Deciphering the formation of granules by n-DAMO and Anammox microorganisms.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) process is a promising wastewater treatment technology, but the slow microbial growth rate greatly hinders its practical application. Although high-level nitrogen removal and excellent biomass accumulation have been achieved in n-DAMO granule process, the formation mechanism of n-DAMO granules remains unresolved. To elucidate the role of functional microbes in granulation, this study attempted to cultivate granules dominated by n-DAMO microorganisms and granules coupling n-DAMO with anaerobic ammonium oxidation (Anammox). After long-term operation, dense granules were developed in the two systems where both n-DAMO archaea and n-DAMO bacteria were enriched, whereas granulation did not occur in the other system dominated by n-DAMO bacteria. Extracellular polymeric substances (EPS) measurement indicated the critical role of EPS production in the granulation of n-DAMO process. Metagenomic and metatranscriptomic analyses revealed that n-DAMO archaea and Anammox bacteria were active in EPS biosynthesis, while n-DAMO bacteria were inactive. Consequently, more EPS were produced in the systems containing n-DAMO archaea and Anammox bacteria, leading to the successful development of n-DAMO granules. Furthermore, EPS biosynthesis in n-DAMO systems is potentially regulated by acyl-homoserine lactones and c-di-GMP. These findings not only provide new insights into the mechanism of granule formation in n-DAMO systems, but also hint at potential strategies for management of the granule-based n-DAMO process.

Deep insights into the biofilm formation mechanism and nitrogen-transformation network in a nitrate-dependent anaerobic methane oxidation biofilm.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) process offers a promising solution for simultaneously achieving methane emissions reduction and efficient nitrogen removal in wastewater treatment. Although nitrogen removal at a practical rate has been achieved by n-DAMO biofilm process, the mechanisms of biofilm formation and nitrogen transformation remain to be elucidated. In this study, n-DAMO biofilms were successfully developed in the membrane aerated moving bed biofilm reactor (MAMBBR) and removed nitrate at a rate of 159 mg NO3--N L-1 d-1. The obvious increase in the content of extracellular polymeric substances (EPS) indicated that EPS production was important for biofilm development. n-DAMO microorganisms dominated the microbial community, and n-DAMO bacteria were the most abundant microorganisms. However, the expression of biosynthesis genes for proteins and polysaccharides encoded by n-DAMO archaea was significantly more active compared to other microorganisms, suggesting the central role of n-DAMO archaea in EPS production and biofilm formation. In addition to nitrate reduction, n-DAMO archaea were revealed to actively express dissimilatory nitrate reduction to ammonium and nitrogen fixation. The produced ammonium was putatively converted to dinitrogen gas through the joint function of n-DAMO archaea and n-DAMO bacteria. This study revealed the biofilm formation mechanism and nitrogen-transformation network in n-DAMO biofilm systems, shedding new light on promoting the application of n-DAMO process.

Biodegradation of various grades of polyethylene microplastics by Tenebrio molitor and Tenebrio obscurus larvae: Effects on their physiology.

Polyethylene (PE) is the most productive plastic product and includes three major polymers including high-density polyethylene (HDPE), linear low-density polyethylene (LLDPE) and low-density polyethylene (LDPE) variation in the PE depends on the branching of the polymer chain and its crystallinity. Tenebrio obscurus and Tenebrio molitor larvae biodegrade PE. We subsequently tested larval physiology, gut microbiome, oxidative stress, and PE degradation capability and degradation products under high-purity HDPE, LLDPE, and LDPE powders (<300 µm) diets for 21 days at 65 ± 5% humidity and 25 ± 0.5 °C. Our results demonstrated the specific PE consumption rates by T. molitor was 8.04-8.73 mg PE ∙ 100 larvae-1⋅day-1 and by T. obscurus was 7.68-9.31 for LDPE, LLDPE and HDPE, respectively. The larvae digested nearly 40% of the ingested three PE and showed similar survival rates and weight changes but their fat content decreased by 30-50% over 21-day period. All the PE-fed groups exhibited adverse effects, such as increased benzoquinone concentrations, intestinal tissue damage and elevated oxidative stress indicators, compared with bran-fed control. In the current study, the digestive tract or gut microbiome exhibited a high level of adaptability to PE exposure, altering the width of the gut microbial ecological niche and community diversity, revealing notable correlations between Tenebrio species and the physical and chemical properties (PCPs) of PE-MPs, with the gut microbiome and molecular weight change due to biodegradation. An ecotoxicological simulation by T.E.S.T. confirmed that PE degradation products were little ecotoxic to Daphnia magna and Rattus norvegicus providing important novel insights for future investigations into the environmentally-friendly approach of insect-mediated biodegradation of persistent plastics.

