Anammox Granular Sludge Research Articles

Importance of exopolysaccharide branched chains in determining the aggregation ability of anammox sludge

The high aggregation ability of anammox granular sludge is an issue of wide concern; however, the mechanism needs to be further clarified. In this study, selective hydrolysis experiments were performed to determine the role of exopolysaccharide (PS) branched chains and proteins for the aggregation mechanism of anammox granular sludge. The results revealed that selective hydrolysis of proteins hardly affected the granular aggregation while the hydrolysis of PS branched chains led to a decrease in the sludge zeta potential by 17.3% (β-amylase group) and 24.1% (isoamylase group), a decrease of hydrophobicity by 11.6% (β-amylase group) and 17.7% (isoamylase group), an increase of surface free energy by 36.8% (β-amylase group) and 55.1% (isoamylase group) and the deterioration of the PS self-assembly ability. In addition, FTIR and XPS spectra analysis showed that the disruption of PS branched chains resulted in a higher proportion of hydrophilic and electronegative groups, which hindered bacterial aggregation, which was further confirmed by XDLVO theory. The key role of the PS chain structure in sludge aggregation is a critical finding of this work that provides helpful insights for the application of anammox process.

Deciphering the genesis of anammox granular sludge floating from the perspective of microbial community

Sludge floating generally occurred in different anammox reactors, while the microbial difference between settling and floating sludge still unclear. In this study, the comprehensive investigation of microbial features between floating sludge and size-fractionated settling sludge was conducted in a stably operated anammox reactor. Firstly, scanning electron microscope image showed that mounts of long filament and a few gas tunnels were found in floating sludge, while cocci and bacilli were stacked in settling sludge. Microbial diversity was gradually decreased as the sludge aggregation, the microbial community structure and composition of floating sludge (FG) were more similar to large size-fractionated sludge (SG-L), and away from the small size-fractionated sludge (SG-S). Anammox bacteria Candidatus Jettenia tended to enrich in small size-fractionated sludge (accounting for 46.3%), while the large particle size feature and starved condition of floating sludge induced the decrease of Candidatus Jettenia (accounting for 23.5%). Meanwhile, denitrifying bacteria Dok59 rapidly enriched in large size-fractionated sludge and floating sludge, accounting for 8.9% and 9.8%, respectively. Network analysis showed that complex microbial interrelationships bring a robust microbial community in the SG-L sample, while the FG sample in the anammox reactor obtained an unstable microbial community. Moreover, the microbial community of FG has narrow niche breadths based on the low betweenness and node transitivity. These results obtained in this study extended our understanding of sludge floating in the anammox reactor and emphasized the crucial role of microorganisms.

Characteristics of ANAMMOX Granular Sludge and Differences in Microbial Community Structure Under Different Culture Conditions

Anaerobic ammonium oxidation (ANAMMOX) granular sludge was cultured during different operating conditions by an expanded granular sludge bed (EGSB) reactor and up-flow anaerobic sludge bed (UASB) reactors, and the characteristics of the granular sludge and microbial community were compared. The results showed that the flocculent ANAMMOX sludge can be granulated after being operated for 384 days by the EGSB and UASB reactors. The average particle size reached 1.17 mm and 1.21 mm, respectively. The particle size ratio of each range (<0.2, 0.2-1.5, 1.5-3, and>3 mm) was 6.06%, 60.05%, 25.25%, and 8.64% in the EGSB reactor, and 7.40%, 58.90%, 32.04%, and 1.66% in the UASB reactor, respectively. The results of scanning electron microscopy showed that the bacterial flora during different operating conditions were mainly Brevibacterium and Cocci aggregates. High-throughput sequencing results showed that the Shannon index of the EGSB reactor was 7.52, higher than the 7.18 of the UASB reactor on day 384; Proteobacteria was the main phylum of the sludge at each stage, and Planctomycetes increased from 3.30% to 12.30% in the EGSB reactor and 13.30% in the UASB reactor on day 384. The main ANAMMOX genera in the EGSB reactor were Candidatus Brocadia, accounting for 7.53%, followed by Candidatus Kuenenia accounting for 1.61%, whereas in the UASB reactor, Candidatus Kuenenia was the dominant anaerobic ammonia genus, accounting for 7.54%, followed by Candidatus Brocadia, which accounted for 3.69%. The proportion of dominant species was related to the change in environmental factors. The proportion of Candidatus Brocadia was positively correlated with the up-flow rate and nitrogen removal rate (NRR), but negatively correlated with hydraulic retention time (HRT). Candidatus Kuenenia was positively correlated with nitrogen removal efficiency (NRE), NRR, and HRT, but negatively correlated with the up-flow rate.

