The regulation of the accumulation of NO2--N in the partial nitritation system with low ammonia concentration by enhanced light is of great significance for coupling with anammox to form a completely autotrophic biological nitrogen removal process. In this study, the effects of different light wavelengths on the nitrogen conversion performance and functional microbial community in partial nitritation system were investigated. The results indicated that all wavelengths of light can promote the conversion of NH4+-N, among which blue light had the most obvious promoting effect. Both blue light and UVA had stable inhibitory effects on the activity of nitrite-oxidizing bacteria (NOB), leading to an increase in the accumulation of NO2--N. Microorganisms alleviated the oxidative stress exerted by different light wavelengths on the system by increasing the secretion of extracellular polymeric substances (EPS) and the activity of antioxidant enzymes. Notably, both EPS concentration and catalase activity were found to be highest in the blue light treatment. The relative abundances of the nitrosifying bacterial genera Ellin6067 and Nitrosomonas were enhanced by all light wavelengths, whereas the nitrifying bacterial genus Nitrospira constituted only 0.14% in the blue light reactor. The relative abundances of the DNA damage repair gene unrA, the electron transport chain functional gene coxA, and the photosensitive receptor protein gene bluF in the partial nitritation system were found to be upregulated by blue light. Blue light enhanced the stable operation of the partial nitritation system and can be used as the optimal light for regulating NO2--N accumulation in the algal-bacterial symbiotic system.

The current phosphorus recovery process in wastewater treatment plants faces several challenges, including process complexity and low recovery efficiency. This study examined applying the sequencing batch biofilm reactor (SBBR) and fluidized bed crystallization (FBC) processes to achieve high-P enrichment. Subsequently, the key environmental factors for phosphorus removal and recovery by the SBBR process were investigated. The concept of phosphorus storage was introduced, and a novel phosphorus harvesting control strategy was developed. The redox conditions in the phosphorus-rich solution generated by the SBBR process are compatible with those required for vivianite formation in the FBC process. The two processes were successfully integrated, enabling direct vivianite formation in the liquid phase, which led to stable phosphorus crystallization recovery efficiency and high purity of vivianite. This study offers both technical and theoretical foundations for achieving efficient phosphorus recovery from mainstream processes as well as enhanced vivianite recovery.

N,N-dimethylformamide (DMF) is a common toxic industrial solvent. It often enters with low C/N ratio and high NH4+-N wastewater, posing a dual stress of organic matter and toxicity to Anammox process. However, the long-term impact mechanisms and microbial response patterns remain unclear. The effects of gradient DMF stress on the nitrogen removal performance, nitrogen transformation pathways, cellular physiological stress, microbial community succession, and functional gene expression of the Anammox system were investigated, and the system's recovery potential were evaluated. The results showed that low concentrations of DMF, acting as organic matter, enhanced the nitrogen removal performance of the Anammox system. However, DMF was not readily biodegradable by the associated heterotrophic bacteria, and its accumulation exerted a significant toxic effect on Anammox bacteria with a half-inhibition concentration (IC50) of 10.26 mg/L. Low concentrations of DMF slightly stimulated exopolymer secretion and antioxidant enzyme activity; high concentrations of DMF triggered a reactive oxygen species burst, inhibited superoxide dismutase activity, disrupted cell membrane integrity, and reduced ATP content, leading to the collapse of energy metabolism. As the DMF concentration increased, the relative abundance of Candidatus_Brocadia and Candidatus_Jettenia first increased and then decreased, while the relative abundance of Candidatus_Kuenenia decreased continuously. The core genes of AnAOB (hzsA/B/C, hdh) and the autotrophic carbon fixation pathway were downregulated, whereas heterotrophic denitrification genes were upregulated, shifting the nitrogen removal pathway from autotrophic processes to heterotrophic denitrification. The denitrification performance, community structure, and functional genes of the Anammox system partially recovered after the cessation of DMF addition.

