Mechanistic insight into microbial interaction and metabolic pattern of anammox consortia on surface-modified biofilm carrier with extracellular polymeric substances.

The extremely slow growth rate of anaerobic ammonia oxidation (anammox) bacteria limits full-scale application of anammox process worldwide. In this study, extracellular polymeric substances (EPS)-coated polypropylene (PP) carriers were prepared for biofilm formation. The biomass adhesion rate of EPS-PP carrier was 12 times that of PP carrier, and EPS-PP achieved significant enrichment of E. coli BY63. The 120-day continuous flow experiment showed that the EPS-PP carrier accelerated the formation of anammox biofilm, and the nitrogen removal efficiency increased by 10.5 %. In addition, the abundance of Candidatus Kuenenia in EPS-PP biofilm was 27.1%. Simultaneously, amino acids with high synthesis cost and the metabolites of glycerophospholipids related to biofilm formation on EPS-PP biofilm were significantly up-regulated. Therefore, EPS-PP carriers facilitated the rapid formation of anammox biofilm and promoted the metabolic activity of functional bacteria, which further contributed to the environmental and economic sustainability of anammox process.

Quorum sensing mediated response mechanism of anammox consortia to anionic surfactant: Molecular simulation and molecular evidence.

The widespread use of surfactants raise challenges to biological wastewater treatment. Anaerobic ammonium oxidation (anammox) process has the potential to treat wastewater containing anionic surfactants, but the response of anammox consortia at the molecular level under long-term exposure is unclear. Using high-throughput sequencing and gene quantification, combined with molecular docking, the effect of sodium dodecyl sulfonate (SDS) on anammox consortia were investigated. Levels of reactive oxygen species (ROS) might be lower than the threshold of oxidative damage, while the increase of lactate dehydrogenase (LDH) represented the cell membrane damage. Decreased abundance of functional genes (hdh, hzsA and nirS) indicated the decrease of the anammox bacterial abundance. Trace amounts of N-acyl homoserine lactone (AHL, C6-HSL, C8-HSL and C12-HSL) contained in influent could induce endogenous quorum sensing (QS), which could regulate the correlation between functional bacteria to optimize the microbial community and strengthen the resistance of anammox consortia to SDS. In addition, the proliferation of disinfectant resistance genes might increase the environmental pathogenicity of sewage discharge. This work highlights the potential response mechanism of anammox consortium to surfactants and provides a universal microbial-friendly bioenhancement strategy based on QS.

Microbial response and recovery strategy of the anammox process under ciprofloxacin stress from pure strain and consortia perspectives.

Ciprofloxacin (CIP) poses a high risk of resistance development in water environments. Therefore, comprehensive effects and recovery strategies of CIP in anaerobic ammonia oxidation (anammox) process were systematically elucidated from consortia and pure strains perspectives. The anammox consortia was not significantly affected by the stress of 10 mg L-1 CIP, while the higher concentration (20 mg L-1) of CIP caused a dramatic reduction in the nitrogen removal performance of anammox system. Simultaneously, the abundances of dominant functional bacteria and corresponding genes also significantly decreased. Such inhibition could not be mitigated by the recovery strategy of adding hydrazine and hydroxylamine. Reducing nitrogen load rate from 5.1 to 1.4 kg N m-3 d-1 promoted the restoration of three reactors. In addition, the robustness and recovery of anammox systems was evaluated using starvation and shock strategies. Simultaneously, antibiotic resistance genes and key metabolic pathways of anammox consortia were upregulated, such as carbohydrate and energy metabolisms. In addition, 11 pure stains were isolated from the anammox system and identified through phylogenetic analysis, 40 % of which showed multidrug resistance, especially Pseudomonas. These findings provide deep insights into the responding mechanism of anammox consortia to CIP stress and promote the application of anammox process for treating wastewater containing antibiotics.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

Research advances in application of mainstream anammox processes: Roles of quorum sensing and microbial metabolism.

