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Biofilm formation by Shewanella oneidensis has been extensively studied under oxic conditions; however, relatively little is known about biofilm formation under anoxic conditions and how biofilm architecture and composition can positively influence current generation in bioelectrochemical systems. In this study, we utilized a recently developed microfluidic biofilm analysis setup with automated 3D imaging to investigate the effects of extracellular electron acceptors and synthetic modifications to the extracellular polymeric matrix on biofilm formation. Our results with the wild type strain demonstrate robust biofilm formation even under anoxic conditions when fumarate is used as the electron acceptor. However, this pattern shifts when a graphite electrode is employed as the electron acceptor, resulting in biofilm formation falling below the detection limit of the optical coherence tomography imaging system. To manipulate biofilm formation, we aimed to express BpfG with a single amino acid substitution in the catalytic center (C116S) and to overexpress bpfA. Our analyses indicate that, under oxic conditions, overarching mechanisms predominantly influence biofilm development, rather than the specific mutations we investigated. Under anoxic conditions, the bpfG mutation led to a quantitative increase in biofilm formation, but both strains exhibited significant qualitative changes in biofilm architecture compared to the controls. When an anode was used as the sole electron acceptor, both the bpfA and bpfG mutations positively impacted mean current density, yielding a 1.8-fold increase for each mutation. [ABSTRACT FROM AUTHOR]