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788000.9469The synergistic mechanism of β-lactam antibiotic removal between ammonia-oxidizing microorganisms and heterotrophs. Nitrifying system is an effective strategy to remove numerous antibiotics, however, the contribution of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and heterotrophs for antibiotic removal are still unclear. In this study, the mechanism of β-lactam antibiotic (cefalexin, CFX) removal was studied in a nitrifying sludge system. Results showed that CFX was synergistically removed by AOB (Nitrosomonas, played a major role) and AOA (Candidatus_Nitrososphaera) through ammonia monooxygenase-mediated co-metabolism, and by heterotrophs (Pseudofulvimonas, Hydrogenophaga, RB41, Thauera, UTCFX1, Plasticicumulans, Phaeodactylibacter) through antibiotic resistance genes (ARGs)-encoded β-lactamases-mediated hydrolysis. Regardless of increased archaeal and heterotrophic CFX removal with the upregulation of amoA in AOA and ARGs, the system exhibited poorer CFX removal performance at 10 mg/L, mainly due to the inhibition of AOB. This study provides new reference for the important roles of heterotrophs and ARGs, opening the possibilities for the application of ARGs in antibiotic biodegradation.202336174754
787510.9451Phenacetin enhanced the inorganic nitrogen removal performance of anammox bacteria naturally in-situ enriched system. Among the earliest synthetic antipyretic drugs, phenacetin (PNCT) could be used as the novel partial nitrification (PN) inhibitor to effectively inhibit nitrite-oxidizing bacteria (NOB). In practical application, the rapidly starting of PN could provide stable source of nitrite for anaerobic ammonium oxidation (anammox) process. However, impact of PNCT on anaerobic ammonia oxidizing bacteria (AnAOB) and its underlying mechanisms were not clear. In this research, totally 14 times of PNCT aerobic soaking treatment were performed in the AnAOB naturally enrichment system to improve total inorganic nitrogen removal efficiency (TINRE). After once of PNCT treatment, TINRE rose from 61.89 % to 79.93 %. After 14 times of PNCT treatment, NOB Nitrospira relative abundance decreased from 9.82 % to 0.71 %, though Candidatus Brocadia relative abundance also declined, it might gradually adjust to PNCT by converting the leading oligotype species. The activity and relative abundances of NOB were reduced by PNCT via decreasing the abundances of genes amoA and nxrB, enzymes NxrA and NxrB. Moreover, Candidatus Jettenia and Ca. Brocadia might be the potential host of qacH-01 and they played the crucial role in the shaping profile of antibiotic resistance genes (ARGs). The explosive propagation or transmission of ARGs might not take place after PNCT treatment.202439566627
787420.9450Phenacetin promoted the rapid start-up and stable maintenance of partial nitrification: Responses of nitrifiers and antibiotic resistance genes. Phenacetin (PNCT) belongs to one of the earliest synthetic antipyretics. However, impact of PNCT on nitrifying microorganisms in wastewater treatment plants and its potential microbial mechanism was still unclear. In this study, PN could be initiated within six days by PNCT anaerobic soaking treatment (8 mg/L). In order to improve the stable performance of PN, 21 times of PNCT aerobic soaking treatment every three days was conducted and PN was stabilized for 191 days. After PN was damaged, ten times of PNCT aerobic soaking treatment every three days was conducted and PN was recovered after once soaking, maintained over 88 days. Ammonia oxidizing bacteria might change the dominant oligotype to gradually adjust to PNCT, and the increase of abundance and activity of Nitrosomonas promoted the initiation of PN. For nitrite-oxidizing bacteria (NOB), the increase of Candidatus Nitrotoga and Nitrospira destroyed PN, but PN could be recovered after once aerobic soaking illustrating NOB was not resistant to PNCT. KEGG and COG analysis suggested PNCT might disrupt rTCA cycle of Nitrospira, resulting in the decrease of relative abundance of Nitrospira. Moreover, PNCT did not lead to the sharp increase of absolute abundances of antibiotic resistance genes (ARGs), and the risk of ARGs transmission was negligible.202438744392
