## 4 Conclusions  
We investigated _C. pasteuranium_ DSM 525 as a biocatalyst in the anode of an MFC. Although the genus _Clostridium_ commonly occurs in bioanodes, its EET capacity is not fully understood. In the presence of this biocatalyst, the MFC current output almost doubled (1.72 vs. 3.02 uA-cm\({}^{-2}\)), which evidenced direct EET. The EET capacity can be further enhanced by introducing NR or MV as a mediator into the \(\mathrm{MFC_{DSM}}\), either in solution or as a film (FNR and FMV, respectively). Compared to NR, FNR introduction into the \(\mathrm{MFC_{DSM}}\) produced almost twice the current density (17.05 \(\pm\) 2.25 vs. 30.76 \(\pm\) 3.44 uA-cm\({}^{-2}\)). Conversely, introduction of MV or FMV into the \(\mathrm{MFC_{DSM}}\) produced nearly identical current density (8.45 \(\pm\) 2.98 vs. 8.91 \(\pm\) 1.92 uA-cm\({}^{-2}\)), indicating that FMV was a less effective mediator than FNR. Therefore, FMV might interact weakly with the \(\mathrm{MFC_{DSM}}\) anode, possibly  
Figure 4: Representative amperometric _i-t_ tests were conducted for (**A**) FNR at 20 cycles and (**B**) FMV at 10 cycles both in the presence and absence of _C. pasteurinum_ DSM 525 (\(\mathrm{MFC_{DSM}}\) and abiotic control, respectively). CVs comparing the anodic electrodes with (**C**) FNR and with or without C. pasteurinum_ DSM 525 (\(\mathrm{MFC_{DSM}}\) and abiotic control, respectively) at 20 cycles and (**D**) FMV and with or without C. pasteurinum_ DSM 525 (\(\mathrm{MFC_{DSM}}\) and abiotic control, respectively) at 10 cycles (scan rate of 1 mV\(\cdot\)s\({}^{-1}\)). All the experiments were carried out in LP medium (pH = 7) at 25 \(\pm\) 2 \({}^{\circ}\)C.  
due to dimer formation [30]. In turn, FNR appears to be promising for enhancing _C. pastearium_ DSM 525 EET because it potentially facilitates microorganism-film-anode interaction. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/fermentation10100497/s1](https://www.mdpi.com/article/10.3390/fermentation10100497/s1)]([https://www.mdpi.com/article/10.3390/fermentation10100497/s1](https://www.mdpi.com/article/10.3390/fermentation10100497/s1)), Figure S1: Representative amperometric \(i\)-\(t\) tests conducted for (A) NR and (B) MV mediators in the solution at different concentrations. All experiments were carried out in LP medium (pH = 7) at 25 \(\pm\) 2 \({}^{\circ}\)C. An overpotential of 250 mV was applied over the anodic peak indicated by CV. Figure S2: Comparison of current densities of NR and MV at concentrations of 20.0, 50.0, 75.0, 100.0, and 150.0 \(\upmu\)M.; Figure S3: O.D. at 600 nm measured at the beginning (purple bar O.D. = 0.10) and after 16 h of growth (gray bar) bar of _C. pastearium_ DSM 525 in the presence of NR and MV at 150.0 \(\upmu\)M. A control experiment was conducted with only with DSM 525, excluding any mediators. Figure S4: Optimization of the number of electropolymerian cycles for (A) NR and (B) MV by amperometric \(i\)-\(t\) tests in LP medium (pH 7.0), at 25 \(\pm\) 2 \({}^{\circ}\)C. Figure S5: Cyclic voltammograms (CVs) of electropolymerian for (A) NR and (B) MV. Figure S6: Representative amperometric \(i\)-\(t\) tests were conducted for (A) NR and (B) MV in presence and absence of _C. pastearium_ DSM 525 across different electropolymerian cycles. All experiments were carried out in LP medium (pH = 7) at 25 \(\pm\) 2 \({}^{\circ}\)C. An overpotential of 250 mV was applied over the anodic peak indicated by CV. Figure S7: EIS at fixed potential for (A) the electrode modified with FNR (\(-\)0.22 V-red line) and (B) FMV (\(-\)0.16 V-purple line) at 20 and 10 cycles, respectively, and (black line) blank experiment in absence of electromediated film. Conceptualization, A.C.B.Z. and V.R. ; methodology, A.C.B.Z., J.C.d.S., A.R.d.A. and V.R. ; investigation, A.C.B.Z. and J.C.d.S. ; resources, A.R.d.A. and V.R. ; data curation, A.C.B.Z., J.C.d.S., A.R.d.A. and V.R. ; writing--original draft preparation, A.C.B.Z., A.R.d.A. and V.R. ; writing--review and editing, A.C.B.Z., A.R.d.A. and V.R.