A study of the flavin response by Shewanella cultures in carbon-limited environments Jared N. Roy, a Heather R. Luckarift, bc Carolin Lau, a Akinbayowa Falase, a Kristen E. Garcia, a Linnea K. Ista, a Privthiraj Chellamuthu, d Ramaraja P. Ramasamy, b Venkataramana Gadhamshetty, b Greg Wanger, e Yuri A. Gorby, e Kenneth H. Nealson, de Orianna Bretschger, e Glenn R. Johnson b and Plamen Atanassov* a Received 8th August 2012, Accepted 24th August 2012 DOI: 10.1039/c2ra21727a Mediated electron transfer has been implicated as a primary mechanism of extracellular electron transfer to insoluble electron acceptors in anaerobic cultures of the facultative anaerobe Shewanella oneidensis. In this work, planktonic and biofilm cultures of S. oneidensis exposed to carbon-limited environments trigger an electrochemical response thought to be the signature of an electrochemically active metabolite. This metabolite was detected via cyclic voltammetry for S. oneidensis MR-1 biofilms. The observed electrochemical potentials correspond to redox potentials of flavin-containing molecules. Chromatographic techniques were then used to quantify concentrations of riboflavin by the carbon-limited environmental response of planktonic S. oneidensis. Further evidence of flavin redox chemistry was associated with biofilm formation on multi-walled carbon nanotube-modified Toray paper under carbon-starved environments. By encapsulating one such electrode in silica, the encapsulated biofilm exhibits riboflavin redox activity earlier than a non-encapsulated system after media replacement. This work explores the electrochemical nature of riboflavin interaction with an electrode after secretion from S. oneidensis and in comparison to abiotic systems. 1. Introduction Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacteria (DMRB) that is widely utilized as a model organism in microbial fuel cell (MFC) research. 1–4 S. oneidensis appears to use a combination of mechanisms for extracellular electron transfer (EET) to insoluble electron acceptors. 5 These mechan- isms include: (i) direct electron transfer (DET) through outer membrane cytochromes; 6–9 (ii) direct electron conduction via extracellular appendages described as bacterial nanowires, 10–12 and (iii) mediated electron transfer (MET) (or shuttling) through exogenous metabolites. 13,14 Each of these mechanisms has been associated with electrochemical activity and, as such, exploited to produce power in MFCs. MFC power production is a fortuitous result of microbial metabolism in which the fuel cell performance is dictated by microbial EET processes, acting either individually or in concert. The ability to effectively transfer electrons is intrinsically linked to microbial metabolism and DMRB exhibit versatile EET mechanisms that allow the bacterium to respond to changes in physiological conditions. 18 It is unclear, however, how EET mechanisms are controlled by physiological constraints or how environmental conditions may dictate each mode of EET. Elucidating and understanding these relationships may lead to improved MFC systems by: 1) guiding the rational development of engineered surfaces to improve the physical association between microbes and products of metabolism; 2) determining microbial culture conditions that provide reproducible redox processes; and thereby 3) define optimal operational conditions for MFCs. In this work we explore the phenomena of riboflavin produc- tion by MR-1 in electron donor-limited conditions, 21 and investigate the influence of riboflavin redox chemistry within biofilms formed on the electrode. One inherent problem in the study of Shewanella spp. is the apparent rapid detachment of biomass from the biofilm under certain environmental stimuli. 22 A method to artificially bind a culture to an electrode to mimic biofilm formation is investigated by immobilizing a defined population of MR-1 cells to an electrode by means of silica coating. This technique overcomes potential limitations of investigating riboflavin redox reactions within a S. oneidensis biofilm by preventing a loss of biomass during medium exchange. In either case, natural biofilm formation or silica immobilized biofilm formation, the subsequent adsorption of riboflavin onto electrode materials and the biofilm surface is predicted to dominate the electrochemical character of an MR-1 populated bio-anode under operating conditions defined within this study. 23 a Department of Chemical and Nuclear Engineering, Centre for Emerging Energy Technologies, The University of New Mexico, Albuquerque, 87131, NM. E-mail: plamen@unm.edu; Tel: +1 505 277-2640 b Airbase Sciences Branch, Air Force Research Laboratory, Tyndall Air Force Base, FL, 32403 c Universal Technology Corporation, Dayton, OH, 45432 d Department of Earth Science, University of Southern California, Los Angeles, CA, 90089 e The J. Craig Venter Institute, San Diego, CA, 92121 RSC Advances Dynamic Article Links Cite this: DOI: 10.1039/c2ra21727a www.rsc.org/advances PAPER This journal is ß The Royal Society of Chemistry RSC Adv. Downloaded on 24 September 2012 Published on 28 August 2012 on http://pubs.rsc.org | doi:10.1039/C2RA21727A View Online / Journal Homepage