Modulation of Antioxidant Activity Enhances Photoautotrophic Cell Growth of Rhodobacter sphaeroides in Microbial Electrosynthesis

: Global warming is currently accelerating due to an increase in greenhouse gas emissions by industrialization. Microbial electrosynthesis (MES) using electroactive autotrophic microorganisms has recently been reported as a method to reduce carbon dioxide, the main culprit of greenhouse gas. However, there are still few cases of application of MES, and the molecular mechanisms are largely unknown. To investigate the growth characteristics in MES, we carried out growth tests according to reducing power sources in Rhodobacter sphaeroides . The growth rate was signiﬁcantly lower when electrons were directly supplied to cells, compared to when hydrogen was supplied. Through a transcriptome analysis, we found that the expression of reactive oxygen species (ROS)-related genes was meaningfully higher in MES than in normal photoautotrophic conditions. Similarly, endogenous contents of H 2 O 2 were higher and peroxidase activities were lower in MES. The exogenous application of ascorbic acid, a representative biological antioxidant, promotes cell growth by decreasing ROS levels, conﬁrming the inhibitory effects of ROS on MES. Taken together, our observations suggest that reduction of ROS by increasing antioxidant activities is important for enhancing the cell growth and production of CO 2 -converting substances such as carotenoids in MES in R. sphaeroides


Introduction
A climate crisis has arisen as carbon dioxide emissions have increased due to industrialization [1]. Various technologies have been proposed to reduce carbon dioxide emissions, and biological CO 2 fixation is attracting attention as a desirable alternative. Recently, sustainable bioelectrochemical systems have been proposed to convert CO 2 into valuable chemicals by microbial electrosynthesis (MES) using electroactive autotrophs.
In this system, electroactive microorganisms can utilize CO 2 using electrons as a reducing power [2]. Production of high value-added materials such as polyhydroxybutyrate (PHB), lycopene and α-humulene using Cupriavidus necator (C. necator) in an MES reactor has been reported [3][4][5]. Acetate was also continuously produced from CO 2 by a mixed microbial consortium via an MES system [6].

Bacterial Strain and Preparation of Microbial Electrosynthesis Reactor
The Rhodobacter sphaeroides KCTC1434 strain was obtained from Korean Collection for Type Cultures (KCTC) and grown on Sistrom's medium without succinic acid [14]. The precultured cells for the microbial electrosynthesis (MES) reaction were cultured in modified Sistrom's medium under autotrophic conditions purged with 5% CO 2 , 60% H 2 , and 35% argon. Cell growth was estimated by measuring optical density (OD) using a spectrophotometer (BioSpectrometer, Eppendorf, Hamburg, Germany) at 660 nm.
The double-chamber H-type microbial electrosynthesis reactor had the same configuration as described previously [15]. The anode and cathode chambers were joined with a glass arm and separated using a proton exchange membrane (PEM, Nafion 117, DuPont Ltd., Wilmington, DE, USA). The anode and cathode electrodes were, respectively, a 4 cm × 10 cm and 4 cm × 5 cm piece of graphite felt. The thickness of the electrodes was 0.3 cm (GF030, FuelCellStore, College Station, TX, USA) and they were connected to titanium wire. The reference electrode, Ag/AgCl (in 3 M NaCl), was placed in the cathode chamber. The cathode electrode was poised with -0.6 V (vs. Ag/AgCl) using a potentiostat (WMPG1000, WonAtech, Seoul, Korea). The microbial electrosynthesis reaction was conducted under light-anaerobic conditions in batch mode with a gas composition of CO 2 5% and argon 95% at a rate of 17.5 mL/min (0.05 vvm). Ascorbic acid (A92902, Sigma-Aldrich, St. Louis, MO, USA), which was used after adjusting the pH to 7 with KOH, was treated to final concentrations of 5 mM and 10 mM in the MES systems. All MES reactors were placed at 30 • C under white LED lamps and operated in triplicate.

Transcriptome Analysis
Preparation of total RNA for the transcriptome analysis was conducted by using a Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research, CA, USA). RNase-free DNaseI was treated to total RNA to remove any contaminating genomic DNA. Complementary DNA library construction and raw data processing were finished by Macrogen (Seoul, Korea). The cDNA libraries were sequenced with an Illumina HiSeq 2500 (Illumina, CA, USA) in pair-end mode. A differentially expressed genes (DEG) analysis was performed with edgeR. The genes were selected by p-value < 0.05 and fold-change (FC) > 2.

