-strength wastewater by using biogas produced in-situ, thus leading to a significant reduction of external carbon addition in practice.

Granular activated carbon assisted nitrate-dependent anaerobic methane oxidation-membrane bioreactor: Strengthening effect and mechanisms

Eutrophication and global warming are two main urgent environmental problems around the world. Nitrate-dependent Anaerobic Methane Oxidation (NdAMO) is a bioprocess coupling nitrate reduction with anaerobic methane oxidation, which could mitigate of these two environmental issues simultaneously. In this study, a newly granular active carbon-NdAMO-membrane bioreactor (GAC-NdAMO-MBR) system was established to evaluate its nitrogen removal efficiency, membrane fouling property and the probable strengthening mechanism was also uncovered. Results indicated that the nitrate removal rate in GAC-NdAMO-MBR reached 31.85 ± 3.19 mgN·L−1·d−1 while it was only 10.35 ± 2.02 mgN·L−1·d−1 in NdAMO-MBR system (lack of GAC), which was multiplied three-fold. The membrane flux decay rate of GAC- NdAMO -MBR was 0.15 L/m2·h·d while it was 0.49 L/m2·h·d without GAC, and the addition of GAC could extend membrane fouling time for 2.5 times. Notablely, the relative abundance of NdAMO bacteria sharply increased from 27.15% to 56.91% after GAC addition while the NdAMO archaea showed similar variation trend. The physicochemical property of GAC mainly contributed the strengthening effect. The porous structure of GAC absorbed methane and adhered by microorganism, which enhance microorganism amount and metabolic activity. The mechanical strength of GAC scoured membrane surface to mitigate external fouling and pores absorbed EPS to reduce internal fouling. The combined effects could improve NdAMO microorganism growth and metabolism activity and finally improved nitrogen removal performance and controlled membrane fouling. These findings could deep the knowledge of NdAMO process and help extend its application potential in environment science and engineering.

Long-term performance of denitrifying anaerobic methane oxidation under stepwise cooling and ambient temperature conditions

Nitrate-dependent anaerobic methane oxidation (N-DAMO), a bioprocess that couples the oxidation of green gas and the removal of nitrogen oxides in a microbial group, has gained much attention as a potential economical method of biological removal of nitrates and methane from wastewater. Low-temperature (20 °C) operation of N-DAMO would be beneficial to utilize the methane dissolved in the effluent and thus decrease the cost of maintaining the bioreactor temperature in wastewater treatment. Here, the long-term (>350 days) operational activities of N-DAMO were evaluated to assess the performance of N-DAMO from stepwise cooling (30–20 °C) to ambient temperatures (13–38 °C). Under stepwise cooling conditions, the activity of the N-DAMO community was first inhibited and then rapidly adjusted. Notably, a similar N-DAMO activity was observed at 30 °C and 20 °C. Under ambient temperature conditions, the highest nitrate removal rate observed at the beginning of the test was 7.14 mg-N/L/d, which was 5.3 times higher than that recorded at the end of the test. This indicates that the long-term temperature fluctuation irreversibly inhibited N-DAMO activity. 16S rRNA gene sequencing analyses found that the functional archaea were ANME-2D, which has been deemed as the dominant culture in the N-DAMO process. The abundance of ANME-2D on the last day at stepwise cooling temperature conditions was much higher than on day 0, but disappeared after a long period of operation at ambient temperature. It was assumed that N-DAMO would stabilize at stepwise cooling temperature conditions, but not at ambient temperature. Our findings could offer a promising technology for anaerobic wastewater treatment plants (WWTPs) in temperate and warm climate zones.

Presence of diverse nitrate-dependent anaerobic methane oxidizing archaea in sewage sludge.