Domestic Sewage Treatment Using a One-Stage ANAMMOX Process

A one-stage anaerobic ammonium oxidation (ANAMMOX) reactor can be quickly started within 40 days by mixing partial nitrifying sludge with ANAMMOX granular sludge with an average temperature of 30 °C. After 70 days of nitrogen load acclimation, Acinetobacter, including Candidatus Kuenenia, became the dominant strain of the system within the reactor, which exhibited high efficiency and a stable nitrogen removal performance. At an influent chemical oxygen demand (COD), NH4+-N content, total nitrogen (TN) content, hydraulic retention time (HRT), temperature, and reactor dissolved oxygen (DO) content of 100, 60, and 70 mg/L, 6 h, 30 ± 1 °C, and below 0.6 mg/L, respectively, the one-stage ANAMMOX reactor could effectively treat domestic sewage on campus. The removal rates of COD, NH4+-N, and TN were approximately 89%, 96.7%, and 70%, respectively.

The branched chains and branching degree of exopolysaccharides affecting the stability of anammox granular sludge

The effect of extracellular polysaccharides on the structural stability of granular sludge is widely recognized, and determining their mechanism of action on the stability of granules remains challenging. Herein, enzymatic experiments were used to systematically study the stability changes and internal mechanisms of anammox granular sludge following hydrolysis of extracellular proteins and polysaccharides (PS). The results revealed that the selective hydrolysis of the proteins hardly affected the stability of the granules, while the hydrolysis of the PS branched chains caused the granules to disintegrate. The hydrolysis of the PS chains in the EPS matrix decreased the degree of branching, width and height via nuclear magnetic resonance (NMR) spectroscopy and atomic force microscopy (AFM), and these parameters are closely related to granular stability. Moreover, scanning electron microscopy (SEM) showed a large number of pores and cracks on the granules, bacterial adhesion decreased, and the EPS adhered to the surface of the granules dissolved. The changes in the gel characteristics of the granules were studied by rheology, and the mechanical strength and viscosity of the granular sludge decreased. For the surface characteristics of granules, the zeta potential and hydrophobicity both decreased, revealing that changes in the branched-chain configuration of the PS and the degree of branching caused granular disintegration. Spectral analysis showed that the hydrolysis of the branch points and the branched glycosides of PS led to a higher proportion of hydrophilic and electronegative groups in the EPS matrix, which hindered bacterial aggregation and reduced anammox granule stability. This investigation clarifies the impact of the branched-chain configuration of the PS and their degree of branching on anammox granule stability, which will promote the further application of anammox granules.

Impact of COD/N on anammox granular sludge with different biological carriers

The purpose of this study is to investigate the effect of COD/N interference on mature anammox granular sludge formed by different biological carriers. Three anammox granular sludge rectors were established with no biological carriers (R1), GAC (R2) and PVA-gel bead (R3), respectively. As the COD/N ratio increased to 1:2, the activity of anaerobic ammonia oxidizing bacteria in R1 and R2 was significantly inhibited. However, the nitrogen removal effect of R3 did not decrease dramatically, and the nitrogen removal rate in this phase was 1.54 ± 0.05 kg N/m3·d. As the COD/N ratio increased to 1:1.5, the removal of NH4+-N in all reactors gradually decreased. The order of COD resistance of the three reactors in this study was R3 > R2 > R1. It was found that Candidatus Brocadia might be sensitive to the presence of organic matter. The abundance of heterotrophic denitrifying bacteria increased gradually in each reactor under increased influent COD/N ratios.

Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

Resistance of anammox granular sludge to copper nanoparticles and oxytetracycline and restoration of performance

Nanoparticles and antibiotics, the two most frequently detected emerging pollutants from different wastewater sources, are eventually discharged into wastewater treatment plants. In this study, the widely used materials CuNPs and oxytetracycline (OTC) were selected as target pollutants to investigate their joint effects on anaerobic ammonium oxidation (anammox). The results indicated that the environmental concentration slightly inhibited the performance of the reactors, while the performance rapidly deteriorated within a week under high-level combined shocks (5.0 mg L−1 CuNPs and 2.0 mg L−1 OTC). After the second shock (2.5 mg L−1 CuNPs and 2.0 mg L−1 OTC), the resistance of anammox bacteria was enhanced, with an elevated relative abundance of Candidatus Kuenenia and absolute abundance of hzsA, nirS, and hdh. Moreover, the extracellular polymeric substance (EPS) content and specific anammox activity (SAA) showed corresponding changes. Improved sludge resistance was observed with increasing CuNP and OTC doses, which accelerated the recovery of performance.

Steady-state and dynamic analysis of the single-stage anammox granular sludge reactor show that bulk ammonium concentration is a critical control variable to mitigate feeding disturbances

One of the challenges for the application of the ANAMMOX (anaerobic ammonium oxidation) process for nitrogen removal in the mainstream municipal wastewater treatment is the disturbances in the feeding influent, in terms of the organic material, ammonium concentration, flow rate and temperature. Past experimental and mathematical simulation researches were mainly carried out under stable feeding condition. It is not clear how the ANAMMOX responds to the feeding disturbances. In this study, a 1D (one-dimensional) biofilm model was used to examine the behavior of feeding disturbances in the single-stage granule PN-A (partial nitrification –ANAMMOX) process. The results indicated that the feeding disturbances could be mitigated by maintaining a small amount of ammonium in the bulk liquid. A cascade control strategy based on DO (dissolved oxygen) manipulation to derive the preset bulk ammonium set-point value (i.e., 2 mg N/L) was shown to be successful in achieving the maximum TN (total nitrogen) removal percentage of above 95% under dynamic feeding conditions. The long-term simulation showed that the small bulk ammonium accumulation could also suppress the NOB (nitrite-oxidizing bacteria) growth.

Carbon quantum dots derived from the extracellular polymeric substance of anaerobic ammonium oxidation granular sludge for detection of trace Mn(vii) and Cr(vi).

Carbon quantum dots (CQDs) were synthesized via a hydrothermal method, in which extracellular polymeric substance (EPS) from anaerobic ammonium oxidation (anammox) granular sludge was used as a carbon precursor, while citric acid and ethylenediamine were applied as auxiliary carbon source and passivation agent, respectively. The synthesized CQDs, with orderly spherical shape and mean size of 7.15 nm, emitted blue fluorescent light under UV radiation of 365 nm. The CQDs had a high fluorescence yield (40.84%), with good water solubility and excellent spectroscopic properties. In addition, the CQDs exhibited selective, sensitive and distinctive fluorescence quenching behaviors for Cr(vi) and Mn(vii) in a PBS buffer solution (NaH2PO4–Na2HPO4) of pH 7, with a detection limit of 5.8 nM for Cr(vi) and 2.3 nM for Mn(vii). Owing to the nitrogen components from the EPS of anammox granules, the CQDs were well nitrogen-doped, promoting electron-transfer and leading to reduction between the CQDs and Mn(vii)/Cr(vi). These results indicate that CQD-based chemical sensing is a simple and efficient means for the fluorescence detection of Mn(vii) and Cr(vi).

Response of FANIR system to starvation stress: “Dormancy”