Anaerobic ammonium oxidation (anammox) is a low-carbon biological nitrogen removal process, that has been widely applied to treat high-strength wastewater. However, the practical application of mainstream anammox treatment is limited due to the slow growth rate of anammox bacteria (AnAOB). Therefore, it is important to provide a comprehensive summary of the potential impacts and regulatory strategies for system stability. This article systematically reviewed the effects of environmental fluctuations on anammox systems, summarizing the bacterial metabolisms and the relationship between metabolite and microbial functional effects. To address the shortcoming of mainstream anammox process, molecular strategies based on quorum sensing (QS) were proposed. Sludge granulation, gel encapsulation and carrier-based biofilm technologies were adopted to enhance the QS function in microbial aggregation and reduction of biomass loss. Furthermore, this article discussed the application and progress of anammox-coupled processes. Valuable insights were provided for the stable operation and development of mainstream anammox process from the perspectives of QS and microbial metabolism.

Deciphering endogenous and exogenous regulations of anammox consortia in responding to lincomycin by multiomics: quorum sensing and CRISPR system.

The widespread use of antibiotics has created an antibiotic resistance genes (ARGs)-enriched environment, which causes high risks on human and animal health. Although antibiotics can be partially adsorbed and degraded in wastewater treatment processes, striving for a complete understanding of the microbial adaptive mechanism to antibiotic stress remains urgent. Combined with metagenomics and metabolomics, this study revealed that anammox consortia could adapt to lincomycin by spontaneously changing the preference for metabolite utilization and establishing interactions with eukaryotes, such as Ascomycota and Basidiomycota. Specifically, quorum sensing (QS) based microbial regulation and the ARGs transfer mediated by clustered regularly interspaced short palindromic repeats (CRISPR) system and global regulatory genes were the principal adaptive strategies. Western blotting results validated that Cas9 and TrfA were mainly responsible for the alteration of ARGs transfer pathway. These findings highlight the potential adaptative mechanism of microbes to antibiotic stress and fill gaps in horizontal gene transfer pathways in the anammox process, further facilitating the ARGs control through molecular and synthetic biology techniques.

Role of quorum sensing-based regulation in development of anaerobic ammonium oxidation process.

Shortage of anaerobic ammonium oxidation (anammox) sludge greatly limits the extensive full-scale application of anammox-based processes. Although numerous start-up strategies have been proposed, the interaction among microbial consortia and corresponding mechanism during the process development remain unknown. In this study, three reactors were established based on different seed sludges. After 27 days, the anammox process inoculated with anammox granules and activated sludge (1:5) was firstly achieved, and the highest nitrogen removal rate was 1.17 kg N m-3 d-1. Correspondingly, the anammox activity and abundances of related functional genes increased. Notably, the dominant anammox bacteria shifted from Candidatus Kuenenia to Candidatus Brocadia. Metagenomic analysis indicated that quorum sensing-based regulation mainly contributed to the proliferation and accumulation of anammox bacteria. This work provides an insight into the quorum sensing (QS)-regulated microbial interactions in the anammox and activated sludge consortia during the process development.

Mitigating the detrimental effects of salt stress on anammox process: A comparison between glycine betaine and mannitol.

The use of seawater to alleviate water shortages causes an increase of salinity in municipal pipe networks, posing challenges for biological wastewater treatment. The impacts of two compatible solutes on the anammox process under salt stress (20 g L-1) were compared here at the genetic and microbial levels. The findings revealed that both 0.3 mM glycine betaine (GB) and mannitol (MA) could alleviate the salt stress on anammox process, with GB exhibiting a better effect. Specifically, the addition of GB recovered the nitrogen removal efficiency (NRE) from 40 % to >80 % within 13 days. The addition of MA caused the reduction of the absolute abundance of hdh and hzsA, implying that 0.6 mM was not the optimal concentration. Moreover, salt stress induced an increase in the absolute abundance of nitrification functional genes and a decrease in the abundance of denitrification functional genes. Notably, compared with the initial level, the abundance of Candidatus Kuenenia increased by 7.1 % and 4.3 % after adding GB and MA, respectively. According to the network analysis, two compatible solutes promoted the bacterial interactions in anammox systems, which promoted the nitrogen circulation and further the nitrogen removal performance. This work provides a feasible strategy to relieve the salt stress on anammox process and then facilitates its application for treating saline wastewater.

