Chen, XiaojueNitrogen (N) in wastewater presents in the forms of ammonia, nitrite, nitrate, and/or organic N. Discharging of ineffectively treated N-containing wastewater into water bodies could cause eutrophication, threaten the safety of the ecosystem, and impair the water quality for humans. Various Biological Nitrogen Removal (BNR) processes, including nitrification, denitrification, and anaerobic ammonium oxidation (Anammox), have been successfully applied in wastewater treatment plants (WWTP) for N removal. However, the performance and stability of BNR can be adversely impacted by chemical inhibitors present in wastewater. Azoles, classified as emerging organic contaminants (EOCs), are a group of man-made chemicals containing N atoms in the heterocyclic ring systems and have been widely applied as aircraft de-icing agents, in semiconductor manufacturing, and household dishwashing detergents. Various azoles have been detected in wastewater streams and their presence within WWTPs may impact the BNR processes. Azole compounds, such as benzotriazole (BTA), have been used as biocides/fungicides in agriculture. However, limited information is available about the potential inhibition of azoles to BNR processes, while guidelines for preventing BNR processes from azole inhibition and methods for system recovery are also unavailable. In this study, we investigated the short- and long-term impacts of azoles on the major BNR processes, including nitrification, denitrification, and Anammox. Besides, potential inhibitory mechanisms of azoles and resistance/adaptation of BNRs were assessed, aiming to develop an effective strategy to prevent BNRs from system interruption and/or efficiency decreasing caused by azoles present in WWTPs. In short-term impact assessment, both experiment-based lab research and literature review approaches were used to assess the inhibition potential of different azole compounds on major BNR processes. Nine azole compounds (5 diazoles and 4 triazoles) were selected to represent azoles with different structures and physiochemical properties. The concentration (IC50) of azoles needed to inhibit BNR processes by 50% is calculated. Different BNR processes showed various responses to azoles after short-term exposure (<24h). Pyrazole (PA), triazole (TA), BTA, and methyl-benzotriazole (MBTA) at 6 mg /L caused >90% inhibition of nitrification activity, while higher inhibition resistance to these compounds was observed in the denitrification process with the calculated IC50 (mg /L) of 126, 520, 412, and 152, respectively. In comparison, 50% inhibition of Anammox activity was observed at the concentration of BTA (20 mg /L) and MBTA (18 mg /L). The differences in azole inhibition were suspected to be related to BNR processes’ characteristics including the potential chelation of azoles with enzyme-bound copper (e.g., ammonia monooxygenase, AMO) in nitrifiers, high biodiversity of denitrification sludge, and unique cell structure (e.g., annammmoxosome) in Anammox bacteria. In addition, azoles with more functional groups and/or complicated structures (e.g., climbazole at 20 mg /L & fluconazole at 100 mg /L) exhibited less inhibition on the nitrification (PA and TA at 6 mg/L) and Anammox processes (BTA and MBTA at 20 mg/L). In WWTPs, wastewater contains a variety of different compounds and it is more difficult for azoles with complex structures to chelate with the key BNR enzymes (Kalyani Vikas Jog, 2021). More attention should be paid to azoles with smaller molecule sizes and simpler chemical structures such as PA, BTA, TA, and MBTA. Compared with short-term experiments (<24 hours), results from long-term exposure (3 to 9 months) of BNR processes to azole provide further insights into understanding the impacts of azole and the system’s response under a condition that is closer to practical. Azoles, such as BTA and MBTA, have been found to cause inhibition (>50%) of the Anammox process in short-term experiments at 20 and 18 mg/L levels, separately. However, the long-term influence of azoles, whether inhibitory or not in short-term experiments, on the Anammox process is not well studied. Therefore, in the second part of this work, we aimed to investigate the long-term impact of BTA and PA on the Anammox process and the potential mechanisms by which Anammox bacteria adapt to resist azole inhibition. Through long-term acclimation, the Anammox granular sludge could gain higher resistance to BTA below 30 mg/L. However, further exposure to higher BTA concentrations (40 mg/L) led to a gradual decrease in NAA from 70% to 40%. No inhibition ≥ 10% was observed during short-term exposure at 300 mg/L PA, but long-term exposure to 200 mg/L resulted in more inhibition than short-term exposure. While long-term exposure to 100 mg/L PA did not cause any decrease in Anammox activity, 200 mg/L PA led to 65% reversible inhibition in 40 days. To