Enhancement of Succinic Acid Production by Actinobacillus succinogenes in an Electro-Bioreactor

Abstract

This work examines the electrochemically enhanced production of succinic acid using the bacterium Actinobacillus succinogenes. The principal objective is to enhance the metabolic potential of glucose and CO₂ utilization via the C4 pathway in order to synthesize succinic acid. We report on the development of an electro-bioreactor system to increase succinic acid production in a power-2-X approach. The use of activated carbon fibers as electrode surfaces and contact areas allows A. succinogenes to self-initiate biofilm formation. The integration of an electrical potential into the system shifts the redox balance from NAD⁺ to NADH, increasing the efficiency of metabolic processes. Mediators such as neutral red facilitate electron transfer within the system and optimize the redox reactions that are crucial for increased succinic acid production. Furthermore, the role of carbon nanotubes (CNTs) in electron transfer was investigated. The electro-bioreactor system developed here was operated in batch mode for 48 h and showed improvements in succinic acid yield and concentration. In particular, a run with 100 µM neutral red and a voltage of −600 mV achieved a yield of 0.7 g_succinate·g_glucose⁻¹. In the absence of neutral red, a higher yield of 0.72 g_succinate·g_glucose⁻¹ was achieved, which represents an increase of 14% compared to the control. When a potential of −600 mV was used in conjunction with 500 µg∙L⁻¹ CNTs, a 21% increase in succinate concentration was observed after 48 h. An increase of 33% was achieved in the same batch by increasing the stirring speed. These results underscore the potential of the electro-bioreactor system to markedly enhance succinic acid production.

1. Introduction

The sustainable production of succinate via fermentative processes represents an environmentally friendly alternative to the petrochemical synthesis of this chemical [1]. Furthermore, fermentation in an electro-bioreactor provides an effective means of utilizing surplus electricity generated by renewable energy sources, such as wind and solar energy, in an efficient manner [2]. This indicates that there is potential for further enhancement of efficiency and cost-effectiveness [3].

1.1. Succinate

Succinate is a valuable chemical platform molecule and is used in various industries [4]. Succinate is a dibasic carboxylic acid. To date, it has mainly been obtained on an industrial scale from petrochemical raw materials, primarily from maleic anhydride [5]. The biotechnological production of succinate from renewable raw materials offers the potential to not only replace fossil raw materials but also to provide ecological advantages through the use of CO₂ as a substrate [6]. The industrial potential of biotechnological produced succinate was first identified by Zeikus in 1980 [7,8]. Since then, the biotechnological production of succinate has been increasingly regarded as an economically viable alternative to petrochemical production, particularly through the use of microorganisms such as Escherichia coli and Basfia succiniciproducens, which have been genetically modified to achieve high yields and productivities [9,10]. Actinobacillus succinogenes is another well-known microorganism that produces biotechnological succinate, mainly due to its high yield and high productivity [11]. These microorganisms are capable of utilizing inexpensive and abundant biomass sources as substrates, which enhances the economic viability and can contribute to a more sustainable production of succinate [12,13]. The development of new biotechnological processes has further improved the efficiency and productivity of these processes [14]. The bio-based succinate industry was valued at USD 159.9 million in 2023. It is estimated to achieve a compound annual growth rate of 17.8% from 2024 to 2034 and is expected to reach a value of USD 1.7 billion by the end of 2034 [15]. The production costs for bio-based succinic acid are 41% of the production costs for petrochemically produced succinic acid (1.17 USD/kg compared to 2.86 USD/kg). This indicates that biotechnologically produced succinic acid is more cost-effective than petrochemically produced succinic acid [16].

