Microecology of aerobic denitrification system construction driven by cyclic stress of sulfamethoxazole.

The construction of aerobic denitrification (AD) systems in an antibiotic-stressed environment is a serious challenge. This study investigated strategy of cyclic stress with concentration gradient (5-30 mg/L) of sulfamethoxazole (SMX) in a sequencing batch reactor (SBR), to achieve operation of AD. Total nitrogen removal efficiency of system increased from about 10 % to 95 %. Original response of abundant-rare genera to antibiotics was changed by SMX stress, particularly conditionally rare or abundant taxa (CRAT). AD process depends on synergistic effect of heterotrophic nitrifying aerobic denitrification bacteria (Paracoccus, Thauera, Hypomicrobium, etc). AmoABC, napA, and nirK were functionally co-expressed with multiple antibiotic resistance genes (ARGs) (acrR, ereAB, and mdtO), facilitating AD process. ARGs and TCA cycling synergistically enhance the antioxidant and electron transport capacities of AD process. Antibiotic efflux pump mechanism played an important role in operation of AD. The study provides strong support for regulating activated sludge to achieve in situ AD function.

Stable continuous flow CANDAN process transitioning from anammox UASB reactor by facilitating indigenous nitrite-producing denitrification community.

The emerging nitrogen removal process known as CANDAN (Complete Ammonium and Nitrate removal via Denitratation-Anammox over Nitrite) has been developed in Sequencing Batch Reactors (SBRs). Yet, starting up and maintaining stability in continuous-flow reactors remain challenging. This study explores the feasibility of transitioning the CANDAN process from an anammox-dominated process by introducing appropriate external organics to facilitate indigenous nitrite-producing denitrification community in an Upflow Anaerobic Sludge Blanket (UASB) reactor. 150-day operation results indicate that under feeding rates of domestic wastewater at 0.54 L/h and nitrate-containing wastewater at 1.08 L/h, excellent N removal was achieved, with effluent TN below 10.0 mg N/L. Adding external sodium acetate at a COD/NO3--N = 2.0 triggered denitratation, ex-situ denitrification activity tests showed increased nitrite production rates, maintaining the nitrate-to-nitrite transformation ratio (NTR) above 90 %. Consequently, anammox activity was consistently maintained, dominating Total Nitrogen (TN) removal with a contribution as high as 78.3 ± 8.0 %. Anammox functional bacteria, Brocadia and Kuenenia were identified and showed no decrease throughout the operation, indicating the robustness of the anammox process. Notably, the troublesome of sludge flotation, did not occur, also contributing to sustained outstanding performance. In conclusion, this study advances our understanding of the synergistic interplay between anammox and denitrifying bacteria in the Anammox-UASB system, offering technical insights for establishing a stable continuous-flow CANDAN process for simultaneous ammonium and nitrate removal.

Redox mediator chlorophyll accelerates low-temperature biological denitrification with responses of extracellular polymers and changes in microbial community composition.

Low temperatures limit the denitrification wastewater in activated sludge systems, but this can be mitigated by addition of redox mediators (RMs). Here, the effects of chlorophyll (Chl), 1,2-naphthoquinone-4-sulfonic acid (NQS), humic acid (HA), and riboflavin (RF), each tested at three concentrations, were compared for denitrification performance at low temperature, by monitoring the produced extracellular polymeric substances (EPS), and characterizing microbial communities and their metabolic potential. Chl increased the denitrification rate most, namely 4.12-fold compared to the control, followed by NQS (2.62-fold increase) and HA (1.35-fold increase), but RF had an inhibitory effect. Chl promoted the secretion of tryptophan-like and tyrosine-like proteins in the EPS and aided the conversion of protein from tightly bound EPS into loosely bound EPS, which improved the material transfer efficiency. NQS, HA, and RF also altered the EPS components. The four RMs affected the microbial community structure, whereby both conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT) were key taxa. Among them, CRAT members interacted most with the other taxa. Chl promoted Flavobacterium enrichment in low-temperature activated sludge systems. In addition, Chl promoted the abundance of nitrate reduction genes narGHI and napAB and of nitrite reduction genes nirKS, norBC, and nosZ. Moreover, Chl increased abundance of genes involved in acetate metabolism and in the TCA cycle, thereby improving carbon source utilization. This study increases our understanding of the enhancement of low-temperature activated sludge by RMs, and demonstrates positive effects, in particular by Chl.