Response of the mainstream anammox process to the biodegradable carbon sources in the granule-based systems: The difference in self-stratification of the microbial community.

The inevitable introduction of biodegradable carbon sources (such as monosaccharides and volatile fatty acids) originating from pretreatment units might affect the performance of the mainstream anaerobic ammonium oxidation (anammox) process. Two model carbon sources (glucose and acetate) were selected to investigate their effects on granule-based anammox systems under mainstream conditions (70 mg total nitrogen (TN) L-1, 15 °C). At a nitrogen loading rate of 2.87 ± 0.80 kg N m-3 d-1, a satisfactory effluent quality (TN < 10 mg L-1) was achieved in the presence of glucose or acetate at a chemical oxygen demand (COD/N) ratio of 0.5. The contribution of anammox to nitrogen removal decreased with increasing COD/N ratio to 1.0 because the expression of anammox functional genes was inhibited, whereas the expression of denitrifying functional genes was promoted. However, the nitrogen removal efficiency of the two considered reactors was maintained above 80 %. Self-stratification of the microbial community along the reactor height facilitated a functional balance through the retention of anammox bacteria in granules but resulted in washout of denitrifying bacteria in flocs under a high-flow pattern. These findings highlighted the advantages of granule-based systems in the mainstream anammox process due to their inherent biomass self-segregation property and the need for the development of targeted biomass retention strategies.

Removal of extracellular deoxyribonucleic acid increases the permeability and mass transfer of anammox granular sludge with different sizes.

As a key component of extracellular polymeric substances (EPS), extracellular deoxyribonucleic acid (eDNA) acts as a bridge in maintaining the structural stability of granular sludge. However, its ability of carrying antibiotic resistance genes (ARGs) promotes the gene horizontal transfer, raising a high risk for human health. In this study, a series of batch tests were performed to elucidate the response of anammox granular sludge (AnGS) with different sizes (S-AnGS with diameters lower than 0.9 mm and L-AnGS with diameters of 0.9-2 mm) to the removal of eDNA and corresponding mechanism. The results showed that the highest bioactivity of S-AnGS and L-AnGS was achieved by adding DNase I, and the absolute abundance of hzsA in the systems also increased. The dominant microorganism in each sludge was Candidatus Kuenenia, which maintained a higher relative abundance of 24% in S-AnGS. Settling experiments demonstrated that the permeability of AnGS was positively correlated with the addition of DNase I. The permeability index of granular sludge, Г, rose by 58.54% in S-AnGS and 11.79% in L-AnGS. The absence of eDNA is conducive to the increase in the permeability and porosity of AnGS. Similarity in the functional genes and microbial communities of intracellular and extracellular DNA implied the occurrence of gene transmembrane transfer. The findings enrich our knowledge of eDNA in anammox granules and provide a guidance for the specific control of gene transfer through reducing eDNA.

The response of anaerobic ammonium oxidation process to bisphenol-A: Linking reactor performance to microbial community and functional gene.

As a typical endocrine disruptor, bisphenol A (BPA) has been widely detected in various water bodies. Although the influence of BPA on traditional biological treatment system has been investigated, it is not clear whether it has potential impact on anaerobic ammonium oxidation (anammox) process. The short- and long-term influences of BPA on reactor operational performance, sludge characteristics and microbial community were investigated in this study. Results revealed that 1 and 3 mg L-1 BPA exhibited a limited adverse impact on granular sludge reactor performance. However, exposure of sludge under 10 mg L-1 BPA would cause an obvious inhibition on nitrogen removal rate from 10.3 ± 0.2 to 7.6 ± 0.4 kg N m-3 d-1. BPA would affect granular sludge metabolic substance excretion and lead to effluent dissolved organic content increase. Both the microbial community and redundancy analysis showed that BPA exhibited a negative influence on Ca. Kuenenia but a positive correlation with SBR1031. Low BPA concentration appeared a limited impact on functional genes while 10 mg L-1 BPA would cause decline of hzsA and hdh abundances. The results of this work might be valuable for in-depth understanding the potential influence of endocrine disruptor on anammox sludge.