1.2. Actinobacillus succinogenes

A. succinogenes is a Gram-negative, facultatively anaerobic bacterial strain that naturally produces high concentrations of succinate [17]. It belongs to the Pasteurellaceae family and was originally isolated from the rumen of cattle [18]. The fermentation of A. succinogenes for succinate production usually takes place under anaerobic conditions [4,19]. Several metabolic pathways are involved in this process, like the glycolysis, the pentose phosphate pathway (PPP), and the citric acid cycle (TCA cycle). As can be seen in Figure 1, during glycolysis, glucose is converted into pyruvate, producing adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH). Pyruvate is a central metabolite that serves as a starting point for various downstream metabolic pathways [4]. In organisms such as A. succinogenes, PPP is important for providing nicotinamide adenine dinucleotide phosphate (NADPH) for biosynthetic reactions and combating oxidative stress [20]. NADPH is mainly generated by the conversion of NADH to NADPH by transhydrogenase and/or NADP-dependent malate enzymes [4]. The TCA cycle in A. succinogenes is a branched metabolic pathway involving both oxidative and reductive reactions. Under anaerobic conditions, the TCA cycle mainly operates in the reductive mode to produce succinate. This process involves the fixation of CO₂ and the conversion of pyruvate to oxaloacetate, malate, and finally succinate. The process is regulated by different enzymes. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of phosphoenolpyruvate (PEP) and CO₂ to oxaloacetate. Malate dehydrogenase (MDH) reduces oxaloacetate to malate. Fumarate reductase reduces fumarate to succinate [18]. Furthermore, the adjustment of the redox potential in the culture can also influence the NADH/NAD⁺ balance, thereby enhancing the efficiency of succinate production by reducing NAD⁺ to NADH [21]. Increasing the CO₂ concentration in the fermentation medium can promote flux through the reductive TCA cycle, as CO₂ serves as a substrate for PEPCK and MDH. High NaHCO₃ concentrations can improve CO₂ availability and thus increase succinate production [22]. In A. succinogenes, the NAD⁺/NADH ratio plays a crucial role in the metabolic pathway and the production of succinate. Succinate production can be increased by increasing the availability of NADH. This can be achieved, for example, by using carbon sources such as sorbitol, which have a higher reduction potential [23]. In addition to the production of succinate, fermentation also produces by-products such as lactate, formate, ethanol, and acetate, which are formed from pyruvate via alternative metabolic pathways. Lactate dehydrogenase (LDH) converts pyruvate to lactate. In addition to lactate, NADH is reduced to NAD⁺ here. Formate and acetate formation occurs through the cleavage of pyruvate into formate and acetyl-CoA by pyruvate formate lyase (PFL). Acetyl-CoA is subsequently converted to acetate by phosphotransacetylase (PTA) and acetate kinase (ACK), which contributes to energy production and regulates the redox balance [24]. Ethanol formation occurs through the conversion of acetyl-CoA via acetaldehyde to ethanol via acetaldehyde dehydrogenase and alcohol dehydrogenase, with ethanol being produced as a by-product in smaller quantities and playing a role in redox balancing [25]. Providing CO₂ and controlling redox potential are critical to maximizing succinate production and minimizing by-products [26].

1.3. Electron Transfer

In a bioelectrochemical system (BES), A. succinogenes can directly benefit from an electrode potential, which can change the NAD⁺/NADH redox balance and increase production of succinate. The application of an electric potential in BES resulted in the overexpression of key enzymes such as PEPCK and pyrophosphatase, while enzymes such as fumarase and pyruvate kinase were suppressed [3]. The exact mechanisms of cathodic electron flow in microorganisms are still not sufficiently understood. However, there is a consensus among scientists that cathodic extracellular electrons can penetrate the cytoplasm and exert an influence on proton-consuming reactions [3,28]. Neutral red (NR), a redox-active dye, can act as an electron carrier and transfer electrons directly from an electrode to A. succinogenes and, thus, reduce NAD⁺. This has already been demonstrated by Engel et al. for C. acetobutylicum [29]. This method has shown that NR can increase glucose uptake, growth, and succinate production, while acetate production was reduced [30]. By optimizing electron transfer, specific metabolic pathways can be favored. This is particularly important for biotechnological applications where a high yield of target metabolites is desired [31]. Improved regeneration of NAD⁺ helps to maintain the redox balance in the cells [32]. The increasing NADH availability leads to increased production of specific metabolites such as ethanol, succinate, or butanol [33]. A central aspect of electrobiotechnology is electron transfer, which can take place either directly or through mediated processes. In direct electron transfer (DET), electrons are transferred directly between the electrode and the microorganisms via redox proteins such as cytochromes or ferredoxins [34]. Cytochromes are proteins with heme groups that can transfer electrons [35]. Ferredoxins are iron–sulfur proteins that transfer electrons between the electrode and the intracellular redox partners [36]. In mediated electron transfer (MET), electrons are transferred between the electrode and the microorganisms via mediators. These mediators can be natural or artificial compounds that can accept and transfer electrons [34]. Mediators enable faster electron transfer, especially in systems where direct electron transfer is slow [37]. MET can be used in systems where direct contact between microorganisms and electrodes is not possible or efficient [38]. Hydrogen (H₂) can serve as a source of electrons, whereby it is oxidized to protons and electrons by enzymes such as hydrogenases. These electrons can then be introduced into the metabolism of the microorganisms. NR is a redox-active dye that can be reduced electrically and acts as an electron carrier to transfer electrons from the electrode to the microorganisms. In A. succinogenes, it has been shown that electrically reduced NR can increase glucose uptake, growth, and succinate production [30]. Integration of carbon nanotubes (CNTs) into BES offers a promising opportunity to improve the efficiency of electron transfer in microbial biofilms. It can be shown that the integration of multi-walled carbon nanotubes (MWCNTs) in biofilms can lead to a significant improvement in electrical conductivity. A study by Zhang et al. demonstrated that a hybrid biofilm of bacteria and MWCNTs significantly increases current density, power density and coulombic efficiency [39]. CNTs can facilitate DET between electrodes and microorganisms [40]. In 2012, Ma et al. demonstrated that CNTs are also biocompatible and can be employed in biological systems without eliciting toxic effects [41]. Nevertheless, the existing literature describes a variety of aspects related to CNTs and biocompatibility. These effects vary depending on their structural characteristics and functionalization. It has been demonstrated that MWCNTs are biocompatible with bacterial cultures [42]. Forms like single-walled CNTs (SWCNTs) have been observed to act as “nano-darts” capable of physically damaging bacterial cell membranes and ultimately leading to cell death [43]. Therefore, MWCNTs are less toxic to microorganisms than SWCNTs. This discrepancy is ascribed to the physical interactions of CNTs with microbial cells [44]. MWCNTs are capable of causing oxidative stress and cell damage, but have less severe effects on microbial viability compared to SWCNTs [45]. In a BES, microorganisms are instrumental in facilitating the transfer of electrons between electrodes and organic substrates [46]. A technical distinction is made between the single-chamber system and the two-chamber system. Single-chamber systems are simplified BES in which all reactions take place in a single reaction chamber. These systems are particularly useful as they are less complex and less expensive to implement but they have the disadvantage that anodic and cathodic processes can mix [47]. As the name implies, the two-chamber system comprises two stepped chambers, which are separated by an ion-selective membrane. The primary benefit is the effective separation of the anodic and cathodic reactions, which can enhance efficiency. However, these systems are more costly, and the expense of the membrane and its deterioration negatively impact efficiency and service life [48].

