Neutral Red Film Augments Extracellular Electron Transfer Performed by Clostridium pasteurianum DSM 525

Abstract

Extracellular electron transfer (EET) is key to the success of microbial fuel cells (MFCs). Clostridium sp. often occurs in MFC anode communities, but its ability to perform EET remains controversial. We have employed Clostridium pasteurianum DSM 525 as a biocatalyst in a glycerol-fed MFC, designated MFC_DSM. We have also followed the EET of this biocatalyst in the presence of a mediator, namely soluble neutral red (NR), soluble methyl viologen (MV), neutral red film (FNR), or methyl viologen film (FMV). MFC_DSM provided power and current densities (j) of 0.39 μW·cm⁻² and 2.47 μA·cm⁻², respectively, which evidenced that the biocatalyst performs direct electron transfer (DET). Introducing 150.0 µM NR or MV into the MFC_DSM improved the current density by 7.0- and 3.7-fold (17.05 and 8.45 μA·cm⁻²), respectively. After 20 cyclic voltammetry (CV) cycles, the presence of FNR in the MFC_DSM anodic chamber provided an almost twofold higher current density (30.76 µA·cm⁻²) compared to the presence of NR in the MFC_DSM. Introducing MV or FMV into the MFC_DSM anodic chamber gave practically the same current density after 10 CV cycles. The MFC_DSM anodic electrode might interact with FMV weakly than with FNR, so FNR is more promising to enhance C. pasteurianum DSM 525 EET within MFC_DSM.

1. Introduction

The increasing world population has raised energy consumption substantially and motivated the search for cleaner and sustainable technologies [1]. In this context, several studies have attempted to improve bioelectrochemical systems (BESs), including microbial fuel cells (MFCs), to attain energy. Investigating new biocatalysts and modifying electrodes is fundamental to enhance microorganism–electrode interaction and to optimize extracellular electron transfer (EET), which are essential for further developing BESs [1].

MFCs recognizably produce clean energy while simultaneously degrading organic compounds and pollutants [2]. Nowadays, this technology is mostly applied by using mixed cultures or microbial consortia. However, there is a gap in the knowledge about how some microorganisms, especially Gram-positive bacteria, contribute to generating energy in MFCs and their actual EET capacity [3].

Shewanella sp. and Geobacter sp. have been extensively studied as pure cultures in MFCs [3,4]. Given that mixed cultures typically encompass numerous genera, the dominance of these species has prevented the electroactivity of other microorganisms from being explored. Furthermore, although Gram-positive bacteria are widely present in bioanodes, their electrogenic capacity has been poorly investigated. Compared to Gram-negative bacteria, Gram-positive bacteria have thicker cell walls, which may reduce their electron transfer efficiency [5]. This may be the reason why the EET of Gram-positive bacteria has been less frequently researched.

Species belonging to the genus Clostridium often occur in microbial communities of MFC bioanodes, and they act mainly as a fermenter [3,6,7]. However, the EET ability of Clostridium sp. remains poorly explored. By using cyclic voltammetry, in 2014 Choi et al. identified redox peaks for C. pasteurianum DSM 525, which highlighted its significant electroactivity. The authors discussed the asymmetry of the reduction and oxidation peaks, which was comparable to the asymmetry observed in current-producing electroactive microorganisms [8].

EET can occur through direct electron transfer (DET) or mediated electron transfer (MET) [9,10]. Microorganisms that exhibit DET frequently contain redox proteins in their outer membrane, so they transfer electrons directly from these redox-active centers to the electrode. Nevertheless, some microorganisms that lack DET can carry out MET through redox mediator molecules [9]. For instance, Pseudomonas aeruginosa and Shewanella oneidensis are known for their ability to excrete phenazines and flavins, respectively, which can serve as electron mediators and enhance EET [11,12].

