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Anaerobic ammonium oxidation (anammox) is a promising biological nitrogen removal process, due to its advantages of high efficiency and low cost. However, problems remain in the application, such as long startup period and susceptibility to environmental variations. Quorum sensing (QS), as a means of bacterial communication, attracts more attentions in regulating aggregation behavior and microbial density. Many physiological characteristics of anammox bacteria have been confirmed to be associated with QS, including specific anammox activity, growth rate and the production of extracellular polymeric substances (EPS), which directly affect the performance of anammox process. Therefore, a comprehensive understanding of the QS in anammox process is prerequisite. This work systematically reviewed the role and application of QS in the anammox process with the focus on mechanism. Additionally, current challenges and research needs were proposed. [ABSTRACT FROM AUTHOR]

While the application of mesophilic anammox process is currently the state of the art, the feasibility of a thermophilic anammox bioprocess is still unclear. In this study, we investigate whether glycine betaine (GB) addition can enhance the thermotolerance of mesophilic anammox biomass in the upflow anaerobic sludge blanket (UASB) reactors fed with synthetic wastewater at a nitrogen loading of approximately 4 kg N m–3 d−1. The results showed that during a long-term operation at 45°C with GB (0, 0.1, 1, 2 mM) addition, anammox performance became worse with the final effluent concentrations of NO2–N of 145 ± 11.6 mg L−1 and nitrogen removal efficiency decreased from 92.3–6.9%. Specific anammox activity decreased from 392.1 ± 12.1–6.0 ± 0.8 mg N g−1 VSS d−1, which were not significantly higher than those in the control reactor. The content of heme c showed a stronger downward trend in T1 (with GB addition) than in the control reactor T0. The qPCR results showed that the relative abundance of Candidatus Kuenenia decreased in both the experimental (from 53.5–28.8%) and control reactors (from 54.1–35.1%). Overall, continuous addition of exogenous GB did not improve the thermotolerance of mesophilic anammox consortia at 45°C. [ABSTRACT FROM AUTHOR]

• Microbial and genetic responses of anammox process to ETC and SMZ were different. • The combined effects of ETC and SMZ was more significant than that of the single. • Increase in ARGs and EPS probably relieved the selection pressures of antibiotics. • Proteins in EPS provided binding sites for two antibiotics via hydrogen bonds. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • EPS-PP facilitated the E. coli BY63 attachment and sludge immobilization. • EPS-PP carrier promoted anammox biofilm formation and nitrogen removal efficiency. • In R 1 with EPS-PP carriers, the abundance of Candidatus Kuenenia was 27.1%. • Upregulation of amino acid and lipid metabolisms contributed to biofilm formation. 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. [ABSTRACT FROM AUTHOR]

To achieve the efficient treatment of high-strength ammonium wastewaters, a two-stage anammox process was proposed in this study. The nitrogen loading rates (NLR) of two anammox reactors were increased to 60.19 g N L−1 d−1 (R 1) and 24.32 g N L−1 d−1 (R 2), through reducing hydraulic retention time (HRT) and increasing ammonium concentration. Afterwards, the reactors were operated in series at variable temperatures (6.15–34.13 °C) for 240 days to treat the synthetic high-ammonium containing wastewater, with the effluent of R 1 pumping into R 2 as influent. Finally, the advanced treatment of the high-strength ammonium wastewater (490 mg NH 4 +-N L−1 and 980 mg TN L−1) was successfully achieved, and nitrogen removal efficiency (NRE) maintained at 83.25 ± 9.48%. During the whole operational period (400 days), the richness and diversity of microbial community in two reactors generally increased, and Candidatus Kuenenia was identified as the dominant anammox bacterium. In serial-operation phase, the dominant phylum changed from Planctomycetes to Proteobacteria, and the average relative abundance of Proteobacteria 39.61% in R 1 and 48.32% in R 2. Variations in bacterial abundances and distributions were closely related to the substrate concentration and temperature. Correspondingly, the expression levels of most functional genes were reduced by low temperature, and were slightly different in settling, middle and floating sludge. Nitrogen transformation pathway in each phase was also determined based on the gene expression levels, and main pathways in both reactors included anammox and first step of denitrification (NO 3 −→NO 2 −). All findings of this study provide new insights into the potential application of the two-stage anammox process to treat high-strength ammonium wastewater under variable temperature conditions. Image 1 • A novel anammox process was proposed to treat high-strength ammonium wastewater. • The two-stage anammox process could maintain efficient and stable for a long term. • Microbial community composition and distribution changed. • Expression levels of functional genes were slightly affected by low temperature. • Main nitrogen transformation pathways included anammox and NO 3 −.→NO 2 −. [ABSTRACT FROM AUTHOR]

