Conversion of Carbon Dioxide to Metabolites by &amp;lt;i&amp;gt;Clostridium acetobutylicum&amp;lt;/i&amp;gt; KCTC1037 Cultivated with Electrochemical Reducing Power

In this research, metabolic fixation of CO2 by growing cells of C. acetobutylicum cultivated with electrochemical reducing power was tested on the basis of the metabolites production and genes expression. In cyclic voltammetry, electrochemical oxidation and reduction reaction of neutral red (NR) immobilized in intact cells of C. acetobutylicum was stationarily repeated like the soluble one in the condition without CO2 but the electrochemical reduction reaction was selectively increased by addition of CO2. In electrochemical bioreactor, the modified graphite felt cathode with NR (NR-cathode) induced C. acetobutylicum to generate acetate, propionate, and butyrate from CO2 in defined medium. When H2 and CO2 were used as an electron donor and an electron acceptor, respectively, C. acetobutylicum also produced the same metabolites in a defined medium. C. acetobutylicum was not grown in the defined medium without substituted electron donors (H2 or electrochemical reducing power). C. acetobutylicum cultivated with electrochemical reducing power produced more butyrate than acetate in complex medium but produced more acetate than butyrate in defined medium. The genes of encoding the enzymes catalyzing acetyl-CoA in C. acetobutylicum electrochemically cultivated in defined medium than conventionally cultivated in complex medium. These results are a clue that C. acetobutylicum may metabolically convert CO2 to metabolites and produce free energy from the electrochemical reducing power.

section_content: Chemoautotrophs that regenerate reducing power and produce free energy in coupling with oxidation of H 2 can more effectively fix CO 2 than ammonium, nitrite, and ferrous-oxidizing bacteria because redox potential of H 2 /2H + (-0. 42 V vs. NHE) is lower than NAD + /NADH (-0. 32 V vs. NHE) [1]. The reducing power generated in coupling with oxidation of ammonium, nitrite, and ferrous ion can't induce regeneration of NAD(P)H without a reverse electron transport system coupled to consumption of external energy [2] [3] [4] [5]. Experimentally measured redox potential of NR is -0. 325 V (vs. NHE), which is theoretically enough to mediate generation of electrondriving force from electrode to NADH [6]. Practically, electrochemically reduced NR catalyzes NADH regeneration by non-enzymatic catalysis [7]. Theoretically, electrochemically reduced NR may be more effective in reducing power than H 2 on the basis of non-enzymatic catalysis of NADH regeneration. 

C. acetobutylicum is a typical fermentation bacterium that produces acetate, propionate, and butyrate by the coupling redox reaction of reducing power and acetyl-CoA generated from metabolic oxidation of glucose, and also autotrophically produces acetyl-CoA from the coupling redox reaction of H 2 and CO 2 in defined medium with organic nitrogen nutrient [8, 9]. The acetyl-CoA is metabolically oxidized to acetate coupled to regeneration of NADH or reduced to butyrate coupled to oxidation of NADH [10]. Biological production of organic polymers, fatty acids and alcohols from H 2 and CO 2 has been studied in order to decrease global warming and produce renewable energy and useful biomass [11, 12]. H 2 is the most effective electron donor to bacteriologically fix CO 2 on the basis of its redox potential but is less practical to apply to biological system owing to very low solubility in water and possible explosiveness in the process of use, transport and storage. The cost for production of H 2 is not economical in comparison of glucose price. In order to solve the problem caused by the H 2 production cost, an alternative technology has been developed [13, 14]. 

