## 3 Results and Discussion
In particular, ferrocene and its derivatives are renowned due to their biocompatibility, solubility in aqueous phase, and they are easy to conjugate [21]. In this study, cyclic voltammograms of riboflavin and ferrocene derivate were compared to identify an appropriate artificial electron mediator to create an enhanced bioanode with our target bacterium. As shown in Figure 3D, the anodic current of riboflavin, which is a potential endogenous electron shuttle for _C. glutamicum_, was increased by ferrocene derivate (ferrocenecarboxylicdehyde). Consequently, the anode electrode was fabricated with ferrocene-branched chitosan hydrogel in two steps. First, chitosan hydrogel was deposited electrochemically onto a carbon cloth electrode by immersion in an acidic chitosan solution (pH 5.0), and negative voltage was applied to achieve a constant current density (6 A m\({}^{-2}\), 60 s) (Figure 4). By applying a constant current, reduction reactions generated localized high pH adjacent to the electrode, resulting in neutralization of chitosan and triggering its sol-gel transition [13]. Figure 3: Differential pulse voltammetry (DPV) graphs (**A**) from negative to positive potentials (oxidation) and (**B**) from positive to negative potentials (reduction) of glassy carbon electrodes in cell-free medium after 72 h _C. glutamicum_ growth. (**C**) Cyclic voltammogram of riboflavin (2 uM) and (**D**) ferrocenecarboxaldehyde (Fc-CHO, 100 ug mL\({}^{-1}\)) in 0.1 M sodium phosphate buffer (pH 7.0). Scan rate, 10 mV s\({}^{-1}\). As shown in the obtained scanning electron microscope (SEM) images (Figure 5A,B), the chitosan layer covered the carbon cloth surface. After successful deposition, the chitosan-hydrogel-coated electrode was washed with distilled water. To observe attachment of bacterial cells on the bare and modified electrode, two types of electrodes were immersed into the _C. glutamicum_ cultured medium for 72 h. Surface morphologies of _C. glutamicum_ attached electrodes were visualized using a fluorescence microscope after SYTO9 staining (Figure 5C-E). The cells appeared to be more intimately associated with the chitosan-hydrogel coated carbon cloth than the bare electrode. In the second step of anode electrode fabrication, ferrocene was grafted onto the chitosan hydrogel layer by immersion of the chitosan-hydrogel-coated electrode in a solution containing ferrocenecarboxylaldehyde (ferrocene-CHO, 1 mg mL-1) followed by drop-wise addition of NaBH4 solution with continuous stirring. The layer was stable against exposure to a mild reducing agent (NaBH4) for fabrication of the ferrocene-branched chitosan redox-hydrogel (data now shown). Figure 4: (**A**) Electrodeposition of chitosan hydrogel. (**B**) Chronopotentiometry data for chitosan hydrogel formation on carbon cloth anode. Figure 5: SEM images of (**A**) bare carbon cloth and (**B**) chitosan-hydrogel-coated carbon cloth. Scale bar, 1 \(\mu\)m. Fluorescence microscopic images of (**C**) chitosan-hydrogel-coated carbon cloth without bacteria, (**D**) chitosan-hydrogel-coated carbon cloth which were incubated with _C. glutamicum_ for 24 h and (**E**) 72 h. Attached bacteria were stained with SYTO9 dye (5\(\times\) magnification). Figure 1B illustrates the hypothesis of direct electron transfer from _C. glutamicum_ to the anode with the aid of the ferrocene-branched chitosan redox-hydrogel. The redox characteristics of the bare carbon cloth (CC), chitosan hydrogel (CC/CS) and ferrocene-branched chitosan redox-hydrogel (CC/CS/Fc) electrodes were compared by cyclic voltammetry (\(-0.5\) to \(+0.8\) V) in 0.1 M sodium phosphate buffer (pH 7.0) (Figure 6A). The similar capacitance of the chitosan hydrogel electrode (CC/CS) to that of the bare electrode (CC) indicated that a thin layer had been grafted onto the electrode by current-controlled electrodeposition [22]. The onset potential and peak potential of the ferrocene-branched chitosan redox-hydrogel electrode (CC/CS/Fc) were \(-0.13\) V and \(+0.36\) V, respectively. By contrast, when _C.