This study examines the fermentation of succinic acid by A. succinogenes in a cathodic bioelectrochemical system (BES) fermentation in a single-chamber system. To this end, a carbon fiber was introduced into the reactor, serving two functions. Firstly, it is served as a surface for biofilm growth, and secondly, as the cathode. This represents a novel and innovative approach. Furthermore, the combination of BES fermentation with CNTs and A. succinogenes represents a novel and promising approach, thereby contributing to an increase in the yield of succinate for the fermentation of A. succinogenes. It is, therefore, shown that this should serve as an innovative contribution to fermentation in the single-chamber reactor system, with the aim of increasing the yield of succinic acid in A. succinogenes.

2. Materials and Methods

2.1. Growth Conditions

The bacterial strain Actinobacillus succinogenes 130Z (DSM-22257) was obtained from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH in Braunschweig, Germany. The inoculum preparation contained tryptic soy broth (TSB) [49] with the following composition: casein peptone 17 g∙L⁻¹, soy peptone 3 g∙L⁻¹, D-glucose 2.5 g∙L⁻¹, NaCl 5 g∙L⁻¹, and K₂HPO₄ 2.5 g∙L⁻¹, all dissolved in distilled water. The pH value of the medium was adjusted to 6.8. The cultivation of the precultures were carried out in 120 mL serum bottles with a working volume of 50 mL. Before these were inoculated, they were first degassed with N₂ for 20 min to create anaerobic conditions. Subsequently, the bottles were inoculated with A. succinogenes in an anaerobic vinyl chamber (Anaerobic Vinyl Chamber Type C from Coy Laboratory Products Inc., Grass Lake, MI, USA). The inoculum was 10% of the total volume. The inoculated cultures were then incubated for 16 h at 37 °C and 80 rpm in an incubator (KS 4000 i control from IKA from IKA-Werke GmbH & Co KG, Staufen, Germany). This process allowed for the growth and adaptation of A. succinogenes under controlled anaerobic conditions. The pre-cultures were used to inoculate the bioreactor. For this purpose, a medium described by Wang et al. [50] was employed, containing 30 g∙L⁻¹ glucose, 31.5 g∙L⁻¹ NaHPO₄·12H₂O, 10 g∙L⁻¹ NaHCO₃, 8.5 g∙L⁻¹ NaH₂PO₄, and 5 g∙L⁻¹ yeast extract. Work was carried out with 30 g∙L⁻¹ glucose to avoid substrate inhibition in order to be able to observe only the influence of the electrical system. Additionally, for designated runs, 100 µM of neutral red (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and 500 µg∙L⁻¹ of CNTs hydrophobic and hydrophilic (Multi-walled carbon nanotubes, 95% from abcr GmbH, Karlsruhe, Germany) were added. NR and the CNTs were weighed in parallel with the preparation of the medium and added to it before autoclaving. The bioreactor was prepared and then autoclaved for sterilization. After autoclaving, the medium was pumped into the bioreactor under sterile conditions in the microbiological safety cabinet. The working volume of the reactor was set to 2 L with an inoculum of 10% (v/v). The pH was monitored using a pH electrode (from Hamilton Bonaduz AG, Bonaduz, Switzerland) and was maintained at 6.8 by automatic titration with 5 M NaOH. The bioreactor was controlled using a bioprocess controller (BioFlo 120 from Eppendorf SE, Hamburg, Germany), ensuring precise regulation of all critical parameters. The bioreactor was purged overnight with a gas mixture of 80% CO₂ and 20% N₂ at a flow rate of 0.25 vvm. The temperature in the reactor was set to 37 °C and the stirring speed to 50 rpm. After the precultures had been incubated for 16 h, the reactor was inoculated and fermentation started. This setup ensured optimal anaerobic conditions for A. succinogenes to metabolize glucose efficiently and produce succinic acid.