Determination of ROS Levels
The levels of H 2 O 2 and peroxidase activities were quantified using an Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, OR, USA), as described previously [16]. The sonicated samples were prepared in potassium phosphate buffer (pH 7.5). First, 50 µL of sample was mixed with the reaction reagent containing Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) and horseradish peroxidase (HRP), and it was then incubated for 30 min at room temperature. For quantification of peroxidase activities, H 2 O 2 was added instead of horseradish peroxidase. Fluorescence was measured using a SYNERGY H1 microplate reader (BioTek, VT, USA) with excitation/emission of 530/590 nm.
Endogenous reactive oxygen species levels were measured using the fluorescent dye CM-H 2 DCFDA (Invitrogen, MA, USA). The cells were washed and prepared in PBS buffer (pH 7.4). The CM-H 2 DCFDA solution was treated to a final working concentration of 1 uM, and mixtures were incubated for 30 min at 30 • C in dark conditions. Fluorescence signals were read at excitation/emission of 495/527 nm.

Measurement of Total Carotenoid Contents
The measurement of total carotenoid was conducted as described previously [16]. Briefly, the freeze-dried cells were suspended in 1 mL of 3 M HCl and then incubated for 30 min at 30 • C, 100 rpm. The supernatants were discarded after centrifugation and the pellets were resuspended in 1 mL of acetone. The supernatants were harvested after incubation and the absorbance for quantification of carotenoids was read at 480 nm.

Statistical Analysis
The statistical analysis was carried out using a Student's t-test. The data were indicated as mean ± standard deviation. The differences were considered as statistically significant when the p-value was less than 0.05.