Anaerobic ammonium oxidation (Anammox) process has been successfully applied in the nitrogen removal from high-strength wastewaters. However, little information is available for its treatment of low-strength wastewaters. In this study, a Famine Anammox NItrogen Removal (FANIR) system was developed to investigate the effect of long-term substrate starvation at the low nitrogen concentration (the influent total nitrogen was set at ∼1 mg/L). The results showed that the response of FANIR system to the starvation stress took on two phases: the functional decline phase (0–54 day) and the functional stabilization phase (62–116 day). Over the two phases, the Nitrogen Removal Rate (NRR) of anammox reactor firstly dropped sharply; and then came to a constant level. The activity and settleability of Anammox Granular Sludge (AnGS) firstly deteriorated seriously, and then stayed in a stable range. The relative abundance of Anaerobic Ammonium Oxidation Bacteria (AnAOB) firstly decreased markedly, and then approached a steady state with the change of dominant genus from Candidatus Brocadia to Candidatus Kuenenia. The abundance of 16S rRNA gene and hzs gene of AnAOB and their transcription level firstly declined largely as well, and then became stable with the 16S rRNA gene, hzs gene, 16S rRNA and hzs-mRNA of AnAOB at 23.9%, 9.1%, 1.2% and 1.0% of the initial value, respectively. To our delight, the behavior of FANIR system in the functional stabilization phase was proved indeed consistent with the feature for AnAOB to enter the dormancy state. These findings are helpful to understand the physiology of AnAOB over the starvation stress and to promote the extension of anammox process to the treatment of low-strength wastewaters.

Two-stage granular sludge partial nitritation/anammox process for the treatment of digestate from the anaerobic digestion of the organic fraction of municipal solid waste

The increasing amount of source separated organic fraction of municipal solid wastes (OFMSW) treated by anaerobic digestion for energy recovery requires the implementation of cost-efficient processes for the treatment of the produced digestate, especially in terms of nitrogen removal. The autotrophic nitrogen removal process, based on the coupling of two biological processes, partial nitritation (PN) and anammox (A), appears as a suitable solution due to important savings in operational costs compared to conventional treatment processes. However, its application could be hampered by the high salinity and inhibitory potential of this kind of digestate. In this contribution, two lab-scale granular sludge reactors performing the PN and anammox processes, respectively, were used to treat (opportunely diluted) real OFMSW digestate originating from full-scale biogas plants with the aim of assessing their treatment feasibility in a two-stage PN/A configuration. The PN process was implemented in an air-lift granular sludge reactor and was able to treat a nitrogen loading rate of about 1 g N L−1 d−1 at 30 ± 0.5 °C; moreover, its effluent was suitable for the subsequent anammox treatment, with an appropriate effluent NO2−/NH4+ ratio and marginal inhibiting effects. In the anammox granular sludge reactor, the anammox activity was affected by high salinity levels, nonetheless a stable reactor performance at a nitrogen removing rate of 0.83 ± 0.20 and 0.31 ± 0.04 g N L−1 d−1 at 35 ± 0.5 °C, were achieved when treating 50% and 30% diluted real wastewaters at a conductivity in the reactor of 9.1 and 11.2 mS cm−1, respectively.

Novel aerobic granular sludge culture strategy: Using granular sludge Anammox process effluent as a biocatalyst

The effluent from granular sludge Anammox process was used as a biocatalyst to improve the culture rate of aerobic granular sludge, and the internal causes of this effect were studied.In this study, in sequencing batch reactor, the formation and changes of AGS was monitored with granular sludge Anammox process effluent added before and after. The community structure of AGS was analyzed by molecular biology methods. The results showed ammonia utilizing rate increased from 3.41 to 5.96 mgNH4+-N/(g VSS·h), NO2–-N maintained a high accumulation rate, and the denitrification performance remained stable. On 40th day, the diameter of the AGS reached 3.5 mm, and the concentrations of PN and PS reached 330.5 and 62.9 mg/gVSS, respectively. The community structure has changed. Nitrosomonas (31.7%) became the new dominant bacteria. Signal molecules contained in the effluent as inter-species signal molecules could enhance the formation of AGS.

Rapid Cultivation of Anaerobic Ammonium Oxidation Granular Sludge and Inhibition Kinetics of Granular Sludge

For the rapid cultivation of anaerobic ammonium oxidation (ANAMMOX) granular sludge (AGS), a small amount of flocculent ANAMMOX sludge (FAS) was taken as the research object, and a bio-flow separate ball was used as the filler in an up-flow anaerobic sludge bed (UASB) to rapidly begin ANAMMOX and to cultivate granular sludge. In addition, the substrate inhibition kinetic characteristics of the AGS were investigated by using the Haldane model. The results showed that start-up of the ANAMMOX was successfully achieved. The total nitrogen removal rate was more than 85%, and the volume load of total nitrogen was 0.72 kg·(m3·d)-1. AGS with diameters of 1.0-3.0 mm were cultured within 127 days using the Bio-flow Separate Ball. The kinetic studies showed that the maximum reaction rates for ammonia and nitrite of granular sludge were 1.46 kg·(kg·d)-1 and 1.76 kg·(kg·d)-1, with half inhibition constants of ammonia and nitrite at 852.2 mmol·L-1 and 108.2 mmol·L-1, respectively. Compared with FAS, AGS can withstand higher concentrations of ammonia and nitrite and can maintain a higher reaction rate. The placement of the filler has positive significance for starting ANAMMOX and rapidly culturing AGS.