To improve EET, exogenous electron redox shuttles (mediators) can be added alongside microorganisms exhibiting low EET efficiency. The choice of mediator is critical, and criteria such as biological compatibility, stability, and redox potential should be considered [1,13]. At non-toxic concentrations, soluble neutral red (NR), a phenazine-based molecule, and soluble methyl viologen (MV), a viologen-based molecule, can mediate electron transfer [1,14]. Additionally, they provide fast electrochemical responses and efficiently operate at negative potentials, fulfilling the aforementioned criteria.

Herein, we have assessed the ability of the Gram-positive Clostridium pasteurianum strain DSM 525 to act as an electroactive biocatalyst in the anodic chamber of a glycerol-fed MFC, designated MFC_DSM. We will show that this bacterial strain can perform EET. We will also show that adding NR or MV in solution or as a film (FNR and FMV, respectively) to the MFC_DSM anodic chamber, especially FNR, improves the MFC_DSM performance and results in higher current densities.

2. Materials and Methods

2.1. Bacterial Cell Growth and Culture Medium

Clostridium pasteurianum DSM 525 was obtained from the culture collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen. The bacterial cells were maintained in glycerol solution (20%, w·v⁻¹) at −80 °C, in a freezer.

Before the electrochemical tests were conducted, the cells were activated in fresh Reinforced Clostridium Medium (RCM, Sigma-Aldrich, St. Louis, MO, USA). After that, the cells were transferred to a pre-inoculum culture medium (the same medium used in the electrochemical tests) at 35 °C for 15 h. The medium that was used in the tests has been described by Lovley and Phillips (LP medium) and comprises (in mmol·L⁻¹) 28.0 NH₄Cl, 0.50 MgCl₂·6H₂O, 0.40 MgSO₄·7H₂O, 0.68 CaCl₂·2H₂O, 1.30 KCl, 1.70 NaCl, 0.003 NaMoO₄·2H₂O, 30.0 NaHCO₃, 5.20 Na₂PO₄, and 4.30 NaH₂PO₄·H₂O, supplemented with 0.05 g·L⁻¹ yeast extract [15]. Glycerol at 1.0 g·L⁻¹ was added as carbon source. The tests were carried out under anaerobic conditions (N₂ was purged through the medium for 8 min before the tests were initiated); the initial was pH 7.0 (25 ± 2 °C). The sterility of the culture medium and materials was ensured by autoclaving them at 121 °C and 1 atm for 20 min.

Before C. pasteurianum DSM 525 was directly exposed to 150.0 μM NR or MV, O.D. at 600 nm was measured to verify whether there were any inhibitory effects. Initially, the bacterial cells were activated in RCM medium and incubated for 24 h. Subsequently, they were transferred to LP medium, at an initial O.D. of 0.1, in the presence of 150.0 μM NR or MV. Later, O.D. was measured after bacterial growth for 16 h.

2.2. Glycerol-Fed C. pasteurianum DSM 525-Based MFC Setup (MFC_DSM)

MFC_DSM consisted of a double-chamber setup, namely an anodic chamber (36 mL) and a cathodic chamber, connected by an external resistance of 1000 Ω. The anodic chamber was filled with LP medium (pH 7.0) and inoculated with C. pasteurianum DSM 525 (O.D. at 600 nm = 0.2). The anode material was carbon cloth (2.0 × 2.5 cm), and the cathode (open-air cathode) was made of carbon cloth with 40% platinum (A6ELAT/BASF), hot-pressed with a Nafion^® membrane (NRE-212/Sigma-Aldrich) at 130 °C and 35 kgf·cm⁻² for 180 s [16]. All the electrochemical measurements are referenced to the Ag/AgCl_(sat) electrode. After inoculation, C. pasteurianum DSM 525 remained in the MFC for 18 h to ensure that MFC_DSM was active for the electrochemical tests to be conducted.