Revisiting the Engineering Roadmap of Nitrate/Nitrite-Dependent Anaerobic Methane Oxidation.

Nitrate/nitrite-dependent anaerobic oxidation of methane (n-DAMO) is a recently discovered process, which provides a sustainable perspective for simultaneous nitrogen removal and greenhouse gas emission (GHG) mitigation by using methane as an electron donor for denitrification. However, the engineering roadmap of the n-DAMO process is still unclear. This work constitutes a state-of-the-art review on the classical and most recently discovered metabolic mechanisms of the n-DAMO process. The versatile combinations of the n-DAMO process with nitrification, nitritation, and partial nitritation for nitrogen removal are also clearly presented and discussed. Additionally, the recent advances in bioreactor development are systematically reviewed and evaluated comprehensively in terms of methane supply, biomass retention, membrane requirement, startup time, reactor performance, and limitations. The key issues including enrichment and operation strategy for the scaling up of n-DAMO-based processes are also critically addressed. Moreover, the challenges inherent to implementing the n-DAMO process in practical applications, including application scenario recognition, GHG emission mitigation, and operation under realistic conditions, are highlighted. Finally, prospects as well as opportunities for future research are proposed. Overall, this review provides a roadmap for potential applications and further development of the n-DAMO process in the field of wastewater treatment.

Reactivated biofilm coupling n-DAMO with anammox achieved high-rate nitrogen removal in membrane aerated moving bed biofilm reactor.

As a promising technology, the combination of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) with Anammox offers a solution to achieve effective and sustainable wastewater treatment. However, this sustainable process faces challenges to accumulate sufficient biomass for reaching practical nitrogen removal performance. This study developed an innovative membrane aerated moving bed biofilm reactor (MAMBBR), which supported sufficient methane supply and excellent biofilm attachment, for cultivating biofilms coupling n-DAMO with Anammox. Biofilms were developed rapidly on the polyurethane foam with the supply of ammonium and nitrate, achieving the bioreactor performance of 275 g N m-3 d-1 within 102 days. After the preservation at -20 °C for 8 months, the biofilm was successfully reactivated and achieved 315 g N m-3 d-1 after 188 days. After reactivation, MAMBBR was applied to treat synthetic sidestream wastewater. Up to 99.9% of total nitrogen was removed with the bioreactor performance of 4.0 kg N m-3 d-1. Microbial community analysis and mass balance calculation demonstrated that n-DAMO microorganisms and Anammox bacteria collectively contributed to nitrogen removal in MAMBBR. The MAMBBR developed in this study provides an ideal system of integrating n-DAMO with Anammox for sustainable wastewater treatment.

Mainstream Nitrogen and Dissolved Methane Removal through Coupling n-DAMO with Anammox in Granular Sludge at Low Temperature.

Mainstream anaerobic wastewater treatment has received increasing attention for the recovery of methane-rich biogas from biodegradable organics, but subsequent mainstream nitrogen and dissolved methane removal at low temperatures remains a critical challenge in practical applications. In this study, granular sludge coupling n-DAMO with Anammox was employed for mainstream nitrogen removal, and the dissolved methane removal potential of granular sludge at low temperatures was investigated. A stable nitrogen removal rate (0.94 kg N m-3 d-1 at 20 °C) was achieved with a high-level effluent quality (<3.0 mg TN L-1) in a lab-scale membrane granular sludge reactor (MGSR). With decreasing temperature, the nitrogen removal rate dropped to 0.55 kg N m-3 d-1 at 10 °C, while the effluent concentration remained <1.0 mg TN L-1. The granular sludge with an average diameter of 1.8 mm proved to retain sufficient biomass (27 g VSS L-1), which enabled n-DAMO and Anammox activity at a hydraulic retention time as low as 2.16 h even at 10 °C. 16S rRNA gene sequencing and scanning electron microscopy revealed a stable community composition and compact structure of granular sludge during long-term operation. Energy recovery could be maximized by recovering most of the dissolved methane in mainstream anaerobic effluent, as only a small amount of dissolved methane was capable of supporting denitrifying methanotrophs in granular sludge, which enabled high-level nitrogen removal.

