Spatial Heterogeneity in Electrochemically Active Biofilms: Mechanism, Measurement, and Future Perspectives

Electrochemically active biofilms (EABs) are a class of conductive assemblies composed of electroactive microorganisms and their extracellular polymers. They have shown potential for diverse environmental applications, including bioremediation, energy conversion, and biosensing. Despite growing evidence that the hierarchical architecture and heterogeneity of EABs critically determine electron transport efficiency and biofilm function, a comprehensive review addressing these spatial features remains lacking. This paper reviews the spatial heterogeneity of EABs, discussing in detail the physicochemical microenvironment gradients, biofilm structural composition, electron transport mechanisms, and metabolic activity heterogeneity. By analyzing existing studies, the ambiguity surrounding current measurements and understanding of the heterogeneity of EABs is clarified. Finally, future research directions are proposed, including the application of advanced imaging technology and gene-editing approaches, the elucidation of the dynamic changes of biofilm heterogeneity, and the in-depth investigation of complex matrices in other ecological systems, which offer new insights into the heterogeneity and architecture of EABs. A deeper understanding of this aspect will facilitate the optimization of the microbial electrode design, configuration, and operational strategies to advance the applications of EABs.