2.3. Electrochemical Measurements in MFC_DSM

To assess how C. pasteurianum DSM 525 performed in MFC_DSM, polarization/power curves and electrochemical impedance spectroscopy (EIS) were carried out in an AUTOLAB PGSTAT 30 potentiostat/galvanostat (NOVA 2.1 software).

Initially, the polarization curves were obtained at a low scan rate (1 mV·s⁻¹) starting from the open circuit potential (OCP) down to zero. OCP denotes the maximum operating voltage of the system, where the current is zero. MFC_DSM operated without an external resistance until OCP stabilized. Subsequently, the power curve was derived from the polarization curves according to Equation (1).

$$P={\displaystyle \frac{i U}{A}}$$

(1)

where “P” is the power density (W m⁻²), “i” is the current (A), “U” is the voltage (V), and “A” is the anode geometric area (m²).

EIS was performed at OCP, from 100 kHz to 0.01 Hz. Ten frequency points per decade with a root mean square (r.m.s.) sinusoidal perturbation were employed; the amplitude was 10 mV.

2.4. NR or MV as Mediator

NR or MV was introduced into the MFC_DSM anodic chamber to verify whether they promoted C. pasteurianum DSM 525 EET. Specifically, 4 g·L⁻¹ NR or 5 g·L⁻¹ MV, prepared in 0.025 M K₂HPO₄/KH₂PO₄ buffer (pH = 7.0) in 0.10 M KNO₃, was added to the MFC_DSM anodic chamber.

The kinetic study was conducted in LP medium (support electrolyte); a 50 mL single-chamber cell was employed. The electrochemical cell included three electrodes: a carbon cloth (2.0 × 2.5 cm) as working electrode, Ag/AgCl_(sat) as reference electrode, and a platinum wire as counter electrode. All the solutions were deaerated with N₂ gas. Kinetics were monitored by cyclic voltammetry (CV) at various scan rates, from 1 to 50 mV·s⁻¹, for 100 µM NR or 200 µM MV. An AUTOLAB PGSTAT 30 potentiostat/galvanostat (NOVA 2.1 software) was used. The potential window ranged from −0.9 to −0.1 V vs. Ag/AgCl_(sat) and −1.1 to 0.2 V vs. Ag/AgCl_(sat) for NR and MV, respectively.

2.5. FNR or FMV Deposition on the MFC_DSM Anode

FNR or FMV was deposited on the MFC_DSM anode according to the methodology described by Ghica and Brett [17]. The same electrochemical cell employed in Section 2.4 was used. All the solutions were deaerated with N₂ gas.

To obtain the MFC_DSM anode modified with FNR, first a 0.025 M K₂HPO₄/KH₂PO₄ buffer solution (pH = 6) was prepared in 0.10 M KNO₃ containing 1 mM NR. The film was formed by conducting CV from −1.0 to 1.3 V vs. Ag/AgCl_(sat) at 50 mV·s⁻¹. Different numbers of cycles (10, 15, 20, or 30) were evaluated.

The MFC_DSM anode modified with FMV was prepared under similar conditions. First, a 0.025 M K₂HPO₄/KH₂PO₄ buffer solution (pH = 7) in 0.10 M KNO₃ containing 1 mM MV was prepared. Then, CV was carried out from −1.1 to 0.2 V vs. Ag/AgCl_(sat) at 50 mV·s⁻¹. The number of cycles (10, 15, 20, or 30) was evaluated, as well.

EIS was performed to compare the internal resistance of the unmodified MFC_DSM anode with the internal resistance of the MFC_DSM anode modified with FNR or FMV. Frequency ranges from 100 kHz to 0.01 Hz and ten frequency points per decade with a root mean square (r.m.s.) sinusoidal perturbation were employed; the amplitude was 10 mV. For the unmodified MFC_DSM anode, OCP was used; for the MFC_DSM anode modified with FNR or FMV, the potential was −0.22 V or −0.16 V vs. Ag/AgCl_(sat), respectively.