811030.9450Removal of chlortetracycline and antibiotic resistance genes in soil by earthworms (epigeic Eisenia fetida and endogeic Metaphire guillelmi). The impacts of two ecological earthworms on the removal of chlortetracycline (CTC, 0.5 and 15 mg kg(-1)) and antibiotic resistance genes (ARGs) in soil were explored through the soil column experiments. The findings showed that earthworm could significantly accelerate the degradation of CTC and its metabolites (ECTC) in soil (P < 0.05), with epigeic Eisenia fetida promoting degradation rapidly and endogeic Metaphire guillelmi exhibiting a slightly better elimination effect. Earthworms alleviated the abundances of tetR, tetD, tetPB, tetG, tetA, sul1, TnpA, ttgB and intI1 in soil, with the total relative abundances of ARGs decreasing by 35.0-44.2% in earthworm treatments at the 28th day of cultivation. High throughput sequencing results displayed that the structure of soil bacteria community was modified apparently with earthworm added, and some possible CTC degraders, Aeromonas, Flavobacterium and Luteolibacter, were promoted by two kinds of earthworms. Redundancy analysis demonstrated that the reduction of CTC residues, Actinobacteria, Acidobacteria and Gemmatimonadetes owing to earthworm stimulation was responsible for the removal of ARGs and intI1 in soil. Additionally, intI1 declined obviously in earthworm treatments, which could weaken the risk of horizontal transmission of ARGs. Therefore, earthworm could restore the CTC-contaminated soil via enhancing the removal of CTC, its metabolites and ARGs.202133798888
775340.9449Sludge biolysis pretreatment to reduce antibiotic resistance genes (ARGs): Insight into the relationship between potential ARGs hosts and BALOs' preferred prey. As an important reservoir of antibiotic resistance genes (ARGs), the sludge discharged from wastewater treatment plants is the key intermediate for ARG transport into the environment. Bdellovibrio-and-like organisms (BALOs) are predatory bacteria that are expected to attack antibiotic-resistant bacteria (ARB). In this study, the screened BALOs (C3 & D15) were mixed with the sludge for biolysis to achieve the satisfying removal efficiencies of six tet genes, two sul genes, and one mobile genetic element (intl 1). Among them, tet(Q) demonstrated the highest reduction rate in relative abundance at 87.3 ± 1.0 %, while tet(X) displayed the lowest of 11.7 ± 0.2 %. The microorganisms, including Longilinea, Methanobacterium, Acetobacterium, Sulfurimonas, allobaculum, Gaiella, AAP99, Ellin6067, Rhodoferax, Ferruginibacter and Thermomonas, were expected to play a dual role in the reduction of ARGs by serving as ARB and BALOs' preferred prey. Meanwhile, BALOs consortium improved ARGs reduction efficiency via the expansion of the prey profile. Additionally, BALOs decreased the relative abundance of not only pathogens (Shinella, Rickettsia, Burkholderia, Acinetobacter, Aeromonas, Clostridium, Klebsiella and Pseudomonas), but also the ARGs' host pathogens (Mycobacterium, Plesiocystis, Burkholderia, and Bacteroides). Therefore, the application of BALOs for sludge biolysis are promising to decrease the sludge's public health risks via limiting the spread of ARGs and pathogens into the environment.202438901315
811350.9447Fate of antibiotic resistance genes in mesophilic and thermophilic anaerobic digestion of chemically enhanced primary treatment (CEPT) sludge. Anaerobic digestion (AD) of chemically enhanced primary treatment (CEPT) sludge and non-CEPT (conventional sedimentation) sludge were comparatively operated under mesophilic and thermophilic conditions. The highest methane yield (692.46±0.46mL CH(4)/g VS(removed) in CEPT sludge) was observed in mesophilic AD of CEPT sludge. Meanwhile, thermophilic conditions were more favorable for the removal of total antibiotic resistance genes (ARGs). In this study, no measurable difference in the fates and removal of ARGs and class 1 integrin-integrase gene (intI1) was observed between treated non-CEPT and CEPT sludge. However, redundancy analysis indicated that shifts in bacterial community were primarily accountable for the variations in ARGs and intI1. Network analysis further revealed potential host bacteria for ARGs and intI1.201728797965