Selective genes expression and metabolites transformation drive a robust nitrite accumulation during nitrate reduction under alternating feast-famine condition.

Nitrite production via denitrification has been regarded as a key approach for survival of anaerobic ammonium oxidation (anammox) bacteria. Despite the important carbon substrate, little is known about the role of differential genes expression and extracellular metabolite regulation among diverse microbial communities. In this study, a novel alternating feast-famine strategy was proposed and demonstrated to efficiently accumulate nitrite in a low-nitrogen loading rate (NLR) (0.2∼0.8 kg N/m3/d) denitrification system. Highly selective expression of denitrifying genes was revealed as key regulators. Interestingly, in absence of carbon source (ACS) condition, the expression of narG and narI/V genes responsible for reduction of nitrate to nitrite jumped to 2.5 and 5.1 times higher than that in presence of carbon source (PCS) condition with carbon to nitrate ratio of 3.0. This fortunately facilitated a rapid nitrite accumulation once acetate was added, despite a significantly down-regulated narG and narI/narV and up-regulated nirS/nirK. This strategy selected Thauera as the most dominant denitrifier (50.2 %) with the highest contribution to narG and narI/narV genes, responsible for the high nitrite accumulation. Additionally, extracellular xylose, pyruvate, and glucose jointly promoted carbon-central metabolic pathway of key denitrifiers in ACS stage, playing an important role in the process of self-growth and selective enrichment of functional bacteria. The relatively rapid establishment and robust performance obtained in this study shows an engineering-feasible and economically-favorable solution for the regulation of partial denitrification in practical application.

Unraveling potential mechanism of different metal ions effect on anammox through big data analysis, molecular docking and molecular dynamics simulation.

Heavy metals (HMs) have been widely reported to pose an adverse effect on anaerobic ammonia oxidation (anammox) bacteria, yet the underlying mechanisms remain unclear. This study provides new insights into the potential mechanisms of interaction between HMs and functional enzymes through big date analysis, molecular docking and molecular dynamics simulation. The statistical analysis indicated that 10 mg/L Cu(II) and Cd(II) reduced nitrogen removal rate (NRR) by 85% and 43%, while 5 mg/L Fe(II) enhanced NRR by 29%. Additionally, the results of molecular simulations provided a microscopic interpretation for these macroscopic data. Molecular docking revealed that Hg(II) formed a distinctive binding site on ferritin, while other HMs resided at iron oxidation sites. Furthermore, HMs exhibited distinct binding sites on hydrazine dehydrogenase. Concurrently, the molecular dynamics simulation results further substantiated their capacity to form complexes. Cu(II) displayed the strongest binding affinity with ferritin for -1576 ± 79 kJ/mol in binding free energy calculation. Moreover, Cd(II) bound to ferritin and HDH for -1052.67 ± 58.49 kJ/mol, -290.02 ± 49.68 kJ/mol, respectively. This research addressed a crucial knowledge gap, shedding light on potential applications for remediating heavy metal-laden industrial wastewater.

Reactivation performance and sludge transformation after long-term storage of Partial denitrification/Anammox (PD/A) process.

This study explores the startup of innovative Partial denitrification/Anammox (PD/A) process using long-term stored sludge (>2 years at 4 °C). Results indicate a swift recovery performance, characterized by a progressive increase in the activity of functional microorganisms with improved nitrogen volumetric loading rate during operation. Stable nitrogen removal efficiency of 99.6 % was attained at 14.2 °C under influent nitrate and ammonium of 120 and 100 mg/L, respectively. A distinctive transformation was observed as the initially black seeding sludge transitioned to brownish-red, accompanied by rapid sludge granulation with size notably increased from 263.1 µm (day 4) to 1255.0 µm (day 128), significantly contributing to the rapid PD/A performance recovery. Microbial community analysis revealed substantial increases in functional bacteria, Thauera (0.09 %-10.4 %) and Candidatus Brocadia (0.003 %-1.98 %), coinciding with enhanced nitrogen removal performance. Overall, this study underscores the viability of long-term stored PD/A sludge as a seed for rapid reactor startup, offering useful technical support to advance practical PD/A process implementation.

Granulation of partial denitrification sludge: Advances in mechanism understanding, technologies development and perspectives.