Comparison of Growth Behavior According to Reducing Source in R. sphaeroides
For CO 2 biological fixation using electroactive microorganisms in MES, an appropriate reducing power, such as organic compounds, hydrogen or electricity, is required. Although several studies on MES have been reported, there has been no direct comparison of growth behavior according to reducing source, such as hydrogen or electricity [17][18][19]. Thus, we first examined the cell growth of R. sphaeroides according to the supply of different electron donors in the presence of constant light and CO 2 ( Figure 1). As a result, it was observed that cell growth was significantly slower when electricity was used as an electron donor than when hydrogen was used. These results indicate that bacterial cell growth is strongly influenced by the type of electron source. Hydrogen is generally one of the most preferred electron donors for autotrophic microorganisms. It readily generates intracellular proton motive force (PMF) to induce sufficient ATP synthesis and promote CO 2 transport. This proton gradient caused by hydrogen ion also promotes the generation of reducing cofactor, such as NADPH 2 [7,20,21]. NADPH 2 is an important biochemical redox cofactor responsible for the transfer of electrons and protons. It is used for reductive biosynthesis, such as biosynthesis of fatty acids and some amino acids. Moreover, it functions as an oxidative stress defense because NADPH 2 is essential for the functions of several antioxidant enzymes, including glutathione peroxidase, glutathione S-transferase, and catalase [22][23][24]. When the hydrogen is used as a reducing source, the supply of NADPH 2 and ATP are sufficient for CO 2 fixation. However, when electricity is used as a reducing source, the supply of NADPH 2 and ATP is insufficient due to only electrons are provided and appropriate proton gradient is not formed. Thus, we suspected that the deficiency of ATP and NADPH 2 is one of the important factors limiting the bacterial cell growth in MES. For this reason, the direct supply of electrons or the indirect supply of permeated hydrogen ions after water decomposition at the anode is estimated to have lower utilization efficiency in R. sphaeroides compared to the direct supply of hydrogen.
transport. This proton gradient caused by hydrogen ion also promotes the generatio reducing cofactor, such as NADPH2 [7,20,21]. NADPH2 is an important biochemical r cofactor responsible for the transfer of electrons and protons. It is used for reductive synthesis, such as biosynthesis of fatty acids and some amino acids. Moreover, it func as an oxidative stress defense because NADPH2 is essential for the functions of sev antioxidant enzymes, including glutathione peroxidase, glutathione S-transferase, catalase [22][23][24]. When the hydrogen is used as a reducing source, the supply of NAD and ATP are sufficient for CO2 fixation. However, when electricity is used as a redu source, the supply of NADPH2 and ATP is insufficient due to only electrons are prov and appropriate proton gradient is not formed. Thus, we suspected that the deficien ATP and NADPH2 is one of the important factors limiting the bacterial cell growth in M For this reason, the direct supply of electrons or the indirect supply of permeated hy gen ions after water decomposition at the anode is estimated to have lower utiliza efficiency in R. sphaeroides compared to the direct supply of hydrogen. We next performed a transcriptomic analysis to understand the variation of the lecular mechanism in MES systems compared to normal photoautotrophic conditions ble 1). Consistent with slower cell growth in MES, the expression of hydrogenase (hu gene for uptake of H2 and the Calvin-Benson-Bassham (CBB) cycle (cbbSL, cfxA, fbp1, cbbR) genes for the uptake of CO2 were downregulated in MES [25,26]. PHB is a re sentative carbon storage material that accumulates in cells and the expression of PH osynthesis (phaA, phaB, phaC1, and phaC2) genes reported to be proportional to cell gro also decreased overall in MES [16]. It is speculated that this is due to the upregulatio the PHB synthesis repressor (PhaR), encoded by RSP_0380 [27]. The transcript leve genes involved in flagellar biosynthesis (flhAB, flgALK1I1HE, fliEF1KPRD, and mo were significantly increased when electricity was used as an electron donor [28]. In systems, electron transfer occurs either directly or indirectly through an extracellular tron uptake mechanism. The flagellar mediate direct electron transfer by adhesion to electrode, and this mechanism has been identified in Desulfovibrio [29,30]. Based on th is hypothesized that the upregulation of flagellar biosynthetic gene expression in M closely associated with direct electron uptake in R. sphaeroides. We next performed a transcriptomic analysis to understand the variation of the molecular mechanism in MES systems compared to normal photoautotrophic conditions (Table 1). Consistent with slower cell growth in MES, the expression of hydrogenase (hupSL) gene for uptake of H 2 and the Calvin-Benson-Bassham (CBB) cycle (cbbSL, cfxA, fbp1, and cbbR) genes