Long-term effects of Cu(II) on denitrification in hydrogen-based membrane biofilm reactor: Performance, extracellular...

Divalent copper (Cu(II)) frequently coexists with nitrate (NO₃^-) in industrial wastewater and the effect of Cu(II) on the autotrophic denitrification system using H₂ as the electron donor remains unknown. In this study, the hydrogen-based membrane biofilm reactor (H₂-MBfR) was operated continuously over 150 days to explore the effect of Cu(II) on the performance of autotrophic denitrification system and understand the key roles of EPS and microbial community. More than 95% of 20 mg-N/L NO₃^- was removed at 1-5 mg/L Cu(II), and the removal rate of NO₃^--N was stabilized to 82% at 10 mg/L Cu(II) after a short period, while NH₄⁺ and NO₂^- in effluent were hardly detected, indicated that high concentration of Cu(II) did not permanently inhibit the denitrification performance in H₂-MBfR. Colorimetric determination showed that Cu(II) stimulated the secretion of EPS, in which the protein (PN) content was much higher than polysaccharide (PS). The PN/PS ratios increased from 0.93 to 1.99, and the PN was more sensitive to copper invasion. The results of three-dimensional excitation-emission matrix illustrated that tryptophan was the main component of EPS chelating Cu(II) to reduce toxicity. The results of Fourier-transform infrared demonstrated that hydroxyl, carboxyl, and protein amide groups bound and reduced Cu(II). Furthermore, Cu(II) was effectively removed (>80%), and the results of distribution and morphology analysis of Cu(II) show that the electron-dense deposits of monovalent copper (Cu(I)) were found in EPS and biofilms and the reduction of Cu(II) to Cu(I) was an obvious self-defense reaction of biofilm to copper stress. The microbial richness and diversity decreased with the long-term exposure to Cu(II), while the relative abundance of denitrifiers Azospira and Dechloromonas increased. This study provides a scientific basis for the optimal design of treatment system for removal of nitrate and recovery of heavy metals simultaneously.