The high-rate and stably efficient nitrite generation is vital and still challenges the wide application of partial denitrification (PD) and anammox technology. Increasing attention has been drawn to the granulation of PD biomass. However, the knowledge of PD granular sludge is still limited in terms of granules characterization and mechanisms of biomass aggregation for high nitrite accumulation. This work reviewed the performance and granulation of PD biomass for high nitrite accumulation via nitrate reduction, including the system start-up, influential factors, granular characteristics, hypothetical mechanism, challenges and perspectives in future application. The physiochemical characterization and key influential factors were summarized in view of nitrite production, morphology analysis, extracellular polymer substance structure, as well as microbial mechanisms. The PD granules exhibit potential advantages of a high biomass density, good settleability, high hydraulic loading rates, and strong shock resistance. A novel granular sludge-based PD combined with anammox process was proposed to enhance the capability of nitrogen removal. In the future, PD granules utilizing different electron donors is a promising way to broaden the application of anammox technology in both municipal and industrial wastewater treatment.

Hydroxyapatite (HAP) formation in acetate-driven partial denitrification process: Enhancing sludge granulation and phosphorus removal.

Partial denitrification/anammox (PD/A) processes have emerged as a promising technology for efficient nitrogen removal from wastewater. However, these processes fail to remove phosphorus (P), a key pollutant that contributes to water eutrophication. To address this issue, the potential of inducing hydroxyapatite (HAP) precipitation in PD processes to achieve simultaneous P removal was investigated for the first time. Specifically, three SBRs (R1-R3) for PD were operated with adding varying concentrations of external Ca (30, 60, and 120 mg/L, respectively). Results demonstrated significant P reduction in all three SBRs, particularly in R3 with high Ca, which achieved an 80 % removal efficiency. Notably, sludge granulation was observed during operation, with the granule size in R3 with high Ca reaching 906.1 µm during the stable period, exceeding those in R2 (788.7 µm) and R1 (707.1 µm). This led to good settle ability of the PD sludge, as demonstrated by the lowest SVI5 (20 mL/g MLSS). Moreover, the decrease in the MLVSS/MLSS ratio suggested that the inorganic content accumulated, as observed by confocal laser scanning microscopy in the interior of the granules. Elemental composition analysis suggested that PD granules contained high P and Ca, while the X-ray diffraction (XRD) results confirmed the formation of HAP. Overall, this study demonstrated that PD-HAP coupled granular sludge process has potential as a robust and efficient method for nitrite production, as well as effective P removal and recovery, thereby advancing the application of anammox processes in wastewater treatment.

Spatiotemporal Assembly and Immigration of Heterotrophic and Anammox Bacteria Allow a Robust Synergy for High-Rate Nitrogen Removal.

The novel partial denitrification-driven anammox (PD/A) is an energy-efficient method for nitrogen removal from wastewater. However, its stability and efficiency are impeded by the competition between heterotrophic denitrifying bacteria and relatively slow-growing anammox bacteria. In this study, a PD/A granular sludge system was developed, which achieved a nitrogen removal efficiency of 94% with 98% anammox contribution, even as the temperature dropped to 9.6 °C. Analysis of bacterial activity in aggregates of different sizes revealed that the largest granules (>2.0 mm) exhibited the highest anammox activity, 2.8 times that of flocs (<0.2 mm), while the flocs showed significantly higher nitrite production rates of PD, more than six times that of the largest granules. Interestingly, fluorescent in situ hybridization (FISH) combined with confocal laser scanning microscopy (CLSM) revealed a nest-shaped structure of PD/A granules. The Thauera genus, a key contributor to PD, was highly enriched at the outer edge, providing substrate nitrite for anammox bacteria inside the granules. As temperature decreased, the flocs transformed into small granules to efficiently retain anammox bacteria. This study provides multidimensional insights into the spatiotemporal assembly and immigration of heterotrophic and autotrophic bacteria for stable and high-rate nitrogen removal.

In a quest for high-efficiency mainstream partial nitritation-anammox (PN/A) implementation: One-stage or two-stage?