CVs were also run for the MFC anode modified with FNR or FMV in the presence and absence of C. pasteurianum DSM 525. The potential window of −0.9 to −0.1 V vs. Ag/AgCl_(sat) was used for C. pasteurianum DSM 525 and FNR, 20 cycles, whereas the potential window of −1.1 to 0.2 V vs. Ag/AgCl_(sat) was employed for C. pasteurianum DSM 525 and FMV, 10 cycles.

2.6. Electrochemical Setup and Measurements Obtained by Using C. pasteurianum DSM 525 Alone or in the Presence of NR, MV, FNR, or FMV

The electrochemical measurements were conducted by using the electrochemical cell setup detailed in Section 2.4. Amperometric i-t tests were performed to evaluate the current density of C. pasteurianum DSM 525 under various conditions: in the absence of a mediator, in the presence of NR or MV, and in the presence of FNR or FMV. These measurements were carried out at a constant potential for 960 s. An anodic overpotential (η) of 250 mV was applied for both mediators. Specifically, tests regarding NR were subjected to −0.22 V vs. Ag/AgCl_(sat), while tests involving MV were maintained at a constant potential of −0.16 V vs. Ag/AgCl_(sat). The current density was obtained at the end of the amperometric i-t test at 960 s.

Initially, 20.0, 50.0, 75.0, 100.0, or 150.0 μM NR or MV was evaluated in LP medium at pH 7.0 (25 ± 2 °C). As for the tests involving FNR or FMV, four CV cycles were employed to obtain the film deposited on the anode. The methodology outlined in Section 2.4 was followed. Subsequently, the anode was transferred to fresh LP medium at pH 7.0 (25 ± 2 °C). Before each test, the solutions were deaerated with N₂.

For the tests involving C. pasteurianum DSM 525 and NR, MV, FNR, or FMV, DSM 525 was initially activated in RCM medium. After bacterial growth, the cells were transferred to a pre-inoculum containing LP medium, where they remained for 15 h. Then, an aliquot was transferred to another LP medium and incubated for 18 h. The bacterial cell culture was standardized to an O.D. at 600 nm of 0.2. After the O.D. was standardized, the supernatant was removed by centrifugation at 7000 rpm for 5 min and discarded. The resulting cell pellet was added to the assays containing 150.0 μM NR or MV and to the assays containing FNR or FMV in all cycles.

3. Results and Discussion

3.1. C. pasteurianum Electroactivity in MFC_DSM

Figure 1A and Figure 1B, respectively, show the power/polarization and EIS curves recorded for C. pasteurianum DSM 525 operating in an MFC with 1 g·L⁻¹ glycerol added (MFC_DSM).

The maximum power achieved with MFC_DSM and the abiotic control was 0.39 and 0.22 μW·cm⁻², respectively. The maximum current density achieved with MFC_DSM and the abiotic control was 3.02 and 1.72 μA·cm⁻², respectively. In other words, the current density almost doubled in the presence of C. pasteurianum DSM 525, indicating that this biocatalyst can perform direct EET to the electrode, effectively transferring electrons through the external circuit.

Although the genus Clostridium is typically known for its fermentative ability, it has also been demonstrated to transfer electrons to electrodes through ferredoxin-mediated processes [18]. However, the electroactive activity of C. pasteurianum remains considerably limited and poorly understood. Only in 2014 did Choi et al. suggest that C. pasteurianum is electroactive. These authors showed that C. pasteurianum DSM 525 can use both the electrode and substrate (glycerol and glucose) as electron donors through DET in a BES. By employing CV, the authors observed well-defined but asymmetric redox peaks for C. pasteurianum DSM 525, which indicates a quasi-reversible reaction. The authors highlighted similarities between these asymmetric peaks and the peaks observed for other electroactive microorganisms that can reduce Fe (III) and produce current [8].

