Partial nitritation/anammox (PN/A) enables energy-efficient nitrogen removal in industrial wastewater treatment. This study provides a comprehensive survey of water chemistry and microbiomes in six full-scale, single-stage plug-flow PN/A systems treating five industrial wastewater types. Amplicon sequencing resolved community profiles for attached biofilms and suspended flocs. A two-year time series from one plant (JM) confirmed baseline consistency of community composition and structure under comparable operating conditions, supporting the representativeness of the sampling window. Following inoculation with seed biofilm (SB), evolved biofilms (EB) that developed under identical conditions exhibited no significant structural divergence from SB, and new biofilms (NB) formed on blank carriers converged toward EB. These patterns suggest that local operating conditions can exert stronger control than inoculum history on attached community assembly. Across PN/A systems, the anammox bacteria (AnAOB) was dominated by Candidatus_Brocadia, Candidatus_Jettenia and Candidatus_Kuenenia, with an average relative abundance of 24.18 +/- 14.75% in biofilms. Correlation analyses, permutation-based random forests, and ordinations identified nitrogen species, chemical oxygen demand (COD), and conductivity (Con) as principal correlates of diversity and AnAOB distribution. Operating-preference and tolerance-range analyses further indicated distinct environmental associations within the anammox guild across the investigated full-scale operating envelopes. Candidatus_Kuenenia was more frequently associated with higher nitrogen loading and COD, Candidatus_Jettenia was more strongly associated with Con, and Candidatus_Brocadia was more prevalent under lower COD, Con, and pH conditions. These findings provide an evidence base to support seed-biofilm selection for full-scale PN/A start-up, with targeted preference-guided matching and multi-genus AnAOB seeding as complementary strategies.

As a sustainable nitrogen removal technology, anammox granular sludge (AnGS) has been widely used in treating various wastewater due to its high nitrogen removal rate and resilience to hydraulic and loading disturbances. However, the relatively slow granulation rate of AnGS is a major bottleneck for its broader applications. Nitric oxide (NO), a key nitrogen-cycle intermediate and microbial signaling molecule, can directly serve as an electron acceptor for anammox bacteria, potentially enhancing metabolic activity. Furthermore, direct gas sparging provides shear force to AnGS that can facilitate biomass aggregation and granule formation. This study demonstrated that NO sparging could accelerate AnGS granulation by coupling metabolic stimulation with shear-induced aggregation. Through long-term cultivation and adaption, the NO-fed reactor (R1) achieved highly efficient and stable NO removal, with a maximum NO removal efficiency of 93.6%. In addition, NO sparging increased the proportions of large-size (∼38%) and medium-size (∼48%) granules in R1, compared to 30% and 25%, respectively, in the control group (R2) without NO sparging (2000 ppm). The AnGS in R1 exhibited enhanced structural stability and elevated metabolic activity. 'Candidatus Kuenenia' was identified as the most abundant in medium-size granules (with a relative abudance of 5.5%). Metatranscriptomic analysis further validated that NO sparging upregulated genes involved in nitrogen metabolisms (e.g., hdh, hzsB) and fundamental energy metabolism (e.g., cooS) within the granular sludge system. Overall, this study demonstrates that NO can be leveraged as a dual metabolic and physical control strategy to accelerate anammox granulation, providing new insights for the integrated management of gaseous and aqueous pollutants.

Partial nitritation/anammox (PN/A) enables energy-efficient nitrogen removal, yet the ecological drivers of stability and resilience in full-scale systems remain insufficiently characterized. Here, this study integrated 13-month performance monitoring with multiomics analyses to characterize spatial heterogeneity and recovery patterns following a dissolved oxygen (DO) shock in a full-scale plug-flow PN/A system. During stable operation, the system achieved a total nitrogen removal efficiency of 84.5 ± 4.8%, with the upstream zone accounting for 61.3 ± 10.1% of total removal. Along the decreasing nutrient gradient, community composition shifted markedly, and metatranscriptomes indicated spatially differentiated nitrogen-cycling transcription, with anammox-related transcripts enriched upstream. A 3-day DO shock (>0.5 mg/L) reduced the nitrogen removal rate (NRR) from 0.16 ± 0.03 to 0.07 ± 0.04 kg N/(m3·d). Although NRR recovered to baseline within three months, community structure and total bacterial 16S rRNA gene copies did not return to preshock levels, indicating a decoupling between microbiome and functional recovery. Across zones, higher bacterial diversity and nitrogen cycling functional redundancy were positively associated with faster recovery trends, and upstream microbiome stability was most representable of whole-system resilience. These findings provide measurable ecological indicators and an upstream-focused control target to strengthen full-scale PN/A operation under dynamic aeration disturbances.