2.2. Construction BES

For the anaerobic BES used in this study, a glass stirred-tank reactor (Eppendorf Q-series from Eppendorf SE, Hamburg, Germany) with a working volume of 2 L was employed. A carbon fiber electrode (ACC-5092-20 from Kynol Europa GmbH, Hamburg, Germany) with a surface area of 414 cm² was installed in the reactor. The carbon fiber serves as a cathode for the BES and also as a surface for the formation of the biofilm in the reactor. The carbon fiber was then connected to the potentiostat (MultiPalmSens4 from PalmSens, Houten, The Netherlands) via a platinum wire. An Ag/AgCl reference electrode (SE11NSK7-4 from Meinsberg Sensortechnik, Xylem Analytics Germany Sales GmbH & Co. KG, Weilheim, Germany) was used to maintain a stable reference potential. The counter electrode (anode) was a graphite electrode with a diameter of 5 mm. Samples were collected through a safelock sampling valve, ensuring sterile and contamination-free sample retrieval. A schematic overview of the electrode arrangement and the BES setup is shown in Figure 2.

2.3. Electrochemical Conditions

Initially, cyclic voltammetry (CV) measurements were performed using the laboratory potentiostat to investigate the redox properties of the medium. For these measurements, a 0.1 M PBS buffer containing 100 µM neutral red was used. A platinum wire was used as a working electrode and as a counter electrode. A reference electrode (Ag/AgCl) was used to maintain a stable reference potential. The analysis of the CVs was performed to understand the electrochemical behavior under experimental conditions. These preliminary measurements helped determine the optimal redox potential for subsequent reactor runs. During the fermentations in the BES, a constant potential of −600 mV was applied. This potential was selected based on the results from the cyclic voltammetry measurements, ensuring that it was suitable for enhancing the metabolic activity of A. succinogenes and optimizing succinic acid production real-time monitoring and control of the electrochemical parameters were conducted via the potentiostat interface, ensuring regulation of the applied potential and allowing for adjustments as needed. The carbon fiber served as the cathode and was strategically positioned along the reactor wall to provide a surface for biofilm formation by A. succinogenes.

2.4. Analytical Procedures

The high-pressure liquid chromatography (HPLC) analysis of the liquid samples from hydrolysis and cultivation was performed using an HPLC system (Infinity II 1100/1260 Series from Agilent Technologies Inc., Santa Clara, CA, USA). The analysis methods used in this study were already presented by our group in 2024 and are explained in more detail below [51]. The HPLC analysis was conducted with a Repromer H⁺ column (Dr. Maisch HPLC GmbH, Ammerbuch, Germany), with a size of 300 × 8 mm. Throughout the analysis, the temperature of the column was meticulously maintained at 30 °C. A sample injection volume of 5 μL was used for each analysis. A total of 5 mM sulfuric acid was used as mobile phase, chosen to enhance the separation efficiency of the analytes under investigation. Detection of the analytes was carried out using an Infinity II Refractive Index Detector (Agilent Technologies Inc., Santa Clara, CA, USA). The run time for each chromatographic analysis was set at 30 min, a time sufficient to allow complete resolution of all components (glucose, succinate, lactate, formate, acetate, ethanol). The flow rate of the mobile phase was set at 0.6 mL∙min⁻¹.

2.5. Statistical Analysis

To statistically analyze the final fermentation concentrations presented in this manuscript, an unpaired t-test was performed using GraphPad Prism software (Version 10.3.0). To perform the analysis, the mean values of the different fermentation conditions were compared with those of the control group. The t-test was performed on an individual basis for each condition, with the three measurements of a technical triplicate considered as independent samples. Given that it was not assumed that the variances between the groups were equal, a Welch’s t-test was employed to adjust for unequal variances between the groups. This method ensures that the requisite level of statistical significance is maintained under these conditions. The significance levels *, **, ***, and ns indicate the likelihood that a statistical effect is not due to chance. One star (*) indicates a p-value of less than or equal to 0.05, two stars (**) a p-value of less than or equal to 0.01, and three stars (***) a p-value of less than or equal to 0.001; ‘ns’ indicates that the effect is not significant (p > 0.05).

3. Results and Discussion

The objective of this study was to examine the impact of diverse fermentation processes on succinate production in A. succinogenes. The influence of direct potential and in combination with the addition of NR and CNTs was investigated. The analysis focused on increasing succinate production and on the characterization of other by-products. The electro-bioreactor established in this work is a BES configured as a single-chamber system. The BES developed here is a single-chamber system. It was shown in preliminary tests that A. succinogenes is still able to form succinate even under a fumigation of 50% CO₂ and 50% compressed air and that the possible formation of O₂ at the anode has no influence on product formation. This differs from the findings of Li et al. 2010, in which succinate was initially produced with A. succinogenes under anerobic conditions and then the product formation could be shifted towards lactate by O₂ fumigation [52].