Anaerobic Oxidation of Methane Coupled with Dissimilatory Nitrate Reduction to Ammonium Fuels Anaerobic Ammonium Oxidation.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) is critical for mitigating methane emission and returning reactive nitrogen to the atmosphere. The genomes of n-DAMO archaea show that they have the potential to couple anaerobic oxidation of methane to dissimilatory nitrate reduction to ammonium (DNRA). However, physiological details of DNRA for n-DAMO archaea were not reported yet. This work demonstrated n-DAMO archaea coupling the anaerobic oxidation of methane to DNRA, which fueled Anammox in a methane-fed membrane biofilm reactor with nitrate as only electron acceptor. Microelectrode analysis revealed that ammonium accumulated where nitrite built up in the biofilm. Ammonium production and significant upregulation of gene expression for DNRA were detected in suspended n-DAMO culture with nitrite exposure, indicating that nitrite triggered DNRA by n-DAMO archaea. 15N-labeling batch experiments revealed that n-DAMO archaea produced ammonium from nitrate rather than from external nitrite. Localized gradients of nitrite produced by n-DAMO archaea in biofilms induced ammonium production via the DNRA process, which promoted nitrite consumption by Anammox bacteria and in turn helped n-DAMO archaea resist stress from nitrite. As biofilms predominate in various ecosystems, anaerobic oxidation of methane coupled with DNRA could be an important link between the global carbon and nitrogen cycles that should be investigated in future research.

Accelerated start-up for photo-fermentative hydrogen production in biofilm reactor by adding waste effluent.

The difficulty and long duration of start-up wastes numerous costs, labors and time and a little fluctuate during the process might fail it. However, studies dealing with the problem hindering accelerated start-up are still insufficient. Current research focused to develop a method for accelerated start-up in an efficient way. This work outlined a novel alternative for accelerated start-up. This joint method, adding waste effluent with applying biofilm reactor, could successfully start up hydrogen production in the first 24 h via increasing ability of hydrogen producers while the control group produced little hydrogen. The two factors, biofilm formation and addition of waste effluent, expressed the combined effects on accelerated start-up. This study suggested that little molecules like quorum sensing system factors and indoles might be the crucial regulating and stimulating factors and express the accelerated start-up ability only in biofilm reactors.

Nitrate/nitrite dependent anaerobic methane oxidation coupling with anammox in membrane biotrickling filter for nitrogen removal.

Combining nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) and anaerobic ammonium oxidation (Anammox) is a promising sustainable wastewater treatment technology, which simultaneously achieve nitrogen removal and methane emission mitigation. However, the practical application of n-DAMO has been greatly limited by its extremely slow growth-rate and low reaction rate. This work proposes an innovative Membrane BioTrickling Filter (MBTF), which consist of hollow fiber membrane for effective methane supplementation and polyurethane sponge as support media for the attachment and growth of biofilm coupling n-DAMO with Anammox. When steady state with a hydraulic retention time (HRT) of 6.00 h was reached, above 99.9% of nitrogen was removed from synthetic sidestream wastewater at a rate of 3.99 g N L-1 d-1. This system presented robust capacity to withstand unstable partial nitritation effluent, achieving complete nitrogen removal at a varied nitrite to ammonium ratio in the range of 1.10-1.40. It is confirmed that n-DAMO and Anammox microorganisms jointly dominated the microbial community by pyrosequencing technology. The complete nitrogen removal potential at high-rate and efficient biomass retention (12.4 g VSS L-1) of MBTF offers promising alternative for sustainable wastewater treatment by the combination of n-DAMO and Anammox.

Sulfate dependent ammonium oxidation: A microbial process linked nitrogen with sulfur cycle and potential application.

Sulfate dependent ammonium oxidation (Sulfammox) is a potential microbial process coupling ammonium oxidation with sulfate reduction under anaerobic conditions, which provides a novel link between nitrogen and sulfur cycle. Recently, Sulfammox was detected in wastewater treatments and was confirmed to occur in natural environments, especially in marine sediments. However, knowledge gaps in the mechanism of Sulfammox, functional bacteria, and their metabolic pathway, make it challenging to estimate its environmental significance and potential applications. This review provides an overview of recent advances in Sulfammox, including possible mechanisms, functional bacteria, and main influential factors, and discusses future challenges and opportunities. Future perspectives are outlined and discussed, such as exploration of microbial community structure and metabolic pathways, possible interactions with other microbes, environmental significance, and potential applications for nitrogen and sulfate removal, to inspire more researches on the Sulfammox process.