811260.9447Fate of antibiotic resistance bacteria and genes during enhanced anaerobic digestion of sewage sludge by microwave pretreatment. The fate of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) were investigated during the sludge anaerobic digestion (AD) with microwave-acid (MW-H), microwave (MW) and microwave-H2O2-alkaline (MW-H2O2) pretreatments. Results showed that combined MW pretreatment especially for the MW-H pretreatment could efficiently reduce the ARB concentration, and most ARG concentrations tended to attenuate during the pretreatment. The subsequent AD showed evident removal of the ARB, but most ARGs were enriched after AD. Only the concentration of tetX kept continuous declination during the whole sludge treatment. The total ARGs concentration showed significant correlation with 16S rRNA during the pretreatment and AD. Compared with unpretreated sludge, the AD of MW and MW-H2O2 pretreated sludge presented slightly better ARB and ARGs reduction efficiency.201626970692
678970.9444Metagenomic insights on promoting the removal of resistome in aerobic composting pig manure by lightly burned modified magnesite. The antibiotic resistance genes (ARGs) have become a serious issue facing public health. In this study, light-burned magnesite with a high specific surface area at 650 °C (MS650) was used for aerobic composting, evaluating its effect on the resistome during pig manure composting. Different concentrations of MS650 reduced the abundance of the resistome, including seven high-risk ARGs, class two metal and biocide resistance genes (MBRGs), and human pathogenic bacteria (HPBs). The addition of 2.5 % MS650 (L1) in the composting had the best reduction effect on ARGs, MBRGs and HPBs. ARG and microbial community assembly are deterministic processes. Proteobacteria and Actinobacteria was the main factor associated with the decrease in ARGs, followed by virulence factor genes (VFGs, 44.2 %). The reduction in MBRGs by MS650 mainly suppressed HGT by reducing the Isfinder abundance. To summarize, MS650 is an effective method to improve emission reduction of ARGs and MBRGs. This study provided a theoretical basis for improving the engineering application potential of MS650.202439490844
794080.9442Microplastics affect the ammonia oxidation performance of aerobic granular sludge and enrich the intracellular and extracellular antibiotic resistance genes. Microplastics (MPs) and antibiotic resistance genes (ARGs), as emerging pollutants, are frequently detected in wastewater treatment plants, and their threats to the environment have received extensive attentions. However, the effects of MPs on the nitrification of aerobic granular sludge (AGS) and the spread patterns of intracellular and extracellular ARGs (iARGs and eARGs) in AGS were still unknown. In this study, the responses of AGS to the exposure of 1, 10 and 100 mg/L of typical MPs (polyvinyl chloride (PVC), polyamide (PA), polystyrene (PS) and polyethylene (PE)) and tetracycline were focused on in 3 L nitrifying sequencing batch reactors. 10 mg/L MPs decreased the nitrification function, but nitrification could recover. Furthermore, MPs inhibited ammonia-oxidizing bacteria and enriched nitrite-oxidizing bacteria, leading partial nitrification to losing stability. PVC, PA and PS stimulated the secretion of extracellular polymeric substances and reactive oxygen species. PE had less negative effect on AGS than PVC, PA and PS. The abundances of iARGs and eARGs (tetW, tetE and intI1) increased significantly and the intracellular and extracellular microbial communities obviously shifted in AGS system under MPs stress. Potential pathogenic bacteria might be the common hosts of iARGs and eARGs in AGS system and were enriched in AGS and MPs biofilms.202133387747