Municipal wastewater holds substantial potential for phosphorus recovery, and mainstream biofilm phosphorus recovery has emerged as a promising strategy. However, this process often requires upstream removal of organics and ammonia to create conditions favorable for polyphosphate-accumulating organisms (PAOs). Therefore, developing a front-end process that can achieve advanced nitrogen removal while maximizing liquid-phase phosphorus retention is critical. Unlike conventional activated sludge (CAS) systems, in which high sludge production leads to significant phosphorus loss, this study evaluated a pilot-scale integrated fixed-film activated sludge (IFAS) system as a front-end configuration to enhance phosphorus retention for downstream recovery. Compared with CAS, IFAS increased phosphorus retention efficiency to 78.6%, representing a 1.7 times improvement, while maintaining 83.6% nitrogen removal under low-temperature conditions. Mass balance analysis showed that IFAS reduced both sludge production and sludge phosphorus content, thereby weakening sludge-associated phosphorus removal and increasing phosphorus in the liquid phase. Microbial and functional analyses further indicated that canonical EBPR-associated PAOs were not identified as dominant taxa in either system, whereas IFAS favored a denitrifying community with broader metabolic adaptability. Intracellular polymer and functional gene patterns suggested that carbon was preferentially stored and subsequently mobilized for endogenous denitrification rather than phosphorus uptake. These results demonstrate the technical potential of IFAS as an energy-efficient and environmentally friendly strategy for upgrading WWTPs toward resource recovery.

Thiourea can significantly inhibit the aerobic ammonia oxidation process, yet it can also serve as a multi‑electron donor in denitrification. Understanding the perturbational influence of thiourea on nitrogen removal in anaerobic ammonium oxidation (Anammox) systems is crucial for its application in treating high NH4+-N wastewater containing thiourea. Therefore, the present inquiry investigated its effects on nitrogen degradation pathways and microbial metabolism in Anammox systems. The findings reveal that when the influent thiourea concentration remains below 139.7 mg/L, both sulfur-oxidizing autotrophic denitrifiers (SOB) and their heterotrophic denitrifiers residing in the Anammox reactor fully metabolize this compound. They use NO2--N/NO3--N as electron acceptors, converting thiourea into SO42- and NH4+-N. The activity of Anammox bacteria (AnAOB) was not significantly affected. However, the activity of the associated ammonia-oxidizing bacteria (AOB) was significantly inhibited, and nitrite-oxidizing bacteria (NOB) took over to mitigate the dissolved oxygen toxicity in the influent. The denitrifying microbial community in the Anammox system, originally dominated by AnAOB, shifted toward a multifunctional denitrifying community dominated by heterotrophic denitrifiers, SOB, and AnAOB. As thiourea concentrations increase, denitrifying microorganisms struggle to grow synergistically, leading to thiourea accumulation. Oxidative stress in the system rises, and catalase (CAT) activity is inhibited, resulting in cellular damage. Accumulated thiourea reduced the relative abundances of core genes hzs and hdh in AnAOB and sqr in SOB. Although the relative abundances of fccA/fccB in SOB and nirS in denitrifiers were upregulated, these potential functional changes could not counteract thiourea toxicity. AnAOB activity is restricted and cannot be restored in the short-term. Thiobacillus and Candidatus_Brocadia, as functional genera of SOB and AnAOB, respectively, exhibit abundance that varies with changes in nitrogen-sulfur transformation performance. Enhancing sulfur-autotrophic and heterotrophic denitrification to prevent thiourea accumulation is a key strategy for alleviating the denitrification stress imposed on the Anammox system.

