Balancing electron competition in nitrogen oxide reduction via membrane-aerated electroactive biofilms for electron flow regulation and N₂O mitigation

Nitrogen removal in bioelectrochemical systems relies on electrons derived from anodic ammonia oxidation and their utilization for nitrogen oxides reduction at the cathode. However, incomplete anodic oxidation often results in electron competition between NO₂^- and N₂O reduction, even under an applied potential difference. Here, a counter-diffusion electroactive biofilm was constructed to synergistically regulate intracellular carbon metabolism and extracellular electron allocation. Specifically, the electric field activated the microbial glyoxylate shunt (GS) by suppressing isocitrate dehydrogenase (icd) while upregulating isocitrate lyase (aceA), thereby avoiding carbon loss via oxidative decarboxylation and conserving reducing equivalents. Meanwhile, membrane aeration facilitated rapid NO₂^- removal, decreasing its concentration from 11.57 to 2.39 mg/L, which effectively alleviated electron competition between nitrite reductase (Nir) and nitrous oxide reductase (NosZ). As a result, N₂O emissions were reduced by 6.6-fold. In addition, the electric field decreased polysaccharide content to 3.71 μg/mg, enhancing biofilm conductivity and electron transfer efficiency. The thermodynamic properties of the biofilm were also improved, as indicated by a 39.5 to 74.5% reduction in hydrophobic interaction energy (U_AB). Overall, the synergy between the electric field and membrane aeration simultaneously optimized intracellular redox balance, enabling efficient nitrogen removal with mitigated N₂O emissions.