The potential application of anaerobic ammonium oxidation (anammox) to treat antibiotic-containing wastewaters has attracted public attentions. The responses of anammox to a typical antibiotic (spiramycin, SPM) with different exposure strategies (elevating-concentration strategy in R 1 and repeated-exposure strategy in R 2) were evaluated on the aspects of general performance, microbial community and genes. Results showed that anammox bacteria could adapt to low concentrations (≤1 mg L−1) of SPM, while high-concentration (≥3 mg L−1) SPM showed inhibitory influences. The nitrogen removal efficiency (NRE) quickly decreased from 90.21% to 76.29% when R 1 was exposed to 5 mg L−1 SPM, and then returned to a higher level within a short period. On the contrary, R 2 could not recover with the inhibition of 5 mg L−1 SPM, and the abundance of Planctomycetes declined from 18.96% to 15.41% on Day 105. Combined with the changes in key genes and functional bacteria, R 1 had a relatively stable performance and higher resistance to SPM, indicating the elevating-concentration strategy might be more effective to gain the tolerance of anammox process to high-concentration antibiotics. The present study provides a guide for the stable operation of anammox process treating antibiotic-containing wastewaters. Image 1 • High-concentration (≥3 mg L−1) spiramycin (SPM) could inhibit anammox activity. • SPM changed the microbial community structure in the anammox systems. • Abundances of antibiotic resistance genes changed with the SPM concentrations. • Elevating concentration strategy was more efficient to gain the SPM resistance. • Anammox process could be applied to treat the SPM-containing wastewaters. [ABSTRACT FROM AUTHOR]

The feasibility of using anaerobic ammonium oxidation (anammox) process to treat wastewaters containing antibiotics and heavy metals was evaluated in this study. The nitrogen removal performance and characteristic parameters were monitored during the whole experimental period of 258 d. The single and joint effects of zinc and tetracycline on the microbial community were studied in upflow anaerobic sludge blanket (UASB) reactors. The anammox performance remained at levels comparable with the initial state at the lower inhibitor concentrations (zinc, 0–2.26 mg L−1; tetracycline, 0–0.5 mg L−1). When the concentrations of zinc and tetracycline increased to 3.39 mg L−1 in R 1 and 1.0 mg L−1 in R 2 , an obvious deterioration in performance was observed. Dual inhibitors with a total concentration of ≥3 mg L−1 caused dramatic decreases in the nitrogen removal efficiency of R 3. The quantification results showed that the abundances of eight antibiotic resistance genes (ARGs), czc A and int I1 in the experimental reactors generally increased under stress from metals or/and antibiotics, with final values higher than in the control, while the functional gene abundances were lower. Moreover, most genes exhibited significant correlations. Microbial community analysis indicated that Planctomycetes (represented by Candidatus Kuenenia) was inhibited by both zinc and tetracycline, but still held the dominant position. Furthermore, Caldilinea (belonging to Chloroflexi) maintained a higher abundance during the inhibitory period, implying its potential resistance to both inhibitors. These findings suggested that anammox could be inhibited by metals and antibiotics, but it has the potential to remove nitrogen from wastewaters containing both of them within the concentration threshold. • The inhibition thresholds of zinc and tetracycline on anammox were 3.39 and 1 mg L−1, respectively. • The joint inhibition of zinc and tetracycline on anammox was stronger than the single inhibition. • Caldilinea was the potential resistance bacteria. • Anammox has the potential to treat wastewaters containing small amounts of metals and antibiotics. [ABSTRACT FROM AUTHOR]

Drug resistance poses a high risk to human health. Extensive use of non-antibiotic drugs contributes to antibiotic resistance genes (ARGs) transfer. However, how they affect the spread of broad-host plasmids in complex biological systems remains unknown. This study investigated the effect of metoprolol on the transfer frequency and host range of ARGs in both intrageneric and intergeneric pure culture systems, as well as in anammox microbiome. The results showed that environmental concentrations of metoprolol significantly promoted the intrageneric and intergeneric conjugative transfer. Initially, metoprolol induced excessive oxidative stress, resulting in high cell membrane permeability and bacterial SOS response. Meanwhile, more pili formation increased the adhesion and contact between bacteria, and the abundance of conjugation-related genes also increased significantly. Activation of the electron transport chain provided more ATP for this energy-consuming process. The underlying mechanism was further verified in the complex anammox conjugative system. Metoprolol induced the enrichment of ARGs and mobile genetic elements. The enhanced bacterial interaction and energy generation facilitated the high conjugative transfer frequency of ARGs. In addition, plasmid-borne ARGs tended to transfer to opportunistic pathogens. This work raises public concerns about the health and ecological risks of non-antibiotic drugs. [Display omitted] ● Metoprolol significantly enhanced the intra- and intergeneric conjugative transfer. ● More intercellular contacts like pili promoted the conjugative transfer of ARGs. ● Bioavailable energy provided driving force for ARGs conjugative transfer. ● Mechanism in pure-culture system was suitable for ARGs transfer in anammox consortia. ● The plasmid-borne ARGs tended to transfer to pathogenic bacteria. [ABSTRACT FROM AUTHOR]