Granular Sludge Coupling Nitrate/Nitrite Dependent Anaerobic Methane Oxidation with Anammox: from Proof-of-Concept to High Rate Nitrogen Removal.

This work developed a novel Membrane Granular Sludge Reactor (MGSR) equipped with a gas permeable membrane module for efficient methane delivery to cultivate nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) microorganisms in granular sludge. As proof of concept, the MGSR was fed with synthetic wastewater containing nitrate and ammonium to facilitate the growth of n-DAMO microorganisms. The granular sludge of n-DAMO and Anammox was gradually developed and achieved a nitrogen removal rate of 1.08 g NO3--N L-1 d-1 and 0.81 g NH4+-N L-1 d-1. Finally, enriched granular sludge was successfully applied for nitrogen removal from the synthetic partial nitritation effluent. The combined dominance of n-DAMO archaea, Anammox bacteria, and n-DAMO bacteria in the microbial community was confirmed by 16S rRNA amplicon sequencing. Fluorescence in situ hybridization revealed that a layered structure was formed in the granular sludge with Anammox bacteria in the outer layer and n-DAMO microorganisms in the inner layer when granules were fed with nitrite and ammonium. The high performance of nitrogen removal (16.53 kg N m-3 d-1) with satisfactory effluent quality (∼8 mg N L-1) and excellent biomass retention capacity (43 g VSS L-1) make the MGSR promising for the practical application of n-DAMO and Anammox in wastewater treatment.

Development of granular sludge coupling n-DAMO and Anammox in membrane granular sludge reactor for high rate nitrogen removal.

The integration of nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) and anaerobic ammonium oxidation (Anammox) provides sustainable solution to simultaneously remove nitrate, nitrite and ammonium. This study demonstrated the sludge granulation process coupling n-DAMO and Anammox from mixed inoculum including river sediment, return activated sludge and crushed anaerobic granule sludge in a novel membrane granular sludge reactor (MGSR). Flocculent biomass gradually turned into compact aggregates and retained as granular sludge with an average diameter of 2.2 mm in MGSR after 684 days' operation. When steady state with a hydraulic retention time of 1.19 days was reached, the MGSR achieved a nitrogen removal rate of 1.77 g N L-1 d-1. Granules with density of 1.043 g mL-1, settling velocity of 72 m h-1 and sludge volume index of 22 mL g-1 leaded to excellent biomass retention (42 g VSS L-1). Pyrosequencing analysis revealed that two dominant microbial groups, n-DAMO archaea and Anammox bacteria, in the microbial community of the granule were enriched to 31.09% and 12.45%. Fluorescence in situ hybridization revealed a homogenous distribution of n-DAMO archaea and Anammox bacteria throughout the granule. The granular sludge coupling n-DAMO and Anammox microorganisms provides significant potential for high rate nitrogen removal from wastewater.

Integrated biogas upgrading and medium-chain fatty acids production for more efficient resource recovery.

The conversion of carbon dioxide (CO2) from biogas into medium-chain fatty acids (MCFAs) represents an eco-friendly resource recovery approach to reduce dependence on fossil fuels and combat global climate change. This study presented the novel concept of integrated resource recovery by coupling biogas upgrading and MCFAs production for the first time. Initially, the impact of different initial ethanol concentrations on chain elongation was examined, determining that an ethanol concentration of 160 mmol/L maximized MCFAs yield at 45.7 mmol/L. Subsequently, using this optimal ethanol supply, the integrated strategy was implemented by connecting two bioreactors in series and maintaining continuous operation for 160-day. The results were noteworthy: upgraded bio-methane purity reached 97.6 %, MCFAs production rate and carbon-flow distribution reached 24.5 mmol/L d-1 and 76.1 %, respectively. In summary, these promising outcomes pioneer a resource recovery approach, enabling the high-value utilization of biogas and the conversion of CO2 into valuable bio-chemicals.

Anaerobic ammonium oxidation coupled with sulfate reduction links nitrogen with sulfur cycle.