3.1. Analysis of Selected Fermentation Runs in the BES

The initial step will be to examine the processes of selected fermentations, after which other parameters will be considered and compared. First, a control was performed in which only the main culture medium was inoculated with the inoculum. Figure 3 shows the progression of substrate consumption and product formation over time for the reactor run with A. succinogenes, A. succinogenes with potential of −600 mV, and A. succinogenes with CNTs 150 rpm and potential of −600 mV. Figure 3a shows the control run, which was conducted without the addition of mediators or the creation of potential. It shows the concentrations of glucose, succinate, formate, and acetate over a period of 0 to 48 h. The glucose concentration decreases continuously, reaching 24.33 g∙L⁻¹ after 8 h. From the 24 h mark, the glucose concentration rapidly decreases until it reaches 6.50 g∙L⁻¹ after 48 h. The concentration of succinate increases steadily. After 8 h, the concentration is 1.79 g∙L⁻¹ and reaches 13.84 g∙L⁻¹ after 48 h. Formate is not detectable in the first 8 h. From the 24 h mark, it begins to increase and reaches a concentration of 4.27 g∙L⁻¹ after 48 h. At the start of fermentation, the acetate concentration is 2.89 g∙L⁻¹. The reason for this lies in the production of the fermentation medium. Acetic acid was used to adjust the pH value to a value of 6.8 during the production of the medium. The concentration of acetate increases slowly and reaches 3.75 g∙L⁻¹ after 8 h. After 24 h, acetate shows a clear increase and reaches a concentration of 8.62 g∙L⁻¹ after 48 h. The formation of formate and acetate is contingent upon the availability of NADH. Two moles of NADH are necessary for the generation of one mole of succinate, but the glycolysis process yields a mere two moles of NADH per mole of glucose. Consequently, in order to produce two moles of succinate from one mole of glucose, the organism requires four moles of NADH. The deficit in NADH is compensated for by the formation of by-products, namely formate and acetate [53]. Furthermore, it was observed that the OD600 increased to a value of 1.8 ± 0.17 during the initial 26 h of fermentation, subsequently declining as the fermentation process continued and succinate was produced. One potential explanation for this phenomenon is the formation of a biofilm on the carbon fiber. Figure 3b illustrates the course of fermentation during a run with a potential of −600 mV in the BES. The initial starting concentration of glucose is 28.29 g∙L⁻¹. Over the course of the fermentation, the glucose concentration decreases continuously. After 8 h, the concentration is 24.11 g∙L⁻¹. From the 24 h mark, the glucose concentration decreases rapidly until it reaches 3.98 g∙L⁻¹ after 48 h. Concurrently, the concentration of succinate begins to rise from the 8 h mark and reaches 9.34 g∙L⁻¹ after 24 h. After 48 h, the concentration reaches 15.79 g∙L⁻¹. Formate is not detectable in the first 4 h. The concentration of acetate begins to rise from the 8 h mark and reaches a concentration of 4.18 g∙L⁻¹ after 48 h. As with the control, the initial acetate concentration is 1.56 g∙L⁻¹. This is also due to the use of acetic acid to adjust the pH value of the medium to 6.8 during production. The concentration of acetate increases slowly and reaches 2.74 g∙L⁻¹ after 8 h. After 24 h, the concentration of acetate increases, reaching 8.12 g∙L⁻¹ after 48 h. It can thus be shown that even without the use of mediators, the application of a potential of −600 mV can achieve an increase in succinate yield, which is consistent with the work of Pateraki et al. [3]. It is also noteworthy that an OD600 of 1.95 ± 0.17 was observed after 24 h. Similarly, the measured OD declines as fermentation progresses, while succinate production continues to increase as previously described. As illustrated in Figure 3c, the reactor run curves are plotted here by operating with a potential of −600 mV and adding 500 µg∙mL⁻¹ CNTs. Furthermore, the stirring speed was increased from 50 rpm to 150 rpm. The concentration of glucose exhibited a continuous decline. After 8 h, the concentration was found to be 26.29 g∙L⁻¹. From the 24 h mark, the glucose concentration declines rapidly until it reaches 0.96 g∙L⁻¹ after 48 h. The concentration of succinate begins to increase from the 4 h mark, reaching 10.29 g∙L⁻¹ after 24 h. After 48 h, the concentration reaches 18.42 g∙L⁻¹. The concentration of formate is below the limit of detection for the first 2 h. A gradual increase is observed from the 4 h mark, reaching a concentration of 3.73 g∙L⁻¹ after 48 h. The concentration of acetate increases gradually and reaches 0.97 g∙L⁻¹ after 8 h. After 24 h, acetate concentration was at 4.87 g∙L⁻¹ and reaches 6.52 g∙L⁻¹ after 48 h. The OD₆₀₀ reached a maximum of 1.57 ± 0.19 after 26 h. It is noteworthy that this value is lower than that observed in the runs already mentioned above. However, the succinate concentration at this point is already 10.57 g∙L⁻¹ and, thus, represents the highest value, whereas the OD₆₀₀ is lower in comparison to the runs already mentioned. This is due to the formation of the biofilm on the carbon fiber, which means that fewer free cells are found in the medium, but primarily on the carbon fiber. It can be observed that the application of an electric potential of −600 mV results in a 14% increase in succinate yield. The addition of 500 µg∙mL⁻¹ CNTs and the simultaneous application of a potential of −600 mV resulted in a 33% increase in succinate yield, reaching a concentration of 18.42 g∙L⁻¹. Consequently, it represents the greatest concentration of succinate following 48 h of fermentation. It can be assumed that better mixing could be achieved by increasing the stirrer speed. It can also be assumed that electron transport can be achieved via the CNTs. The CNTs were seen to integrate into the biofilm [39,40].

