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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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. 15 N isotope tracing, gene abundance quantification, and external H 2 O 2 addition experiments showed that photoinduced trace H 2 O 2 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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 + N 2 H 4, 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 (R 1 ) 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 R 1 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, R 1 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 R 1 compared with that in the control suggested that the R 1 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier Ltd.)

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 copA triggered by intI1 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 hzsA, hdh and nirS 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 NO 2 - 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 T 1 (with GB addition) than in the control reactor T 0 . 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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The extensive application of antibiotics, and the occurrence and spread of antibiotic resistance genes (ARGs) shade health risks to human and animal. The long-term effects of sulfamethoxazole (SMX) and tetracycline (TC) on denitrification process were evaluated in this study, with the focus on nitrogen removal performance, microbial community and ARGs. Results showed that low-concentration SMX and TC (<0.2 mg L -1 ) initially caused a deterioration in nitrogen removal performance, while higher concentrations (0.4-20 mg L -1 ) of both antibiotics had no further inhibitory influences. The abundances of ARGs in both systems generally increased during the whole period, and most of them had significant correlations with intI1, especially efflux-pump genes. Castellaniella, which was the dominant genus under antibiotic pressure, might be potential resistant bacteria. These findings provide an insight into the toxic effects of different antibiotics on denitrification process, and guides future efforts to control antibiotics pollution in ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (hzsA, hdh, nirK and nirS) 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Persulfate based advanced oxidation process is a promising technology for refractory contaminants removal. Cobalt is considered as the most efficient metal in catalyzing peroxymonosulfate decomposition. Although different cobalt based nanomaterials have been developed, easy aggregation and metal ion leaching during catalytic reaction would result in its deficiency. To address the above issue, in this work, carbon supported Co/CoO core-shell nanocomposite was in-situ fabricated by using polyphenol-metal coordinate as precursor. Results indicated that cobalt nanoparticle with size of 10 nm was successfully prepared and well dispersed within the carbon matrix. By using as-prepared material as catalyst, 50 mg/L orange II was completely removed under the condition of 0.2 g/L peroxymonosulfate, 0.05 g/L catalyst, pH = 4.0-10.0. Both sulfate and hydroxyl radicals were formed during peroxymonosulfate decomposition, while sulfate radical dominated the pollutant removal. Mechanism study revealed that the cobalt was the key site for catalyzing peroxymonosulfate decomposition. This work might provide valuable information in designing and fabricating metal anchored carbon composite catalyst for efficiently and cost-effectively activate peroxymonosulfate., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Candidatus Microthrix parvicella has been frequently detected as the dominant filamentous bacteria in bulking sludge and thus seriously affects the stable operation of activated sludge processes. The extremely low growth rate of Ca. M. parvicella and its sensitivity to environmental variations greatly limit the development of effective techniques to control filamentous bulking. Based on previous investigations, a variety of restrictive substrates, operating and culture conditions, environmental factors and other potential inhibitors have varying degrees of impact on the growth of this microorganism. This review systematically summarizes the key factors affecting Ca. M. parvicella growth with a focus on the influencing mechanism. Recent filamentous bulking control strategies are also critically reviewed and discussed. Additionally, research needs for the next few years are proposed with the aim of establishing effective and specific control strategies for filamentous sludge bulking., (Copyright © 2019. Published by Elsevier Ltd.)

The sustainable management and recirculation of phosphorus resources are essential to our human lives. In this work, phosphorus removal and recovery from secondary effluent were achieved using municipal wastewater-derived materials as adsorbents. Through modification with 0.5 M NaOH for 30 min, iron containing sludge that originated from the coagulation pretreatment of municipal wastewater was successfully converted to phosphorus adsorbent. The maximal adsorption capacity of the prepared adsorbent was estimated to be 22 mg-P/g, and the adsorption performance remained stable in the pH range of 5-8. FeO(OH) was identified as the key adsorption site, and the ligand exchange mediated chemical adsorption was the main mechanism for phosphorus removal by the prepared material. Moreover, a laboratory-scale continuous-flow adsorption column experiment showed that the surplus phosphorus in secondary effluent could be readily reduced to <0.1 mg/L. By pyrolysis of P-laden alkali-treated iron sludge under oxygen limited conditions, the phosphorus was recovered and successfully applied to support wheat growth. This work provides valuable information for both the sustainable management of phosphorus streams in wastewater and cyclic utilization of waste sludge., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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), czcA and intI1 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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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), 2.55kgm -3 d -1 nitrogen removal rate (NRR), and 95% sulfide removal efficiency (SRE) were obtained at the influent total nitrogen of 280mgL -1 and sulfide of 221.5mgL -1 , and the final effluent nitrate concentration was as low as 8mgL -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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The increasing release of engineered nanoparticles (NPs) from consumer products has raised great concerns about their impacts on biological wastewater treatment. In this study, the widely-used ZnO NP was selected as a model NP to investigate its impact on high-rate denitrifying granular sludge in terms of sludge properties and community structure. A hormesis effect was observed during short-term exposure, in which the specific denitrification activity (SDA) was stimulated by 10% at 1 mg L -1 ZnO NPs, but inhibited by 23% at 5.0 mg L -1 ZnO NPs. When continuously exposed to 2.5 mg L -1 ZnO NPs, the nitrogen removal capacity of the denitrification reactor was nearly deprived within 15 days, and the relative abundance of the dominant denitrifying bacterium (Castellaniella) was decreased from 51.0 to 8.0%. Meanwhile, the dehydrogenase activity (DHA) and the content of extracellular polymeric substance (EPS) significantly decreased to 22.3 and 61.1%, respectively. Nevertheless, the presence of phosphate substantially weakened the adverse effects of ZnO NPs on the SDA, EPS, DHA and the relative abundance of functional genes even exposed to 6.25 mg L -1 ZnO NPs, which was associated with the fact that the level of Zn(II) released from ZnO NPs was significantly reduced in the presence of phosphate. Therefore, the toxicity of ZnO NPs may be mainly attributed to the release of toxic Zn(II) and could be attenuated in the presence of phosphate. Overall, this study provided further reference and meaningful insights into the impact of engineered NPs on biological wastewater treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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%., (Copyright © 2019. Published by Elsevier Ltd.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018. Published by Elsevier B.V.)