Energy harvesting by microorganisms is typically associated with the formation of biofilms, but the precise physiological conditions for these processes is not fully elucidated, particularly for the facultative anaerobe Shewanella oneidensis MR-1 when cultured under anaerobic conditions. In this study, electrodes were incubated with anaerobic cultures of S. oneidensis MR-1 while being exposed to applied potential. Cell-to-cell signaling may be utilized by Shewanella to form biofilms, triggered by the release of specific metabolites, such as riboflavin, under changes in environmental conditions. Riboflavin may be retained at the electrode surface and thereby create aredox gradients that directs cell attachment to insoluble electron acceptors. A prolific biofilm developed on the electrode when it was a subjected to an applied potential of -0.3 V vs Ag/AgCl; corresponding to the redox potential for surface-bound oxidized riboflavin. These biofilms display a non-reversible oxidation at 0.2 V determined to be the terminal heme-protein complex in the Mtr pathway by using a double deletion mutant, Delta MtrC/OmcA, (C) 2013 The Electrochemical Society. All rights reserved.