3.2. Comparison Different Fermentation Conditions in the BES

In Figure 4, the reactor runs carried out in this work are compared with each other. The concentrations of succinate after 48 h of fermentation are plotted in Figure 4a and the yields of succinate in g in relation to the glucose consumed in g are plotted in Figure 4b. Another essential analysis is the comparison of the proportions of fermentation products. The main aim here is to shift the distribution towards succinate. By providing more NADH, the metabolism should be shifted in the direction of the C4 metabolism while fixing CO₂. This should increase the ratio of succinate compared to the other fermentation products [27]. Figure 5 shows the percentage distributions of the fermentation products. The analysis of the fermentation products is certainly limited to succinate, formate, and acetate, as neither lactate nor ethanol were formed in any of the runs.

A line has been inserted here to enable a clear comparison with the control. The succinate concentration in the control run was 13.84 ± 0.41 g∙L⁻¹. With a yield of 0.673 ± 0.04 g_succinate·g_glucose⁻¹. These values serve as the basis for the comparison. A total productivity of 0.288 g∙L⁻¹∙h⁻¹ was determined. The control displays the distribution of fermentation products formed without additional treatments or modifications. The proportion of succinate is 58.1%, that of formate 17.9%, and that of acetate 24%. The control thus demonstrates the distribution of the products in their unmodified state. In the literature, higher quantities of succinate were sometimes formed, mainly due to the high initial glucose concentrations [54]. As we primarily wanted to compare 48 h batch runs, the initial glucose concentration of 30 g∙L⁻¹ was sufficient for the fermentation period considered here, as can be seen in Figure 3. An objective comparison of BES effects should be performed via yield comparison. Our results show a yield higher compared to a recent publication of Pateraki et al., where the yield in non-BES was 0.66 g_succinate·g_glucose⁻¹, which can be attributed to enhanced reaction conditions [3]. The reactor run with 100 µM neutral red showed a very similar succinate concentration to the control 13.75 ± 0.36 g∙L⁻¹. The yield here was 0.668 ± 0.03 g_succinate·g_glucose⁻¹ and is, therefore, slightly lower than the control, as is the final concentration achieved. This is mainly due to the slight toxicity of NR in non-reduced state for the cells. As previously stated, the addition of 100 µM neutral red results in a slight decrease in the proportion of succinate and formate, while the proportion of acetate increases slightly. The proportion of succinate is 56.5%, that of formate is 17.8%, and that of acetate is 25.7%. The succinate yield is observed to decrease in comparison to the control, while the concentration of acetate is found to increase by 1.7%. The results of unregulated batch fermentations demonstrate that the yield at 100 µM NR was 5% lower than in the control. At 250 µM NR, the yield exhibited a further decrease of 15%, while at 500 µM NR, it decreased by 23%. These findings suggest that NR in its non-reduced form may exert an inhibitory effect on the growth of A. succinogenes. Park et al. have previously reached analogous conclusions. The results demonstrate that non-reduced NR can exert toxic effects and impede cell growth and metabolite production. [55]. Additionally, Park and Zeikus demonstrated that non-reduced NR can interact with cell membranes, impeding electron transfer and resulting in the loss of energy and disruption of cellular processes. This ultimately leads to a reduction in cell viability and functionality [30]. This finding is also consistent with the ratio of succinate formed, which is 56.5% compared to 58.1% in the control group. The availability of non-reduced NR results in oxidative stress, which, in turn, leads to a reduction in NADH levels. This subsequently inhibits the formation of succinate. With the combination of 100 µM neutral red + potential −600 mV, succinate concentrations of 15.04 ± 0.72 g∙L⁻¹ with a yield of 0.700 ± 0.03 g_succinate·g_glucose⁻¹ were measured. This represents an increase of 9% in the succinate formed. The application of a potential of −600 mV in conjunction with the addition of 100 µM neutral red yielded comparable results to those observed with 100 µM neutral red alone, albeit with a marginal increase in succinate content. This rises to 57.4%, which is still slightly below that of the control. The concentration of formate is 17% and that of acetate is 24.7%, which is slightly higher than in the control. Park and Zeikus achieved an increase in succinate production of 20%, whereas the current study reports a 9% increase [30]. One potential explanation for this discrepancy is the difference in experimental setup. Park and Zeikus employed a two-chamber system, whereas the current study utilized a single-chamber system. The proximity of the cathode and anode within the same chamber allows for the reduction of NR at the cathode and subsequent oxidation at the anode. This phenomenon may result in the accumulation of non-reduced NR within the medium, which can impede cellular activity. Consequently, the observed increase is only 9%, in contrast to the 20% reported by Park and Zeikus. In the study by Park and Zeikus, potentials of 1.5 V for system I and 1.0 to 10.0 mA, and 2.0 V for system II were employed [30]. Hydrogen electrolysis theoretically occurs at a potential of −1.23 V and practically around −1.4 V [56]. This suggests that external factors may influence the results, making the data from Park and Zeikus less comparable. Moreover, considering potential process upscale, the higher costs associated with a two-chamber system, due to larger volume and membrane use, make a single-chamber system more appealing [57]. A further increase of 14% in succinate formation was achieved by applying a potential of −600 mV, a succinate concentration of 15.79 ± 0.29 g∙L⁻¹ was measured, with a yield of 0.720 ± 0.03 g_succinate·g_glucose⁻¹. A total productivity of 0.329 g∙L⁻¹∙h⁻¹ was determined. This shows that an increase could be achieved even without mediators. The application of a potential of −600 mV resulted in a higher proportion of succinate and a decrease in the proportion of formate, while the proportion of acetate remained stable. The succinate content is 59.5%, which represents a 1.4% increase relative to the control. The proportion of formate decreases to 15.8%, while that of acetate remains at 24.7%. As Pateraki et al. state, the yield could be increased by 7% by directly applying the potential [3]. This indicates that direct electron transfer to A. succinogenes can effectively enhance metabolic pathways without external mediators, probably due to the direct effects on the cellular redox state and electron transport chain [58]. The results of this study underline the potential of electrochemical improvement to increase succinic acid production using A. succinogenes. Higher succinate concentrations were achieved by adding 500 µg∙mL⁻¹ CNTs. In the fermentation 500 µg∙mL⁻¹ CNTs + potential −600 mV, a succinate concentration of 16.72 ± 0.12 g∙L⁻¹ was achieved and was, thus, approx. 21% higher than in the control run. A total productivity of 0.348 g∙L⁻¹∙h⁻¹ was determined. A concentration of 500 µg∙mL⁻¹ CNTs at a potential of −600 mV results in an increase in the proportion of succinate and a decrease in the proportion of formate and acetate. The succinate content is 63.3%, which represents a 5.3% increase relative to the control. The concentration of formate decreased by 4.3% to 13.6%, while the acetate content increased by 23.1%. By increasing the stirring speed, the highest succinate concentration of this test series was achieved in CNTs 500 µg∙mL⁻¹ + potential −600 mV + 150 rpm, with a concentration of 18.42 ± 0.11 g∙L⁻¹, which indicates the best condition for succinate production. This is an increase of 33% compared to the control fermentation, which is the largest increase here. The yield is 0.702 ± 0.01 g_succinate·g_glucose⁻¹. A total productivity of 0.384 g∙L⁻¹∙h⁻¹ was determined, which represents the highest productivity in this work. Increasing the stirring speed from 50 rpm to 150 rpm at 500 µg∙mL⁻¹ CNTs and −600 mV potential resulted in a further increase in succinate content and a reduction in formate and acetate content. The succinate content was found to be 64.3%, representing the most favorable result of this investigation. The proportion of formate is at a minimum, at 13%, as is that of acetate, which is 22.7%. It can be posited that the transfer of electrons in A. succinogenes was efficacious, as evidenced by the increased formation of succinate concomitant with a reduction in by-products, namely formate and acetate. This can be attributed to the diminished necessity for NADH regeneration via these pathways [53]. In addition, increasing the agitation speed can lead to a better distribution of nutrients and CO₂. Improved mixing ensures a more even distribution of CO₂ and substrates, which promotes cell growth and favors the reduction in by-products [59]. As previously stated, the incorporation of hydrophilic CNTs resulted in the highest yield, yet the distribution remained below that of conventional CNTs. The presence of CNTs likely provided an improved conductive environment that facilitated more efficient electron transfer, thus enhancing the metabolic conversion of glucose to succinic acid [60].