for the uptake of CO 2 were downregulated in MES [25,26]. PHB is a representative carbon storage material that accumulates in cells and the expression of PHB biosynthesis (phaA, phaB, phaC1, and phaC2) genes reported to be proportional to cell growth also decreased overall in MES [16]. It is speculated that this is due to the upregulation of the PHB synthesis repressor (PhaR), encoded by RSP_0380 [27]. The transcript levels of genes involved in flagellar biosynthesis (flhAB, flgALK1I1HE, fliEF1KPRD, and motAB) were significantly increased when electricity was used as an electron donor [28]. In MES systems, electron transfer occurs either directly or indirectly through an extracellular electron uptake mechanism. The flagellar mediate direct electron transfer by adhesion to the electrode, and this mechanism has been identified in Desulfovibrio [29,30]. Based on this, it is hypothesized that the upregulation of flagellar biosynthetic gene expression in MES is closely associated with direct electron uptake in R. sphaeroides. In particular, the genes related to ROS signaling (oxyR, rpoE, chrR, and phrA), redoxins (grxC, RSP_2953, trxA, RSP_0725), and superoxide dismutase (sodC) were highly expressed in MES conditions [31]. In contrast, the expression of catalase gene (katC) involved in hydrogen peroxide scavenging and the carotenoid biosynthetic genes (crtBCDEFI), which were reported to be involved in antioxidant activity, was notably decreased. Overall, the genes related to ROS signaling were upregulated, while the genes related to ROSscavenging mechanisms were downregulated except for the sodC gene. It is confirmed that expression of the genes related to ROS and antioxidant activity were critically influenced by the direct supply of electricity. These transcriptome analysis results suggest that when CO 2 is used as a carbon source, cellular metabolisms are greatly changed depending on the type of reducing power. In particular, it causes a large difference in antioxidant activity, and the mechanism regulating it is very complex, and thus, further studies are needed to elucidate the change in MES.
Many studies have reported that ROS metabolism is very sensitive to changes of environmental growth conditions. In our transcriptome analysis data, it was confirmed that the ROS signaling was strictly associated with the reducing power under photoautotrophic growth conditions. To identify whether changes in transcript levels affected the generation of ROS, we next evaluated the endogenous levels of ROS in MES compared to those under normal photoautotrophic conditions (Figure 2a,b). The levels of endogenous hydrogen peroxide were relatively higher under MES systems than under photoautotrophic conditions. Consistent with this, higher fluorescence values were also observed in MES when the levels of ROS were measured by the detection of fluorescence caused by oxidation of H 2 DCFDA, an indicator of general oxidative stress. We subsequently measured the activities of peroxidase, which is related to scavenging of hydrogen peroxide (Figure 2c). The peroxidase activities were 4.9-fold lower when electricity was utilized as the electron source than when H 2 was utilized. Generally, when the level of endogenous ROS exceeds the detoxification capabilities of microorganisms, cell damage and bacterial cell death occur. It has been reported that the production of endogenous ROS predicted by an ensemble approach of genome-scale increased the susceptibility of E. coli to oxidative attack, quickly leading to cell death [32]. Furthermore, cytotoxic ROS are generated in electrochemical systems through water splitting side reactions. It was also reported that the growth of C. necator in the hybrid microbial-water-splitting catalyst system is inhibited by ROS generated from the electrode in MES when the voltage is less than 2.3 V or higher than 4.0 V. In this system, carbon dioxide converts into biomass and isopropanol along with hydrogen and oxygen produced from water splitting. Since the minimum thermodynamic potential for water splitting is 1.23 V, the relatively high potential for microorganisms was applied in this system [33]. Afterward, to avoid the toxic effect of ROS against microbes and to decrease the potential for water splitting, the biocompatible catalyst system which used an ROS-resistant cobalt-phosphorus alloy cathodic electrode was reported [34]. It was also reported that ROS generated in the inorganic electrocatalytic system, such as O 2 − and NO, has a toxicity to C. necator, resulting in the inhibition of cell growth and yield of the products [35]. To evade the toxicity of ROS to bacterial cell growth, it has been reported that the two-chamber MES reactor is efficient. This configuration, which separates the cathodic and anodic chamber by a proton exchange membrane (PEM) and has an applied potential at −0.6 V, is similar to our MES reactor [36]. Both the present and the previous observations support that ROS generated in MES systems unfavorably influence cell growth, indicating that the regulation of endogenous ROS is a key element for the improvement of growth in MES.