[Display omitted] • The anammox process could tolerate to the stress of 10 mg/L ciprofloxacin (CIP). • The performance of anammox system deteriorated under 20 mg/L CIP. • The strategy of starvation and shock was effective to mitigate the CIP inhibition. • Upregulating ARG expression and key metabolic pathways promoted process recovery. • Isolated ARB showed multidrug resistance, particularly Pseudomonas. 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Propranolol and doxycycline triggered oxidative stress and MGE enrichment. • Doxycycline induced the higher frequency of HGT than propranolol. • Propranolol could also promote HGT by upregulating relevant functional genes. • Anammox consortia upregulated amino acid metabolism in responding to propranolol stress. Given that antibiotic resistance genes (ARGs) become emerging pollutants, their accumulation and transfer caused by extensive use of antibiotic and non-antibiotic drugs raise concerns. In contrast, the impacts of non-antibiotic drugs have been largely overlooked, and their regulation mechanism in anammox system remains unclear. In this study, the single and combined effects of typical antibiotic (doxycycline) and non-antibiotic drug (propranolol) on anammox consortia were evaluated and compared from process performance, community succession, metabolic and gene regulation perspectives. Significant reduction in the nitrogen removal efficiency (49.0 ± 0.5%) was only observed in the anammox process stressed by propranolol and doxycycline, but it was reversible. Metabolome results showed that anammox consortia upregulated amino acid metabolism under propranolol stress, while the abundance of pyrimidine and purine metabolic pathways significantly increased under doxycycline and multidrug stress. Microorganisms adapted to these stresses by changing the utilization preference for metabolites. Metagenomic analyses indicated that doxycycline and propranolol induced the high frequency of gene transfer through increasing oxidative stress level, membrane permeability, pili and efflux pumps. Significant correlations between ARGs and mobile genetic elements (MGEs), especially int I1, demonstrated the conjugative transfer of ARGs. Furthermore, structural equation models showed that microbial community, bacterial metabolism, MGEs, reactive oxygen species (ROS) and corresponding SOS response were key driving factors for ARGs transfer in the anammox systems. These findings fill the gap in the effect of non-antibiotic drugs on anammox system and their regulation mechanism, which further provides a theoretical guidance for anammox-based process to treat multidrug-containing wastewater. [ABSTRACT FROM AUTHOR]

Anammox and autotrophic desulfurization-denitrification (AADD) process is feasible for the nitrogen and sulfide removal in the same reactor, and the influence of excess nitrate produced by anammox could also be alleviated simultaneously. This study firstly proposed a novel strategy with inoculating single anammox sludge to start up the AADD process. Results demonstrated that the 90% nitrogen removal efficiency (NRE), 2.55 kg m−3 d−1 nitrogen removal rate (NRR), and 95% sulfide removal efficiency (SRE) were obtained at the influent total nitrogen of 280 mg L−1 and sulfide of 221.5 mg L−1, and the final effluent nitrate concentration was as low as 8 mg L−1 under the appropriate operation conditions. Tryptophan-like and protein-like substances were characterized as the main components in bound EPS. Thiobacillus (35.68%) and Pseudoxanthomonas (11.61%) were identified as the predominant genera. This study will pave a potential avenue to promote the treatment of high concentration nitrogen and sulfide in wastewater in the future. Unlabelled Image • AADD process could be successfully started up by merely seeding the anammox sludge. • Nitrate concentration was under 8 mg L−1 in the final effluent. • Tryptophan-like and protein-like were the main components in bound EPS. • Thiobacillus and Pseudoxanthomonas were the predominant genus in AADD process. [ABSTRACT FROM AUTHOR]

The anaerobic ammonium oxidation (anammox) process has the advantages of a high nitrogen removal rate, low operational cost, and small footprint and has been successfully implemented to treat high-content ammonium wastewater. However, very little is known about the toxicity of the heavy metal element Ni(II) to the anammox process. In this study, the short- and long-term effects of Ni(II) on the anammox process in an upflow anaerobic sludge blanket (UASB) reactor were revealed. The results of the short-term batch test showed that the half maximal inhibitory concentration (IC 50) of Ni(II) on anammox biomass was 14.6 mg L−1. A continuous-flow experiment was performed for 150 days of operation, and the results illustrated that after domestication, the achieved nitrogen removal efficiency was up to 93±0.03% at 10 mg L−1 Ni(II). The settling velocity, specific anammox activity and EPS content decreased as the Ni(II) concentration increased. Nevertheless, the content of heme c increased as the Ni(II) increased. These results indicate that short-term exposure to Ni(II) has an adverse impact on anammox process, but the anammox system could tolerate 10 mg L−1 Ni(II) stress after acclimation during continuous-flow operation for 150 days. High-throughput sequencing results indicated that the presence of Ni(II) had an impact on the microbial community composition in the anammox reactor, especially Candidatus Kuenenia. At Ni(II) concentrations of 0-10 mg L−1, the relative abundance of Candidatus Kuenenia decreased from 36.23% to 28.46%. • The IC50 of Ni(II) on anammox biomass was 14.64 mg L−1 in short-term assay. • The effect of Ni(II) on substrate removal kinetic behavior of anammox was studied. • The long-term effects of Ni(II) in anammox UASB reactor were evaluated. [ABSTRACT FROM AUTHOR]

