Long-term Effects Of Copper Stress On Denitrification In Hydrogen-based Membrane Biofilm Reactor

Due to disordered discharge of industrial wastewater,bivalent copper（Cu（Ⅱ））has become one of the common heavy metals in nitrate wastewater,which has a significant impact on various denitrification systems.At present,compared with bio logical heterotrophic nitrogen removal,the researches about the effect of Cu（Ⅱ）on biological autotrophic nitrogen removal system are particularly limited.In this study,hydrogen-based membrane biofilm reactor（H₂-MBfR）as a new biological autotrophi c denitrification process was operated continuously for more than 150 days.Based on exploring the long-term effects of Cu（Ⅱ）on the denitrification performance of the reactor,the protective response mechanism of extracellular polymeric substances（EPS）and autotrophic microbial community in the system was analyzed,and this study provides a scientific basis for the application of this technology to the efficient denitrification of wastewater containing copper.The effect of Cu（Ⅱ）on the denitrification performance of H₂-MBfR showed that with the increased of Cu（Ⅱ）concentration,the inhibition effect on the removal of NO₃^--N increased.1～5 mg/L Cu（Ⅱ）had a slight inhibition effect on the system,and the average removal rate and removal flux of 20 mg/L NO₃^--N were 95%and 425 mg m^-2d^-1,respectively.The instantaneous impact brought by 10 mg/L Cu（Ⅱ）made the removal rate of NO₃^--N drop sharply,but it recovered to 80.2%within 3 days.Reduced the concentration of Cu（Ⅱ）to 5 mg/L to explore the self recovery ability of the system,the removal rate gradually increased and stabilized to 98%,and the removal flux was 43 1 mg m^-2 d^-1,indicating that the reactor showed strong resistance and adaptability to the toxic effect of Cu（Ⅱ）,and the inhibition effect was reversible.The determination of EPS content showed that Cu（Ⅱ）promoted the secretion of EPS,the content of protein（PN）was much higher than that of polysaccharide（PS）,and the PN/PS increased from 0.93 to 1.99,indicating that PN was more sensitive to copper invasion.The results of three-dimensional excitation-emission matrix（3D-EEM）illustrated that tryptophan was the main component of EPS chelating Cu（Ⅱ）to reduce toxicity.The results of Fourier-transform infrared（FTIR）demonstrated that-OH、C=O、C-O and C-N bound and reduced Cu（Ⅱ）.The biofilm was characterized by scanning electron microscopy combined with X-ray energy dispersive spectroscopy（SEM-EDS）,transmission electron microscopy（TEM）and X-ray photoelectron spectroscopy（XPS）,and founded the electron-dense deposits of monovalent copper（Cu（Ⅰ））were found in EPS and bio films.High throughput sequencing analysis showed that long-term exposure to Cu（Ⅱ）would reduce the richness and diversity of microorganisms,and with the long-term selection of Cu（Ⅱ）toxicity,Azospira and Dechloromonas became the dominant denitrifying bacteria in the reactor.This study provides a new research perspective and theoretical reference for the process optimization design of efficient nitrate removal and copper recovery from high nitrogen wastewater containing copper.