Electro-driven aerobic biological nitrogen fixation for protein and ammonium production by Azotobacter vinelandii...

The major challenges to the large-scale application of biological nitrogen fixation are its high energy demand and the acute oxygen sensitivity of nitrogenase. Bioelectrochemical nitrogen fixation (e-BNF) presents a promising strategy to circumvent these energetic constraints, however, extant e-BNF systems, largely restricted to anaerobic or microaerobic conditions, remain unable to reconcile efficient nitrogen fixation with high-density cell cultivation and industrial aerobic fermentation. Herein, a novel aerobic e-BNF system was developed, which elicited significant enhancements in nitrogen fixation performance of Azotobacter vinelandii under aerobic conditions with sucrose as the sole carbon source: biomass and ammonium yield increased by 2.73- and 9.36-fold, respectively. Mechanistic investigations revealed that exogenous electricity specifically activated the Mo‒Fe nitrogenase system, orchestrated through extensive metabolic reprogramming and augmented oxidative phosphorylation. A dual electron transfer mechanism was identified-primarily reinforcing the Rnf1-NifF pathway (>90% activity) with a newly discovered alternative electron transfer bypass restoring ∼ 9.3% activity. Metabolic engineering amplified these effects: heterologous OmcS nanowires increased electrically driven ammonium secretion 7.23-fold; phbB deletion eliminated competing PHB synthesis, reducing carbon consumption by 57.30% and raising cellular protein to 66.72%. This work elucidates synergistic mechanisms of aerobic e-BNF and provides a renewable-energy prototype for green ammonia and single-cell protein production.