LncRNA B4GALT1-AS1 promotes non-small cell lung cancer cell growth via increasing ZEB1 level by sponging miR-144-3p.

Background: Long noncoding RNAs (lncRNAs) are emerging as key players in the development and progression of cancer. Several malignancies involve dysregulated long noncoding ribonucleic acids (lncRNAs) in non-small cell lung cancer cell growth and their aggressive phenotypes. LncRNA B4GALT1-AS1 is important in the advancement of various malignancies, although its contribution to non-small cell lung cancer (NSCLC) remains unexplored. Methods: LncRNA B4GALT1-AS1 in NSCLC tissues was detected and further validated in a cohort of non-small cell lung cancer tissues. The effects of lncRNA B4GALT1-AS1 on proliferation were determined by in vitro experiments. The B4GALT1-AS1-miR-144-3p-ZEB1 axis was assessed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Furthermore, the mechanism of B4GALT1-AS1 was investigated using loss-of-function assays in vitro. Results: We showed significant upregulation of B4GALT1-AS1 in cell lines and tissues of NSCLC. B4GALT1-AS1 knockdown impeded the in vitro proliferation-related characteristics of the NSCLC cells. The demonstration of the binding capacity of B4GALT1-AS1 and miR-144-3p was predicted by bioinformatics and luciferase reporter activity assay. The B4GALT1-AS1 and miR-144-3p interaction was shown by using rescue experiments. NSCLC has a positive association with its target, zinc finger e-box binding homeobox 1 (ZEB1). Conclusions: In summary, the progression of NSCLC was facilitated by lncRNA B4GALT1-AS1 via interaction with miR-144-3p and positive regulation of ZEB1 expression.

Intracellular and extracellular protective mechanisms of the anammox consortia against exogenous sulfadimidine.

Antibiotics and antibiotic resistance genes (ARGs) have been recognized as emerging high-risk pollutants for human and animal health. This study systematically investigated the comprehensive effects of a typical antibiotic (sulfadimidine, SDM) in livestock and poultry breeding wastewater on the anammox process, with the aim of elucidating the intracellular and extracellular protective mechanisms of the anammox consortia to the antibiotic stress. Results revealed that the high-concentration SDM significantly reduced the specific anammox activity (SAA) by 37.8%. Changes in the abundance of Candidatus Kuenenia showed a similar trend with that of SAA, while other nitrogen-related microorganisms (e.g., Nitrosomonas and Nitrospira) contributed to the nitrogen removal especially during the inhibitory period. Resistance of the anammox consortia to SDM mainly depended on the protection of ARGs and EPS. Network analysis revealed the host range of eARGs was relatively larger than that of iARGs, and intI1 was closely associated with representative denitrifiers. In addition, metaproteomic analysis and molecular docking results indicated that abundant proteins in EPS could detain SDM in the extracellular matrix through forming complex via hydrogen bond. These findings provide a guidance for the stable operation of anammox process and ARGs transfer controlling.

Anammox sludge preservation: Preservative agents, temperature and substrate.

The difficulties of enrichment and preservation of anaerobic ammonium oxidation bacteria (AnAOB) greatly limit their application in practice. Herein, traditional and emerging preservative agents (e.g., EPS + N2H4, betaine, glycerol and trehalose) were evaluated for their preservation of AnAOB-dominant sludge at different temperatures (e.g., 4 °C and room temperature). In addition, the effects of substrates on preservation were also considered. The results showed that adding betaine or glycerol at 4 °C was the optimal strategy for preserving anammox granular sludge. The relative anammox activities (rAA) increased by 145.26% and 158.30% at the recovery phase, respectively. Moreover, the absolute abundances of functional gene hzsA increased by 339% and 46%, respectively. Although the granular properties and microbial community structures changed during the preservation, the general performance of anammox granules could effectively restored. Collectively, this study provides the optimal strategies for anammox sludge preservation at low temperatures.

A review on characterizing the metabolite property of anammox sludge by spectroscopy.