further investigate the potential mechanisms by which Anammox granular sludge adapts to resist azole inhibition, starvation experiments, ATP content analysis, and microbial composition analysis were conducted. BTA and MBTA were selected as representatives of azoles in the starvation experiment due to their reported inhibitory effects on Anammox granular sludge. Over a 28-day starvation experiment, Anammox activity decreased, and the lag phase increased with starvation time at substrate conditions of 75 mg/L NH4+ -N and 100 mg/L NO2- -N. However, the presence of BTA and MBTA in the starved Anammox sludge did not result in a further reduction of Anammox activity and change in Anammox sludge’s ATP content levels, suggesting that the short-term energy-required mitigation method may not be the major defense mechanism for Anammox bacteria resisting BTA and MBTA inhibition. Overall, the results indicate that a stepwise acclimation strategy could enhance Anammox resistance to azole inhibition. As an important pre-step to conventional (nitrification/denitrification) and advanced (Anammox) BNR, the long-term impacts of azoles on the nitrification process were studied using lab-scale sequencing batch reactors (SBR). BTA and PA were selected as two representatives of the azoles due to their wide usage, detection in wastewater, and their structure as benzene-containing triazoles and diazole. Adapted nitrification sludge had better performance in treating wastewater containing azoles. Before acclimation, the addition of 2.5 mg/L BTA and PA decreased 45% and >90% nitrification activity, respectively. After the acclimation with the stepwise increase strategy, no ammonium and nitrite accumulation was observed in the effluent when 2.5 mg/L BTA and 2.7 mg/L PA were added into the system. The normalized nitrification activity (NNA) of the BTA and PA groups were 72.5 ± 2.5% and 70.2 ± 2.5%, respectively, which were also higher than the non-adapted nitrification sludge in short-term tests. The nitrification sludge developed BTA degradation ability and high degradation rates were achieved during the acclimation process. The removal of N loading didn’t impact the BTA degradation process. The addition of an extra 50 mg/L COD increased the BTA removal rate while the extra 200 mg/L COD decreased the BTA removal rate. According to the results, the heterotrophic bacteria that existed in the nitrification sludge may contribute to the BTA degradation. Reported aromatic compounds/organic compound degraders such as Dechloromonas and Zoogloea were identified in the microbial community. Azoles showed a variable inhibition to denitrifying microorganisms in activated sludge batch tests. In the last part of this work, the long-term inhibition potentials of BTA and PA to the denitrification process were investigated. A stable denitrification process was observed with BTA and PA addition at 2.5 and 2.7 mg/L, respectively, suggesting the low risk of azole inhibiting the denitrification process in WWTPs. No BTA and PA degradation was observed within the 12-hour operation cycle. However, after increasing the reaction time to 7 days, more than 85% BTA and 45% PA degradation are observed during the process. Furthermore, all the denitrification reactors (Control, BTA, and PA) were found to be able to degrade the BTA and PA at similar degradation rates. The removal of N and organic content (acetate as COD source) loading didn’t impact the BTA and PA degradation process. Multiple potential aromatic compound/organic compound degraders were found in the denitrification reactor such as Thauera, Azoarcus, Georgfuchsia, and Dechloromonas. Further investigation is needed to examine the contribution of individual species to azole degradation and their synergistic relationship. The results of this work indicate that the presence of azoles in wastewater has the potential to adversely impact the BNRs in WWTPs, in particular those to which azoles are new. However, proper implementation of the acclimation strategy can enhance the resistance of BNR bacteria to azole inhibition, preventing the system from interruption, even failure, caused by azoles. The synergistic effect of the microbial community may contribute to the attenuation of the azole inhibition to the system. The results provide new insights into understanding how the BNRs respond to chemicals of emerging concerns and are expected to assist WWTP operators in developing effective strategies to recover the system from azole inhibition. This work could provide suggestions for WWTPs to maintain activity and efficiency when treating azole-containing wastewater, identify azole types with high potential risks, and understand which part of the wastewater treatment systems may be impacted.enDissertationEnvironmental engineeringBiological nitrogen removalBiological reactorsEmerging organic contaminantsInhibition adaptationWastewater treatment