Enhancement of Bacterial Cell Growth and Carotenoid Production by Modulating ROS in MES
Based on our experimental results so far, when electricity was used as an electron source, we confirmed that the cell growth is slower and the endogenous ROS content is higher compared to when hydrogen is used as an electron source (Figures 1 and 2). Through this, we hypothesized that cell growth is poor in MES due to increased levels of ROS. Furthermore, the growth of microbial cells, including attached cells and planktonic cells, is an important factor in increasing the production of high-value added materials in MES. To improve the cell growth by modulation of ROS while confirming our hypothesis, we introduced ascorbic acid, which is known as a powerful biological antioxidant. The cell growth was observed in MES with the addition of 5 mM and 10 mM ascorbic acid, respectively (Figure 3a). During 10 days of cultivation, the bacterial cell growth was significantly improved by the addition of ascorbic acid. When 5 mM or 10 mM of ascorbic acid was added, the cell density increased by 161% and 131%, respectively. Dosing of more than 10 mM ascorbic acid appears undesirable in MES because excess ascorbic acid can act as an oxidizing agent and inhibit cell growth. To confirm the effect of ascorbic acid on endogenous ROS, we subsequently analyzed the contents of H 2 O 2 and the levels of endogenous ROS (Figure 3b,c). Compared to the control, the levels of H 2 O 2 were reduced proportionally to the concentration of treated ascorbic acid. When ascorbic acid was supplied at 5 mM and 10 mM, the fluorescence value indicating the endogenous contents of ROS was also decreased by 60% and 75%, respectively. In addition, activities of peroxidase were improved 2.9-fold and 2.1-fold in the presence of 5 mM or 10 mM ascorbic acid, respectively (Figure 3d). These observations suggest that supplementation of exogenous antioxidants successfully reduced endogenous levels of ROS, which have a significant impact on the bacterial cell growth. and has an applied potential at −0.6 V, is similar to our MES reactor [36]. Both the present and the previous observations support that ROS generated in MES systems unfavorably influence cell growth, indicating that the regulation of endogenous ROS is a key element for the improvement of growth in MES.

