Integrating mechanistic modeling and microbial kinetics to diagnose electron allocation in a biofilm electrode reactor treating real low-COD/N municipal wastewater.

Biofilm electrode reactors (BERs) hold promise for nitrate removal of low-COD/N wastewater, yet their application to real municipal wastewater is hindered byunpredictable accumulation of denitrification intermediates (NO2-, N2O). In this study, a BER treating real municipal wastewater was operated and integrated with a hydrogenotrophic-heterotrophic denitrification model to diagnose the electron-allocation bottlenecks underlying intermediate accumulation. The BER achieved an average nitrate removal efficiency of 76.62 %, compared with 44.69 % for the conventional biofilm reactor, corresponding to a relative improvement of 71.44 %. This improvement was attributed to sustained cathodic H2 generation that maintained electron flux after organic carbon depletion. However, NO2- and N2O accumulated despite enhanced nitrate reduction, indicating that the denitrification bottleneck shifted from nitrate activation to downstream intermediate reduction. ETSA and enzyme analyses indicated that NO2- accumulation was associated with insufficient downstream nitrite-reduction capacity relative to nitrate reduction and unbalanced electron competition, which may have been further exacerbated by the limited bioavailability of complex organic carbon in real wastewater. In contrast, N2O accumulation was more closely associated with limited upstream electron supply and reduced NADH availability than with insufficient nitrous oxide reductase (NOS) capacity. The model captured nitrogen transformation and intermediate accumulation under both synthetic and real wastewater conditions, while deviations between model predictions and real-wastewater observations provided a quantitative indicator of the kinetic heterogeneity of real dissolved organic matter (DOM). Pathway-level flux analysis showed the mean total electron flux decreased from 6.56 mmol e-/cycle (Stage I) to 5.28 mmol e-/cycle (Stage II), and then partially recovered to 5.64 mmol e-/cycle in Stage III. These findings reveal electron-allocation bottlenecks in BERs treating real low-COD/N wastewater and provide a mechanistic diagnostic tool for optimizing bioelectrochemical denitrification toward coordinated reduction of denitrification intermediates under realistic wastewater conditions.