787690.9441Sulfamethoxazole impact on pollutant removal and microbial community of aerobic granular sludge with filamentous bacteria. In this study, sulfamethoxazole (SMX) was employed to investigate its impact on the process of aerobic granule sludge with filamentous bacteria (FAGS). FAGS has shown great tolerance ability. FAGS in a continuous flow reactor (CFR) could keep stable with 2 μg/L of SMX addition during long-term operation. The NH(4)(+), chemical oxygen demand (COD), and SMX removal efficiencies kept higher than 80%, 85%, and 80%, respectively. Both adsorption and biodegradation play important roles in SMX removal for FAGS. The extracellular polymeric substances (EPS) might play important role in SMX removal and FAGS tolerance to SMX. The EPS content increased from 157.84 mg/g VSS to 328.22 mg/g VSS with SMX addition. SMX has slightly affected on microorganism community. A high abundance of Rhodobacter, Gemmobacter, and Sphaerotilus of FAGS may positively correlate to SMX. The SMX addition has led to the increase in the abundance of the four sulfonamide resistance genes in FAGS.202336871701
7848100.9440Simultaneous Removal of Antibiotic Resistant Bacteria, Antibiotic Resistance Genes, and Micropollutants by FeS(2)@GO-Based Heterogeneous Photo-Fenton Process. The co-occurrence of various chemical and biological contaminants of emerging concerns has hindered the application of water recycling. This study aims to develop a heterogeneous photo-Fenton treatment by fabricating nano pyrite (FeS(2)) on graphene oxide (FeS(2)@GO) to simultaneously remove antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs), and micropollutants (MPs). A facile and solvothermal process was used to synthesize new pyrite-based composites. The GO coated layer forms a strong chemical bond with nano pyrite, which enables to prevent the oxidation and photocorrosion of pyrite and promote the transfer of charge carriers. Low reagent doses of FeS(2)@GO catalyst (0.25 mg/L) and H(2)O(2) (1.0 mM) were found to be efficient for removing 6-log of ARB and 7-log of extracellular ARG (e-ARG) after 30 and 7.5 min treatment, respectively, in synthetic wastewater. Bacterial regrowth was not observed even after a two-day incubation. Moreover, four recalcitrant MPs (sulfamethoxazole, carbamazepine, diclofenac, and mecoprop at an environmentally relevant concentration of 10 μg/L each) were completely removed after 10 min of treatment. The stable and recyclable composite generated more reactive species, including hydroxyl radicals (HO(•)), superoxide radicals (O(2)(• -)), singlet oxygen ((1)O(2)). These findings highlight that the synthesized FeS(2)@GO catalyst is a promising heterogeneous photo-Fenton catalyst for the removal of emerging contaminants.202235759741
8054110.9438Effects of nanoscale zero-valent iron on the performance and the fate of antibiotic resistance genes during thermophilic and mesophilic anaerobic digestion of food waste. The effects of nanoscale zero-valent iron (nZVI) on the performance of food waste anaerobic digestion and the fate of antibiotic resistance genes (ARGs) were investigated in thermophilic (TR) and mesophilic (MR) reactors. Results showed that nZVI enhanced biogas production and facilitated ARGs reduction. The maximum CH(4) production was 212.00 ± 4.77 ml/gVS with 5 g/L of nZVI in MR. The highest ARGs removal ratio was 86.64 ± 0.72% obtained in TR at nZVI of 2 g/L. nZVI corrosion products and their contribution on AD performance were analyzed. The abundance of tetracycline genes reduced significantly in nZVI amended digesters. Firmicutes, Chloroflexi, Proteobacteria and Spirochaetes showed significant positive correlations with various ARGs (p < 0.05) in MR and TR. Redundancy analysis indicated that microbial community was the main factor that influenced the fate of ARGs. nZVI changed microbial communities, with decreasing the abundance bacteria belonging to Firmicutes and resulting in the reduction of ARGs.201931505392
8117120.9437Composting of oxytetracycline fermentation residue in combination with hydrothermal pretreatment for reducing antibiotic resistance genes enrichment. Hydrothermal pretreatment can efficiently remove the residual antibiotics in oxytetracycline fermentation residue (OFR), but its effect on antibiotic resistance genes (ARGs) during composting remains unclear. This study compared the shifts in bacterial community and evolutions in ARGs and integrons during different composting processes of OFRs with and without hydrothermal pretreatment. The results demonstrated that hydrothermal pretreatment increased the bacterial alpha diversity at the initial phase, and increased the relative abundances of Proteobacteria and Actinobacteria but decreased that of Bacteroidetes at the final phase by inactivating mycelia and removing residual oxytetracycline. Composting process inevitably elevated the abundance and relative abundance of ARGs. However, the increase in ARGs was significantly reduced by hydrothermal pretreatment, because the removal of oxytetracycline decreased their potential host bacteria and inhibited their horizontal gene transfer. The results demonstrated that hydrothermal pretreatment is an efficient strategy to reduce the enrichment of ARGs during the OFR composting.202033099099
