Challenging the Diffusion Barrier Paradigm: Biofilms Promote Flavin-mediated Electron Shuttling in Shewanella oneidensis

Abstract

Flavins are ubiquitous diffusible redox mediators that are crucial in enabling extracellular electron transfer (EET) in electroactive bacteria that often form biofilms in both natural and engineered environments. However, the behavior of flavin diffusion within biofilms remains poorly understood. In this study, we developed a colony-based electrochemical platform using interdigitated electrode arrays to quantify the diffusion coefficients of flavins within bacterial biofilms. We found that flavin diffusivity was about 11-fold enhanced in Shewanella oneidensis MR-1 biofilms than in the bulk solution. However, this enhancement was abolished in the gene deletion mutant that lacked the membrane-associated flavin-binding c-type cytochrome OmcA, which suggests that the flavin-OmcA interaction on cell surfaces facilitates flavin diffusion within the biofilm. Notably, the diffusion coefficients of other redox molecules, such as methylene blue and safranin, scarcely improved in biofilms, which validates the inference that flavin-specific interaction-accelerated diffusion occurs in biofilm environments. These findings uncover a phenomenon promoting long-range electron transfer, overturning the prevailing assumption that shuttling-based EET is hindered by slow molecular diffusion in biofilms. Our study highlights the functional importance of cell-surface cytochromes in overcoming the kinetic limitation of diffusion-based electron transfer, thereby shaping the bioenergetics in biofilms.