Electron Flow Shifts from Anode Respiration to Nitrate Reduction During Electroactive Biofilm Thickening

As electrons generated through substrate oxidation compete with electrodes, dissimilatory nitrate reduction to ammonium (DNRA), denitrification in bioelectrochemical systems in the presence of nitrate, and nitrate reduction through an electroactive biofilm (EAB) are unpredictable. We find that pathways of nitrate reduction are related to EAB thickness and that 76 +/- 2 pm is the critical thickness of a biofilm at which both the inner and outer layers simultaneously include DNRA, leading to a maximum level of DNRA efficiency of 42%. Fractions of electrons flowing during nitrate reduction are relatively stable, but their distributions between DNRA and denitrification vary with biofilm thickness. Electrons prefer denitrification in an EAB that is 66 +/- 2 mu m, while DNRA reversely surpasses denitrification when the thickness increases in the range of 76 +/- 2 to 210 +/- 2 mu m. Biofilm thickening enhances the DNRA of all biofilms close to solution, where nirK remains constant and nrfA is significantly upregulated. However, nrfA is downregulated in layers close to the electrode when the biofilm is thicker than 76 +/- 2 mu m. These findings reveal the spatially heterogeneous reduction of nitrate in thick EABs, highlighting the importance of biofilm thickness to the regulation of end products of nitrate reduction.