7859130.9436Abatement of antibiotics and resistance genes during catalytic ozonation enhanced sludge dewatering process: Synchronized in volume and hazardousness reduction. Based on the efficiency of the catalytic ozonation techniques (HDWS+O(3) and MnFe(2)O(4) @SBC+O(3)) in enhancing the sludge dewaterability, the effectiveness in synchronized abatement antibiotics and antibiotic resistance genes (ARGs) was conducted to determine. The results revealed that catalytic ozonation conditioning altered the distribution of target antibiotics (tetracycline (TC), oxytetracycline (OTC), norfloxacin (NOR), ofloxacin (OFL)) in the dewatered filtrate, the dewatered sludge cake and the extra-microcolony/cellular polymers (EMPS/ECPS) layers, achieving the redistribution from solid-phase adsorption to liquid-phase dissolution. The total degradation rate was over 90% for TC and OTC, 72-78% for NOR and OFL; the abatement efficiency of eleven ARGs reached 1.47-3.01 log and 1.64-3.59 log, respectively, and more than four eARGs were eliminated. The effective abatement of the absolute abundance of Mobile genetic elements (MGEs) (0.91-1.89 log) demonstrated that catalytic ozonation conditioning could also significantly inhibit horizontal gene transfer (HGT). The abundance of resistant bacteria was greatly reduced and the signal transduction of the typical ARGs host bacteria was inhibited. The highly reactive oxidation species (ROS) generated were responsible for the abatement of antibiotics and ARGs. These findings provided new insights into the sludge conditioning for ideal and synchronized reduction in volume and hazardousness by catalytic ozonation processes in sludge treatment.202437944236
8056140.9435Antibiotic resistance gene profiles and evolutions in composting regulated by reactive oxygen species generated via nano ZVI loaded on biochar. In this study, nano zero-valent iron loaded on biochar (BC-nZVI) was analyzed for its effects on antibiotic resistance genes (ARGs) in composting. The results showed that BC-nZVI increased reactive oxygen species (ROS) production, and the peak values of H(2)O(2) and OH were 22.95 % and 55.30 % higher than those of the control group, respectively. After 65 days, the relative abundances of representative ARGs decreased by 56.12 % in the nZVI group (with BC-nZVI added). An analysis of bacterial communities and networks revealed that Actinobacteria, Proteobacteria, and Firmicutes were the main hosts for ARGs, and BC-nZVI weakened the link between ARGs and host bacteria. Distance-based redundancy analysis showed that BC-nZVI altered the microbial community structure through environmental factors and that most ARGs were negatively correlated with ROS, suggesting that ROS significantly affected the relative abundance of ARGs. According to these results, BC-nZVI showed potential for decreasing the relative abundance of ARGs in composting.202337611721
8125150.9435The removal performances and evaluation of heavy metals, antibiotics, and resistomes driven by peroxydisulfate amendment during composting. This study aimed to explore the effect of peroxydisulfate on the removal of heavy metals, antibiotics, heavy metal resistance genes (HMRGs), and antibiotic resistance genes (ARGs) during composting. The results showed that peroxydisulfate achieved the passivation of Fe, Mn, Zn, and Cu by promoting their speciation variations, thus reducing their bioavailability. And the residual antibiotics were better degraded by peroxydisulfate. In addition, metagenomics analysis indicated that the relative abundance of most HMRGs, ARGs, and MGEs was more effectively down-regulated by peroxydisulfate. Network analysis confirmed Thermobifida and Streptomyces were dominant potential host bacteria of HMRGs and ARGs, whose relative abundance was also effectively down-regulated by peroxydisulfate. Finally, mantel test showed the significant effect of the evolution of microbial communities and strong oxidation of peroxydisulfate on the removal of pollutants. These results suggested that heavy metals, antibiotics, HMRGs, and ARGs shared a joint fate of being removed driven by peroxydisulfate during composting.202337307729