Abstract Both nickel and salinity are often detected in the environment. Especially electroplating wastewater contains some salt and nickel, which affects microbial activity in biological wastewater treatment process. In this study, the effects of sustaining addition of a high-concentration salinity and Ni(II) on the anaerobic ammonium oxidation (anammox) process were examined. The results indicated that the anammox system had an acclimation ability to <0.2 mg L−1 Ni(II) and 20 g L−1 NaCl. After a recovery phase of approximately 70 days, the nitrogen removal efficiency and rate reached at 77.1% and 1.18 kg N m−3 d−1, respectively. The three-dimensional excitation-emission matrix fluorescence and Fourier transform infrared spectra results showed that the introduction of NaCl and Ni(II) caused a substantial variation in the quantity and composition of the bound EPS in the surface of anammox granules. The present study is the first to document the long-term effect of co-existence of salinity and Ni(II) on the performance of the freshwater-derived anammox bacteria in the upflow anaerobic sludge blanket reactor and to provide a reference for the stable operation of anammox bioreactors for the treatment of sulfonamide-containing wastewater. Graphical abstract Unlabelled Image Highlights • Long-term exposure to NaCl and Ni(II) resulted in co-inhibition on the anammox UASB system. • The sludge characteristics changed after chronic exposure to NaCl and Ni(II). • 3D-EEM analyses showed a change in organic compositions in B-EPS. • FTIR analyses suggested a shift in functional groups in B-EPS. • The deleterious impact of NaCl and Ni(II) was reversible. [ABSTRACT FROM AUTHOR]

Abstract The short- and long-term influence of quinoline on the properties of anaerobic ammonium oxidation (anammox) biogranules was evaluated. During batch tests, the bioactivity of anammox granules in the presence of different quinoline concentrations was monitored, and the IC 50 of quinoline was calculated to be 13.1 mg L−1 using a non-competitive inhibition model. The response of anammox granules to pre-exposure to quinoline was dependent on metabolic status, and the presence of both quinoline and NO 2 −-N had a rapid detrimental effect, resulting in a 64.5% decrease within 12 h. During continuous-flow experiments, the nitrogen removal rate (NRR) of the reactor decreased sharply within 3 days in the presence of 10 mg L−1 quinoline and then was restored to 2.6 kg N m−3 d−1. In the presence of quinoline-induced stress, the specific anammox activity and levels of extracellular polymeric substance and heme c were decreased, while settling velocity persistently increased. After cultivation and acclimation obtained by adding a medium level of quinoline to the influent, the anammox granule sludge was able to tolerate 10 mg L−1 quinoline in 178 days. Graphical abstract Unlabelled Image Highlights • The inhibitory effects of quinoline on anammox were investigated for the first time. • Gradient domestication was applied and toleration of 10 mg L−1 quinoline was achieved. • The content of EPS, heme c and diameter decreased after quinoline inhibition. [ABSTRACT FROM AUTHOR]

Abstract Given the increasing applications of NiO nanoparticles (NPs) in battery products, the potential effects of NiO NPs on anaerobic ammonium oxidation (anammox) systems were studied for the first time. The results showed that the anammox system performance obviously differed under the stresses of different NiO NPs concentrations. After the withdrawal of NiO NPs, the nitrogen removal performance of the anammox reactor returned to nearly that of the initial phase within 35 days. Compared with 0 mg L−1 NiO NPs, the specific anammox activity first increased and then decreased to the minimum value of 116.8 ± 13.8 mg TN g−1 VSS d−1 at 60 mg L−1 NiO NPs. The variations in the heme c contents and extracellular polymeric substance amounts were similar to the variations in the specific anammox activity throughout the whole experiment. Additionally, the relative abundance of the dominant bacteria (Candidatus kuenenia) increased from 20.44% at 60 mg L−1 NiO NPs to 23.14% at the end of the last phase. Thus, the potential effects of NiO NPs on anammox systems should be a cause for great concern. Graphical abstract Unlabelled Image Highlights • Long-term effects of NiO NPs on the anammox system were firstly investigated. • The anammox performance was enhanced below 10 mg L−1 NiO NPs and suppressed by 10–60 mg L−1 NiO NPs. • The stability of the reactor operation could be controlled by the (F/M)/SAA ratio. • "Ca. kuenenia " dominated the community in the presence of 1–60 mg L−1 NiO NPs. • The performance of the anammox reactor could recover after withdrawing the NiO NPs. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The environmental factors affecting anammox process were introduced. • Metabolic feedback and quorum sensing (QS) effects of anammox bacteria were discussed. • Biomass retention strategies and microbial aggregation under QS effects were reviewed. • Anammox coupled processes in mainstream was practical and promising. [ABSTRACT FROM AUTHOR]