Interactions between tetracycline and extracellular polymeric substances in anammox granular sludge

The effects of antibiotics on extracellular polymeric substances (EPS) using tetracycline as the model chemical were analyzed in terms of molecular property and structure. Results showed that three components, tryptophan, tryptophan type-proteins and polysaccharides in EPS of granular sludge from anaerobic ammonium oxidation (anammox) reactor can interacted with tetracycline, detected by the static quenching via the endogenous fluorescence quenching and transient fluorescence spectroscopy. Thermodynamic experiment confirmed that their interaction was dominated by the hydrophobic force. Combined with the synchronous fluorescence spectroscopy, it was found that tetracycline facilitated the extension degree of peptide chains in tryptophan type-proteins, leading to the enhancement of hydrodynamic diameter of the macromolecules in EPS when binding with tetracycline. EPS in AnGS demonstrated the resistance ability to tetracycline by converting from gel to sol state in rheological term. With the increase of tetracycline concentration, the stability of elastic structures in EPS declined, influencing the AnGS stability.

Hydrogels formed by anammox extracellular polymeric substances: structural and mechanical insights

The recovery of biopolymers from the waste sludge produced in wastewater treatments and their application in other industrial sectors, would substantially increase the environmental and economical sustainability of the process, promoting the development of a circular economy. In this study, extracellular polymeric substances (EPS) extracted from anammox granular waste sludge, were investigated and characterized. Rheological and differential scanning calorimetry measurements on EPS aqueous dispersions indicate the formation of an extended 3-D network above a threshold concentration, with a clear dependence of the mechanical and water retention properties on EPS content. The structural characterization, performed with transmission electron microscopy and small angle X-ray scattering, reveals the presence of functional amyloids as putative structural units, observed for the first time in an EPS-based hydrogel. As a proof of concept of the applicative potential, we explored the water and grease resistance provided to paper by an EPS coating. These results shed light on the structural details of EPS-based hydrogels, and pave the way for the possible use of EPS-based materials as a cheap, eco-friendly alternative to commonly adopted paper coatings, in line with a circular economy pattern for wastewater treatment.

Solubilization and characterization of extracellular proteins from anammox granular sludge

Elucidating the extracellular polymeric substances (EPS) of anammox granular sludge is important for stable nitrogen removal processes in wastewater treatment. However, due to a lack of standardized methods for extraction and characterization, the composition of anammox granule EPS remains mostly unknown. In this study, alkaline (NaOH) and ionic liquid (IL) extractions were compared in terms of the proteins they extracted from different “Candidatus Brocadia” cultures. We aimed to identify structural proteins and evaluated to which extend these extraction methods bias the outcome of EPS characterization. Extraction was focussed on solubilization of the EPS matrix, and the NaOH and IL extraction recovered on average 20% and 26% of the VSS, respectively. Using two extraction methods targeting different intermolecular interactions increased the possibility of identifying structural extracellular proteins. Of the extracted proteins, ∼40% were common between the extraction methods. The high number of common abundant proteins between the extraction methods, illustrated how extraction biases can be reduced when solubility of the granular sludge is enhanced. Physicochemical analyses of the granules indicated that extracellular structural matrix proteins likely have β-sheet dominated secondary structures. These β-sheet structures were measured in EPS extracted with both methods. The high number of uncharacterized proteins and possible moonlighting proteins confounded identifying structural (i.e. β-sheet dominant) proteins. Nonetheless, new candidates for structural matrix proteins are described. Further current bottlenecks in assigning specific proteins to key extracellular functions in anammox granular sludge are discussed.

