Biofilm formation by Shewanella oneidensis MR-1 towards fundamental understanding of anode respiring communities

Microbial fuel cells are bioelectrochemical devices where, on the anode, microorganisms oxidize complex carbon sources and reduce the electrode via complex mechanisms of extra-cellular electron transfer (EET). To date, Geobacter sulfurreducens has been identified as producing not only the largest current density but also large population density biofilms, of the known model organisms commonly used within the field. Another common model organism associated with microbial fuel cells is Shewanella oneidensis, containing multiple EET mechanisms and an apparent inability to form multi-layered biofilms in anaerobic culturing, i.e. microbial fuel cell operating conditions. In contrast to G. sulfurreducens, S. oneidensis biofilm formation appears to coincide with oxygen metabolism, with removal of such, prompting biomass detachment. In this study, to overcome obstacles in biofilm formation a method was employed to encapsulate biomass onto the surface of an electrode. This method used the chemical vapor deposition (CVD) of the silica precursor, tetramethyl orthosilicate (TMOS), causing SiO 2 to form a porous thin film effectively binding the culture to the electrode surface and acting as an artificial exopolysaccharide (EPS) biofilm binder. As a demonstration of proof, this methodology was applied to novel biocompatible electrode materials (PHBV-CF) and combined with a laccase catalyzed oxygen reduction cathode in a hybrid biological fuel cell. In comparison to the encapsulated anodes, additional experiments were performed to optimize the growth conditions at which S. oneidensis will naturally form biofilms on PHBV-CF. Observations indicated initial biofilm formation occurred at the onset of stationary phase growth in mirco-aerobic and anaerobic cultures, indicating a biofilm response to carbon substrate limitation. This observation was explored further and led to results indicating the importance of intra-cellular carbon fluxes and the subsequent metabolic response of riboflavin production by S. oneidensis. It was also observed that riboflavin production occurred in micro-aerobic and anaerobic environments at the onset of carbon limitation but no riboflavin was detectable in anaerobic cultures. As riboflavin has been indicated in many studies as one primary mechanism employed in EET via mediated electron transfer (MET; results herein contradict the idea of riboflavin MET due to the lack of carbon required to produce riboflavin, with carbon being essential to drive metabolic reduction of the electrode. Therefore, the hypotheses within this study explores the idea of riboflavin acting as an electrochemical signal, leading S. oneidensis cells to populate and form a biofilm on the surface of an electrode as the culture transitions from aerobic to anaerobic metabolism. To test this, S. oneidensis cells in anaerobic media were exposed to electrodes with externally applied potentials (-0.4 , -0.3, +0.1 and open circuit vs Ag/AgCl). Results indicated only the electrode with an applied potential of -0.3 V (corresponding to accumulation of oxidized riboflavin on the surface) retained biomass in the form of a biofilm. Furthermore, this biofilm exhibited a nonreversible oxidation species centered at +0.2 V vs Ag/AgCl, which was identified as outer membrane cytochromes. This novel culturing method provides for the first time, anaerobically formed biofilms of S. oneidensis engaged in direct electron transfer (DET) of the anode. Based on this methodology, S. oneidensis based anodes were made with current densities comparable to those reported within the literature for G. sulfurreducens. While providing novel S. oneidensis based anodes, a methodology is also described herein for statistical quantification of essential electrochemical characteristics for comparison between these electrodes, a first such description for microbial fuel cells.