As one of the most promising autotrophic biological nitrogen removal technology, anaerobic ammonia oxidation (anammox) has gained intense attention for the past decades and several full-scale facilities have been implemented worldwide. However, anammox bacteria are easily affected by disturbed external environmental factors, which commonly leads to the fluctuations in reactor performance. The response of anammox sludge to external stress results in changes in components and structural characteristics of intracellular and extracellular polymer substances. Real-time and convenient spectral analysis of anammox sludge metabolites can give early warning of performance deterioration under external stresses, which is of great significance to the stable operation of bioreactor. This review summarized the research progress on characterizing the intracellular and extracellular metabolites of anammox sludge through spectroscopic techniques. The correlation between anammox sludge activity and its key metabolites was analyzed. Also, the limitations and future prospects of applying spectral analytical techniques for anammox bioreactor monitoring were discussed and outlooked. This review may provide valuable information for both scientific study and engineering application of anammox based nitrogen removal technology.

Curcumin increases crizotinib sensitivity through the inactivation of autophagy via epigenetic modulation of the miR-142-5p/Ulk1 axis in non-small cell lung cancer.

Drug resistance is a critical factor responsible for the recurrence of non-small cell lung cancer (NSCLC). Previous studies suggest that curcumin acts as a chemosensitizer and radiosensitizer in human malignancies, but the underlying mechanism remains elusive. In the present study, we explored how curcumin regulates the expression of miR-142-5p and sensitizes NSCLC cells to crizotinib. We found that miR-142-5p is significantly downregulated in NSCLC tissue samples and cell lines. Curcumin could increase crizotinib cytotoxicity by epigenetically restoring the expression of miR-142-5p. Furthermore, curcumin treatment suppressed the expression of DNA methylation-related enzymes, including DNMT1, DNMT3A, and DNMT3B, in NSCLC cells. In addition, the upregulation of miR-142-5p expression increased crizotinib cytotoxicity and induced apoptosis in tumor cells in a similar manner to that of curcumin. Strikingly, miR-142-5p overexpression suppressed crizotinib-induced autophagy in A549 and H460 cells. Mechanistically, miR-142-5p inhibited autophagy in lung cancer cells by targeting Ulk1. Overexpression of Ulk1 abrogated the miR-142-5p-induced elevation of crizotinib cytotoxicity in A549 and H460 cells. Collectively, our findings demonstrate that curcumin sensitizes NSCLC cells to crizotinib by inactivating autophagy through the regulation of miR-142-5p and its target Ulk1.

How anammox process resists the multi-antibiotic stress: Resistance gene accumulation and microbial community evolution.

The effects of multiple antibiotics on the anaerobic ammonia oxidation (anammox) process were investigated. The resistance of the anammox system to high-concentration antibiotics was also demonstrated through gradual acclimation experiments. Inhibition of the anammox process (R1) occurred when the concentrations of erythromycin (ERY), sulfamethoxazole (SMX) and tetracycline (TC) were 0.1, 5.0 and 0.1 mg L-1, respectively. The nitrogen removal efficiency (NRE) of R1 was reduced from 97.2% to 60.7% within 12 days and then recovered to 88.9 ± 9.5% when the nitrogen loading declined from 4.52 ± 0.69 to 2.11 ± 0.58 kg N m-3 d-1. Even when the concentrations of ERY, SMX and TC were as high as 1.0, 15.0 and 1.0 mg L-1, respectively, R1 maintained stable operation. The increases in the abundance of antibiotic resistance genes (ARGs) and in extracellular polymeric substances (EPS) content showed that the anammox process alleviated stress from multiple antibiotics mainly by producing ARGs and secreting EPS. The molecular docking simulation results illustrated the potential binding sites between ammonium transporter and different antibiotics. The upregulation of functional gene expression and the stable abundance of Candidatus Kuenenia in R1 compared with that in the control suggested that the R1 reactor generally maintained more stable long-term operation. This work provides a new understanding of the application of the anammox process to treat wastewater containing multiple antibiotics.

Inhibition of wastewater pollutants on the anammox process: A review.