[Display omitted] • Inhibition of roxarsone on the anammox process was relieved through acclimation. • Partial removal of roxarsone was achieved by the anammox process. • Arsenic biotransformation alleviated the side effect of oxidative stress. • Potential mechanism of anammox consortia in responding to roxarsone was elucidated. Roxarsone is a common organoarsenic feed additive used in livestock and poultry breeding, while the ecotoxicity of organic arsenic remains a concern. Therefore, the effects of roxarsone on the anaerobic ammonium oxidation (anammox) process and responding mechanism were investigated in this study. The minimum nitrogen removal efficiency (NRE) of anammox process was 64.0% under roxarsone stress. Meanwhile, the reactive oxygen species (ROS) content increased by 1.5–2.3 times, and electron transport system activity (ETSA) decreased by 22.9±5.0%. The total antioxidant capacity (T-AOC) level first increased and then decreased, resulting in an imbalance between ROS production and antioxidant defense. The copy numbers of arsenic resistance genes significantly increased, indicating that roxarsone was transformed in the anammox system. Co-occurrence networks manifested that arsenic resistance genes were significantly positively correlated with dominant genera. The detoxification mediated by biotransformation mitigated the toxicity of roxarsone and the side effects of oxidative stress. This work provides insights into the response and mechanism of anammox consortia to roxarsone and promotes the efficient treatment of wastewater containing organic arsenic by the anammox process. [ABSTRACT FROM AUTHOR]

• Microorganisms adapted to lincomycin through endogenous and exogenous regulations. • Microbial interactions contributed to their adaptations to lincomycin. • Quorum sensing regulated the microbial preference for amino acids. • CRISPR/Cas mediated the changes in gene transfer pathway of anammox consortia. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

• PFOA had no significant effect on the nitrogen removal efficiency of anammox system. • PFOA could bind with proteins in EPS with multiple binding sites. • PFOA reduced the absolute abundances of functional bacteria and genes. • PFOA changed the metabolic pathways of anammox consortia. Perfluorooctanoic acid (PFOA) is ubiquitous in aquatic environment, posing serious threats to human health, while the potential of anaerobic ammonium oxidation (anammox) process in treating PFOA-containing wastewater and the response mechanism of anammox consortia to PFOA remain unknown. In this study, the comprehensive effects of PFOA on anammox granules and their interaction mechanism were investigated based on molecular simulation and metabolomic analysis. The environmental concentrations (≤2.0 mg/L) of PFOA had no impact on the anammox process performance. However, microbial consortia structure and functional gene abundances changed under the exposure of PFOA. By contrast, Prokaryotes were more sensitive to PFOA than eukaryotes. In addition, PFOA regulated microbial metabolic pathways, including amino acid metabolism and nucleotide metabolism, by changing the cell energy allocation strategy. Molecular docking simulation suggested that the effects of PFOA on key proteins in anammox bacteria were significantly different. These findings provide insights into the interaction between anammox consortia and PFOA, and further promote the implementation of anammox process to treat fluorine-containing wastewater. [ABSTRACT FROM AUTHOR]

• PA caused oxidative stress in the anammox system. • PA exacerbated the CFX inhibition on anammox consortia. • Most antibiotics were adsorbed by PA in the anammox system. • The most dominant bacterium changed from Candidatus Kuenenia to norank_o_SBR1031. Emerging pollutants pose high risks to human health, and also put huge pressures on wastewater treatment. Their interactions would also affect the activity of functional microorganisms. This study investigated the response and mechanism of anaerobic ammonium oxidation (anammox) consortia to the long-term stress of common microplastics (polyamide, PA) and a broad-spectrum antibiotic (cephalexin, CFX). Although 0.1 g L−1 PA microplastics (MP) did not significantly inhibit the nitrogen removal performance, adsorption assays revealed that large amounts of cephalexin were adsorbed by PA. Thus, 0.5 mg L−1 CFX and 0.1–0.2 g L−1 PA caused a deterioration of anammox process. Simultaneously, the most dominant genus changed from Candidatus Kuenenia to norank o SBR1031 , and expressional levels of functional genes dramatically decreased. In addition, the abundance of gene encoding superoxide dismutase (SOD) increased by 10.7 %, verifying that PA also caused the oxidative stress of anammox bacteria. PA promoted the accumulation of CFX in the anammox system through adsorption and extended its side effects on anammox consortia. This study provides a new direction into the implementation of anammox process in complex wastewater treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fast start-up of anammox process was achieved with a mixture of activated sludge. • The maximum nitrogen removal rate of anammox reactor was 1.14 g N L−1 d−1. • Candidatus Brocadia was more competitive under high-strength substrate condition. • Quorum sensing potentially contributed to the start-up of anammox 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. [ABSTRACT FROM AUTHOR]