Surface convexity of anammox granular sludge: Digital characterization, state indication and formation mechanism

Anammox granular sludge system is a promising biotechnology for nitrogen removal from wastewaters. The anammox granules possess the distinctive morphological features which can be developed as visible indicators for anammox process monitoring. In this study, the surface convexity of anammox granular sludge (AnGS) was systematically investigated for the first time. The AnGS was withdrawn from four long-term operated anammox bioreactors at different nitrogen loading rates. Firstly, the spherical convexity and gap concavity with a diameter of 50–100 μm were observed to be distributed on the granules surface under the microscopic observation. Then, the surface convexity was determined by image processing technology and the statistical analysis showed that the convexity had a significant difference (p = 0.003) among bioreactors and the average surface convexity decreased from 0.937 ± 0.030 to 0.899 ± 0.034, and then rose to 0.914 ± 0.035 which had a significant correlation with the volumetric gas production rate of bioreactor (r = −0.873, p < 0.05). An optical method was further developed to fast characterize the surface convexity using relative lightness (LSCE⁎/LSCI⁎) as the index. At last, the composition and structure of AnGS were investigated to deduce the formation mechanism of surface convexity. The formation could be attributed to the outward growth of zoogloea led to the surface protrusion (convexity); the periodical extrusion of microbubbles caused the striping of surface zoogloea (concavity) and the gas-driven collision and friction between granules which finally shaped the surface convexity. The produced dinitrogen gas links the metabolic activity with the formation of surface convexity and concavity of AnGS. This finding provided an alternative visible performance indictor of anammox process.

Impacts of salt shocking and the selection of a suitable reversal agent on anammox.

In this study, an anaerobic ammonium oxidation (anammox) reactor, which was inhibited by a salinity of 50 g NaCl L-1 during a long-term experiment, was rapidly restarted by decreasing the salinity to 20 g NaCl L-1 and adding biomass. The effects of exposure time and shock concentrations on the anammox reactor indicate that anammox granular sludge has a high tolerance to salinity and strong ability for self-recovery. The nitrogen removal efficiency was higher than 50% after exposure to 50 g NaCl L-1 for 66 h. To shorten the time taken for effluent nitrogen concentrations to attain national standards (GB18918-2002) after the anammox reactor was shocked with NaCl, reactor performance (i.e., recovery) after the addition of K+, glycine betaine, Fe2+, and hydroxylamine were compared after the reactor was inhibited by 80 g NaCl L-1. The results indicate that hydroxylamine was the best reversal agent. The recovery time of the anammox reactor could be shortened by 50% following the addition of hydroxylamine. The most favorable NH2OH-N/NO2--N concentration ratio for improving nitrogen removal of anammox was 1:11. The abundances of Planctomycetes and its genera Candidatus Kuenenia and Brocadiaceae_g_unclassified increased after repeated salinity shock-recovery phases, indicating that Candidatus Kuenenia and Brocadiaceae_g_unclassified are able to adapt to NaCl shocking and recovery.

The performance and microbial community in response to MnO2 nanoparticles in anammox granular sludge

The response of anaerobic ammonium oxidation (anammox) process and granular sludge in the upflow anaerobic sludge blanket reactor was analyzed under long-term exposure to MnO2 nanoparticles (NPs) (1–200 mg L−1). The addition of 200 mg L−1 MnO2 NPs had a significantly positive effect on nitrogen removal and this system exhibited excellent performance, with a total nitrogen removal efficiency of 93.1%. Moreover, the specific anammox activity enhanced with increasing concentrations of MnO2 NPs up to the maximum value of 657.3 ± 9.3 mg TN g−1 VSS d−1 under MnO2 NPs concentration of 200 mg L−1. This value was approximately 1.6-fold higher than that of the reactor in the absence of MnO2 NPs. The extracellular polymeric substances and settling velocity were both increased with MnO2 NPs addition. Meanwhile, the high-throughput sequencing results revealed that MnO2 NPs increased the relative abundance of dominant bacteria (Candidatus Kuenenia) from 17.3% at the absence of MnO2 NPs to 23.9% at 200 mg L−1 MnO2 NPs, which resulted in a higher efficiency of biological nitrogen removal on the anammox system. These results indicated that MnO2 NPs enhanced nitrogen removal performance of anammox process.