This study systematically investigated the phosphorus (P) adsorption and release behaviors of biofilm extracellular polymeric substances (EPS) in a sequencing batch biofilm reactor (SBBR). Batch experiments, enzyme activity assays, P forms, and spectral characterizations (3D-EEM, FTIR, XPS) were integrated to elucidate the independent role of EPS in biofilm P metabolism. The results quantified the independent P adsorption and release capacities of EPS as 1.68 mg/g and 2.46 mg/g, respectively. Different from cell-dependent biological P metabolism, EPS-mediated P transformation was insensitive to dissolved oxygen variation and carbon addition, and was mainly dominated by physicochemical adsorption. EPS-derived orthophosphate (Orth-P) and polyphosphate (Poly-P) contributed over 67% and less than 12% to the total Orth-P and Poly-P metabolism of biofilms, respectively. Although polyphosphate kinase (PPK) and polyphosphate hydrolase (PPX) activities were detected in EPS, the lack of effective carbon utilization capacity restricted their involvement in Poly-P transformation. Metal-mediated complexation served as the core immobilization pathway, in which Ca2⁺ and Mg2⁺ bound with phosphate groups, as well as carboxyl and amino functional groups of tryptophan- and tyrosine-rich proteins in EPS. Beyond the inherent physicochemical adsorption properties of EPS, microbial cells further regulated EPS content by aerobic biosynthesis and anaerobic biodegradation. Such microbial regulation synergistically optimized the P adsorption-release performance of EPS, verifying that EPS acts as the dominant functional component responsible for P transformation in biofilm systems. This study clarifies the intrinsic mechanisms underlying EPS-mediated P adsorption and release, and provides a theoretical basis for the development of low-carbon and high-efficiency P recovery technologies.

As a primary pathway for mitigating the global phosphorus pollution crisis, widely used conventional activated sludge phosphorus recovery processes in municipal wastewater treatment plants (WWTPs) are facing increasing challenges due to continuously declining influent carbon and phosphorus concentrations. This review systematically compares activated sludge processes with emerging biofilm processes, highlighting a conceptual shift from the biomass growth driven enhanced biological phosphorus removal theory to the novel DAM theory centered on microbial metabolism in biofilms. The DAM theory conceptualizes the biofilm as a reusable, tunable phosphorus reservoir, where periodic accumulation in biofilms promotes liquid phase recovery even under low carbon source conditions. In terms of mechanism, extracellular polymeric substances are examined for their roles in phosphorus transfer, storage, and transformation. Microbial interactions are analyzed, with particular emphasis on the functional roles of glycogen-accumulating organisms in different processes and their implications for system stability. Ultimately, this review proposes a novel phosphorus recovery paradigm based on biofilm processes to circumvent the operational bottlenecks associated with insufficient influent carbon in WWTPs, providing a theoretical framework and technical guidance for sustainable nutrient management.

The process of preparing a flocculant using concentrated organic matter has demonstrated effective results in enriching organic matter in municipal wastewater. The basic preparation process involves a combination of hydrothermal treatment and chemical modification. To enhance the enrichment effect of organic matter, the optimal hydrothermal conditions and their influence on the characteristics of liquid products and coarse flocculant were investigated. The results indicated that liquid products obtained at 140 degrees C for 30 min yielded a high concentration of active groups and exhibited a significantly higher molecular weight, making them suitable as precursors for the coarse flocculant. When the dosage of the coarse flocculant was set at 10 mg/L, the turbidity removal rate of the kaolin suspension reached 93 %. In the treatment of municipal domestic sewage, a COD removal rate of approximately 62 % was achieved at a dosage of 10 mg/L. Within the experimental range, a decrease in hydrothermal time and temperature favored the production of more reactive groups from the dissolved polymeric substances (DPS), thereby enhancing the charge density of the coarse flocculant. The intrinsic viscosity of the coarse flocculant was influenced by both the number of reactive groups and the molecular weight of DPS. Specifically, obtaining a molecular weight distribution of 8-9.5 kDa favored the production of a coarse flocculant with high characteristic viscosities.

