This study investigates bacterial proliferation across anode surfaces in MFCs using a novel multi-electrode configuration. The research aims to provide novel insights into the temporal and spatial dynamics of biofilm formation and its impact on system performance.

Microbial fuel cells (MFCs) offer a promising alternative for sustainable wastewater treatment and energy recovery. However, the mechanisms underpinning electrogenic biofilm formation remain poorly understood. This study investigates the spatial and temporal dynamics of microbial community assembly using a novel multi-electrode MFC design under two substrate conditions: acetate and starch. Pre-inoculation of three select electrodes led to successful current generation within 110 hours in both MFCs, while a dispersed inoculation strategy failed to establish electrogenic biofilms despite equivalent inoculum volume. Electrode positioning significantly influenced start-up, with vertical alignment above inoculated electrodes facilitating faster colonisation and current generation than lateral spacing. Notably, starch-fed MFCs exhibited more rapid and widespread biofilm proliferation, suggesting complex microbial consortia may disperse more efficiently than single-function electrogens. Community sequencing revealed spatial heterogeneity and a shift from diverse to more specialised anodic communities over time. Geobacter initially dominated, but community succession was shaped by substrate complexity, competition, and spatial structure. Interestingly, non-inoculated electrodes often outperformed inoculated ones, indicating that deterministic selection pressures favoured more efficient biofilms. However, long-term current production declined, particularly under batch conditions, suggesting that population drift and limited microbial renewal limited sustained performance. This study is the first to characterise electrogenic biofilm assembly in a multi-electrode MFC, highlighting the interplay between stochastic dispersal and deterministic selection. These findings underscore the importance of inoculation strategy, substrate selection, and continuous microbial replenishment for optimising MFC performance and real-world applicability.