Inoculum-dependent biofilm assembly shapes electron uptake in microbial electrochemical denitrification

Microbial electrochemical denitrification systems (MEDS) offer a promising route for nitrate removal from low C/N waters without external organic carbon addition, yet their practical application is limited by slow startup and low reaction rates. Here, we investigated how inoculum-derived initial conditions shape cathodic biofilm assembly and electron uptake behavior in MEDS by comparing organic-rich and organic-poor mixed inocula. Organic-rich inocula promoted rapid biofilm establishment, with biomass reaching 22.79 ± 0.56 μg·cm^-2 and total nitrogen (TN) removal efficiencies exceeding 90%, whereas organic-poor inocula formed sparse biofilms and achieved TN removal below 60%. Control experiments that removed or compensated for inoculum-derived soluble organics showed that these differences were not explained solely by residual substrates, but were strongly linked to inoculum-dependent biofilm assembly capacity. Electrochemical analyses, inhibitor assays, mediator measurements, and cytochrome-associated characterization collectively indicated that sludge-derived biofilms exhibited a greater contribution of surface-coupled electron uptake, whereas biofilms derived from organic-poor inocula showed a greater relative contribution of diffusible/mediator-assisted electron transfer. This inoculum-dependent performance trend persisted in real low C/N water and was accompanied by lower N₂O yield in sludge-derived systems after stable biofilm establishment. Mechanistically, the results show that inoculum-derived carbon and microbial legacy regulate early-stage biofilm formation, which in turn shapes electron uptake behavior and system-level denitrification performance. These findings provide a mechanistic framework for understanding startup limitation in mixed-culture electrotrophic denitrification and offer practical guidance for designing more robust nitrate-removal strategies in carbon-limited waters.