Iron not only influences the activity of anammox bacteria (AnAOB) but also participates in complex Fe-N cycles. In this study, the advanced 15N isotope tracing method was set up to quantify the potential rates of full nitrogen metabolic pathways under different ferrous iron (Fe2+ and FeO) within two identical anammox granular reactors. The results indicated that both Fe2+ and FeO enhanced AnAOB activity. However, compared to Fe2+, which readily precipitates and oxidizes, the system supplemented with FeO exhibited higher Fe-N metabolic activity and greater metabolic diversity. This is attributed to the gradual release of Fe2+ from FeO, providing a sustainable and stable supply of Fe2+ for microorganisms. Furthermore, Subgroup_10 and Paludibaculum were identified as potential functional bacteria for feammox, while Denitratisoma, I-8 and Arenimonas were for NDFO. These results suggest that FeO addition is more beneficial for the construction of a Fe-N coupling system. Overall, this study enhances our understanding of how with exogenous iron can strengthen the anammox system, laying a theoretical foundation for the development of anammox-dominant Fe-N coupling systems.

The membrane-aerated biofilm reactor (MABR) is naturally suitable for partial nitritation-anammox (PN/A) because aerobic and anaerobic microorganisms can grow in different biofilm layers. However, suppressing nitrite-oxidizing bacteria (NOB) in the oxygen-rich inner MABR biofilms remains challenging, while anammox bacteria (AnAOB) in the outer layer are more vulnerable to many harsh treatments. This study demonstrated the strategy of applying hydroxylamine (NH2OH) to achieve stable PN/A in MABR. Over 250 days, long-term experiments showed that low dissolved oxygen (0.06-0.20 mg L-1) could not suppress NOB, while both continuous and intermittent 10 mg L-1 NH2OH addition not only achieved effective NOB suppression but also significantly promoted ammonia-oxidizing bacteria and AnAOB. Consequently, the nitrogen removal efficiency increased from 74.0 ± 1.1 % to 91.7 ± 1.6 %. Fundamentally, NH2OH addition rapidly changed the transcription levels of key functional genes in membrane biofilms, resulting in a significant decrease in nxrB transcription level by 68.6 ± 3.8 % and an increase in those of amoA and hzsB by 1835.8 ± 307.2 % and 314.2 ± 112.7 %, respectively. NH2OH addition resulted in the temporary accumulation of hydrazine and nitric oxide at low levels, which collectively contributed to NOB suppression. Particularly, Nitrospira, "Ca. Nitrotoga" and Nitrolancea (all three distinct NOB genera present in MABR biofilm) relative abundances decreased by 48.6 ± 7.5 %, 10.8 ± 2.5 %, and 75.0 ± 6.8 % respectively, which alleviated NOB adaptation risk. Therefore, NH2OH can be used to support NOB suppression in MABR.

Enhancing the activity of key enzymes has been recognized as an effective strategy to improve anammox performance. Neutral red (NR), a potent redox-active electron carrier, has been shown to boost various enzyme activities and microbial reaction rates. However, its potential to enhance anammox performance remains underexplored. This study aimed to investigate the effects of different NR concentrations on anammox nitrogen removal efficiency and gene transcription levels. The results revealed that anammox activity increased with NR doses in the lower concentration range (0.05-0.3 g L-1). The optimal dosage at 0.1 g L-1 significantly increased specific anammox activity (SAA) by 16.73 ± 2.68% (p ≤ 0.001), compared to the control without NR addition. Moreover, the total EPS concentration increased by 16.87 ± 1.20% (p ≤ 0.01). Conversely, NR concentrations exceeding the optimal range inhibited anammox activity. Metatranscriptomic analysis showed that appropriate NR supplementation upregulated the expression of cofactor modules related to electron transfer and functional genes (hdh and hzsB) involved in anammox nitrogen removal, thereby enhancing overall performance. Moreover, the mild oxidative stress induced by low NR doses was mitigated through the upregulation of antioxidant genes. In contrast, excessive NR (0.5-1.0 g L-1) led to an accumulation of reactive oxygen species (ROS) that overwhelmed the antioxidant defense system, resulting in impaired electron transfer and reduced metabolic activity. Specifically, when the NR concentration was increased to 1.0 g L-1, SAA decreased significantly by 26.45 ± 2.55% (p ≤ 0.001). These findings indicate that appropriately controlled NR supplementation can improve anammox activity, providing a promising strategy for rapid start-up and improved nitrogen removal in practical anammox systems.