Sulfate-dependent ammonium oxidation (Sulfammox) is a critical process linking nitrogen and sulfur cycles. However, the metabolic pathway of microbes driven Sulfammox is still in suspense. The study demonstrated that ammonium was not consumed with sulfate as the sole electron acceptor during long-term enrichment, probably due to inhibition from sulfide accumulation, while ammonium was removed at âˆ¼ 10 mg N/L/d with sulfate and nitrate as electron acceptors. Ammonium and sulfate were converted into nitrogen gas, sulfide, and elemental sulfur. Sulfammox was mainly performed by Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida, both of which encoded ammonium oxidation pathway and dissimilatory sulfate reduction pathway. Not sulfide-driven autotrophic denitrifiers but Candidatus Kuenenia stuttgartiensis converted nitrate to nitrite with sulfide. The results of this study reveal the specialized metabolism of Sulfammox bacteria (Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida) and provide insight into microbial relationships during the nitrogen and sulfur cycles.

Microbial interactions facilitating efficient methane driven denitrification via in-situ utilization of short chain fatty acids.

High nitrate pollution in agriculture and industry poses a challenge to emerging methane oxidation coupled denitrification. In this study, an efficient nitrate removal efficiency of 100 % was achieved at an influent loading rate of 400 mg-N/L·d, accompanied by the production of short chain fatty acids (SCFAs) with a maximum value of 80.9 mg/L. Batch tests confirmed that methane was initially converted to acetate, which then served as a carbon source for denitrification. Microbial community characterization revealed the dominance of heterotrophic denitrifiers, including Simplicispira (22.8 %), Stappia (4.9 %), and the high­nitrogen-tolerant heterotrophic denitrifier Diaphorobacter (19.0 %), at the nitrate removal rate of 400 mg-N/L·d. Notably, the low abundance of methanotrophs ranging from 0.24 % to 3.75 % across all operational stages does not fully align with the abundance of pmoA genes, suggesting the presence of other functional microorganisms capable of methane oxidation and SCFAs production. These findings could facilitate highly efficient denitrification driven by methane and contributed to the development of denitrification using methane as an electron donor.

Large language model for horizontal transfer of resistance gene: From resistance gene prevalence detection to plasmid conjugation rate evaluation.

The burgeoning issue of plasmid-mediated resistance genes (ARGs) dissemination poses a significant threat to environmental integrity. However, the prediction of ARGs prevalence is overlooked, especially for emerging ARGs that are potentially evolving gene exchange hotspot. Here, we explored to classify plasmid or chromosome sequences and detect resistance gene prevalence by using DNABERT. Initially, the DNABERT fine-tuned in plasmid and chromosome sequences followed by multilayer perceptron (MLP) classifier could achieve 0.764 AUC (Area under curve) on external datasets across 23 genera, outperforming 0.02 AUC than traditional statistic-based model. Furthermore, Escherichia, Pseudomonas single genera based model were also be trained to explore its predict performance to ARGs prevalence detection. By integrating K-mer frequency attributes, our model could boost the performance to predict the prevalence of ARGs in an external dataset in Escherichia with 0.0281-0.0615 AUC and Pseudomonas with 0.0196-0.0928 AUC. Finally, we established a random forest model aimed at forecasting the relative conjugation transfer rate of plasmids with 0.7956 AUC, drawing on data from existing literature. It identifies the plasmid's repression status, cellular density, and temperature as the most important factors influencing transfer frequency. With these two models combined, they provide useful reference for quick and low-cost integrated evaluation of resistance gene transfer, accelerating the process of computer-assisted quantitative risk assessment of ARGs transfer in environmental field.

Simultaneous biogas upgrading and medium-chain fatty acids production using a dual membrane biofilm reactor.

Utilizing H2-assisted ex-situ biogas upgrading and acetate recovery holds great promise for achieving high value utilization of biogas. However, it faces a significant challenge due to acetate's high solubility and limited economic value. To address this challenge, we propose an innovative strategy for simultaneous upgrading of biogas and the production of medium-chain fatty acids (MCFAs). A series of batch tests evaluated the strategy's efficiency under varying initial gas ratios (v/v) of H2, CH4, CO2, along with varying ethanol concentrations. The results identified the optimal conditions as initial gas ratios of 3H2:3CH4:2CO2 and an ethanol concentration of 241.2 mmol L-1, leading to maximum CH4 purity (97.2 %), MCFAs yield (54.2 ± 2.1 mmol L-1), and MCFAs carbon-flow distribution (62.3 %). Additionally, an analysis of the microbial community's response to varying conditions highlighted the crucial roles played by microorganisms such as Clostridium, Proteiniphilum, Sporanaerobacter, and Bacteroides in synergistically assimilating H2 and CO2 for MCFAs production. Furthermore, a 160-day continuous operation using a dual-membrane aerated biofilm reactor (dMBfR) was conducted. Remarkable achievements were made at a hydraulic retention time of 2 days, including an upgraded CH4 content of 96.4 ± 0.3 %, ethanol utilization ratio (URethanol) of 95.7 %, MCFAs production rate of 28.8 ± 0.3 mmol L-1 d-1, and MCFAs carbon-flow distribution of 70 ± 0.8 %. This enhancement is proved to be an efficient in biogas upgrading and MCFAs production. These results lay the foundation for maximizing the value of biogas, reducing CO2 emissions, and providing valuable insights into resource recovery.