Partial nitritation-anammox (PN/A) process is known as an energy-efficient technology for wastewater nitrogen removal, which possesses a great potential to bring wastewater treatment plants close to energy neutrality with reduced carbon footprint. To achieve this goal, various PN/A processes implemented in a single reactor configuration (one-stage system) or two separately dedicated reactors configurations (two-stage system) were explored over the past decades. Nevertheless, large-scale implementation of these PN/A processes for low-strength municipal wastewater treatment has a long way to go owing to the low efficiency and effectiveness in nitrogen removal. In this work, we provided a comprehensive analysis of one-stage and two-stage PN/A processes with a focus on evaluating their engineering application potential towards mainstream implementation. The difficulty for nitrite-oxidizing bacteria (NOB) out-selection was revealed as the critical operational challenge to achieve the desired effluent quality. Additionally, the operational strategies of low oxygen commonly adopted in one-stage systems for NOB suppression and facilitating anammox bacteria growth results in a low nitrogen removal rate (NRR). Introducing denitrification into anammox system was found to be necessary to improve the nitrogen removal efficiency (NRE) by reducing the produced nitrate with in-situ utilizing the organics from wastewater itself. However, this may lead to part of organics oxidized with additional oxygen consumed in one-stage system, further compromising the NRR. By applying a relatively high dissolved oxygen in PN reactor with residual ammonium control, and followed by a granules-based anammox reactor feeding with a small portion of raw municipal wastewater, it appeared that two-stage system could achieve a good effluent quality as well as a high NRR. In contrast to the widely studied one-stage system, this work provided a unique perspective that more effort should be devoted to developing a two-stage PN/A process to evaluate its application potential of high efficiency and economic benefits towards mainstream implementation.

Robustness and stability of acetate-driven partial denitrification (PD) in response to high COD/NO3--N.

Partial Denitrification (PD) producing nitrite for anammox may face the issue of relatively high chemical oxygen demand (COD) loading (i.e., COD/NO3--N) due to real wastewater being changed in substrate concentration and flowrate. In this study, three PD systems (R1, R2, R3) with sodium acetate providing electrons were developed to investigate the influence of the relatively high COD/NO3--N ratios (4.0, 6.0, and 8.0) on NO2--N production and the subsequent recoverability. It was found that a relatively high NO2--N production with nitrate-to-nitrite transformation ratio (NTR) of 74.0% could be still obtained despite COD/NO3--N even improving to 8.0 under limited reaction time (10 min) with small nitrate remaining. However, a deteriorated nitrite production was observed with sufficient reaction time (15 min) with NTR being lowered to 19.2%. Delightedly, when reducing influent COD/NO3--N to a normal level of 3.0, PD with high nitrite production was rapidly achieved after suffering from a relatively high COD/NO3--N (4.0-8.0) for 130 cycles. Besides, it was found the relatively high COD/NO3--N had a minor influence on the recoverability of PD, as evidenced by the close NTRs. Microbial analysis revealed the relative abundance of PD functional bacteria, Thauera, decreased under high COD/NO3--N, while it is still highly dominated in the systems, varying from 75.1% in R1 to 62.8% in R3 after around 110-cycles recovery. Furthermore, it appeared that the high pH (9.1-9.2) induced by sodium acetate also likely played a role in maintaining the excellent PD. Overall, this study demonstrated the robustness and stability of acetate-driven PD in response to high COD/NO3--N, further informing the technological superiority of PD in supplying stable and efficient nitrite, which provided solid technical support to apply it with anammox for high-efficient N removal.

Extending reaction duration has minor influence on nitrite production in partial-denitrification process.

Partial denitrification (PD) is another important pathway producing nitrite for anammox, however, whether its performance is affected by overlong reaction time, a situation that often takes place is still unknown. Three sequencing batch reactors were operated for PD to evaluate this factor on nitrite production. Results indicated effluent nitrite was very close despite reaction time even extending to four times longer than control (i.e., nitrate-to-nitrite transformation ratio (NTR) of 94.4%-89.8%). Meanwhile, it was found PD could recover to the normal after suffering from high organics shocking. Cycle studies suggested produced nitrite would not be further reduced with prolonged time, as indicated by changing trend of pH and alkalinity. Microbial analysis revealed PD functional bacteria, Thauera, slightly decreased with prolonged reaction, while it was always predominated. Taken together, this study indicated overlong reaction time had minor influence on PD, demonstrating its robustness with great technological superiority in supplying nitrite for anammox.

Potential causes of partial-denitrification (PD) granular sludge breakdown under high nitrate loading rates.