Machine learning (ML) was employed to simultaneously predict nitrogen removal rate (NRR) and functional microbial abundance of single-stage partial nitrification and anammox (PNA) system. Shapley additive explanations (SHAP) and causal inference were used to analyze the impact of key factors and their optimal ranges. Artificial neural network (ANN) and extreme gradient boosting (XGBoost) have strong predictive abilities for NRR (R2 = 0.94) and functional microbial abundance (R2 ≥ 0.57), respectively. pH and free ammonia (FA) are important factors affecting NRR. To inhibit nitrite oxidizing bacteria (NOB), it was recommended that FA be maintained above 5 mg/L, while O2 be kept below 0.4 mg/L. Candidatus Brocadia-dominated sludge is recommended under low nitrogen (NH4+-Ninf < 200 mg/L) or O2 fluctuation environments, while Candidatus Kuenenia-dominated sludge is recommended under high nitrogen (NH4+-Ninf > 400 mg/L), low temperature (20-30°C), or pH fluctuations (7.4-8.4). These models provide prospects and references for the application of PNA technology.

A wide range of trace pollutants persist in aquatic environments, prompting concerns for public health and raising heightened attention. Therefore, the detection of toxic and nonbiodegradable pollutants in aquatic environments at ppm and ppb level is of great significance for measuring waste disposal and monitoring water purity. Among various reported sensors, metal-organic frameworks (MOFs), owning to their customizable structures and adjustable binding sites, have emerged as promising sensing materials in various sensing systems. Herein, this review explores the sensing mechanisms and design strategies for MOF-based fluorescent, electrochemical and photoelectrochemical sensors, followed by noteworthy cases demonstrating their sensitive determination of inorganic anions, metal ions, and organic matter in water. Notably, this review comprehensively summarizes, for the first time, the monitoring performance and sensing system of laboratory-prepared MOFs in real water samples. This comprehensive review may further guide the development of MOF-based sensors and promote their practical applications for sensing aqueous environmental pollutants.

The partial nitritation-anammox process based on a membrane-aerated biofilm reactor (MABR) faces several challenges, such as difficulty in suppressing nitrite-oxidizing bacteria (NOB), excessive effluent nitrate, and ineffective synergy between denitrification and anammox bacteria. Therefore, a novel partitioned granular sludge coupling with MABR (G-MABR) was constructed. The chemical oxygen demand (COD) and nitrogen removal efficiency were 88.8 f 1.8 %-92.6 f 1.2 % and 88.8 f 1.5 %-93.6 f 0.7%, respectively. The COD was mainly lowered in the lower granular sludge-zone, while nitrogen was removed in the upper MABR-zone. NOB was significantly suppressed in the MABR-zone due to competition for substrate with denitrifying bacteria and anammox bacteria. This partitioned configuration reduced the C/N ratio in the MABR-zone, thus facilitating autotrophic nitrogen removal. Both partial nitrification and denitrification provided nitrite for anammox bacteria in granular sludge, whereas partial nitrification mainly supplied nitrite to the anammox bacteria in membrane biofilms.

Rapid recognition and timely management of the emergent situation in wastewater treatment are crucial to maintaining the stable operation of anammox process. In this study, the feasibility of pH, conductivity (Cond) and oxidation reduction potential (ORP) profiles for monitoring and controlling anammox process for synthetic wastewater treatment was evaluated, and the practicability of the method was further verified by using real wastewater. The results showed that the characteristic values of these parameter profiles exhibited high accuracy and reproducibility in indicating the endpoint of the anammox reaction. Moreover, the positive correlations between TN removal and ΔpH, ΔCond and ΔORP were found. Nevertheless, only the slope of the Cond curve was found to be significantly linearly correlated with the specific anammox activity, which was further validated by the Haldane inhibition kinetic model, suggesting that the Cond curve can be used as an immediate feedback signal on whether anammox activity was inhibited. Overall, this study presents a fast, convenient and accurate strategy based on online real-time monitoring of instrument parameters, which was conducive to tracking the nitrogen removal process dynamics and performing the necessary operations in a timely manner, and to improving the stability of anammox process in wastewater treatment.

