Enhancing nitrogen and phosphorus removal in plant-biochar-pyrite stormwater bioretention systems: Impact of temperature and high-frequency heavy rainfall.

Global climate change and rapid urbanization have resulted in more frequent and intense rainfall events in urban areas, raising concerns about the effectiveness of stormwater bioretention systems. In this study, we optimized the design by constructing a multi-layer filler structure, including plant layer, biochar layer, and pyrite layer, and evaluated its performance in nitrogen (N) and phosphorus (P) removal under different temperatures (5-18 °C and 24-43 °C), rainfall intensity (47.06 mm rainfall depth), and frequency (1-5 days rainfall intervals) conditions. The findings indicate that over 775 days, the plant system consistently removed 62.3% of total nitrogen (TN) and 97.0% of total phosphorus (TP) from 103 intense rainfall events. Temperature fluctuations had minimal impact on nitrate nitrogen (NO3--N) and TP removal, with differences in removal rates of only 1.0% and 0.6%, respectively, among plant groups. Across the multi-layer structure, plant roots mitigated the impact of temperature differences on NO3--N removal, while high-frequency rainfall fluctuated the stability of NO3--N removal. Dense plant roots reinforced N and P removal by facilitating denitrification in the vadose zone (biochar) and strengthening denitrification processes. Biochar and pyrite contributed to stable microenvironments and diverse ecological functions, enhancing NO3--N and PO43- removal. In summary, the synergistic effects of the multi-layer filler structure improved and stabilized N and P removal, providing valuable insights for addressing runoff pollution in bioretention systems amidst rapid urbanization and climate change challenges.

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

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

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.

Novel Use of a Ferric Salt to Enhance Mainstream Nitrogen Removal from Anaerobically Pretreated Wastewater.

This study aims to demonstrate a new technology roadmap to support the ongoing paradigm shift in wastewater management from pollutant removal to resource recovery. This is achieved by developing a novel use of an iron salt (i.e., FeCl3) in an integrated anaerobic wastewater treatment and mainstream anammox process. FeCl3 was chosen to be dosed in a proposed sidestream unit rather than in a primary settler or a mainstream reactor. This causes acidification of returned activated sludge and enables stable suppression of nitrite-oxidizing bacterial activity and excess sludge reduction. A laboratory-scale system, which comprised an anaerobic baffled reactor, a continuous-flow anoxic-aerobic (A/O) reactor, and a secondary settler, was designed to treat real domestic wastewater, with the performance of the system comprehensively monitored under a steady-state condition. The experimental assessments showed that the system had good effluent quality, with total nitrogen and phosphorus concentrations of 12.6 ± 1.3 mg N/L and 0.34 ± 0.05 mg P/L, respectively. It efficiently retained phosphorus in excess sludge (0.18 ± 0.03 g P/g dry sludge), suggesting its potential for further recovery. About half of influent organic carbon was recovered in the form of bioenergy (i.e., methane). This together with low energy consumption revealed that the system could produce a net energy of about 0.11 kWh/m3-wastewater, assessed by an energy balance analysis.

Pyrogenic Carbon Promotes Anaerobic Oxidation of Methane Coupled with Iron Reduction via the Redox-Cycling Mechanism.

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

Mycolicibacter acidiphilus sp. nov., an extremely acid-tolerant member of the genus Mycolicibacter.

A nonmotile, facultatively anaerobic and rod-shaped bacterial strain, designated M1T was isolated from a bioreactor being operated at pH ~2 at Brisbane, Australia. Colonies appeared to be convex and white. Phylogenetic analysis of its genome revealed an affiliation with the genus Mycolicibacter and its closest species based on 16S rRNA gene analysis were Mycolicibacter algericus DSM 45454T (98.8 % similarity) and Mycolicibacter terrae CIP 104321T (98.8 %) with which strain M1T shared average nucleotide identity of 81.2 % and digital DNA-DNA hybridization similarity of 23.8 %. Strain M1T grew optimally at 0 % NaCl, at pH 6 and at between 30-33 °C. The polar lipid profile of strain M1T consisted of diphosphatidylglycerol, aminophosphoglycolipid, phosphatidylcholine, phospholipid, aminolipid, phosphoglyolipid, phosphatidylglycerol, two unidentified glycolipids and four unidentified lipids. The dominant cellular fatty acids (>10 %) were C16 : 0 and C18 : 1 ω9c and summed feature 7 (C19 : 1 ω7c and/or C19 : 1 ω6c). The DNA G+C content of strain M1T was 69.1 mol%. Based on in silico phylogenomic analysis coupled with physiological and chemotaxonomic characterizations, we classify strain M1T as representing a novel species within the genus Mycolicibacter, for which the name Mycolicibacter acidiphilus nov. is proposed. The type strain is M1T (=MCCC 1H00416T=KCTC 49392T).

