Revisiting methane-dependent denitrification.

Methane-dependent denitrification links the global nitrogen and methane cycles. Since its initial discovery in 2006, this process has been understood to involve a division of labor between an archaeal group and a bacterial group, which sequentially perform nitrate and nitrite reduction, respectively. Yao et al. have now revised this paradigm by identifying a Methylomirabilis bacterium capable of performing methane-dependent complete denitrification on its own.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal.

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20-50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Role of n-DAMO in Mitigating Methane Emissions from Intertidal Wetlands Is Regulated by Saltmarsh Vegetations.

Coastal wetlands are hotspots for methane (CH4) production, reducing their potential for global warming mitigation. Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) plays a crucial role in bridging carbon and nitrogen cycles, contributing significantly to CH4 consumption. However, the role of n-DAMO in reducing CH4 emissions in coastal wetlands is poorly understood. Here, the ecological functions of the n-DAMO process in different saltmarsh vegetation habitats as well as bare mudflats were quantified, and the underlying microbial mechanisms were explored. Results showed that n-DAMO rates were significantly higher in vegetated habitats (Scirpus mariqueter and Spartina alterniflora) than those in bare mudflats (P < 0.05), leading to an enhanced contribution to CH4 consumption. Compared with other habitats, the contribution of n-DAMO to the total anaerobic CH4 oxidation was significantly lower in the Phragmites australis wetland (15.0%), where the anaerobic CH4 oxidation was primarily driven by ferric iron (Fe3+). Genetic and statistical analyses suggested that the different roles of n-DAMO in various saltmarsh wetlands may be related to divergent n-DAMO microbial communities as well as environmental parameters such as sediment pH and total organic carbon. This study provides an important scientific basis for a more accurate estimation of the role of coastal wetlands in mitigating climate change.

Microbial Stratification Affects Conversions of Nitrogen and Methane in Biofilms Coupling Anammox and n-DAMO Processes.

A methane-based membrane biofilm reactor (MBfR) has a suitable configuration to incorporate anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) processes because of its high gas-transfer efficiency and efficient biomass retention. In this study, the spatial distribution of microorganisms along with the biofilm depth in methane-based MBfRs was experimentally revealed, showing the dominance of anammox bacteria, n-DAMO bacteria, and n-DAMO archaea in the outer layer, middle layer, and inner layer of biofilms, respectively. The long-term and short-term experimental investigations in conjunction with mathematical modeling collectively revealed that microorganisms living in the outer layer of biofilms tend to use substrates from wastewater, while microorganisms inhabiting the inner layer of biofilms tend to use substrates originating from biofilm substratum. Specifically, anammox bacteria dominating the biofilm surface preferentially removed the nitrite provided from wastewater, while n-DAMO bacteria mostly utilized the nitrite generated from n-DAMO archaea as these two methane-related populations spatially clustered together inside the biofilm. Likewise, the methane supplied from the membrane was mostly consumed by n-DAMO archaea, while the dissolved methane in wastewater would be primarily utilized by n-DAMO bacteria. This study offers novel insights into the impacts of microbial stratification in biofilm systems, not only expanding the fundamental understanding of biofilms and microbial interactions therein but also providing a rationale for the potential applications of methane-based MBfRs in sewage treatment.

Enhancement of nitrate-dependent anaerobic methane oxidation via granular activated carbon.

Denitrifying anaerobic methane oxidation (DAMO) is a bioprocess utilizing methane as the electron source to remove nitrate or nitrite, but denitrification rate especially for nitrate-dependent DAMO is usually limited due to the low methane mass transfer efficiency. In this research, granular active carbon (GAC) was added to enhance the nitrate-dependent DAMO process. The results showed that the maximum nitrate removal rate of GAC assisted DAMO system reached as high as 61.17 mg L-1 d-1, 8 times higher than that of non-amended control SBR. The porous structure of GAC can not only adsorb methane, but also keep the internal DAMO archaea from being washed out, and thus benefits for DAMO archaea enrichment. The relative abundance of DAMO archaea accounted for 96.3% in GAC-SBR, which was significantly higher than that of non-amended control SBR system (29.9%). Furthermore, GAC amendment up-regulated metabolic status of denitrification and methane oxidation based on gene sequence composition. The absolute abundances of function genes (NC10 pmoA and ANME mcrA) in GAC-SBR were almost 20 times higher than that of non-amended control SBR. This study provides a novel technique to stimulate the nitrate-dependent DAMO process.

Efficient nitrogen removal from mainstream wastewater through coupling Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (PNAM).

The application of partial nitritation and anammox to remove nitrogen from mainstream wastewater is of great interest because of the potential to reduce energy cost and carbon dosage. However, this process confronts a dilemma of relatively high effluent nitrogen concentration (>10 mg N/L), owning to the unwanted prevalence of nitrite-oxidizing bacteria (NOB) and the intrinsic nitrate production by anammox bacteria. Here, a novel technology, named the one-stage PNAM, that integrates Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction reactions, was developed in a single membrane biofilm reactor (MBfR). With feeding of synthetic mainstream wastewater containing ∼50 mg NH4+-N/L at a hydraulic retention time of 12 h, more than 95% nitrogen was removed in the established one-stage PNAM process at a practically useful rate of 0.1 kg N/m3/d. Microbial community characterization and in-situ batch tests revealed a sophisticated microbial structure consisting of ammonia-oxidizing bacteria (AOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) bacteria and archaea, and a small fraction of NOB and aerobic methanotrophs. The role of methane in removing nitrate was confirmed by switching on/off the methane supply, which relaxed the requirement for NOB suppression. In addition, the established system was relatively robust against temperature variations, evidenced by a total nitrogen removal efficiency above 80% at temperature as low as 14 â„ƒ. The results provide a promising alternative for efficient nitrogen removal from domestic wastewater using methane as the sole carbon source.