The partial nitritation anammox (PN/A) process has been widely used in wastewater treatment owing to its notable advantages, including a low aeration rate and the non-requirement of an additional carbon source. In practical implementation, the accumulation of nitrite affects the nitrogen-removal efficiency and the amount of N2O released during the PN/A process. By implementing wastewater reflux, the concentration of nitrite can be decreased, thereby achieving a balance between the nitrogen-removal efficiency and N2O release. In this study, the CANON process was conducted with varying reflux ratios in the range of 0 to 300% and ~300 mg/L ammonium in the influent. The highest removal efficiency of ammonium and total nitrogen (98.2±0.8 and 77.8±2.3%, respectively) could be achieved at a reflux ratio of 200%. Further, a reflux ratio of 200% led to the lowest N2O emission factor (2.21%), with 31.74% reduction in N2O emission compared to the process without refluxing. Additionally, the reactor at a reflux ratio of 200% presented the highest relative abundance of anaerobic ammonium-oxidizing bacteria (30.98%) and the lowest proportion of ammonium-oxidizing bacteria (9.57%). This study aimed to elucidate the impact of the reflux ratio on the nitrogen-removal efficiency of the CANON process and to theoretically explain the influence of different reflux ratios on N2O release. These findings provide a theoretical framework for enhancing the nitrogen-removal efficiency and mitigating carbon emissions in practical applications of the CANON process.

Anaerobic ammonium oxidation（Anammox） process is considered as an efficient, economical and environmentally friendly biological nitrogen removal technology, which is an ideal alternative to the conventional biological nitrogen removal process. However, anaerobic ammonia oxidizing bacteria（AnAOB）, as the functional microbes in Anammox process, has slow growth rate, resulting in a long reactor start-up time, which hinders the practical application and promotion of the process. Therefore, it is urgent to enhance the AnAOB activity and further reduce the reactor start-up time. Redox mediators（RMs）, as electron carriers, can enhance the activity and metabolic performance of AnAOB nitrogenconverting enzymes by accelerating the electron transfer process, thus improving the overall nitrogen removal effect. This paper discussed the role of RMs in enhancing the Anammox performance, introduced the characteristics and types of RMs, as well as their basic principles, reviewed the effects of RMs on the Anammox process in terms of Anammox performance, extracellular polymeric substances production,nitrogen-converting enzymes activity and functional bacterial abundance, as well as analyzed and summarized the potential mechanism, with a view to providing theoretical basis and reference value for future practical applications.

Nutrient availability significantly impacts microbial biosynthesis, cell growth, and cell cycle progression. In this study, a full-scale plug-flow partial nitritation/anammox (PN/A) system was used to investigate variations in the microbial community structure in both immobilized carriers and flocs, as well as a gradual decrease in nutrient availability from upstream to downstream. We found that reduced ammonia nitrogen (from 150.4 to 30.6 mg/L) and organic carbon (from 415.7 to 342.8 mg/L) availability significantly lowered microbial diversity and altered microbial communities in biofilms other than flocs from upstream to downstream. The abundance of all anammox bacteria increased by 1.97 times, from 3.25 x 1010 to 6.40 x 1010 copies per gram of wet sludge, in the biofilm core microbiome. Furthermore, from upstream to downstream, taxa with lower ribosomal RNA operon copy numbers were consistently enriched in both biofilm and floc communities, indicating that slow-growing microorganisms are more likely to be enriched in low-nutrient environments. Rare taxa with a relative abundance of less than 0.1% exhibited unique metabolic functions, including amino acid, carbohydrate, cofactor, and vitamin metabolisms, which was inferred by PICRUST2 and persisted across the nutrient gradient in both the biofilm and floc communities. Despite their low abundance, they may play important roles in mediating the stability and function of the PN/A system. Overall, the results demonstrate the impact of a naturally formed ammonia nitrogen and organic carbon gradient in a full-scale plug-flow PN/A installation on nutrient availability and its effects on microbial diversity, community composition, and microbial interactions, which expands our fundamental understanding of this energy-efficient and promising biotechnology for treating high-strength ammonium wastewater.