A 20-Year Journey of Partial Nitritation and Anammox (PN/A): from Sidestream toward Mainstream.

Anaerobic ammonium oxidation (anammox) was discovered as a new microbial reaction in the late 1990s, which led to the development of an innovative energy- and carbon-efficient technology─partial nitritation and anammox (PN/A)─for nitrogen removal. PN/A was first applied to remove the nitrogen from high-strength wastewaters, e.g., anaerobic digestion liquor (i.e., sidestream), and further expanded to the main line of wastewater treatment plants (i.e., mainstream). While sidestream PN/A has been well-established with extensive full-scale installations worldwide, practical application of PN/A in mainstream treatment has been proven extremely challenging to date. A key challenge is achieving stable suppression of nitrite-oxidizing bacteria (NOB). This study examines the progress of NOB suppression in both sidestream- and mainstream PN/A over the past two decades. The successful NOB suppression in sidestream PN/A was reviewed, and these successes were evaluated in terms of their transferability into mainstream PN/A. Drawing on the learning over the past decades, we anticipate that a hybrid process, comprised of biofilm and floccular sludge, bears great potential to achieve efficient mainstream PN/A, while a combination of strategies is entailed for stable NOB suppression. Furthermore, the recent discovery of novel nitrifiers would trigger new opportunities and new challenges for mainstream PN/A.

Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply.

The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02-0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR.

Determining Factors for Nitrite Accumulation in an Acidic Nitrifying System: Influent Ammonium Concentration, Operational pH, and Ammonia-Oxidizing Community.

Acidic nitrification is attracting wide attention because it can enable robust suppression of nitrite-oxidizing bacteria (NOB) in wastewater treatment. This study reports a comprehensive assessment of the novel acidic nitrification process to identify the key factors that govern stable nitrite accumulation. A laboratory-scale moving-bed biofilm reactor receiving low-alkalinity wastewater was continuously operated under acidic conditions (pH < 6) for around two years, including nine stages varying influent and operational conditions. The results revealed that nitrite accumulation was related to three factors, i.e., influent ammonium concentration, operating pH, and ammonia-oxidizing microbial community. These three factors impact nitrite accumulation by altering the in situ concentration of free nitrous acid (FNA), which is a potent inhibitor of NOB. The critical FNA concentration is approximately one part per million (ppm, ∼1 mg HNO2-N/L), above which nitrite accumulation is stably maintained in an acidic nitrifying system. The findings of this study suggest that stable nitrite accumulation via acidic ammonia oxidation can be maintained under a range of influent and operational conditions, as long as a ppm-level of FNA is established. Taking low-strength mainstream wastewater (40-50 mg NH4+-N/L) with limited alkalinity as an example, stable nitrite accumulation was experimentally demonstrated at a pH of 4.35, under which an in situ FNA of 2.3 ± 0.6 mg HNO2-N/L was attained. Under these conditions, Candidatus Nitrosoglobus became the only ammonia oxidizer detectable by 16S rRNA gene sequencing. The results of this study deepen our understanding of acidic nitrifying systems, informing further development of novel wastewater treatment technologies.

Characterizing and comparing microbial community and biofilm structure in three nitrifying moving bed biofilm reactors.

This study investigated biofilm establishment, biofilm structure, and microbial community composition of biofilms in three laboratory-scale moving bed biofilm reactors. These reactors were filled with three types of plastic carriers with varied depths of living space for microbial growth. The reactors were operated under the same influent and operational conditions. Along with the operation, the results showed that carriers with grids of 50 µm in height delayed the biofilm development and formed the thinnest biofilm and a carpet-like structure with the lowest α-diversity. In comparison, another two carriers with grids of 200 and 400 µm in height formed thick biofilms and large colonies with more voids and channels. Quantified properties of biofilm thickness, biomass, heterogeneity, portion of the biofilm exposed to the nutrient, and maximum diffusion distance were examined, and the results demonstrated that they almost (except for heterogeneity) strongly correlated to the α-diversity of microbial community. These illustrate that depth of living space, as an important parameter for carrier, could drive the formation of biofilm structure and community composition. It improves understanding of influencing factors on biofilm establishment, structure and its microbial community, and would be helpful for the design of biofilm processes.