Furthermore, the polarization curves obtained for MFC_DSM and the abiotic control showed an about 1.7-fold lower MFC internal resistance in the former case (9933 and 17,022 Ω, respectively). Polarization curves are a valuable tool to understand how an MFC behaves because they allow the main domains of potential losses limiting performance to be identified [19,20].

Also, we used EIS to describe how MFC_DSM and the abiotic control behave. We conducted the EIS tests at OCP (MFC_DSM = −0.265 V; abiotic control = −0.331 V vs. Ag/AgCl_(sat)). From these values, we generated a Nyquist plot (Figure 1B), which provided information about the resistance of the solution (R_s), namely 96.30 and 16.10 Ω for MFC_DSM and the abiotic control, respectively. We attributed the higher Rs obtained for MFC_DSM to the presence of microorganisms in the solution [21].

Additionally, total impedance (R_ct), which is associated with the charge transfer process, was 17-fold lower for MFC_DSM compared to the abiotic control (65 and 1128 Ω, respectively). This indicated that C. pasteurianum DSM 525 enhanced the efficiency of the overall electron transfer process. At low frequencies, a straight line characteristic of limited diffusion processes arose [21]. Moreover, the double layer capacitance (C_dl) of MFC_DSM was 2.2-fold higher (C_dl = 3.8 μF·cm⁻²) compared to the abiotic control (C_dl = 1.7 μF·cm⁻²). The C. pasteurianum DSM 525 present on the electrode probably helped to increase C_dl.

3.2. NR and MV Electrochemical Characterization

Figure 2A and Figure 2B, respectively, show the electrochemical behavior of NR and MV in LP medium in the presence of glycerol (1 g·L⁻¹) at pH 7.0.

NR is a phenazine-type molecule bearing a nitrogen-heterocyclic ring in its structure [22]. As mentioned earlier, many microorganisms lack redox-active proteins for DET on their cell surfaces, but they can perform MET by producing their own mediators, such as pyocyanin [11]. Alternatively, molecules of this class, which have well-known redox properties, can be added as mediators in BESs [17].

Figure 2A displays the CV curves obtained for 100.0 μM NR at different scan rates. A redox couple centered around −0.47 and −0.53 V vs. Ag/AgCl_(sat) emerged. Pauliukaite et al. (2016) studied the formation of poly(neutral red) films and reported a pair of redox peaks centered at −0.6 and −0.5 V vs. SCE, in phosphate buffer, which they attributed to NR-leuco–NR reduction–oxidation processes [23].

Furthermore, CV measurements at various scan rates ranging from 1 to 50 mV·s⁻¹ provided insights into the electrochemical behavior of NR. I_ap was linearly related with the square root of the scan rate, described by the equation $I_{ap}=5.3290\times {10}^{-4}v-7.4421\times {10}^{-4}$ (R² = 0.9514). Similarly, I_cp increased linearly with the square root of the scan rate, following the equation $I_{cp}=-5.1927\times {10}^{-4}v-8.1165\times {10}^{-4}$ (R² = 0.9409). This response, shown in the graph inserted in Figure 2A, indicated a diffusion-controlled process [24].

Overall, the electrochemical behavior of NR in LP medium closely resembled the way it behaves in supporting electrolytes such as phosphate buffer solutions [23] and MOPS buffer with 10 mM MgCl₂ and 100 mM glucose [22].

MV, a viologen-based mediator, plays a significant role as an electron relay. It exhibits rapid electrochemical response at negative potentials, which allows it to be applied in diverse areas such as batteries and as a redox mediator in numerous enzymatic reactions [14,25]. Moreover, in other applications, MV has been shown to redirect the electron flow from Clostridium acetobutylicum by controlling the NADH/NAD⁺ balance [26].