The anaerobic ammonium oxidation (anammox) process has been recognized as an efficient nitrogen removal technology. However, anammox bacteria are susceptible to surrounding environments and different pollutants, which limits the extensive application of the anammox process worldwide. Numerous researchers investigate the effects of various pollutants on the anammox process or bacteria, and related findings have also been reviewed with the focused on their inhibitory effects on process performance and microbial community. This review systemically summarized the recent advances in the inhibition, mechanism and recovery process of traditional and emerging pollutants on the anammox process over a decade, such as organics, metals, antibiotics, nanoparticles, etc. Generally, low-concentration pollutants exhibited a promotion on the anammox activity, while high-concentration pollutants showed inhibitory effects. The inhibitory threshold concentration of different pollutants varied. The combined effects of multipollutant also attracts more attentions, including synergistic, antagonistic and independent effects. Additionally, remaining problems and research needs are further proposed. This review provides a foundation for future research on the inhibition in anammox process, and promotes the proper operation of anammox processes treating different types of wastewaters.

Unveiling the circRNA-Mediated Immune Responses of Western Honey Bee Larvae to Ascosphaera apis Invasion.

Western honey bee (Apis mellifera), a eusocial insect with a superior economic and ecological value, is widely used in the beekeeping industry throughout the world. As a new class of non-coding RNAs (ncRNAs), circular RNAs (circRNAs) participate in the modulation of considerable biological processes, such as the immune response via diverse manners. Here, the identification, characteristic investigation, and molecular verification of circRNAs in the Apis mellifera ligustica larval guts were conducted, and the expression pattern of larval circRNAs during the Ascosphaera apis infection was analyzed, followed by the exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 2083 circRNAs in the larval guts of A. m. ligustcia were identified, with a length distribution ranging from 106 nt to 92,798 nt. Among these, exonic circRNAs were the most abundant type and LG1 was the most distributed chromosome. Additionally, 25, 14, and 30 up-regulated circRNAs as well as 26, 25, and 62 down-regulated ones were identified in the A. apis-inoculated 4-, 5-, and 6-day-old larval guts in comparison with the corresponding un-inoculated larval guts. These DEcircRNAs were predicted to target 35, 70, and 129 source genes, which were relative to 12, 23, and 20 GO terms as well as 11, 10, and 27 KEGG pathways, including 5 cellular and humoral immune pathways containing apoptosis, autophagy, endocytosis, MAPK, Toll, and Imd signaling pathways. Furthermore, complex competing endogenous RNA (ceRNA) regulatory networks were detected to be formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The Target DEmRNAs were engaged in 24, 20, and 25 functional terms as well as 62, 80, and 159 pathways, including several vital immune defense-associated pathways, namely the lysosome, endocytosis, phagosome, autophagy, apoptosis, MAPK, Jak-STAT, Toll, and Imd signaling pathways. Finally, back-splicing sites within 15 circRNAs and the difference in the 9 DEcircRNAs' expression between un-inoculated and A. apis-inoculated larval guts were confirmed utilizing molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions, but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. m. ligustica larvae against A. apis invasion.

Resistance genes and extracellular proteins relieve antibiotic stress on the anammox process.

The anaerobic ammonium oxidation (anammox) process is regarded as a promising approach to treat antibiotic-containing wastewater. Therefore, it is urgent to elucidate the effects of various antibiotics on the anammox process. Moreover, the mechanism of extracellular polymeric substance (EPS) as protective barriers to relieve antibiotic stress remain unclear. Therefore, the single and combined effects of erythromycin (ETC) and sulfamethoxazole (SMZ), and interactions between EPS and antibiotics were investigated in this study. Based on a 228-day continuous flow experiment, high concentrations of ETC and SMZ had significant inhibitory effects on the nitrogen removal performance of the anammox process, with the abundances of corresponding antibiotic resistance genes (ARGs) increasing. In addition, the combined inhibitory effect of the two antibiotics on the anammox process was more significant and longer-lasting than that of the single. However, the anammox process was able to quickly recover from deterioration. The tolerance of anammox granules to the stress of low-concentration antibiotics was probably attributed to the increase in ARGs and secretion of EPS. Molecular docking simulation results showed that proteins in EPS could directly bind with SMZ and ETC at the sites of GLU-307, HYS-191, ASP-318 and THR-32, respectively. These findings improved our understanding of various antibiotic effects on the anammox process and the interaction mechanism between antibiotics and proteins in EPS.