## 3 Results and Discussion
### NR and MV as Redox Mediators for C. pasteurianum DSM 525  
We started by testing five concentrations of each mediator, 20.0, 50.0, 75.0, 100.0, and 150.0 \(\upmu\)M, under anaerobic conditions (Figures S1 and S2). We selected the highest concentration (150.0 \(\upmu\)M) for tests with MFC\({}_{\text{DSM}}\) because it provided optimal \(j\)-values regardless of the mediator. In addition, preliminary tests had shown that adding 150.0 \(\upmu\)M NR or MV does not inhibit _C. pasteurianum_ DSM 525 growth (Figure S3).  
We selected the potential for recording the amperometric \(i\)-\(t\) curves on the basis of the oxidation potential of each mediator determined by CV. We applied an anodic overpotential (n) of 250 mV. Thus, we measured the current density at a fixed potential of \(-0.22\) V vs. Ag/AgCl and \(-0.16\) V vs. Ag/AgCl\({}_{\text{(stat)}}\) for MFC\({}_{\text{DSM}}\) in the presence of NR or MV in the anodic chamber, respectively. Furthermore, we obtained amperometric \(i\)-\(t\) curves for _C. pasteurianum_ DSM 525 without a mediator (MFC\({}_{\text{DSM}}\)) in the same experimental conditions (Figure 3).  
For the electrochemical tests, MFC\({}_{\text{DSM}}\) (_C. pasteurianum_ DSM 525, O.D. = 0.2) in the presence of NR or MV in the anodic chamber gave a current density of 17.05 \(\pm\) 2.25 and 8.45 \(\pm\) 2.98 \(\upmu\)A\(\cdot\)cm\({}^{-2}\), respectively. The combined system improved the _C. pasteurianum_ DSM 525 catalytic performance by 7.0- and 3.7-fold for NR and MV, respectively, compared to MFC\({}_{\text{DSM}}\) in the absence of a mediator (2.47 \(\pm\) 0.53 and 2.29 \(\pm\) 0.69 \(\upmu\)A\(\cdot\)cm\({}^{-2}\) by applying \(-0.22\) and \(-0.16\) V vs. Ag/AgCl\({}_{\text{(stat)}}\), respectively).  
Introducing mediators can enhance the _C. pasteurianum_ DSM 525 EET ability given that the MFC\({}_{\text{DSM}}\)\(j\)-values improved compared to the abiotic control. Mediators are frequently used with _C. pasteurianum_ in traditional fermentation and electrofermentation systems. However, studies focusing on EET enhancement are scarce. Many mediators can alter the NADH/NAD\({}^{+}\) redox ratio and modulate the profile of the desired products [28]. This metabolic shift occurs because substrate conversion into products relies on a specific balance between electron donors and acceptors that is unique to each microorganism [29]. Additional research is needed to investigate the use of mediators to facilitate EET in _C. pasteurianum_.  
Figure 3: Representative amperometric \(i\)-\(t\) tests were conducted for (**A**) NR and (**B**) MV, in solution at 150.0 \(\upmu\)M, in presence and absence of _C. pasteurianum_ DSM 525. All experiments were carried out in LP medium (pH = 7) at 25 \(\pm\) 2 \({}^{\circ}\)C.  
Nevertheless, the presence of _C. pasteurianum_ DSM 525 in MF\({}_{\text{DSM}}\) diminished its \(j\)-values compared to the presence of a mediator alone in solution (Figure 3). Mediators can form radicals that may react with cellular components, to decrease the electronic efficiency and to increase electronic losses. Conversely, compared to _C. pasteurianum_ DSM 525 alone, the catalytic efficiency improved, indicating that the _C. pasteurianum_ DSM 525, mediator, and anode interacted.