Unveiling Phase-Dependent Fe–O Bond-Guided Nitrate Reduction at Biocathodes: Balancing Interfacial Reactivity and Iron Bioavailability

Abstract Iron oxides are widespread in anoxic water environments, yet how their structures regulate microbial nitrate reduction remains unclear. Here, we reveal that phase-dependent Fe–O bond characteristics guide denitrification in bioelectrochemical system (BES) by balancing lattice-iron bioavailability and interfacial electrochemical reactivity at biocathodes. Density functional theory (DFT) calculations revealed that surface Fe–O bond covalency was associated with the thermodynamic barrier to lattice-iron release. α-Fe2O3, with the most negative ICOHP, exhibited the strongest Fe–O bonding and restricted iron release, thereby limiting microbial iron acquisition and downstream Fe–S cluster assembly. In contrast, γ-Fe2O3 and γ-FeOOH exhibited higher surface electroactivity but suffered from structural instability, evidenced by phase transformation, cell encrustation, and elevated microbial mortality. With mixed valence and balanced Fe–O bonding characteristics, Fe3O4 achieved the highest denitrification efficiency (17.8-fold enhancement over its abiotic control), which enabled direct Fe(II) uptake via the FeoAB system, bypassing siderophore-dependent acquisition and simultaneously activating both TonB-mediated iron acquisition and Fur-flavin-regulated respiratory pathways. This dual-pathway activation supported enhancement in biomass growth, intracellular electron transfer, and Fe–S cluster-dependent enzymatic activity. These findings help establish Fe–O bond characteristics as a key structural factor for Fe–N coupling and offer a structural perspective for rational design of iron-oxide-based nitrate-removal technologies.