Robust Nitritation Sustained by Acid-Tolerant Ammonia-Oxidizing Bacteria.

Oxidation of ammonium to nitrite rather than nitrate, i.e., nitritation, is critical for autotrophic nitrogen removal. This study demonstrates a robust nitritation process in treating low-strength wastewater, obtained from a mixture of real mainstream sewage with sidestream anaerobic digestion liquor. This is achieved through cultivating acid-tolerant ammonia-oxidizing bacteria (AOB) in a laboratory nitrifying bioreactor at pH 4.5-5.0. It was shown that nitrite accumulation with a high NO2-/(NO2- + NO3-) ratio of 95 ± 5% was stably maintained for more than 300 days, and the obtained volumetric NH4+ removal rate (i.e., 188 ± 14 mg N L-1 d-1) was practically useful. 16S rRNA gene sequencing analyses indicated the dominance of new AOB, "Candidatus Nitrosoglobus," in the nitrifying guild (i.e., 1.90 ± 0.08% in the total community), with the disappearance of typical activated sludge nitrifying microorganisms, including Nitrosomonas, Nitrospira, and Nitrobacter. This is the first identification of Ca. Nitrosoglobus as key ammonia oxidizers in a wastewater treatment system. It was found that Ca. Nitrosoglobus can tolerate low pH (<5.0), and free nitrous acid (FNA) at levels that inhibit AOB and nitrite-oxidizing bacteria (NOB) commonly found in wastewater treatment processes. The in situ inhibition of NOB leads to accumulation of nitrite (NO2-), which along with protons (H+) also produced in ammonium oxidation generates and sustains FNA at 3.0 ± 1.4 mg HNO2-N L-1. As such, robust PN was achieved under acidic conditions, with a complete absence of NOB. Compared to previous nitritation systems, this acidic nitritation process is featured by a higher nitric oxide (NO) but a lower nitrous oxide (N2O) emission level, with the emission factors estimated at 1.57 ± 0.08 and 0.57 ± 0.03%, respectively, of influent ammonium nitrogen load.

Critical Factors Facilitating Candidatus Nitrotoga To Be Prevalent Nitrite-Oxidizing Bacteria in Activated Sludge.

Nitrite oxidation is the primary pathway that generates nitrate in engineered systems. However, little is known about the role of a novel nitrite-oxidizing bacteria (NOB) genus Candidatus Nitrotoga in activated sludge systems. To elucidate key factors that impact NOB community composition, laboratory-scale sequencing batch reactors (SBRs) were designed and operated under the same conditions as real wastewater treatment plants to achieve considerable nitrogen removal and similar community; then, different conditions including temperature (T), dissolved oxygen (DO), free nitrous acid (FNA), and free ammonia (FA) were applied. The 16S rRNA gene-based PCR and sequence analysis illustrated that Ca. Nitrotoga were abundant even at ambient temperature, thus further challenging the previous conception of them being solely cold-adapted. Ca. Nitrotoga are less competitive than Nitrospira during oxygen deficiency, indicating its lower affinity to dissolved oxygen. Ca. Nitrotoga are the dominant nitrite oxidizers under regular exposure to FNA and FA due to their relatively higher resistance than other NOB toward these two effective biocides. Therefore, this study demonstrates that Ca. Nitrotoga can play an important role in biological nitrogen removal and also highlights the need for multiple strategies for NOB suppression for the next-generation, shortcut nitrogen removal.

Simultaneous Removal of Dissolved Methane and Nitrogen from Synthetic Mainstream Anaerobic Effluent.

Anaerobic technologies have been proposed as a promising solution to enhance bioenergy recovery and to transform a wastewater treatment plant (WWTP) from an energy consumer to an energy exporter. However, 20-60% of the methane produced remains dissolved in the anaerobically treated effluent, which is a potent greenhouse gas and is easily stripped out in the aeration tank. This study aims to develop a solution using dissolved methane to support denitrification, thus simultaneously enhancing nitrogen removal and achieving beneficial use of dissolved methane. By coupling anaerobic ammonium oxidation (anammox) with nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO), up to 85% of dissolved methane and more than 99% of nitrogen were removed in parallel in a biofilm system. Mass balance was conducted during both long-term operation and short-term batch tests, which indicated that n-DAMO bacteria and n-DAMO archaea indeed contributed jointly to the methane removal. The 16S rRNA gene amplicon sequencing further showed the co-presence of n-DAMO bacteria and n-DAMO archaea, while anammox bacteria were detected with a low relative abundance. This proposed technology can potentially be applied to reduce the carbon footprint and to save the organic carbon consumption in WWTPs.