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 int I1 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. [Display omitted] • Exogenous 20 mg L−1 sulfadimidine (SDM) affected the anammox process performance. • Intracellular and extracellular protection depended on ARGs and EPS, respectively. • The absolute abundances of iARGs and eARGs increased under the stress of SDM. • Typical denitrifiers were the poteintal host of ARGs. • Extracellular proteins detained SDM through forming complex via hydrogen bond. [ABSTRACT FROM AUTHOR]

[Display omitted] • High concentrations of As(III) and SMX had negative but reversible effects on the anammox process. • Exogenous As(III) increased the permeability of extracellular polymeric substances. • As(III) had direct toxic effects on anammox bacteria. • The denitrifying bacteria became dominant when 3 mg L−1 SMX was added. • The host of arsenic resistance genes was changed under SMX pressure. Anaerobic ammonium oxidation (anammox) process exhibits advantages in treating high-ammonium livestock wastewater, while the anammox bacteria is vulnerable to heavy metal and antibiotics. Arsenic and sulfonamides extensively existed in livestock wastewater; unfortunately, their effects on the anammox process have not yet been investigated. This work comprehensively evaluated the single and joint effects of As(III) and sulfamethoxazole (SMX) on the anammox process and revealed the related inhibitory mechanism. Anammox had extraordinary resistance to As(III) stress (0–50 mg L−1), while nitrogen removal sharply deteriorated under the stress from 50 mg L−1 As(III) and 3 mg L−1 SMX. Exogenous As(III) damaged the protection of extracellular polymeric substance (EPS) and aggravated the inhibition of SMX. As(III) had direct toxic effects on anammox bacteria, while enhanced denitrifying capacity was the reason for inhibition of anammox by SMX. Moreover, the host arsenic resistance genes were changed under SMX inhibition. The findings of this study enrich our knowledge of the anammox response to various pollutants, and provide the operational guidance for anammox process to treat wastewater containing antibiotic and heavy metal. [ABSTRACT FROM AUTHOR]

[Display omitted] • The response of anammox process to STM was revealed for the first time. • The anammox process had higher STM tolerance than SPM. • The int I1 and corresponding ARGs generally increased under stress from antibiotics. • The collaboration of ARGs implied the multiple resistance of anammox process. The issues of antibiotics and corresponding resistance genes (ARGs) have attracted public attention due to their environmental destructiveness. Herein, the response of the anammox process to the typical antibiotics streptomycin (STM) and spiramycin (SPM) was systematically explored. In terms of process performance, the anammox system was highly resistant to STM (50 mg L−1), while 3 mg L−1 SPM led to a significant inhibition. Exposure to STM resulted in the selective cultivation of Candidatus Kuenenia, while SPM significantly reduced its abundance. In addition, the relative abundance of bacteria belonging to Chloroflexi increased significantly during the SPM feeding phase, implying its potential resistance. The int I1 and corresponding ARGs (STM resistance genes: aad A, aad B, and aph 3ib; SPM resistance genes: ere A, erm F and mph A) generally increased under stress from antibiotics. Co-occurrence network analysis showed that the collaboration among multiple ARGs might be the strategy used to relieve STM stress in the anammox system. This study systematically revealed the response of the anammox process to successive exposure to STM and SPM, and provides guidance for the treatment of wastewater multiple antibiotics containing. [ABSTRACT FROM AUTHOR]

[Display omitted] • The responses of two different anammox granules to CuNPs were investigated. • Antibiotic-exposed anammox granules were more sensitive to the CuNPs stress. • CuNPs induced the co-selection of multiple resistance genes in the anammox system. • The mechanism through which CuNPs affected the anammox granules was speculated. Two types of anaerobic ammonium oxidation (anammox) seed sludge were selected to evaluate their responses to copper nanoparticles (CuNPs) exposure. Antibiotic-exposed anammox granules (R 1) were more likely to be inhibited by 5.0 mg L-1 CuNPs than the normal anammox granules (C 1). The nitrogen removal efficiency (NRE) of C 1 decreased by 9.00% after two weeks of exposure to CuNPs, whereas that of R 1 decreased by 20.32%. Simultaneously, the abundance of Candidatus. Kuenenia decreased by 27.65% and 36.02% in C 1 and R 1 under CuNPs stress conditions, respectively. Generally, R 1 was more susceptible to CuNPs than C 1. The correlation analysis indicated that the horizontal transfer of antibiotic resistance genes and cop A triggered by int I1 facilitated the generation of multiresistance in the anammox process. Moreover, the potential multiresistance mechanism of anammox bacteria was hypothesized based on previous results. The results will generate new ideas for the treatment of complex wastewater using the anammox process. [ABSTRACT FROM AUTHOR]