Biodegradation of polyethylene terephthalate by Tenebrio molitor: Insights for polymer chain size, gut metabolome and host genes.

Polyethylene terephthalate (PET or polyester) is a commonly used plastic and also contributes to the majority of plastic wastes. Mealworms (Tenebrio molitor larvae) are capable of biodegrading major plastic polymers but their degrading ability for PET has not been characterized based on polymer chain size molecular size, gut microbiome, metabolome and transcriptome. We verified biodegradation of commercial PET by T. molitor larvae in a previous report. Here, we reported that biodegradation of commercial PET (Mw 29.43 kDa) was further confirmed by using the δ13C signature as an indication of bioreaction, which was increased from - 27.50‰ to - 26.05‰. Under antibiotic suppression of gut microbes, the PET was still depolymerized, indicating that the host digestive enzymes could degrade PET independently. Biodegradation of high purity PET with low, medium, and high molecular weights (MW), i.e., Mw values of 1.10, 27.10, and 63.50 kDa with crystallinity 53.66%, 33.43%, and 4.25%, respectively, showed a mass reduction of > 95%, 86%, and 74% via broad depolymerization. Microbiome analyses indicated that PET diets shifted gut microbiota to three distinct structures, depending on the low, medium, and high MW. Metagenome sequencing, transcriptomic, and metabolic analyses indicated symbiotic biodegradation of PET by the host and gut microbiota. After PET was fed, the host's genes encoding degradation enzymes were upregulated, including genes encoding oxidizing, hydrolyzing, and non-specific CYP450 enzymes. Gut bacterial genes for biodegrading intermediates and nitrogen fixation also upregulated. The multiple-functional metabolic pathways for PET biodegradation ensured rapid biodegradation resulting in a half-life of PET less than 4 h with less negative impact by PET MW and crystallinity.

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.

Deep insights into the population shift of n-DAMO and Anammox in granular sludge: From sidestream to mainstream.

Granular sludge combined n-DAMO and Anammox (n-D/A) is an energy-efficient biotechnique for the simultaneous removal of nitrogen and dissolved methane from wastewater. However, the lack of knowledge so far about the metabolic interactions between n-DAMO and Anammox in response to operation condition in granular sludge restrains the development of this biotechnology. To address this gap, three independent membrane granular sludge reactors (MGSRs) were designed to carry out the granule-based n-D/A process under different conditions. We provided the first deep insights into the metabolic interactions between n-DAMO and Anammox in granular sludge via combined metagenomic and metatranscriptomic analyses. Our study unveiled a clear population shift of n-DAMO community from Candidatus Methanoperedens to Candidatus Methylomirabilis from sidestream to mainstream. Candidatus Methanoperedens with relative abundance of 25.2% played the major role in nitrate reduction and methane oxidation under sidestream condition, indicated by the high expression activities of mcrA and narG. Candidatus Methylomirabilis dominated the microbial community under mainstream condition with relative abundance of 32.1%, supported by the high expression activities of pmoA and hao. Furthermore, a transition of Anammox population from Candidatus Kuenenia to Candidatus Brocadia was also observed from sidestream to mainstream. Candidatus Kuenenia and Candidatus Brocadia jointly contributed to the primary anaerobic ammonium oxidation suggested by the high expression value of hdh and hzs. Candidatus Methylomirabilis was speculated to perform ammonium oxidation mediated by pMMO under mainstream condition. These findings might help to reveal the microbial interactions and ecological niches of n-DAMO and Anammox microorganisms, shedding light on the optimization and management of the granule-based n-D/A system.