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.

Deciphering the roles of attached and suspended sludges in simultaneous nitrogen and phosphorus removal in an IFAS system based on metagenomic analysis.

Integrated fixed-film activated sludge (IFAS) system, an improvement of the activated sludge process, combines the advantages of both attached sludge (AS) and suspended sludge (SS). This study aimed to fully decipher the roles of AS and SS in simultaneous N and P removal in an IFAS system through metagenomic analysis. It was found that AS contributed about 84.04%, 97%, and 95.12% to exogenous NO3--N reduction, endogenous NO3--N reduction, and endogenous NO2--N reduction, respectively. Compared with AS, SS exhibited a greater contribution to anaerobic P release (69.06%) and aerobic P uptake (73.48%). Nitrate and nitrite reductase enzymes showed higher activities in AS, while the activities of exopolyphosphatase and alkaline phosphatase D were more active in SS. P content further indicated that in AS, only a small amount of P was stored in EPS, with most presented intracellularly. In SS, the amount of P stored in EPS was found to be higher. Metagenomic analysis revealed genes related to the synthesis and degradation of endogenous carbon were higher in AS, whereas the TCA cycle exhibited higher activity in SS. P removal-related genes (such as ppk2, ppx, and adk) was significantly higher in SS than in AS. The alteration of genes associated with nitrogen metabolism suggested that the microbes in AS had a higher capacity for nitrification and denitrification. In summary, the discrepancy in the roles of AS and SS in N and P removal in IFAS can be attributed to variations in enzyme activity, P storage in EPS, microbial community composition, and functional gene abundance.

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.

A critical review on graphene oxide membrane for industrial wastewater treatment.

An important way to promote the environmental industry's goal of carbon reduction is to promote the recycling of resources. Membrane separation technology has unique advantages in resource recovery and advanced treatment of industrial wastewater. However, the great promise of traditional organic membrane is hampered by challenges associated with organic solvent tolerance, lack of oxidation resistance, and serious membrane fouling control. Moreover, the high concentrations of organic matter and inorganic salts in the membrane filtration concentrate also hinder the wider application of the membrane separation technology. The emerging cost-effective graphene oxide (GO)-based membrane with excellent resistance to organic solvents and oxidants, more hydrophilicity, lower membrane fouling, better separation performance has been expected to contribute more in industrial wastewater treatment. Herein, we provide comprehensive insights into the preparation and characteristic of GO membranes, as well as current research status and problems related to its future application in industrial wastewater treatment. Finally, concluding remarks and future perspectives have been deduced and recommended for the GO membrane separation technology application for industrial wastewater treatment, which leads to realizing sustainable wastewater recycling and a nearly "zero discharge" water treatment process.

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.

Synergistic analysis of performance, functional genes, and microbial community assembly in SNDPR process under Zn(II) stress.

One of the most prevalent heavy metals found in rural sewage is Zn(II), while its effect on simultaneous nitrification, denitrification and phosphorus removal (SNDPR) remains unclear. In this work, the responses of SNDPR performance to long-term Zn(II) stress were investigated in a cross-flow honeycomb bionic carrier biofilm system. The results indicated that Zn(II) stress at 1 and 5 mg L-1 could increase nitrogen removal. Maximum ammonia nitrogen, total nitrogen, and phosphorus removal efficiencies of up to 88.54%, 83.19%, and 83.65% were obtained at Zn(II) concentration of 5 mg L-1. The functional genes, such as archaeal amoA, bacterial amoA, NarG, NirS, NapA, and NirK, also reached the highest value at 5 mg L-1 Zn(II), with the absolute abundances of 7.73 × 105, 1.57 × 106, 6.68 × 108, 1.05 × 109, 1.79 × 108, and 2.09 × 108 copies·g-1 dry weight, respectively. The neutral community model demonstrated that deterministic selection was responsible for the system's microbial community assembly. Additionally, response regimes with extracellular polymeric substances and cooperation among microorganisms facilitated the stability of the reactor effluent. Overall, the findings of this paper contribute to improving the efficiency of wastewater treatment.

Biofilm inhibition based on controlling the transmembrane transport and extracellular accumulation of quorum sensing signals.