7941160.9434Microplastics accelerate nitrification, shape the microbial community, and alter antibiotic resistance during the nitrifying process. Microplastics (MPs) and antibiotic resistance genes (ARGs) are both emerging pollutants that are frequently detected in wastewater treatment plants. In this study, the effects of various MPs, including polyethylene (PE), polyvinyl chloride (PVC), and biodegradable polylactic acid (PLA), on nitrification performance, dominant microbial communities, and antibiotic resistance during nitrification were investigated. The results revealed that the addition of MPs increased the specific ammonia oxidation rate and specific nitrate production rate by 15.2 % - 15.5 % and 8.0 % - 11.6 %, respectively, via enrichment of nitrifying microorganisms, Nitrospira and Nitrosomonas. Moreover, ARGs were selectively enriched in nitrifying sludge and microplastic biofilms under stress from different MPs. Compared with PE-MPs (23.9 %) and PVC-MPs (21.4 %), exposure to PLA-MPs significantly increased intI1 abundance by 51.6 %. The results of the variance decomposition analysis implied that MPs and the microbial community play important roles in the behavior of ARGs. Network analysis indicated that Nitrosomonas and potentially pathogenic bacteria emerged as possible hosts, harboring ARGs and intI1 genes in the nitrifying sludge and microplastic biofilms. Critically, PLA-MPs were found to enrich both ARGs and potential pathogenic bacteria during nitrification, which should be considered in their promotion of application processes due to their biodegradability.202539740624
8105170.9433Refluxing mature compost to replace bulking agents: A low-cost solution for suppressing antibiotic resistance genes rebound in sewage sludge composting. Antibiotic resistance genes (ARGs) rebounding during composting cooling phase is a critical bottleneck in composting technology that increased ARGs dissemination and application risk of compost products. In this study, mature compost (MR) was used as a substitute for rice husk (RH) to mitigate the rebound of ARGs and mobile genetic elements (MGEs) during the cooling phase of sewage sludge composting, and the relationship among ARGs, MGEs, bacterial community and environmental factors was investigated to explore the key factor influencing ARGs rebound. The results showed that aadD, blaCTX-M02, ermF, ermB, tetX and vanHB significantly increased 4.76-32.41 times, and the MGEs rebounded by 38.60% in the cooling phase of RH composting. Conversely, MR reduced aadD, tetM, ermF and ermB concentrations by 59.49-98.58%, and reduced the total abundance of ARGs in the compost product by 49.32% compared to RH, which significantly restrained ARGs rebound. MR promoted secondary high temperature inactivation of potential host bacteria, including Ornithinibacter, Rhizobiales and Caldicoprobacter, which could harbor aadE, blaCTX-M02, and blaVEB. It also reduced the abundance of lignocellulose degrading bacteria of Firmicutes, which were potential hosts of aadD, tetX, ermF and vanHB. Moreover, MR reduced moisture and increased oxidation reduction potential (ORP) that promoted aadE, tetQ, tetW abatement. Furthermore, MR reduced 97.36% of total MGEs including Tn916/1545, IS613, Tp614 and intI3, which alleviated ARGs horizontal transfer. Overall finding proposed mature compost reflux as bulking agent was a simple method to suppress ARGs rebound and horizontal transfer, improve ARGs removal and reduce composting plant cost.202539798649
7888180.9432Microecology 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.202438710419
7886190.9432Resistance 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.202032244076