mZVI‐Enhanced Mixed Nitrogen Removal in Klebsiella oxytoca via Coordinated Electron Transfer and Metabolic Reprogramming

Co‐occurrence of ammonium (NH 4 + ‐N) and nitrate (NO 3 − ‐N) in wastewaters complicates biological treatment because of opposing oxygen demands and frequent nitrite (NO 2 − ‐N) accumulation. This study evaluates how microscale zero‐valent iron (mZVI) influences mixed‐nitrogen removal by Klebsiella strain under anaerobic and anoxic conditions. In synthetic mixed‐nitrogen tests (NH 4 + ‐N/NO 3 − ‐N = 35/15 mg L −1 ), mZVI supplementation accelerated NO 2 − ‐N turnover and increased total nitrogen (TN) removal to ~83% within 48 h; in real wastewater (after C/N adjustment), TN removal rose to ~92%. Electrochemical measurements demonstrated enhanced extracellular electron transfer (EET) in mZVI‐amended systems, while fluorescence spectroscopy showed elevated secretion of humic‐like substances and flavin‐like compounds. Transcriptomic analysis revealed upregulation of pathways and genes associated with nitrate reduction, oxidoreductase activity, cofactor (flavins) biosynthesis, and oxidative phosphorylation, consistent with increased electron flux and energy supply. An integrated mechanism is proposed whereby mZVI stimulates EPS‐flavin‐mediated EET, coupling extracellular redox enhancement with intracellular metabolic reprogramming to promote efficient denitrification and NH 4 + ‐N assimilation. These results clarify electron‐mediated controls on microbial denitrification and support the potential of mZVI as a cost‐effective adjunct for treating complex nitrogen‐laden wastewaters.