The simultaneous nitrification-denitrification (SND) process has been widely studied for cost-effective nitrogen (N) removal. However, integrating SND with the phosphorus (P) enrichment process in the one-stage system remains challenging due to the conflicting conditions (low vs. high DO) and design principles. Herein, we demonstrated this integration in a single biofilm reactor under alternating anaerobic/aerobic conditions, achieving 94 % of NH4+-N and 93 % of TN removal under high-DO operation (5-6 mg/L) and obtaining 75.7 mg/L P-enriched solution in the anaerobic stage. The dynamic test in situ and stoichiometric evaluations showed that SND was the main N removal pathway at high DO conditions, which was rate-limited by the nitridation process. The ex-situ denitrification batch tests revealed that NO3--N was the primary electron acceptor in the aerobic denitrification process, which was mainly attributed to the DGAOs using the intracellular carbon source, as well as the DPAOs. The latter cooperated with the APAOs and contributed to P removal. The macro-genome sequencing further revealed that DGAOs (Pseudoxanthomonas and Sulfuritalea et al.) were the dominant denitrifiers in the P enrichment system due to a series of denitrifying genes' high expression. That implied the DGAOs have a higher tolerance to high DO concentration, which was crucial to performing SND in the P enrichment system. These findings shed light on the positive roles of DGAOs in the denitrification process and lay an experimental basis for developing a new feasible technical route for simultaneous N removal and P enrichment in a single-stage system.

Phosphorus (P) recovery in municipal sewage from sludge via enhanced biological P removal process is an effective solution to P shortage. However, low influent P concentration (Pinfluent) makes it difficult to obtain highconcentration P-enriched liquid from sludge. This study examined applying a sequencing batch biofilm reactor (SBBR) to remove and enrich low-concentration P from municipal wastewater concurrently. The results demonstrated that the SBBR could efficiently remove and enrich low-concentration P with low carbon consumption. When the phosphate (PO4 3--P) concentration in the influent was 2.5 mg/L, the aerobic effluent met discharge standards. After anaerobic recycling enrichment, the average PO4 3--P concentration of the recovered solution reached 75.8 mg/L, with an enrichment factor of 30.3 times. The average total P recovery rate was 72.8 %, with a carbon consumption of 37.3 mg-P/mg-COD. This is mainly attributed to the recycling operation mode of the P-enriched recovery solution. Even when the Pinfluent is low, it can create conditions for the biofilm to be exposed to a high-P environment. This situation ensures the P absorption by extracellular polymeric substances, enabling the P storage capacity reached 55 mg-P/g-MLSS, and the P mainly exists in the form of PO4 3--P, which is the key to achieving high-efficiency P enrichment in the recovery solution under low carbon consumption. In addition, the decrease in Pinfluent did not alter the dominant role of polyphosphate-accumulating organisms in the biofilm. This study provides theoretical support and a technical approach for the simultaneous removal and enrichment of low-concentration phosphate in municipal wastewater.

Vivianite is a high-value product for phosphorus (P) recovery from municipal wastewater. To obtain vivianite with higher crystallization ratio and larger particle size, excessive iron source was often dosed during the crystallization process, which not only increases the cost of reagents but also leads to excessive iron concentration in the effluent. In this study, by analyzing the influence of pH and Fe/P ratio on the saturation index value, a "cyclic crystallization mode" was proposed to reduce the iron source dosage in fluidized bed crystallization process to realize the phosphorus recovery from municipal wastewater. With which, the iron dosage was reduced by 20 %, the d50 was increased by 94 %, and the hydraulic retention time was reduced by 0.5 h. This study provides theoretical basis and method to reduce the iron source cost in the vivianite crystallization technology for the recovery of phosphorus.