MV displays three main oxidation states [27]. Figure 2B depicts the CVs recorded for 200 μM MV at scan rates ranging from 1 to 50 mV·s⁻¹. The CV curves exhibited two redox pairs: one at −0.41 and −0.48 V vs. Ag/AgCl_(sat), denoted 1 and 1′, respectively, and another at −0.65 and −0.83 V vs. Ag/AgCl_(sat), denoted 2 and 2′, respectively. According to Shpilevaya and Foord, reduction peak 1′ corresponds to MV⁺ formation, which is consistent with reaction 1. Reduction peak 2′ refers to formation of the neutral molecule in MV⁰ solution (reaction 2). The oxidation peaks labelled 2 and 1 correspond to reactions 3 and 4, respectively [27]

$$\begin{gathered} \mathrm{Reaction}1: M{V}^{2+}+e^{-}\to M{V}^{+} \left(peak 1\prime \right) \\ \mathrm{Reaction}2:M{V}^{+}+e^{-}\to M{V}^0 \left(peak 2\prime \right) \\ \mathrm{Reaction}3:M{V}^0\to M{V}^{+}+e^{-}\left(peak 2\right) \\ \mathrm{Reaction}4:M{V}^{+}\to M{V}^{2+}+e^{-} \left(peak 1\right) \end{gathered}$$

Generally, MV²⁺ undergoes reduction through a reversible reaction involving an electron, to give a blue cation radical. This radical can undergo further reduction to its neutral form (MV⁰), which typically adsorbs onto the electrode surface [25].

The graph inserted in Figure 2B shows the profile of I_ap and I_cp vs. the square root of the scan rate for the redox couple 1/1′. I_ap behaved according to the equation $I_{ap}=0.001790 v-0.001666$ (R² = 0.984317), while I_cp followed the equation $I_{cp}=-6.68305\times {10}^{-4}v+3.653847\times {10}^{-4}$ (R² = 0.979345). The linear increase suggested diffusion-controlled processes [24,27].

3.3. 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 μM, under anaerobic conditions (Figures S1 and S2). We selected the highest concentration (150.0 μM) for tests with MFC_DSM because it provided optimal j-values regardless of the mediator. In addition, preliminary tests had shown that adding 150.0 μM 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 (η) 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_(sat) for MFC_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_DSM) in the same experimental conditions (Figure 3).

For the electrochemical tests, MFC_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 ± 2.25 and 8.45 ± 2.98 μA·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_DSM in the absence of a mediator (2.47 ± 0.53 and 2.29 ± 0.69 μA·cm⁻² by applying −0.22 and −0.16 V vs. Ag/AgCl_(sat), respectively).

Introducing mediators can enhance the C. pasteurianum DSM 525 EET ability given that the MFC_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.

Nevertheless, the presence of C. pasteurianum DSM 525 in MFC_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.

3.4. FNR or FMV Deposited on the MFC_DSM Anode as Mediator

Due to the results obtained in the previous Section 3.3, we investigated whether FNR or FMV enhances electron collection in MFC_DSM. Upon electropolymerization, NR forms poly(neutral red), known as FNR, which has been extensively investigated for biosensor applications [23]. As mentioned earlier, FNR can react with biological components (e.g., NADH, proteins, and enzymes) [23].

In turn, immobilized viologen-based mediators have been successfully employed to interact with biological components [25]. However, unlike FNR, FMV interacted weakly with the MFC_DSM anodic electrode, so the carbon chain did not increase effectively. This effect has already been described, and the fact that the reduced species MV⁺ is prone to dimerization has been reported [30].

First, we evaluated the optimal number of CV cycles for FNR and FMV (Figure S4) in the absence of C. pasteurianum DSM 525 (abiotic control). In the case of FNR, the current density increased between 10 and 20 cycles and decreased thereafter. Therefore, for FNR, we adopted 20 cycles for FNR in further investigations. Concerning MV, the current density started to decrease after 10 cycles, so we limited assays with FMV to 10 cycles (Figure S5). In the latter case, decreasing current density upon increasing number of cycles was probably due to MV⁺ species forming dimers, which can further disproportionate into MV²⁺ and insoluble MV⁰ [30].