Antibiotic pollution is becoming increasingly severe due to its extensive use. The potential application of the anaerobic ammonium oxidation (anammox) process in the treatment of wastewater containing antibiotics has attracted much attention. As common antibiotics, spiramycin (SPM) and streptomycin (STM) are widely used to treat human and animal diseases. However, their combined effects on the anammox process remain unknown. Therefore, this study systematically evaluated the response of the anammox process to both antibiotics. The half maximal inhibitory concentrations of SPM and STM were determined. The continuous-flow anammox system could adapt to SPM and STM at low concentrations, while antibiotics at high concentrations exhibited inhibitory effects. When the concentrations reached 5 mg L−1 SPM and 50 mg L−1 STM, the nitrogen removal efficiency dramatically decreased and then rapidly recovered within 8 days. Correspondingly, the abundances of dominant bacteria and genes also changed with antibiotic concentrations. In general, the anammox process showed a stable performance and a high resistance to SPM and STM, suggesting that acclimatization by elevating the concentrations was beneficial for the anammox process to obtain resistance to different antibiotics with high concentrations. This study provides guidance for the stable operation of anammox-based biological treatment of antibiotics containing wastewater. [Display omitted] • Combined effects of two different antibiotics on anammox process were investigated. • Spiramycin and streptomycin at high concentrations inhibited anammox activity. • Correlations between dominant bacteria and key genes were evaluated. • The mechanism of multi-antibiotic resistance was illustrated based on results. [ABSTRACT FROM AUTHOR]

The wide existence of saline wastewater has attracted public attention due to its environmental destructiveness. The potential of anammox to treat saline wastewater was systematically evaluated in this study. Three bioreactors with different salinity increasing strategies (R 1 : inhibition kinetic; R 2 : gradient increasing; R 3 : pulsed increasing) were operated to identify the optimal acclimation mode. The results showed that the inhibition of anammox activity by salinity was mainly caused by the loss of enzyme activity. Under the condition of 25.0 g NaCl L−1, the highest nitrogen removal rate of R 3 (2.36 ± 0.14 kg N m−3 d−1) indicated that the pulsed strategy might be optimal. Changes in microbial community might be the primary reason lead to different acclimatization results. The relative abundance of anammox bacteria increased by 37.19% in R 1 and by 46.81% in R 3 , but remained stable in R 2 with increasing salinity. Dynamic varations in bacterial interactions, proteins, and functional genes revealed the potential resistance mechanisms of bacteria to salinity. The present work provides a novel approach and guidance for the treatment of nitrogen-rich saline wastewater by the anammox process. Image 1 • Three acclimation strategies for salt-tolerant anammox process were proposed for the first time. • The inhibition of FAB by salinity was mainly caused by the loss of enzyme activity. • High abundance of anammox bacteria led to the excellent acclimatization results. • Corresponding strategies can be selected based on the wastewater characteristics. [ABSTRACT FROM AUTHOR]

The preservation of anammox granules is of great significance for the rapid start-up of the anammox process and improvement of performance stability. Therefore, it is necessary to explore an economical and stable preservation strategy. Exogenous extracellular polymeric substances (EPS) were used as protective agents for the preservation of anammox granules in this study. In brief, EPS from anammox sludge (A-EPS) and denitrifying sludge (D-EPS) were added to preserve anammox sludge at 4 °C and room temperature (15–20 °C). The results showed that A-EPS addition at 4 °C was the optimal condition for the preservation of anammox granules. After 90 days of preservation, the specific anammox activity (SAA) of the anammox granules remained at 92.7 ± 2.2 mg N g−1 VSS day−1 (remaining ratio of 33.4%), while that of the sludge with D-EPS addition at the same temperature was only 77.1 ± 3.2 mg N g−1 VSS day−1 (remaining ratio of 27.8%). The nitrogen removal efficiency of the experimental group with D-EPS at room temperature was 85.9%, and that of the A-EPS group reached 90.6% under the same temperature conditions. The abundance of the functional genes hzs A, hdh and nir S of the sludge (4 °C; A-EPS addition) after recovery were 138.5%, 317.1%, and 375.9%, respectively, of those of sludge from the D-EPS-added group at the same temperature. RDA revealed the contribution of proteins to the preservation process. Overall, this study provides an economical and robust strategy for the preservation of anammox granules. Unlabelled Image • Exogenous EPS was used as protective agents for the anammox sludge preservation. • Addition of exogenous EPS significantly increased anammox activity in short term. • Adding A-EPS at 4 °C led to the optimal recovery performance. • The protein in EPS is a potential factor in optimizing preservation performance. [ABSTRACT FROM AUTHOR]