The granule instability has been frequently reported during the operation of high loading rates. While, there no research was performed on the recently developed anoxic partial-denitrification (PD) granules, a novel pathway in producing nitrite from nitrate for anammox process. Herein, this work, for the first time, investigated the influence of nitrate loading rates on the instability of PD granules and identified the key causes. Two lab-scale sequencing batch reactors (SBRs) were operated with nitrate loading rates (NLR) increased from 0.48 to 3.84 kg N/m3/d (R1, 8 cycles/d), and 0.96 to 7.68 kg N/m3/d (R2, 16 cycles/d) by gradually elevating the influent nitrate concentration. Results showed that nitrite production rates increased with the NLRs, with a maximal value of 5.26 kg N/m3/d obtained. However, the compact regular PD granules were not stable and broke down when NLR was above 3.84 kg N/m3/d, which resulted in serious sludge washing out from SBR. The high NLRs led to the extracellular polymeric substances (EPS) transformation in terms of its composition and structure, which the protein content in the EPS and the tightly bound EPS (T-EPS) fraction was significantly decreased, this was supposed to be the major reason causing the breakdown of PD granules. Besides, it was found the PD granule in R2 was more deteriorated than that in R1 under the same high NLR, suggesting the short starvation (idle) times in SBR cycle was likely another reason impairing the stability of PD granules. Overall, this research provides useful information in development of granule-based PD systems and sheds light on achieving high-rate nitrite production in SBR with great stability.

Beyond an Applicable Rate in Low-Strength Wastewater Treatment by Anammox: Motivated Labor at an Extremely Short Hydraulic Retention Time.

The application of anammox technology in low-strength wastewater treatment is still challenging due to unstable nitrite (NO2--N) generation. Partial denitrification (PD) of nitrate (NO3--N) reduction ending with NO2--N provides a promising solution. However, little is known about the feasibility of accelerating nitrogen removal toward the practical application of anammox combined with heterotrophic denitrification. In this work, an ultrafast, highly stable, and impressive nitrogen removal performance was demonstrated in the PD coupling with an anammox (PD/A) system. With a low-strength influent [50 mg/L each of ammonia (NH4+-N) and NO3--N] at a low chemical oxygen demand/NO3--N ratio of 2.2, the hydraulic retention time could be shortened from 16.0 to 1.0 h. Remarkable nitrogen removal rates of 1.28 kg N/(m3 d) and excellent total nitrogen removal efficiency of 94.1% were achieved, far exceeding the applicable capacity for mainstream treatment. Stimulated enzymatic reaction activity of anammox was obtained due to the fast NO2--N jump followed by a famine condition with limited organic carbon utilization. This high-rate PD/A system exhibited efficient renewal of bacteria with a short sludge retention time. The 16S rRNA sequencing unraveled the rapid growth of the genus Thauera, possibly responsible for the incomplete reduction of NO3--N to NO2--N and a decreasing abundance of anammox bacteria. This provides new insights into the practical application of the PD/A process in the energy-efficient treatment of low-strength wastewater with less land occupancy and desirable effluent quality.

Integrated thermal hydrolysis pretreated anaerobic digestion centrate and municipal wastewater treatment via partial nitritation/anammox process: A promising approach to alleviate inhibitory effects and enhance nitrogen removal.

Two-stage Partial nitritation/Anammox (PN/A) was firstly performed for recalcitrant organics (RO)-rich thermal hydrolysis pretreated anaerobic digestion (THP-AD) centrate treatment with municipal wastewater (MW) as co-substrate. Results indicated the inhibitory effects of RO was alleviated and high nitrate issue in PN/A effluent was addressed by cotreatment strategy. Stable PN with nitrite accumulation ratio of 95% and N removal efficiency of 97.1% were well maintained at MW of 80%. Nevertheless, nitrate accumulation and anammox activity loss were observed with lowering MW proportion owing to the weakened denitrification activity and aggravated inhibitory effect. Microbial analysis revealed Nitrosomonas was the major ammonium oxidizing bacteria and the ideal PN performance was due to the effective out-selection of nitrite oxidizing bacteria. Candidatus Kuenenia was identified as the primary bacteria for nitrogen removal (82.7%), and the controlled abundance of heterotrophic denitrifiers in anammox system ensured the enhanced nitrogen removal regardless of high COD loading from THP-AD centrate.

Mitigation of inhibitory effect of THP-AD centrate on partial nitritation and anammox: Insights into ozone pretreatment.