Enhancement of Bacterial Cell Growth and Carotenoid Production by Modulating ROS in MES
Based on our experimental results so far, when electricity was used as an electron source, we confirmed that the cell growth is slower and the endogenous ROS content is higher compared to when hydrogen is used as an electron source (Figures 1 and 2). Through this, we hypothesized that cell growth is poor in MES due to increased levels of ROS. Furthermore, the growth of microbial cells, including attached cells and planktonic cells, is an important factor in increasing the production of high-value added materials in MES. To improve the cell growth by modulation of ROS while confirming our hypothesis, we introduced ascorbic acid, which is known as a powerful biological antioxidant. The cell growth was observed in MES with the addition of 5 mM and 10 mM ascorbic acid, respectively (Figure 3a). During 10 days of cultivation, the bacterial cell growth was significantly improved by the addition of ascorbic acid. When 5 mM or 10 mM of ascorbic acid was added, the cell density increased by 161% and 131%, respectively. Dosing of more than 10 mM ascorbic acid appears undesirable in MES because excess ascorbic acid can act as an oxidizing agent and inhibit cell growth. To confirm the effect of ascorbic acid on endogenous ROS, we subsequently analyzed the contents of H2O2 and the levels of endogenous ROS (Figure 3b,c). Compared to the control, the levels of H2O2 were reduced proportionally to the concentration of treated ascorbic acid. When ascorbic acid was supplied at 5 mM and 10 mM, the fluorescence value indicating the endogenous contents of ROS was also decreased by 60% and 75%, respectively. In addition, activities of peroxidase were improved 2.9-fold and 2.1-fold in the presence of 5 mM or 10 mM ascorbic acid, respectively (Figure 3d). These observations suggest that supplementation of exogenous Ascorbic acid is one of the most important antioxidants present in cells. It prevents oxidative damage caused by ROS and mediates diverse cell metabolism. In plants, ascorbate serves as a cofactor of multiple enzymatic reactions and supports photosynthesis and flowering [37]. Ascorbic acid is able to scavenge hydrogen peroxide, which occurs through the photosynthetic electron transport chain, and thereby contributes to the control of photosynthetic efficiency in plants [38]. Furthermore, the addition of ascorbic acid also has various effects on microbial metabolism. In R. sphaeroides, the activity of light-dependent ATPase involved in ATP synthesis is usually observed to be lower under aerobic conditions than under anaerobic conditions. However, additional supply of ascorbic acid reduced ROS toxicity and recovered ATPase activity to anaerobic levels [39]. The photosynthesis of R. sphaeroides is mediated by the reaction centers (RCs). As the concentration of ascorbate increases, the generation of singlet oxygen ( 1 O 2 ) decreases in the reaction centers [40]. These findings indicate that the supplement of ascorbic acid modulates the production of ROS during photosynthesis, affecting the microbial CO 2 fixation efficiency. According to both the present and previous results, ROS regulation by the supply of antioxidants meaningfully improves bacterial cell growth in MES. In order to better understand the effect of regulating antioxidant activity on microorganisms, it will be of great help if we further analyze changes of gene expression patterns and related mechanisms in the future. Ascorbic acid is one of the most important antioxidants present in cells. It prevents oxidative damage caused by ROS and mediates diverse cell metabolism. In plants, ascorbate serves as a cofactor of multiple enzymatic reactions and supports photosynthesis and flowering [37]. Ascorbic acid is able to scavenge hydrogen peroxide, which occurs through the photosynthetic electron transport chain, and thereby contributes to the control of photosynthetic efficiency in plants [38]. Furthermore, the addition of ascorbic acid also has various effects on microbial metabolism. In R. sphaeroides, the activity of light-dependent ATPase involved in ATP synthesis is usually observed to be lower under aerobic conditions than under anaerobic conditions. However, additional supply of ascorbic acid reduced ROS toxicity and recovered ATPase activity to anaerobic levels [39]. The photosynthesis of R. sphaeroides is mediated by the reaction centers (RCs). As the concentration of ascorbate increases, the generation of singlet oxygen ( 1 O2) decreases in the reaction centers [40]. These findings indicate that the supplement of ascorbic acid modulates the production of ROS during photosynthesis, affecting the microbial CO2 fixation efficiency. According to both the present and previous results, ROS regulation by the supply of antioxidants meaningfully improves bacterial cell growth in MES. In order to better understand The valorization of CO 2 into high value-added products is an important aspect of expanding the application of MES. In the context, we next investigated the production of carotenoids, which are naturally accumulated in R. sphaeroides and possess antioxidant activity, under MES systems (Figure 4). When 5 mM and 10 mM of ascorbic acid was added, the contents of total carotenoids were 2.7-fold and 3.3-fold elevated in cells, respectively. These results indicate that the production of total carotenoids in R. sphaeroides is increased in proportion to the concentration of ascorbic acid. Consistent with this, it was reported that a moderate positive correlation was observed between the levels of ascorbate and the contents of total carotenoid in spinach [41]. However, since plants and microorganisms have very different metabolic processes, it is necessary to conduct further research to elucidate the molecular mechanism between ascorbic acid and carotenoid production in R. sphaeroides. Moreover, the antioxidant activity of carotenoids is expected to be beneficial in utilization of CO 2 under MES conditions. Previous studies reported that the carotenoid pigments assist the modulation of photosynthetic electron transport and protect against the damage induced by light during photosynthesis [38]. If a synergistic effect between carotenoid biosynthesis and its antioxidant activity could be achieved, then a vigorous Energies 2022, 15, 935 9 of 11 electricity-driven cell factory could be established for sustainable carotenoid production in MES [4]. Taken together, the present results show that modulation of ROS by an exogenous antioxidant promoted the production of carotenoids as well as cell growth. These observations suggest that the enhancement of antioxidant activity can be used as a novel strategy to increase the bacterial cell growth and the yield and productivity of valuable products under MES systems.
research to elucidate the molecular mechanism between ascorbic acid and carotenoid production in R. sphaeroides. Moreover, the antioxidant activity of carotenoids is expected to be beneficial in utilization of CO2 under MES conditions. Previous studies reported that the carotenoid pigments assist the modulation of photosynthetic electron transport and protect against the damage induced by light during photosynthesis [38]. If a synergistic effect between carotenoid biosynthesis and its antioxidant activity could be achieved, then a vigorous electricity-driven cell factory could be established for sustainable carotenoid production in MES [4]. Taken together, the present results show that modulation of ROS by an exogenous antioxidant promoted the production of carotenoids as well as cell growth. These observations suggest that the enhancement of antioxidant activity can be used as a novel strategy to increase the bacterial cell growth and the yield and productivity of valuable products under MES systems. Figure 4. Measurement of total carotenoid contents in the presence of ascorbic acid under MES conditions. Asc, ascorbic acid. Experiments were performed in triplicate and error bars indicate standard deviation of mean. Asterisk represent statistically significant difference, as determined by a Student's t-test (* p < 0.05).

Conclusions
Improving bacterial cell growth is a crucial factor in converting CO2 into valuable materials under MES systems. However, the cell growth in MES has been much slower than that under normal photoautotrophic conditions due to the generation of large amounts of endogenous ROS. To overcome this, we applied on exogenous antioxidant to MES. When ascorbic acid was treated, the cell growth and carotenoid accumulation were significantly increased. Furthermore, while the levels of endogenous ROS were decreased, the activity of ROS scavenging enzyme was increased. Altogether, our results suggest that

Conclusions
Improving bacterial cell growth is a crucial factor in converting CO 2 into valuable materials under MES systems. However, the cell growth in MES has been much slower than that under normal photoautotrophic conditions due to the generation of large amounts of endogenous ROS. To overcome this, we applied on exogenous antioxidant to MES. When ascorbic acid was treated, the cell growth and carotenoid accumulation were significantly increased. Furthermore, while the levels of endogenous ROS were decreased, the activity of ROS scavenging enzyme was increased. Altogether, our results suggest that modulation of ROS is very important to promote bacterial cell growth and carotenoid production under MES conditions.