Realizing the full-scale treatment of aged landfill leachate with high NH4+-N, low carbon-to-nitrogen (C/N) ratio and containing a large amount of refractory organic matter is an urgent problem to be solved. Constructed a partial denitrification/anammox (PD/A)-partial nitrification/anammox (PN/A)-alternating electrochemical oxidation/denitrification process to explore the feasibility of efficiently utilizing all organic carbon source of raw water to achieve aged landfill leachate full-scale treatment. The results show that the PN/A system could efficiently remove NH4+-N from wastewater without relying on carbon sources. Meanwhile, it utilizes biodegradable organic matter (BOD) in raw water to convert the NO3--N produced into NO2--N for reuse by anammox. When BOD/TN is 0.5, the total nitrogen (TN) removal rate of PD/A-PN/A system reaches >90 %. Under the conditions of a pH of 7 and a current density of 40 mA/cm(2), the BOD5/COD ratio of refractory organic matter was further increased through electrochemical oxidation, achieving deep denitrification removal of nitrogen from PD/A-PN/A effluent, and reducing COD and TN to approximately 100 and 20 mg/L, respectively. The low-consumption and high-efficiency treatment of aged landfill leachate is achieved without the addition of organic matter or the discharge of concentrated liquid.

Thiourea (CH4N2S) is a widely used industrial reagent and is frequently detected in both sewage and industrial wastewater. However, treating thiourea-containing wastewater remains challenging due to its toxicity, high ammonium concentration, and low C/N ratio. In this study, a novel integrated autotrophic-heterotrophic denitrification (IAHD)- completely autotrophic nitrogen removal over nitrite (CANON) process was developed. The degradation pathway of toxic compounds, nitrogen, and sulfur release and transformation, as well as variations in functional genes were comprehensively examined. The results show that by incorporating an IAHD unit, prior to CANON, toxic thiourea was effectively degraded by the recycled nitrate from CANON. The released sulfur and organic carbon served as electron donors facilitating efficient NO3--N reduction. The optimal thiourea/NO3--N ratio for IAHD operation was determined to be 4:1 (m:m), achieving NO3- and thiourea removal efficiencies of 90 % and 99 %, respectively. Additionally, NH4+-N and SO42--S concentrations increased by 199.9 mg/L and 201.9 mg/L, respectively. Approximately 53.3 % of thiourea was converted into high-molecular-weight biological metabolites in the IAHD unit, which were subsequently and completely degraded in the CANON unit, where a robust nitrite-shunt and anammox process occurred. 16S rRNA amplicon sequencing revealed that Thiobacillus (with a relative abundance of 39.9 %) was the dominant genera in the IAHD unit, followed by Arenimonas (10.8 %) and norank_o_1013-28-CG33 (12.4 %), indicating that sulfur autotrophic denitrification was the primary pathway for thiourea degradation. Metagenomic analysis further confirmed that thiourea, acting as an electron donor, stimulated the expression of key functional genes involved in denitrification, sulfur oxidation, dissimilatory nitrate reduction, hydrolytic oxidation, and amino acid synthesis and transport pathways. These processes contributed to the active biological transformation of carbon, nitrogen and sulfur in the IAHD unit. This study demonstrates that implementing a prior autotrophic-heterotrophic denitrification unit effectively degrades toxic thiourea, thereby ensuring the subsequent nitrogen removal performance of CANON. This approach offers a new paradigm for the treatment of thiourea-containing wastewater, promoting a more efficient and low-carbon process.

Current mainstream sequencing batch biofilm reactor (SBBR) processes has the problem of that the concentration of phosphate recovery liquid (PRL) is difficult to be improved after reaching the highest. This study found that only the three-step carbon dosing strategy could increase the concentration of PRL after comparing the regulation of the dissolved oxygen (DO), hydraulic retention time (HRT), pH, P concentration in influent and temperature. After treatment with the three-step carbon dosing strategy, PRL concentration were improved by 10.23 mg/L with shorter harvest cycle and lower carbon source dosing. The COD/Prel decreased by 28.4 % in each phosphate harvest cycle, indicating that the optimal regulation strategy improved the economic benefits of phosphorus recovery by SBBR. The carbon source stimulation of the optimal regulatory strategy not only activated the enzyme activities of phosphate kinase (PPK) and exopolyphosphatase (PPX) to enhance the Poly-P metabolism of cells, but also reduced the extracellular polymeric substances (EPS) content in the anaerobic phase to facilitate the Orth-P desorption and apatite phosphorus (AP) shedding from EPS, so as to realize the simultaneous enhancement of phosphorus absorption and release of cells and EPS. This study explored the mechanism of optimal carbon source regulation strategy to improve the efficiency of phosphorus recovery, and provided regulatory guidance for the economical and efficient phosphorus recovery of biofilm process.

