## 3 Results and Discussion  
After _C. glutamicum_ cells were inoculated into a reactor containing a glassy carbon electrode, cyclic voltammograms were analyzed. As shown in Figure 2A, both the anodic (oxidation) and cathodic (reduction) currents after 72 h of bacterial growth (red) were highly increased relative to 24 h (black). The cyclic voltammogram, after removing bacterial cells (cell-free medium) after 72 h growth, still maintained its original capacitance (dashed red). Different cyclic voltammetry patterns were also measured at abiotic (medium only) and biotic condition (with bacteria) (Figure S1). Chronoamperometry data (Figure S2) and the mean current densities (Figure 2B) after 72 h growth with or without bacterial cells (cell-free medium) and poised at +0.4 V (vs. Ag/AgCl) were measured to ca. \(205.5\pm 14.7\) nA cm\({}^{-2}\) (\(n=3\)). The results reflect the electrical interaction between the bacteria and the electrode through the extracellular components. According to previous studies, flavins are known to act as an endogenous electron shuttle in electrici _Shewanella_ species [15,16]. Interestingly, _C. glutamicum_ has relative metabolic pathways for endogenous biosynthesis of flavins including riboflavin (vitamin B\({}_{2}\)) and flavin 5\({}^{\prime}\)-monophosphate (FMN) [17]. To determine if extracellular electron transfer from _C. glutamicum_ to the anode had occurred, reverse-phase LC-MS coupled with secondary MS analysis was performed with supernatant (cell-free medium) after 72 h growth of _C. glutamicum_. The results showed that riboflavin had accumulated to \(1.67\pm 0.08\) uM, whereas FMN had accumulated only to \(0.07\pm 0.04\) uM (data not shown). To explore the electrochemical activity of the extracellular components, the redox peaks of the supernatant (cell-free medium) after 72 h growth of _C. glutamicum_ were scanned by differential pulse voltammetry (DPV). When scanning from negative to positive (Figure 3A) and from positive to negative (Figure 3B), anodic and cathodic peaks centered around \(-0.4\) V were detected. The cyclic voltammogram of riboflavin (2 uM in 0.1 M sodium phosphate, pH 7.0), which was a dominant extracellular electron shuttle candidate in this work, also showed anodic (\(E_{\text{pa}}\)) and cathodic (\(E_{\text{pc}}\)) peak potentials around \(-0.4\) V (at \(-0.42\) and \(-0.45\) V, respectively, Figure 3C). Besides, a similar shaped voltammogram appeared for flavin-producing microorganisms such as _Bacillus_ sp. WS-WY1 and _Pichia stipites_[18]. Besides, redox peaks at around 0 V were presumed that the reduced quinone pool in the respiratory chain in the bacterial cytoplasmic membrane is involved in the extracellular electron transfer [19]. _C. glutamicum_ also possesses a branched electron transport chain via menaquinone in its cytoplasmic membrane [9]. Taken together, these results imply that indirect or mediated electron transfer between _C. glutamicum_ and the electrode can occur via extracellular electron shuttles across dense cellular walls [15]. Figure 2: Evidence for secretion of electron shuttle by _C. glutamicum_. (**A**) Cyclic voltammograms of glassy carbon working electrode with _C. glutamicum_ growth. (**B**) Total oxidation current by established _C. glutamicum_ application (poised at \(+0.4\) V vs. Ag/AgCl). The y-axis represents the mean value of each current density recording for 600 s. The current densities were measured after 24 h (with bacteria) and 72 h (with bacteria or cell-free medium). Due to its non-motile character [7], the capacity of an anode for effective current collection from _C. glutamicum_ should be improved (notwithstanding the possibility of its independent production of extracellular electron shuttles). A number of electroactive chemical compounds used as artificial electron mediators have been used to generate bioelectricity such as neutral red, potassium ferricyanide, AQDS, and so on [20].