The regulation of bacterial quorum sensing (QS) has been used to inhibit biofouling in wastewater treatment plants and the formation of biofilms. In contrast to traditional QS regulation strategies, this study aimed to obstruct the transmembrane transport process of QS signals to decrease their extracellular accumulation. Three phytochemicals, astragaloside IV, eugenol, and baicalin were selected, their effects on biofilm formation by Pseudomonas aeruginosa PA14 were studied, and the mechanisms determined. The inhibition efficiency of biofilm formation by 50 mg/L astragaloside IV, 1 mg/L eugenol, and 1 mg/L baicalin were 37%, 26%, and 26%, respectively. Confocal laser scanning microscopy and analysis of extracellular polymeric substances indicated that the three inhibitors affected the structure and composition of the biofilms. Furthermore, bacterial motility and a variety of QS-related virulence factors were suppressed by the inhibitor treatment due to changes in bacterial QS. Notably, the three inhibitors all decreased the extracellular concentration of the QS signaling molecule 3-oxo-C12-homoseine lactone by affecting the function of efflux pump MexAB-OprM. This indirectly interfered with the bacterial QS system and thus inhibited biofilm formation. In conclusion, this study revealed the inhibitory effects and inhibition mechanism of three phytochemicals on efflux pump and QS of P. aeruginosa and realized the inhibition on biofilm formation. We update the efflux pump inhibitor library and provide a new way for biofilm contamination control.

How Nitrogen and Sulfur Doping Modified Material Structure, Transformed Oxidation Pathways, and Improved Degradation Performance in Peroxymonosulfate Activation.

Current research has widely applied heteroatom doping for the promotion of catalyst activity in peroxymonosulfate (PMS) systems; however, the relationship between heteroatom doping and stimulated activation mechanism transformation is not fully understood. Herein, we introduce nitrogen and sulfur doping into a Co@rGO material for PMS activation to degrade tetracycline (TC) and systematically investigate how heteroatom doping transformed the activation mechanism of the original Co@rGO/PMS system. N was homogeneously inserted into the reduced graphene oxide (rGO) matrix of Co@rGO, inducing a significant increase in the degradation efficiency without affecting the activation mechanism transformation. Additionally, S doping converted Co3O4 to Co4S3 in Co@rGO and transformed the cooperative oxidation pathway into a single non-radical pathway with stronger intensity, which led to a higher stability against environmental interferences. Notably, based on density functional theory (DFT) calculations, we demonstrated that Co4S3 had a higher energy barrier for PMS adsorption and cleavage than Co3O4, and therefore, the radical pathway was not easily stimulated by Co4S3. Overall, this study not only illustrated the improvement due to the heteroatom doping of Co@rGO for TC degradation in a PMS system but also bridged the knowledge gap between the catalyst structure and degradation performance through activation mechanism transformation drawn from theoretical and experimental analyses.

Simultaneous removal of NOX and SO2 from flue gas in an integrated FGD-CABR system by sulfur cycling-mediated Fe(II)EDTA regeneration.

Chemical absorption-biological reduction (CABR) process is an attractive method for NOX removal and Fe(II)EDTA regeneration is important to sustain high NOX removal. In this study a sustainable and eco-friendly sulfur cycling-mediated Fe(II)EDTA regeneration method was incorporated in the integrated biological flue gas desulfurization (FGD)-CABR system. Here, we investigated the NOX and SO2 removal efficiency of the system under three different flue gas flows (100 mL/min, 500 mL/min, and 1000 mL/min) and evaluated the feasibility of chemical Fe(III)EDTA reduction by sulfide in series of batch tests. Our results showed that complete SO2 removal was achieved at all the tested scenarios with sulfide, thiosulfate and S0 accumulation in the solution. Meanwhile, the total removal efficiency of NOX achieved ∼100% in the system, of which 3.2%-23.3% was removed in spray scrubber and 76.7%-96.5% in EGSB reactor along with no N2O emission. The optimal pH and S2-/Fe(III)EDTA for Fe(II)EDTA regeneration and S0 recovery was 8.0 and 1:2. The microbial community analysis results showed that the cooperation of heterotrophic denitrifier (Saprospiraceae_uncultured and Dechloromonas) and iron-reducing bacteria (Klebsiella and Petrimonas) in EGSB reactor and sulfide-oxidizing, nitrate-reducing bacteria (Azoarcus and Pseudarcobacter) in spray scrubber contributed to the efficient removal of NOX in flue gas.

Runoff control simulation and comprehensive benefit evaluation of low-impact development strategies in a typical cold climate area.

With the acceleration of urbanization, the proportion of surface imperviousness is increasing continuously in cities, resulting in frequent waterlogging disasters. In this context, storm water management, based on the low-impact development (LID) concept, offers an effective measure for the management of urban storm waters. First, the storm water management model (SWMM) was built for a typical cold climate city (Changchun) in China. Next, the two-stage calibrated model was employed to explore the surface runoff and storm sewer control effects of four LID combination plans. Finally, these plans were put through a "cost-benefit" evaluation through an analytic hierarchy process. According to the results, after using four LID plans, the reduction rates of peak runoff exceeded 40% and the problem of overflow load of the storm sewage was significantly mitigated. The infiltration-oriented Plan I proved to be the optimal plan, with the lowest proportions of the overflow nodes and full-load pipe sections in each return period, as well as with maximum overall performance. This study offers technical and conformed methodological support to cold cities for the prevention and control of waterlogging disasters and recycling of rainwater resources.

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.

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.

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.

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.