The use of hydrophilic CNTs was also considered, as the miscibility of the CNTs used so far was more difficult and they could not be distributed homogeneously in the medium. Therefore, a further run with hydrophilic CNTs was carried out. The conditions were CNTs (hydrophilic) 500 µg∙mL⁻¹ + potential −600 mV + 150 rpm. This condition also showed a high succinate concentration 17.02 ± 0.06 g∙L⁻¹ with a yield of 0.762 ± 0.02 g_succinate·g_glucose⁻¹. This increased the yield by approx. 13% with the control run. The utilization of hydrophilic CNTs under identical conditions results in a reduction in succinate content in comparison to normal CNTs, accompanied by an elevated formate content. The proportion of succinate is 62.3%, that of formate 14.4%, and that of acetate 23.3%. The results demonstrate that the addition of CNTs, particularly at higher concentrations and with stirring, favors the production of succinate while reducing the formation of formate and acetate. It could be shown that with CNTs (hydrophilic) 500 µg∙mL⁻¹ + potential − 600 mV + 150 rpm, a yield of 0.762 ± 0.02 g_succinate·g_glucose⁻¹ could be achieved, which corresponds to an increase of 13% compared to the control. Thus, the results of Pateraki et al. with direct electron transfer could be further exceeded [3]. The consistent absence of ethanol and lactate production in all experimental setups indicates a specific channeling of metabolic flux to succinic acid and its associated by-products (acetate and formate). This specificity is crucial for industrial applications where the aim is to maximize the yield of a target product and minimize by-products [61]. In addition, in the experiments where only −600 mV potential were used, the distribution of the products could be distributed in the direction of succinate. For example, the proportion of succinate in the −600 mV product stream increased to 59.5% up to 64.3% in the experiment with CNTs 500 µg∙mL⁻¹ + potential − 600 mV + 150 rpm. It can, therefore, be said that the product-to-by-product ratio is consistent with this increase in metabolic efficiency [1]. An additional rationale for the enhanced outcomes observed with CNTs may be attributed to the expansion in surface area within the reactor, which could potentially facilitate augmented biofilm formation [62,63]. The biofilm exhibits a higher metabolic activity than freely suspended cells, which become metabolically inactive over time. This illustrates the advantage of a biofilm for long fermentation times [64]. However, the reduction in by-product formation is inconsistent with this hypothesis. It may be inferred that NADH was reduced by the potential, and that this reduction did not occur via the formation of by-products [53]. Studies should address the issue of the underestimation of these effects. In light of these readily apparent outcomes, the open-circuit run was deemed unnecessary.

The results of this study demonstrate that the production of succinate by A. succinogenes can be enhanced by the application of an electrical potential of −600 mV in the single-chamber system developed here. The utilization of 100 µM NR with an electrical potential of −600 mV resulted in a 9% enhancement in succinate production. Nevertheless, this increase is below the levels previously described in the literature. This can be attributed to the single-chamber system, whereby the oxidized form of NR was also present in the reactor. This resulted in the cells being subjected to oxidative stress, which prevented them from utilizing the full potential of the reduced NR [30]. Furthermore, it was demonstrated that DET is feasible in A. succinogenes in conjunction with the electrode utilized in this study (carbon fiber). The production of succinate was observed to increase by 14%. It was demonstrated that the addition of 500 µg∙mL⁻¹ CNTs resulted in an increase of 21% or up to 33% when the stirring speed was increased. These findings are also consistent with those reported in the literature, which have demonstrated that the incorporation of CNTs into the biofilm enhances conductivity and facilitates electron transfer. It could thus be shown that the CNTs integrate into the biofilm that forms on the cathode (on the carbon fiber) and, thus, induce a conductive biofilm. Furthermore, future research should endeavor to analyze the conductivity in the biofilm with greater precision by incorporating CNTs. The combination of CNTs with electrofermentation for the production of succinate with A. succinogenes represents a promising new avenue of research. Subsequent analysis and optimization of further reactor parameters may be conducted at a future date. In light of these promising results, further work and the implementation of these results on a large scale appear to be warranted. In summary, the integration of an electro-bioreactor system improves succinic acid production by optimizing electron transfer processes. The combination of electrical potential and advanced materials such as CNTs offers a promising approach to enhance the metabolic capabilities of A. succinogenes. Future research will focus on further understanding these conditions and exploring the scalability of this system for industrial applications.

4. Conclusions

It can be demonstrated that the utilization of NR in combination with a potential of −600 mV in the single-chamber system may only yield a moderate improvement. Moreover, it has been demonstrated that A. succinogenes is susceptible to direct electron influence, forming a biofilm on the carbon fiber resulted in an increase of 14%. The results substantiate the efficacy of integrating electrochemical enhancements with carbon nanotubes (CNTs) to enhance succinate production. In particular, the use of higher concentrations of CNTs and increased stirring speeds proved to be especially beneficial. A 33% increase in succinate production was observed. Furthermore, the proportion of succinate in the product stream increased from 58.1% compared to the control to 64.3%. Concurrently, the formation of formate and acetate decreased, which also indicates a positive influence of the electrical potential on the metabolism. A yield of 0.673 ± 0.04 g_succinate·g_glucose⁻¹ was achieved in the control. The highest yield achieved in this study was 0.762 ± 0.02 g_succinate·g_glucose⁻¹ when hydrophilic CNTs were used. These results demonstrate the potential for further development up to an industrial scale, particularly with regard to the utilization of CO₂ from the air and the use of surplus electricity, which is increasingly being generated by renewable energies. This could facilitate the formation of succinates from these two resources, thereby enabling the manufacture of new high-quality products. This work contributes to the further development of electrofermentation of A. succinogenes with the objective of increasing succinate production. The use of CNTs in combination with a potential of −600 mV represents an innovative approach, particularly in view of the potential benefits that this combination may offer.