• Binding of anammox EPS with Cu2+ was endothermic and entropy-increased. • Carboxyl groups of proteins were preferentially responded to Cu2+. • The protein in EPS changed into loose structure after combining with Cu2+. Extracellular polymeric substance (EPS) of anaerobic ammonium oxidation (anammox) sludge plays an important role in resisting the toxicity when treating heavy metal containing wastewater. Understanding the interaction mechanism between EPS and heavy metal is essential for stable and optimal operation of such technology. In this work, the identification and preferential response of the binding sites, as well as the binding thermodynamic and the conformational changes of EPS to Cu2+ were explored. Unlike the activated sludge system, functional groups in EPS would coordinate with Cu2+ to form inner sphere complexes, which is an endothermic and entropy-increasing process. Low concentration of Cu2+ was found to result in the reduced electrostatic repulsion and bridging effect, which promoted the compaction and agglomeration of EPS colloid. Two-dimensional correlation spectroscopy analysis confirmed that carboxyl groups of proteins were preferentially responded to Cu2+ compared to polysaccharides and hydrocarbons. More importantly, the secondary structure of the protein in EPS was disrupted and changed into loose state after combining with Cu2+. This work is of great significance for understanding the binding properties of anammox sludge EPS with Cu2+ and may provide theoretical support for elucidating the resist mechanism of anammox bacteria to heavy metals. [ABSTRACT FROM AUTHOR]

Anaerobic ammonium oxidation (anammox) bacteria are sensitive and susceptible to operating condition fluctuations that can lead to the instability of a bioreactor. Through multivariate spectral analysis, the dynamic changes of intracellular and extracellular metabolites of anammox sludge under the declined temperature stress were characterized. It was found that effluent fluorescence components were positively related to the bacterial activity, and the response of the protein-like substances to the temperature change was more sensitive than that of humic substances. Under the transient disturbance during temperature change from 35 to 15 °C, anammox system tended to considerably excrete extracellular polymeric substances to resist the low temperature inhibition. However, the long-term exposure of the sludge at 10 °C resulted in the considerably inhibition of sludge activity, granular disintegration and heterotrophic denitrification bacteria increase. The two-dimensional correlation analysis further revealed that the humic acid in extracellular polymeric substances was preferentially responded to the temperature change than protein. Anammox bacteria tended to increase the intracellular protein and electron transfer-related reactive substance excretion to counteract the low temperature inhibition. Herein, both the intra- and extra-cellular response characteristics of anammox sludge to temperature variation were successfully resolved via the combined spectra. This work provides a comprehensive understanding on the mechanism of anammox sludge to temperature variation and may be valuable for the development of bioreactor monitoring techniques. Unlabelled Image • The performance of anammox bioreactor was characterized by spectra combination. • 2-dimension correlation spectroscopy was used for extracellular substances analysis. • Effluent fluorescence components were positively related to the bacterial activity. • 15 °C was an inflection temperature for the extracellular substances excretion. [ABSTRACT FROM AUTHOR]

• Sulfide (20 mg L−1) could reduce the specific anammox activity by 61.7%. • Protein-like substances were quenched due to sulfide stress. • Sulfide addition led Candidatus Kuenenia decrease while Thiobacillus increase. • Functional gene abundances significantly decreased with 20 mg L−1 sulfide. Sulfide has attracted increasing attention due to its odor nuisance, toxicity and corrosion. Although variations in the nitrogen removal performance of anammox under sulfide stress have been reported previously, understanding the microorganisms at the molecular level is of greater significance. This study first deciphered the microbial community and functional gene response of anammox sludge to sulfide stress. Results showed that 20 mg L−1 sulfide could reduce specific anammox activity by 61.7%. The protein-like substances within extracellular polymeric substances were quenched at the end of the experiment. Moreover, the relative abundance of Candidatus Kuenenia significantly decreased from 28.7% to 6.4% while Thiobacillus increased from 0 to 7.2% due to sulfide stress. Furthermore, the abundances of functional genes (hzs A, hdh , nir K and nir S) significantly decreased when the sulfide concentration reached 20 mg L−1. These findings provide a further theoretical basis for the anammox process for nitrogen removal from wastewater containing sulfide. [ABSTRACT FROM AUTHOR]

• Cd(II) at 2 mg L−1 enhanced anammox performance. • Cd(II) at 5 mg L−1 inhibited anammox performance. • The relative abundance of Candidatus kuenenia increased at ≤2 mg L−1 Cd(II). • The SAA, EPS and microbial community could be recovered after withdrawing Cd(II). • Anammox biomass displayed a certain adaption to Cd(II) suppression. Cadmium (Cd) can cause the deterioration of biological systems through inhibiting the enzymes activity and disturbing the microbial metabolism. Although the influence of Cd on conventional wastewater treatment process has been studied, the response of anammox to Cd exposure still remains unclear. This study firstly investigated the adaption and restoration of anammox biomass to Cd(II) stress. Results showed that long-term exposure of anammox bacteria to 2 mg L−1 Cd(II) was beneficial for the reactor performance, while 5 mg L−1 Cd(II) would cause the decline of SAA, extracellular polymeric substance content and relative abundance of Candidatus kuenenia by 40%, 25% and 31%, respectively. Furthermore, these indexes could approximately recover to the initial status after withdrawing Cd(II) from the influent. Overall, the anammox biomass exhibited a certain adaption and restoration ability to the suppression of Cd(II). This study may provide key valuable information for the biological treatment of wastewater containing Cd(II). [ABSTRACT FROM AUTHOR]