Temperature-Tolerated Mainstream Nitrogen Removal by Anammox and Nitrite/Nitrate-Dependent Anaerobic Methane Oxidation in a Membrane Biofilm Reactor.

The mainstream anaerobic ammonium oxidation (anammox) process provides strong support to the on-going paradigm shift from energy-negative to energy-neutral in wastewater treatment plants. However, the low temperature (e.g., below 15 °C) represents one of the major challenges for mainstream anammox in practice. In this study, a stable nitrogen removal rate (0.13 kg m-3 day-1), together with a high-level effluent quality (<5.0 mg N L-1), was achieved in a lab-scale upflow membrane biofilm reactor (MBfR) by coupling anammox with nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) microorganisms, at a temperature as low as 10 °C. With the temperature being progressively decreased from 25 to 10 °C, the total nitrogen removal efficiency was maintained in the range of 90-94% at a constant hydraulic retention time of 9 h. The impact of temperature on the biofilm system coupling anammox and n-DAMO reactions increased at a lower temperature range with higher Arrhenius coefficients. Additionally, 16S rRNA gene sequencing results showed that anammox bacteria, n-DAMO bacteria, and n-DAMO archaea jointly dominated the biofilm, and their respective abundances remained relatively stable when the temperature was decreased. The major reason for this temperature-tolerated performance is the overcapacity developed, which is indicated by biofilm thickness measurements and mathematical modeling. The stable performance obtained in this study shows promise for the n-DAMO application in domestic wastewater.

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes.

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.

Biochar-Mediated Anaerobic Oxidation of Methane.

Biochar was recently identified as an effective soil amendment for CH4 capture. Corresponding mechanisms are currently recognized to be from physical properties of biochar, providing a favorable growth environment for aerobic methanotrophs which perform aerobic methane (CH4) oxidation. However, our study shows that the chemical reactivity of biochar can also stimulate anaerobic oxidation of CH4 (AOM) by anaerobic methanotrophic archaea (ANME) of ANME-2d, which proposes another plausible mechanism for CH4 mitigation by biochar amendment in anaerobic environments. It was found that, by adding biochar as the sole electron acceptor in an anaerobic environment, CH4 was biologically oxidized, with CO2 production of 106.3 ± 5.1 µmol g-1 biochar. In contrast, limited CO2 production was observed with chemically reduced biochar amendment. This biological nature of the process was confirmed by mcr gene transcript abundance as well as sustained dominance of ANME-2d in the microbial community during microbial incubations with active biochar amendment. Combined FTIR and XPS analyses demonstrated that the redox activity of biochar is related to its oxygen-based functional groups. On the basis of microbial community evolution as well as intermediate production during incubation, different pathways in terms of direct or indirect interactions between ANME-2d and biochar were proposed for biochar-mediated AOM.

Acetate Production from Anaerobic Oxidation of Methane via Intracellular Storage Compounds.

There is great interest in microbial conversion of methane, an abundant resource, into valuable liquid chemicals. While aerobic bioconversion of methane to liquid chemicals has been reported, studies of anaerobic methane bioconversion to liquid chemicals are rare. Here we show that a microbial culture dominated by Candidatus 'Methanoperedens nitroreducens', an anaerobic methanotrophic archaeon, anaerobically oxidizes methane to produce acetate, indirectly via reaction intermediate(s), when nitrate or nitrite is supplied as an electron acceptor under a rate-limiting condition. Isotopic labeling tests showed that acetate was produced from certain intracellular storage compounds that originated from methane. Fluorescence in situ hybridization and Nile red staining demonstrated that polyhydroxyalkanoate in M. nitroreducens was likely one of the intracellular storage compounds for acetate production, along with glycogen. Acetate is a common substrate for the production of more valuable chemicals. The microbial conversion discovered in this study potentially enables a new approach to the use of methane as a feedstock for the chemical market.

Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage.

