Kinetic Competition Between Extracellular Electron Transport and Upstream Reactions in Microbial Electrode Catalysis

Abstract

Specific microbes are utilized for bioelectrode catalysis, at the anodic or cathodic electrode, in applications such as microbial fuel cells or electrosynthesis. In the anode chamber of microbial fuel cells (MFCs), microbial metabolism oxidizes organic compounds, and the electrons are transferred to the anode by extracellular electron transport (EET) processes. Despite numerous studies on the kinetics of EET to enhance the catalysis rate, the relevance with upstream reactions, including metabolism and intracellular electron transport chain, has scarcely been investigated. Herein, we investigate the rate-determining step for anodic current production (j) for the lactate oxidation of Shewanella oneidensis MR-1 by using riboflavin to specifically enhance the rate of EET via outer membrane c-type cytochromes (OM c-Cyts).1 Microbial current production was measured in an anaerobic, three-electrode system poised at +0.4 V vs. SHE and kept at 303 K with 10 mM sodium lactate as a sole electron donor. Without the addition of flavin, j increases, saturates, and decreases as illustrated in Figure 1a. Upon the addition of 5µM riboflavin, j showed immediate increase in increasing and saturating current phases, though it increased sharper in the former (Figure 1b and c). In contrast, there was little effect of the riboflavin addition when j was decreasing (Figure 1d), suggesting that the RDS for j gradually shifted from EET to other upstream processes. Differential pulse (DP) voltammograms performed in each phases showed three peaks at approximately –230, –100, and +60 mV, which can be assigned to free riboflavin, bound riboflavin on OM c-Cyts, and the heme centers in OM c-Cyts, respectively.2 Compared with the DP voltammogram when j was increasing, the peak current of the bound flavin showed a 50% decrease after j saturation, though the peak current of the heme centers was almost identical, indicating the dissociation of bound flavin from the OM c-Cyts. Given that flavin dissociation is induced by heme oxidation in OM c-Cyts,2,3 the hemes in OM c-Cyts appear to become more oxidized after j saturation. These results further support the observation that the electron supply to OM c-Cyts from the upstream reactions gradually becomes slower than the EET rate. We will present the data for microbial growth, lactate consumption rate and voltammetric analysis in each phase, and the effect of trace metal removal from the medium to discuss the assignment of upstream reactions. Figure legend: Fig. 1. (a) A schematic image of the time course of current production. Area b, c, and d indicate the increasing, saturating, and decreasing current phase, respectively. (b–d) Current production in the presence (black lines) or absence (gray lines) of the addition of 5 µM riboflavin at the increasing (b), saturating (c), and decreasing (d) current phase, respectively. Arrows indicate the time points at which riboflavin was added. Microbes were added at t = 0 h. Reference 1. Dan C., Daniel B. B., Daniel R. B., and Jeffrey A. G.The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis. Journal of Bacteriology, 192, 2: 467–474 (2010). 2. Okamoto, A., Hashimoto, K., Nealson, K. H. and Nakamura, R. Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones. P Natl Acad Sci USA 110, 7856-7861 (2013). 3. Okamoto, A. et al. Cell-secreted Flavins Bound to Membrane Cytochromes Dictate Electron Transfer Reactions to Surfaces with Diverse Charge and pH. Sci Rep-Uk 4, e5628 (2014) Figure 1