Red anaerobic ammonia oxidation (Anammox) granular sludge (AnGS) has been reported in successfully operating Anammox systems, and its color is associated with sludge activity. However, in long-term operating systems, AnGS exhibits different sensory colors, physical structures, community structures, and denitrification performance, but the relationship between them has not yet been elucidated. The AnGS of the Anammox system, which has been in operation for more than a decade, can be divided into two main categories: red and white. The specific Anammox activity (SAA) in conventional red AnGS increased continuously as the particle size increased from <0.51 mm to 6.02 ± 0.84 mm. The SAA of white AnGS were slightly lower than those of red AnGS with similarly-size granules but significantly higher than AnGS with smaller red granules. Compared with red AnGS, the extracellular polymeric substances of white AnGS were significantly reduced, mainly due to the higher intracellular iron content, resulting in lower heme c concentration. Thus, heme c may prove not to be an evaluative tool for measuring Anammox activity. Red and white AnGS, whether through self-aggregation or adsorption by hydroxyl apatite and other carriers, will face the fate of internal voids during particle size growth. White AnGS exhibited a more complex microbial community than red AnGS. Candidatus Brocadia was abundant in red AnGS and the abundance increased with increasing granule size. Candidatus Kuenenia and Candidatus Jettenia made significant contributions to denitrification in white AnGS. This study provides a new perspective on particle selection for anammox engineering applications.

An innovative kind of coarse flocculant employs captured organics from wastewater as a raw material was proposed in our previous research, which is subsequently utilized to capture organics from wastewater in return. Coarse flocculants with different molecular weight, zeta potential and charge density were produced by adjusting modification condition. They were characterized through various methods, while the flocculation effect and structure-activity relationship of the coarse flocculant was also evaluated. The flocculation performance is primarily determined by the charge properties. The molecular weight significantly influences the adsorption-bridging and catching-sweeping effect, thereby affecting floc size and stability. The charge density has important impact on the colloidal particle instability, which in turn impacts floc size. The flocculation mechanisms were discussed based on the structure-activity relationship. Notably, when zeta potential ranges from 7 +/- 0.02-9.5 +/- 0.02 mV and charge density exceeds 1.35 +/- 0.01 mmol & sdot;L-1, the charge neutralization effect is maximized. Large floc particle helps enhance organic matter capturing. The results help to guide the development of the novel flocculant.

This study investigated the impact of different operational sequences on phosphorus removal and enrichment in biofilm phosphorus enrichment system. The research com-pared two distinct operational modes, analyzing phosphorus uptake and release characteristics in cells and extracellular polymeric substances (EPS) over a single cycle, while also examining microbial community composition and associated functional genes. After long-term acclimation, the Ae/An system achieved higher phosphorus concentration (120 mg/L) than the An/Ae system (65 mg/L). However, the An/Ae system showed stronger phosphorus uptake and release capabilities due to higher phosphorus load during the aerobic phase. In both systems, Mg-P and Ca-P dominated in cells and EPS. Compared to the Ae/An system, the An/Ae system stored phosphorus mainly in EPS, with higher orthophosphate content. However, EPS-associated phosphorus is more easily released, explaining the An/Ae system's higher aerobic phosphorus load but lower overall storage capacity. Microbial analysis revealed higher abundance of phosphorus accumulating organisms (PAOs) in the An/Ae system (25.99 % vs. 19.69 %), while glycogen accumulating organisms (GAOs) showed the opposite trend. Candidatus Competibacter was abundant in both systems and correlated with phosphorus metabolism genes. The An/Ae system expressed the pst system more, whereas the Ae/An favored the pit system, suggesting that transfer system variations affect enrichment solution concentration. Lower expression of polyphosphate kinase (ppk1) in the An/Ae system may explain its unsatisfied phosphorus enrichment performance. Mantel analysis confirmed connections among environmental factors, kinetic parameters, phosphorus metabolism genes, and phosphorus morphology in EPS, demonstrating their combined influence on enrichment solution concentration.