We tested MFC_DSM (C. pasteurianum DSM 525, O.D. = 0.2) across all CV cycles for each mediator film. The combination of MFC_DSM with FNR or FMV increased the current density in all the CV cycles (Figure S6). Nevertheless, although FMV interacted with C. pasteurianum DSM 525 on the anode, it did not interact as effectively as FNR.

Figure 4A,B compare the amperometric i-t tests for the highest current density achieved with FNR (20 cycles) and FMV (10 cycles), respectively, at MFC_DSM. After we introduced FNR into the MFC_DSM anodic chamber, the current density increased 12-fold compared to MFC_DSM (30.76 ± 3.44 and 2.47 ± 0.53 μA·cm⁻², respectively). As for FMV, the current density increased 3.9-fold after it was introduced into the MFC_DSM anodic chamber compared to MFC_DSM (8.91 ± 1.92 and 2.29 ± 0.69 μA·cm⁻², respectively).

Regarding FNR introduction into the MFC_DSM anodic chamber, the current density doubled compared to NR introduction into the MFC_DSM anodic chamber (30.76 ± 3.44 and 17.05 ± 2.25 μA·cm⁻², respectively). This effect has not been previously reported for C. pasteurianum DSM 525. This result suggested that the presence of FNR on the electrode surface enhances the electron collection efficiency and reduces electron losses [31]. Polymeric film deposition on electrodes creates effective electrode transfer mediators [31]. In addition, the presence of FNR improves the EET capacity of C. pasteurianum DSM 525, indicating that the film interacts with the microorganism.

On the other hand, FMV or MV introduction into the MFC_DSM anodic chamber elicited similar effects: 3.9- and 3.7-fold increases in the current density compared to MFC_DSM. The MV structure does not favor the formation of polymeric films, so MV remains mostly in the dimeric form [30]. The dimer and monomer must diffuse in a similar way, which could explain the similar increase in current density.

We recorded CV curves at 1 mV·s⁻¹ to determine the anodic peak potentials for each mediated electron transfer system. We conducted the experiments with MFC_DSM and FNR or FMV (Figure 4C,D). We also recorded a control CV without the biocatalyst. The data confirmed the results described above that the presence of FNR in the MFC_DSM anodic chamber increased the current density, while the presence of FMV in the MFC_DSM anodic chamber elicited a less pronounced capacitive behavior.

Figure S7 compares the EIS of the unmodified electrode (carbon cloth) and the modified electrode containing FNR and FMV. In the two latter cases, total impedance decreased significantly. Specifically, that containing FNR and FMV had an R_ct of 30.4 and 48.3 Ω, respectively, which represents a 33.7- and 21.4-fold reduction compared to the unmodified electrode (R_ct = 1025.6 Ω). As for R_s, values were similar (20.5, 18.3, and 21.7 Ω for the unmodified electrode, that containing FNR, and that containing FMV, respectively).

4. Conclusions

We investigated C. pasteurianum 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 μA·cm⁻²), which evidenced direct EET. The EET capacity can be further enhanced by introducing NR or MV as a mediator into the MFC_DSM, either in solution or as a film (FNR and FMV, respectively). Compared to NR, FNR introduction into the MFC_DSM produced almost twice the current density (17.05 ± 2.25 vs. 30.76 ± 3.44 μA·cm⁻²). Conversely, introduction of MV or FMV into the MFC_DSM produced nearly identical current density (8.45 ± 2.98 vs. 8.91 ± 1.92 μA·cm⁻²), indicating that FMV was a less effective mediator than FNR. Therefore, FMV might interact weakly with the MFC_DSM anode, possibly due to dimer formation [30]. In turn, FNR appears to be promising for enhancing C. pasteurianum DSM 525 EET because it potentially facilitates microorganism–film–anode interaction.