Anaerobic digestion centrate produced from thermal hydrolysis pretreated sludge (THP-AD centrate) has serious inhibitory effect on ammonium oxidizing bacteria (AOB) and anammox bacteria. This imposes huge challenge to employ partial nitritation/anammox (PN/A) process to treat THP-AD centrate. This study, for the first time, presented an effective strategy, ozone pretreatment, to alleviate such inhibitory effect. The activities of AOB and anammox bacteria increased with increasing ozone dosage, which were likely related to the transformation of organic compounds including humic acid-like and fulvic acid-like substances as well as high molecular weight (HMW) protein. Long-term operation of PN/A system further demonstrated the improved performance in term of nitrogen removal, organics degradation as well as sludge settleability and effluent solids. Nitrogen removal rate (NRR) of 0.64 Kg N/m3/d was achieved (1.38 g O3/ g COD), which was 42.2% higher compared to treating untreated THP-AD centrate. Effluent nitrate, the by-product of PN/A process, was reduced by 39.7% despite of its release in ozonation. This was due to the enhanced denitrification activity, humic acid-like and fulvic acid-like substances as well as HMW protein were significantly reduced. Overall, this study provides a promising method to improve PN/A performance and final effluent quality when treating organic-rich THP-AD centrate.

NOB suppression strategies in a mainstream membrane aerated biofilm reactor under exceptionally low lumen pressure.

Integrating the aeration-efficient membrane aerated biofilm reactor (MABR) with anaerobic ammonium oxidation (anammox) could yield further reduction in energy in wastewater treatment facilities. However, nitrite oxidizing bacteria (NOB) suppression remained challenging due to the absence of intrinsic inhibition factors in mainstream conditions. This study investigated selective NOB suppression strategies in MABR under <5 kPa lumen pressure. Three MABRs were seeded from different seeding sludge, and operated under various ammonium loading rates, aeration pressure, and temporary inhibitory shock conditions. The three reactors were operated for 170-456 days depending on studied parameters. The results showed that higher ammonium loading could create a substrate-oxygen imbalance and quickly contain emergent NOB activity when aeration pressure was not excessive. In addition, lowering of aeration pressure reversed nitrite oxidizing activities without affecting ammonium oxidizing bacteria (AOB). Cultivating partial nitritation biofilm under zero positive aeration pressure slowed down the growth of NOB yet resulted in self-induced anammox activities. With the aid of temporary free ammonia (FA)/free nitrous acid (FNA) treatment, full-nitrifying biofilm could be transformed to stable partial nitritation biofilm. More than 84% nitrite accumulation ratio (NAR) was sustained during stable operation in each reactor together with an ammonium removal rate of more than 100 mg-N/L/d. Microbial analysis revealed that Nitrosomonas was the main AOB taxon in the three reactors while K-strategist Nitrospira showed presence despite low nitrite oxidizing activities. Under zero positive pressure, proliferation of Nitrospira was much slower while Candidatus Brocadia was self-induced. Furthermore, Nitrospira showed downturn after temporary inhibition treatment.

Challenges of THP-AD centrate treatment using partial nitritation-anammox (PN/A) - inhibition, biomass washout, low alkalinity, recalcitrant and more.

The centrate produced from a thermal hydrolysis pretreatment coupled anaerobic digestion (THP-AD) system is generally characterized by high concentrations of ammonium and recalcitrant organics. In this study, a cost-effective partial nitritation-anammox (PN/A) process was developed to evaluate the potential challenges in THP-AD centrate treatment. The results show ammonium oxidizing bacteria (AOB) and anammox bacteria were seriously inhibited by THP-AD centrate, while long-term acclimation together with aeration optimization can mitigate such inhibition. A nitrogen removal rate (NRR) of 0.55 kg N/m3/d was obtained and maintained with 60% THP-AD centrate as feed. However, 100% THP-AD centrate caused sludge wash-out from PN reactor due to excessive polymer and high solids in influent. The alkalinity deficit also reduced the AOB activity. Moreover, anammox activity and overall NRR also declined (to 0.37 kg N/m3/d). The organics transformation mainly occurred in PN reactor with very low removal efficiency due to their recalcitrant characteristics. The humic acid-like, fulvic acid-like substances and building blocks were revealed as the major organic compounds in THP-AD centrate (51.5-53.8% TOC), which likely contributed to the recalcitrant. Nitrosomonas and Candidatus Brocadia were the major AOB and anammox bacteria in the PN and anammox reactors respectively. With the increased THP-AD centrate proportion in the feed, the abundance of both population declined. Interestingly, Denitratisoma, being the major denitrifying bacteria in anammox reactor, had relatively stable abundance (7.0-7.9%) when THP-AD centrate was improved from 3 and 100%, suggesting the inhibition on anammox bacteria was not due to the overgrowth of denitrifying microorganism despite the high organics loading rate. Overall, this study provides a guide to develop the energy-saving PN/A process for THP-AD centrate treatment by pointing out potential challenges and mitigating strategies.