The aim of this study was to explore the community diversity and abundance of nitrate-dependent anaerobic methane oxidizing archaea, Candidatus Methanoperedens nitroreducens, in sewage sludge from wastewater treatment plants. Seasonal sampling of the sewage sludge was carried out from two wastewater treatment plants (WWTPs) located in the northern and southern parts of China. Through amplicon sequencing using our newly designed primers, a large number of Candidatus Methanoperedens nitroreducens-like (M. nitroreducens) archaeal sequences (638743) were generated. These sequences were assigned into 742 operational protein units (OPUs) at 90% cut-off level and classified as Group B member of M. nitroreducens archaea in the phylogenetic tree. More than 80% of the OPUs were not shared between these two WWTPs, showing the M. nitroreducens-like archaeal community in each WWTP was unique. Quantitative PCR assays also confirmed the presence of M. nitroreducens-like archaea and revealed a higher abundance in autumn and winter than other seasons, indicating that the environmental attributes in these seasons might favour the growth of this archaea. Further redundancy analysis revealed that volatile solid and pH were the significant environmental attributes (P<0·05) in shaping the M. nitroreducens-like archaeal community based on variance inflation factor selection and Monte Carlo permutation test. The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. The results of this study revealed the ecological characteristics of M. nitroreducens-like archaea in sewage sludge that improved our understanding of nitrate-dependent anaerobic methane oxidation process and may be the basis for future application of M. nitroreducens-like archaea for new nitrogen removal in WWTPs.

Microbial chromate reduction coupled with anaerobic oxidation of methane in a membrane biofilm reactor

It has been reported that microbial reduction of sulfate, nitrite/nitrate and iron/manganese could be coupled with anaerobic oxidation of methane (AOM), which plays a significant role in controlling methane emission from anoxic niches. However, little is known about microbial chromate (Cr(VI)) reduction coupling with AOM. In this study, a microbial consortium was enriched via switching nitrate dosing to chromate feeding as the sole electron acceptor under anaerobic condition in a membrane biofilm reactor (MBfR), in which methane was continuously provided as the electron donor through bubble-less hollow fiber membranes. According to long-term reactor operation and chromium speciation analysis, soluble chromate could be reduced into Cr(III) compounds by using methane as electron donor. Fluorescence in situ hybridization and high-throughput 16S rRNA gene amplicon profiling further indicated that after feeding chromate Candidatus ‘Methanoperedens’ (a known nitrate-dependent anaerobic methane oxidation archaeon) became sole anaerobic methanotroph in the biofilm, potentially responsible for the chromate bio-reduction driven by methane. Two potential pathways of the microbial AOM-coupled chromate reduction were proposed: (i) Candidatus ‘Methanoperedens’ independently utilizes chromate as electron acceptor to form Cr(III) compounds, or (ii) Candidatus ‘Methanoperedens’ oxidizes methane to generate intermediates or electrons, which will be utilized to reduce chromate to Cr(III) compounds by unknown chromate reducers synergistically. Our findings suggest a possible link between the biogeochemical chromium and methane cycles.

Denitrifying Anaerobic Methane Oxidation: A Previously Overlooked Methane Sink in Intertidal Zone.

The intertidal zone is an open ecosystem rich in organic matter and plays an important role in global biogeochemical cycles. It was previously considered that methane was mainly removed by sulfate-dependent anaerobic methane oxidation (sulfate-AOM) process in marine ecosystems while other anaerobic methane oxidation processes were ignored. Recent researches have demonstrated that denitrifying anaerobic methane oxidation (DAMO), consisting of nitrite-dependent anaerobic methane oxidation (nitrite-AOM) and nitrate-dependent anaerobic methane oxidation (nitrate-AOM), can also oxidize methane. In this work, the community structure, quantity and potential methane oxidizing rate of DAMO archaea and bacteria in the intertidal zone were studied by high-throughput sequencing, qPCR and stable isotope tracing method. The results showed that nitrate-AOM and nitrite-AOM were both active in the intertidal zone and showed approximate methane oxidation rates. The copy number of 16S rRNA gene of DAMO archaea and DAMO bacteria were 104 ∼ 105 copies g-1 (dry sediment), whereas NC10 bacteria were slightly higher. The contribution rate of DAMO process to total anaerobic methane removal in the intertidal zone reached 65.6% ∼ 100%, which indicates that DAMO process is an important methane sink in intertidal ecosystem. Laboratory incubations also indicated that DAMO archaea were more sensitive to oxygen and preferred a more anoxic environment. These results help us draw a more complete picture of methane and nitrogen cycles in natural habitats.