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

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.