Facing the interference of nitrite-oxidizing bacteria (NOB) and the challenge of low-organic matter consumption in the autotrophic nitrogen removal process of mainstream municipal wastewater, a partial denitrification Anammox (PDA)-partial nitritation/Anammox (PN/A) process with independent sludge zones was constructed. Activated sludge model (ASM) simulation based on the BioWIN platform, the feasibility of achieving stable operation of PN by synergistic regulation of sludge retention time (SRT)- dissolved oxygen (DO) under the mainstream environment and the response mechanism of organic carbon stress and microorganisms in the autotrophic biological nitrogen removal process were explored. Results demonstrated that coupled low-DO and short-SRT effectively suppressed NOB activity, achieving stable NO2- -N accumulation rates exceeding 75 % in PN zones under 15-25 degrees C and high nitrogen loads ranging from 0.4 to 0.8 kg/(m3 center dot d). The effluent total nitrogen (TN) of PDA-PN/A system was lower than 4.5 mg/L, and the TN removal rate reaches 90 %, when the COD/N was 1. Independent sludge partitioning significantly enhanced carbon utilization efficiency and reduced functional/ non-functional microbial competition. PN zone exhibited stable NOB suppression with upregulated nitritation genes (pmoA-amoA, hao). In PDA zone, Thauera (1.68 %) and Candidatus Kuenenia (9.68 %) formed metabolic synergy, showing 542-670 % increased hdh and hzs expression compared to a COD/N of 0.5, alongside coupregulation of narG and nirK. This integrated process achieves efficient nitrogen removal under ultralowcarbon conditions (COD/N of 1), non-temperature-controlled environments, and low-ammonia scenarios, providing a sustainable solution for mainstream municipal wastewater treatment.

Under low temperature and low ammonia nitrogen conditions, the partial nitritation/Anammox (PN/A) process faces challenges such as unstable partial nitritation and insufficient nitrogen removal. Here, a novel two-stage PN/A reactor was developed to investigate nitrite-oxidizing bacteria (NOB) inhibition and nitrogen removal performance in mainstream wastewater treatment. This investigation was conducted with multi-strategy regulation under a high influent ammonia nitrogen loading rate (ANLR) of 0.8-1.3 kg N/(m3·d). The results indicated that when the water temperature was 26-30 °C, combining low DO levels of 0.2 mg/L with a SRT of 25 days achieved an ammonia nitrogen conversion efficiency (ACE) of 65.8 ± 2.1 % and nitrite accumulation efficiency (NiAE) of 80.4 ± 1.8 %. However, when the water temperature dropped below 20 °C, relying solely on low DO levels (0.35-0.40 mg/L) and a shorter SRT (15 days) was ineffective due to the proliferation of NOB. After combining the low-nitrite strategy to selectively inhibit NOB activity, the ACE and NiAE reached approximately 47.4 ± 8.3 % and 79.5 ± 4.6 %, respectively. Finally, in the two-stage PN/A system, the integrated process achieved a nitrogen removal efficiency of 92.8 ± 1.9 % and a nitrogen removal rate of 0.99 ± 0.03 kg/(m3·d) below 20 °C. Microbial community analysis revealed that Bacillus and Nitrosomonas were the dominant functional microorganisms in the PN zone, which confirmed the synergistic nitrogen removal by the denitrification and Anammox pathway. This study offers critical insights for advancing the practical application of the PN/A process in treating mainstream wastewater under high-ANLR and low-temperature conditions.