Anaerobic oxidation of methane (AOM) is critical for controlling the flux of methane from anoxic environments. AOM coupled to iron, manganese and sulphate reduction have been demonstrated in consortia containing anaerobic methanotrophic (ANME) archaea. More recently it has been shown that the bacterium Candidatus 'Methylomirabilis oxyfera' can couple AOM to nitrite reduction through an intra-aerobic methane oxidation pathway. Bioreactors capable of AOM coupled to denitrification have resulted in the enrichment of 'M. oxyfera' and a novel ANME lineage, ANME-2d. However, as 'M. oxyfera' can independently couple AOM to denitrification, the role of ANME-2d in the process is unresolved. Here, a bioreactor fed with nitrate, ammonium and methane was dominated by a single ANME-2d population performing nitrate-driven AOM. Metagenomic, single-cell genomic and metatranscriptomic analyses combined with bioreactor performance and (13)C- and (15)N-labelling experiments show that ANME-2d is capable of independent AOM through reverse methanogenesis using nitrate as the terminal electron acceptor. Comparative analyses reveal that the genes for nitrate reduction were transferred laterally from a bacterial donor, suggesting selection for this novel process within ANME-2d. Nitrite produced by ANME-2d is reduced to dinitrogen gas through a syntrophic relationship with an anaerobic ammonium-oxidizing bacterium, effectively outcompeting 'M. oxyfera' in the system. We propose the name Candidatus 'Methanoperedens nitroreducens' for the ANME-2d population and the family Candidatus 'Methanoperedenaceae' for the ANME-2d lineage. We predict that 'M. nitroreducens' and other members of the 'Methanoperedenaceae' have an important role in linking the global carbon and nitrogen cycles in anoxic environments.

Microbial Selenate Reduction Driven by a Denitrifying Anaerobic Methane Oxidation Biofilm.

Anaerobic oxidation of methane (AOM) plays a crucial role in controlling the flux of methane from anoxic environments. Sulfate-, nitrite-, nitrate-, and iron-dependent methane oxidation processes have been considered to be responsible for the AOM activities in anoxic niches. We report that nitrate-reducing AOM microorganisms, enriched in a membrane biofilm bioreactor, are able to couple selenate reduction to AOM. According to ion chromatography, X-ray photoelectron spectroscopy, and long-term bioreactor performance, we reveal that soluble selenate was reduced to nanoparticle elemental selenium. High-throughput 16S rRNA gene sequencing indicates that Candidatus Methanoperedens and Candidatus Methylomirabilis remained the only known methane-oxidizing microorganisms after nitrate was switched to selenate, suggesting that these organisms could couple anaerobic methane oxidation to selenate reduction. Our findings suggest a possible link between the biogeochemical selenium and methane cycles.

Complete Nitrogen Removal from Synthetic Anaerobic Sludge Digestion Liquor through Integrating Anammox and Denitrifying Anaerobic Methane Oxidation in a Membrane Biofilm Reactor.

Partial nitritation and Anammox processes are increasingly used for nitrogen removal from anaerobic sludge digestion liquor. However, their nitrogen removal efficiency is often limited due to the production of nitrate by the Anammox reaction and the sensitivity to the nitrite to ammonium ratio. This work develops and demonstrates an innovative process that achieves complete nitrogen removal from partially nitrified anaerobic sludge digestion liquor through the use of a membrane biofilm reactor (MBfR), with methane supplied through hollow fiber membranes. When steady state with a hydraulic retention time (HRT) of 1 day was reached, the process achieved complete nitrite and ammonium removal at rates of 560 mg N/L/d and 470 mg N/L/d, respectively, without any nitrate accumulation. The process is relatively insensitive to the nitrite to ammonium ratio, achieving complete nitrogen removal when their ratio in influent varied in the range of 1.125-1.32. Pyrosequencing and fluorescence in situ hybridization analysis revealed that denitrifying anaerobic methane oxidation (DAMO) archaea, Anammox bacteria and DAMO bacteria jointly dominated the microbial community. Mass balance analysis showed that nitrate produced by Anammox (122.2 mg N/L/d) was entirely converted to nitrite by DAMO archaea, while nitrite in the feed and produced by DAMO archaea was jointly removed by Anammox (90%) and DAMO bacteria (10%). The nitrogen removal rate of over 1 kg N/m3/d is comparable to the practical rates reported for side-stream nitrogen removal processes.