| The wide application of nanoparticles(NPs)would inevitably cause their release into the municipal sewer network during their production,transportation,utilization and disposal processes,which potentially obstruct the normal operation of the biological wastewater treatment system.Microbial driven nitrogen transformation process is not only the main pathway of biological nitrogen removal(BNR),but also an important source of greenhouse gas nitrous oxide(N2O)emissions during water treatment process.Hence,the systematic study of the toxic effects and stress mechanisms of NPs on the BNR system is the key to assessing the potential hazards of NPs to wastewater treatment systems and establishing toxic stress regulation strategies.However,there are few studies about the effects of NPs on the nitrogen transformations in the BNR system,especially the N2O emissions during the BNR process.In view of the problems above,this study combined the macro-system water treatment performances and the micro-molecular ecotoxicological analysis to investigate the typical NP(Zn O NPs,Ce O2 NPs,and Ti O2 NPs)stress on the nitrogen transformation processes and N2O emissions of the BNR system;explored the stress response of the sludge flora and extracellular polymeric substances(EPS)in the impaired system and the system's self-recovery regulation mechanisms;and revealed the response and regulation mechanisms of the BNR system to the NP stress under different reaction control conditions.Firstly,the short-term(6 h)stress of the three NPs(1,25 and 50 mg/L)on the nitrogen transformation processes and N2O emissions of the BNR system were evaluated.One mg/L of Zn O NPs or Ce O2 NPs,and all tested concentrations(1-50 mg/L)of Ti O2 NPs did not exert significant effects on the BNR system,but 25 and 50 mg/L of Zn O NPs or Ce O2 NPs had a dose-dependent negative impact on the biological nitrogen transformation process.Zn O NPs mainly interfered with the denitrification process,which led to the reduction of the total nitrogen(TN)removal efficiency and N2O emission flux.The reduction of the TN removal efficiency and the N2O emission flux induced by Zn O NPs may be due to the down-regulation of nitrate(NO3-)reductase gene(nar G)expression and the decrease of nitrite(NO2-)reductase gene/nitric oxide(NO)reductase gene and N2O reductase gene transcription ratios(nir K/nos Z and nor B/nos Z).Ce O2 NPs showed the potential of inhibiting ammonia nitrogen(NH4+-N)oxidation activity and increasing the N2O emission flux.The inhibition of ammonia oxidation activity under the stress of Ce O2 NPs can be attributed to the down-regulation of the ammonia monooxygenase gene(amo A)expression and the inhibition of the ammonia oxidizing bacteria(AOB)Nitrosomonas activity,while the increase of N2O emission was ascribed to the down-regulation of the N2O reductase gene(nos Z)expression and the promotion of the denitrification glycogen-accumulating organism(d GAO)Dechloromonas activity.The Zn O NPs'cytotoxicity were on the account of the release of the soluble Zn2+and the reactive oxygen species(ROS)generation,while the inducements of Ce O2 NPs'cytotoxicity were excessive accumulation of the intracellular ROS.In addition,the long-term stress mechanisms(180 d)of Zn O NPs(1 and 10 mg/L)on the biological nitrogen transformations and N2O emissions of the BNR system were quantified and the system's self-recovery potentials were explored.One mg/L of Zn O NPs did not display significant effects on the BNR system,but 10 mg/L of Zn O NP long-term stress inhibited both nitrification and denitrification processes,and eventually reduced the removal efficiencies of NH4+-N and TN,and the N2O emission fluxes.The suppressed N2O emissions exhibited a positive relationship with the activity ratios of NO2-/NO reductases and N2O reductase(NIR/NOS and NOR/NOS),which was expected to be the result of the inhibited heterotrophic denitrification process.The Zn O NP long-term stress induced the decrease of glucose and nitrogen metabolisms'key enzyme activities,nitrogen metabolism key gene abundances,and denitrification related microbial abundances,which were chiefly responsible for the inhibition of heterotrophic denitrification process and the decrease of N2O emission flux.During the recovery period,the removal efficiencies of NH4+-N and TN were restored to the control levels,while the N2O emission fluxes increased significantly,presumably due to the irreversible NO2-oxidation suppression.Furthermore,the short-term(24 h)stress mechanisms of Zn O NPs(1,25,and 50 mg/L)on the flocculation and sedimentation performances of activated sludge in the BNR system were revealed.All tested concentrations(1-50 mg/L)of Zn O NPs did not dramatically impact the sludge's sedimentation performance,but 25 and 50 mg/L of Zn O NPs exerted a dose-dependent negative effect on the sludge's flocculation performance.It was NPs themselves rather than the dissolved Zn2+that impaired the sludge flocculation performance because the Zn2+alone would not compromise the sludge's flocculation efficiency.The sludge flocculation performance was revealed to be inversely related to the EPS content in the sludge and the direct contacts between Zn O NPs and the cells in the sludge should be the prerequisite to stimulate the secretion of the sludge EPS.The poor sludge flocculation performance could also be caused by the reduction of the ratio of protein(PN)to polysaccharide(PS)and the zeta potential in the loosely bound(LB-EPS)after the sludge exposure to Zn O NPs.Fourier transform-infrared spectra(FT-IR)and three dimensional-excitation emission fluorescence spectra(3D-EEM)analysis further revealed that the decrease of the tyrosine PN-like substance level in the LB-EPS was probably the key reason for the decline of PN/PS ratio and zeta potential in the LB-EPS,which eventually induced the deterioration of the sludge flocculation performance under the Zn O NP stress.Finally,the response and regulation mechanism of the BNR system under different dissolved oxygen(DO)concentrations and influent NH4+-N loads to short-term stress of 50mg/L Zn O NPs were investigated and analyzed.Zn O NPs had no obvious effect on the NH4+-N oxidation activity of the BNR system under high DO concentration.However,the system's ammonia oxidation rate and AMO activity were significantly inhibited under low DO concentration,indicating that ammonia oxidizing microorganisms were more sensitive to Zn O NP stress.Under different aerobic DO concentrations and influent NH4+-N loads,NO3-reductase(NAR)activity,specific NO3-reduction rate(SNARR),TN removal rate,and N2O emission flux were markedly suppressed by 50 mg/L Zn O NPs,but the system's TN removal efficiency and N2O emission flux were more weakly inhibited by Zn O NPs under low DO concentration and high NH4+-N load,which were beneficial to alleviate the NP's toxic effect.Although the DO concentration has no significant effect on the Zn O NP dissolution rate,the soluble Zn2+concentration in the system was positively correlated with the influent NH4+-N load.The increases of superoxide dismutase(SOD)and catalase(CAT)activities under high DO concentration and low NH4+-N loading were beneficial to the system's resistance to the oxidative damage induced by Zn O NPs,while the over-production of EPS under low DO concentration and high NH4+-N loading was crucial for protecting the encapsulated sludge microbes from Zn O NP invasions.In summary,this study investigated the effects of typical NPs on the nitrogen transformations and N2O emissions of the BNR system at the physiological level,metabolic level,and molecular level;discussed the self-recovery potential of the sludge flora and pollutant treatment efficiency;revealed the mechanism of NPs interfering with sludge EPS and flocculation performances;and proposed the reaction conditions that are beneficial to alleviate the toxic effect of NPs.The findings of this study provide the necessary theories for correctly predicting and comprehensively evaluating the potential threats of NPs to the wastewater BNR system,establishing the NP release limits and environmental risk control standards,and promoting the establishment of effective emergency management and regulation strategies for wastewater treatment systems against NP stress. |
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