The partial nitritation/anammox (PN/A) process has been widely used in wastewater treatment owing to its notable advantages, including a low aeration rate and the non-requirement of an additional carbon source. In practical implementation, nitrite accumulation affects the nitrogen-removal efficiency and the amount of N2O released during the PN/A process. By implementing wastewater reflux, the nitrite concentration can be decreased, thereby achieving a balance between the nitrogen-removal efficiency and N2O release. This study conducted the CANON process with varying reflux ratios of 0 to 300 % and ~300 mg/L ammonium in the influent. The highest removal efficiency of ammonium and total nitrogen (98.2 ± 0.8 and 77.8 ± 2.3 %, respectively) could be achieved at a reflux ratio of 200 %. Further, a reflux ratio of 200 % led to the lowest N2O emission factor (2.21 %), with a 31.74 % reduction in N2O emission compared to the process without refluxing. Additionally, the reactor at a reflux ratio of 200 % presented the highest relative abundance of anaerobic ammonium-oxidizing bacteria (30.98 %) and the lowest proportion of ammonium-oxidizing bacteria (9.57 %). This study aimed to elucidate the impact of the reflux ratio on the nitrogen-removal efficiency of the CANON process and to theoretically explain the influence of different reflux ratios on N2O release. These findings provide a theoretical framework for enhancing the nitrogen-removal efficiency and mitigating carbon emissions in practical applications of the CANON process.

Nitrous oxide (N2O), a potent greenhouse gas, significantly contributes to the carbon footprint of wastewater treatment plants (WWTPs) and contributes significantly to global climate change and to the deterioration of the natural environment. Our understanding of N2O generation mechanisms has significantly improved in the last decade, but the development of effective N2O emission mitigation strategies has lagged owing to the complexity of parameter regulation, substandard monitoring activities, and inadequate policy criteria. Based on critically screened published studies on N2O control in full-scale WWTPs, this review elucidates N2O generation pathway identifications and emission mechanisms and summarizes the impact of N2O on the total carbon footprint of WWTPs. In particular, a linear relationship was established between N2O emission factors and total nitrogen removal efficiencies in WWTPs located in China. Promising N2O mitigation options were proposed, which focus on optimizing operating conditions and implementation of innovative treatment processes. Furthermore, the sustainable operation of WWTPs has been anticipated to convert WWTPs into absolute greenhouse gas reducers as a result of the refinement and improvement of on-site monitoring activities, mitigation mechanisms, regulation of operational parameters, modeling, and policies.

The understanding of microbial compositions in different dimensions is essential to achieve the successful design and operation of the partial nitritation/anammox (PN/A) process. This study investigated the microbial communities of different sludge morphologies and spatial distribution in the one-stage PN/A process of treating real coal to ethylene glycol (CtEG) wastewater at a pilot-scale integrated fixed-film activated sludge (IFAS) reactor. The results showed that ammonia-oxidizing bacteria (AOB) was mainly distributed in flocs (13.56 ± 3.16%), whereas anammox bacteria (AnAOB) was dominated in the biofilms (17.88 ± 8.05%). Furthermore, the dominant AnAOB genus in biofilms among the first three chambers was Candidatus Brocadia (6.46 ± 2.14% to 11.82 ± 6.33%), whereas it was unexpectedly transformed to Candidatus Kuenenia (9.47 ± 1.70%) and Candidatus Anammoxoglobus (8.56 ± 4.69%) in the last chamber. This demonstrated that the niche differentiation resulting from morphological (dissolved oxygen) and spatial heterogeneity (gradient distribution of nutrients and toxins) was the main reason for dominant bacterial distribution. Overall, this study presents more comprehensive information on the heterogeneous distribution and transformation of communities in PN/A processes, providing a theoretical basis for targeted culture and selection of microbial communities in practical engineering.

