nZVI-mediated cell membrane repair and respiratory chain reconstruction enhance biological denitrification mechanisms under chlorfenapyr stress

Hydrophobic chlorfenapyr stress can inhibit nitrogen removal by promoting biofilm enrichment, inducing cell membrane damage, and impairing respiratory chain function. Based on this, an nanoscale zero-valent iron (nZVI)-enhanced biofilm reactor was constructed in this study to elucidate the mechanisms by which nZVI alleviates chlorfenapyr stress and maintains nitrogen removal function. The results showed that nZVI significantly improved the system's shock load resistance and recovery resilience. Under chlorfenapyr stress, the system maintained a maximum NH 4 + -N removal efficiency of 83.86% and TN removal efficiency of 80.88%. Multi-omics analysis indicated that nZVI increased the predicted relative abundance of extracellular polymeric substances (EPS)-related functional genes, accompanied by relatively stable EPS characteristics. This process effectively reduced reactive oxygen species (ROS) levels, accompanied by an aqueous-phase chlorfenapyr removal efficiency of approximately 81%. nZVI alleviated the impact of chlorfenapyr stress on the dominant phylum Pseudomonadota and simultaneously induced the directional enrichment of Luteimonas and Truepera with tolerance and degradation potential. Analysis of the respiratory chain and nitrogen metabolism pathways revealed that nZVI increased the predicted relative abundance of genes associated with respiratory chain core complexes including Complex I through Complex V. nZVI also attenuated the chlorfenapyr-induced decline in the predicted relative abundance of denitrification (NAR)-related functional genes by approximately 10%, and increased the inferred functional potential of the dissimilatory nitrate reduction to ammonium (DNRA) pathway. Metabolic correlation analysis further demonstrated that nZVI induced the directional enrichment of the key functional genus Luteimonas and driving its secretion of Lpc (18:1) and Lpc (19:1-Sn1) to strengthen biofilm repair. This work provides theoretical support for the efficient and stable operation of high-toxicity and high NH 4 + -N wastewater treatment.