Rapid formation of granules coupling n-DAMO and anammox microorganisms to remove nitrogen.

Granular sludge exhibits unique features, including rapid settling velocity, high loading rate and relative insensitivity against inhibitors, thus being a favorable platform for the cultivation of slow-growing and vulnerable microorganisms, such as anaerobic ammonium oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) microorganisms. While anammox granules have been widely applied, little is known about how to speed up the granulation process of n-DAMO microorganisms, which grow even slower than anammox bacteria. In this study, we used mature anammox granules as biotic carriers to embed n-DAMO microorganisms, which obtained combined anammox + n-DAMO granules within 6 months. The results of whole-granule 16S rRNA gene amplicon sequencing showed the coexistence of anammox bacteria, n-DAMO bacteria and n-DAMO archaea. The microbial stratification along granule radius was further elucidated by cryosection-16S rRNA gene amplicon sequencing, showing the dominance of n-DAMO archaea and anammox bacteria at inner and outer layers, respectively. Moreover, the images of cryosection-fluorescence in situ hybridization (FISH) verified this stratification and also indicated a shift in microbial stratification. Specifically, n-DAMO bacteria and n-DAMO archaea attached to the anammox granule surface initially, which moved to the inner layer after 4-months operation. On the basis of combined anammox + n-DAMO granules, a practically useful nitrogen removal rate (1.0 kg N/m3/d) was obtained from sidestream wastewater, which provides new avenue to remove nitrogen from wastewater using methane as carbon source.

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.

Model-based investigation of membrane biofilm reactors coupling anammox with nitrite/nitrate-dependent anaerobic methane oxidation.

An innovative process coupling anaerobic ammonium oxidation (anammox) with nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) in membrane biofilm reactors (MBfRs) has been developed to achieve high-level nitrogen removal from both sidestream (i.e., anaerobic digestion liquor) and mainstream (i.e., domestic strength) wastewater. In this study, a 1D biofilm model embedding the n-DAMO and anammox reactions was developed to facilitate further understanding of the process and its optimization. The model was calibrated and validated using comprehensive data sets from two independent MBfRs, treating sidestream- and mainstream-strength wastewater, respectively. Modelling results revealed a unique biofilm stratification. While anammox bacteria dominated throughout the biofilm, n-DAMO archaea (coupling nitrate reduction with anaerobic methane oxidation) only occurred at the inner layer and n-DAMO bacteria (coupling nitrite reduction with anaerobic methane oxidation) spread more evenly with a slightly higher fraction in the outer layer. The established MBfRs were robust against dynamic influent flowrates and nitrite/ammonium ratios. Thicker biofilms were beneficial for not only the total nitrogen (TN) removal but also the system robustness. Additionally, a positive correlation between the nitrogen removal efficiency and the residual methane emission was observed, as a result of higher methane partial pressure required. However, there was a threshold of methane partial pressure, above which the residual methane increased but nitrogen removal efficiency was stable. Meanwhile, thicker biofilms were also favorable to achieve less residual methane emission. Simulation results also suggested the feasibility of methane-based MBfRs to polish mainstream anammox effluent to meet a stringent N discharge standard (e.g., TN < 5 mg/L).

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

AIM: 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. METHODS AND RESULTS: 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 (638 743) 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. CONCLUSIONS: The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Cooccurrence and potential role of nitrite- and nitrate-dependent methanotrophs in freshwater marsh sediments.

Nitrite- and nitrate-dependent anaerobic methane oxidation are mediated by the NC10 bacteria closely related to "Candidatus Methylomirabilis oxyfera" (M. oxyfera) and the ANME-2d archaea closely related to "Candidatus Methanoperedens nitroreducens" (M. nitroreducens), respectively. Here, we investigated the occurrence and activity of both M. oxyfera-like bacteria and M. nitroreducens-like archaea in the sediment of freshwater marshes in Eastern China. The presence of diverse M. oxyfera-like bacteria (>87% identity to M. oxyfera) and M. nitroreducens-like archaea (>96% identity to M. nitroreducens) was confirmed by using Illumina-based total bacterial and archaeal 16S rRNA gene sequencing, respectively. The recovered M. oxyfera-like bacterial sequences accounted for 1.6-4.3% of the total bacterial 16S rRNA pool, and M. nitroreducens-like archaeal sequences accounted for 0.2-1.8% of the total archaeal 16S rRNA pool. The detected numbers of OTUs of the 16S rRNA genes of M. oxyfera-like bacteria and M. nitroreducens-like archaea were 78 and 72, respectively, based on 3% sequence difference. Quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria (6.1 × 106-3.2 × 107 copies g-1 sediment) was 2-4 orders of magnitude higher than that of M. nitroreducens-like archaea (1.4 × 103-3.2 × 104 copies g-1 sediment). Stable isotope experiments showed that the addition of both nitrite and nitrate stimulated the anaerobic methane oxidation, while the stimulation by nitrite is more significant than nitrate. Our results provide the first evidence that the M. oxyfera-like bacteria play a more important role than the M. nitroreducens-like archaea in methane cycling in wetland systems.