The regional distribution of antibiotic resistance genes has been caused by the use and preference of antibiotics. Not only environmental factors, but also the population movement associated with transportation development might have had a great impact, but yet less is known regarding this issue. This research study has investigated and reported that the high-speed railway train was a possible mobile reservoir of bacteria with antibiotic resistance, based on the occurrence, diversity, and abundance of antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs), and mobile gene elements (MGEs) in untreated train wastewater. High-throughput 16S rRNA sequencing analyses have indicated that opportunistic pathogens like Pseudomonas and Enterococcuss were the predominant bacteria in all samples, especially in cultivable multi-antibiotic resistant bacteria. The further isolated Enterococcus faecalis and Enterococcus faecium exhibited multi-antibiotic resistance ability, potentially being an indicator for disinfection proficiency. Positive correlations amongst ARGs and MGEs were observed, such as between intI1 and tetW, tetA, blaTEM, among Tn916/154 and mefA/F, qnrS, implying a broad dissemination of multi-ARGs during transportation. The study findings suggested that the high-speed railway train wastewater encompassed highly abundant antibiotic-resistant pathogens, and the wastewater discharge without effective treatment may pose severe hazards to human health and ecosystem safety.

The completely autotrophic nitrogen-removal over nitrite (CANON) process has merits in energy saving and consumption reducing, thus being considered as an attractive alternative over the common denitrification technology. In this study, the effects of three common heavy metals (Cu2+, Zn2+ and Mn2+) in wastewater to the CANON process were evaluated comprehensively. A central composite design with response surface methodology was utilized to investigate the joint effect of these three metal ions on the nitrogen removal performance of CANON process. In accordance with the determined optimal dosage in batch tests, four bioreactors were established with different amounts of heavy metal dosage in long-term operation, which determined the optimal concentrations for Cu2+, Zn2+ and Mn2+ to be 0.25, 0.81 and 1.00 mg/L, respectively. However, the optimal dosing level determined in batch tests showed no promotion during long-term experiment. This indicated that the actual concentration of heavy metals in bioreactors during long-term operation could be higher than expectation, leading to the difference between short-term tests and long-term experiment. The distribution of metal ions revealed that Mn2+ was mainly absorbed in anammox bacteria cells while Cu2+ and Zn2+ were mostly identified inside AOB cells. Moreover, the addition of heavy metals consistently showed positive effects for the relative abundance of AOB, while only a low level of dosage could promote the abundance of anammox bacteria. Furthermore, a mathematical model was established to simulate the CANON system considering the impacts of heavy metals, which was calibrated and validated using independent dataset in this study.

One‐stage partial nitritation/anammox (PN/A) has been proposed as a sustainable method for removing nitrogen from various wastewater. However, the activities of ammonium‐oxidizing bacteria (AOB) and anammox bacteria are often inhibited by the exposure to salinity, thereby hindering their wide application in treating industrial wastewater with high salinity. This study reports that the addition of glycine betaine (GB), which is a compatible solute, could alleviate the inhibitory effects of salinity on both AOB and anammox, thereby improving nitrogen removal performance in a one‐stage PN/A system. Short‐term tests showed that with an addition of GB higher than 1 mM, the activity of AOB and anammox under salinity of 30 g/L could be increased by at least 45% and 51%, respectively. The half‐inhibitory concentration of AOB and anammox rose with increasing GB concentration, with 1 mM GB being the optimal cost‐effective dosage. Long‐term experiments also demonstrated that 1 mM GB addition could enhance nitrogen removal performance and shorten recovery time by 42.9% under a salinity stress of 30 g/L. Collectively, GB addition was found to be a feasible and effective strategy to the counteract adverse effects of salinity on PN/A process.

This study aims to compare the effects of different Fe stress on anammox (anaerobic ammonium oxidation) process, therefore seven identical reactors were operated under different Fe(II)/Fe(III) concentrations. After 38 days of operation, the anammox activity was highest (10.49 ± 0.41 mg-TN/(g-VSS·h)) under conditions of 5 mg/L-Fe(II), while under 30 mg/L-Fe(III) displayed severe inhibition. The results showed that continuous addition of 30 mg/L-Fe(III) would damage the composition of EPS (extracellular polymeric substances) and make anammox bacteria more sensitive to environmental stress. While high Fe(II) concentrations could result in precipitates encasing granular sludge, affecting substrate utilization. Moreover, the results of ΔNO3--N/ΔNH4+-N indicated that Fe(II)-dependent nitrate reduction was induced in reactors added with Fe(II). OM27_clade and norank_f__Burkholderiaceae might be candidates for this process according to the correlation of genera and functional genes (based on the PICRUSt 2 functional prediction). Overall, this research is expected to provide new ideas to the effects of Fe(II)/Fe(III) on anammox and to the practical application of coupled system based on anammox in wastewater treatment.