Electricity generated from the solar cell can be directly converted to biochemical reducing power in coupling with the redox reaction of NR immobilized in bacterial cell or graphite felt electrode [6]. Bacterial CO 2 fixation induced by biochemical reducing power electrochemically regenerated by the solar cell electricity may correspond to the photosynthesis because O 2 is generated from anode compartment and CO 2 is biochemically assimilated into biomass and converted to metabolites in cathode compartment [15]. Covalently immobilized NR in graphite felt electrode can function as a catalyst for NADH regeneration and a redox carrier for electron transfer from electrode to bacterial cell [16]. Redox potential of NR is -0. 325 volt (vs. NHE), which is 0. 05 volt lower than NAD +. The electrochemical redox reaction of NR can be coupled to biochemical redox reaction as follows: [NR ox + 2e -+ 1H + → NR red ; NR red + NAD + → NR ox + NADH]. Commonly, NR ox and NAD + are reduced to NR red and NADH, respectively by accepting two electrons from electrode and NR red (ox: oxidation; red: reduction). 

In this study, electrochemical reducing power was charged to C. acetobutylicum culture using the NRcathode to induce autotrophic production of acetate and butyrate from CO 2 in a defined medium and increase of butyrate production in a complex medium. The NRcathode, to which -2 V of DC-electricity was charged, may be an optimized habitat for strict anaerobes because the lower oxidation-reduction potential than -300 mV (vs. NHE) is electrochemically generated and the electrochemically reduced NR may catalyze bacterial NADH regeneration.
section_name: Introduction

section_name: Materials and Methods

section_content: Reinforced clostridial (RC) medium (Tryptone 10 g/L, Sodium chloride 5 g/L, Beef extract 10 g/L, Yeast extract 3 g/L, Glucose 20 g/L, Starch 1 g/L, L-cystein hydrochloride 0. 5 g/L, Sodium acetate 3 g/L) was used as a complex medium and for successive cultivation of C. acetobutylicum. M9 mineral medium (Disodium phosphate 6. 8 g/L, Monosodium phosphate 3 g/L, Ammonium chloride 5 g/L, Sodium chloride 0. 5 g/L, Magnesium sulfate 0. 246 g/L, Calcium chloride 0. 0147 g/L) supplemented with sodium bicarbonate (25 mM) and yeast extract (3 g/L) was used as a defined medium. Fifty ml of RC or defined medium was prepared in anaerobic serum vials (total volume 165 ml) whose headspace was filled with 2 atmospheres of oxygen-free N 2 or H 2.
section_name: Medium

section_content: An electrochemical bioreactor that was designed for con-tinuous culture in previous research [16] was partially modified for cultivation of strict anaerobic bacterium in batch culture, as is shown in Figure 1. The electrochemical bioreactor (inner diameter, 80 mm; height, 200 mm; medium volume, 500 ml; electrode volume, 250 ml; total volume, 1000 ml; Pyrex, USA) with a built-in anode compartment was designed to equalize distance between anode and all round of cylindrical cathode. A sintered glass filter (diameter, 50 mm; thickness, 5 mm; pore, 1 -1. 6 μm, Duran, Germany) that was modified with cellulose acetate film (35 μm thickness, Electron Microscopy Sciences, USA) was fixed at the bottom end of the tube-type anode compartment (inner diameter 20 mm; height, 150 mm; working volume, 50 ml). Cellulose acetate film attached to the sintered glass functions as a semipermeable membrane capable of selectively transferring water, gas, and proton. Five hundred ml of the media was prepared in the electrochemical bioreactor (Figure 1 ) to which O 2 -free CO 2 (50 ml•min -1 ) was continuously supplied during cultivation. Inoculation ratio was adjusted to 5% (w/w) of medium volume. DC -2 V of electricity was charged to NR-graphite felt cathode to induce electrochemical reduction reaction of NR for C. acetobutylicum. The defined medium was used as anolyte to avoid generation of osmosis between anode and cathode compartment.
section_name: Electrochemical Bioreactor

section_content: Graphite felt (thickness, 10 mm; height, 200 mm; length, 500 mm; Electrosynthesis, USA) was rolled up to be a cylinder type (internal diameter, 40 mm; external diameter, 75 mm). Neutral red was immobilized to thegraphite felt (10 × 200 × 500 mm, Electrosynthesis, USA) by the covalent bond between neutral red and polyvinyl alcohol (mean molecular weight, 80,000, Sigma, USA) according to the technique used in previous research [16]. The graphite felt modified with NR was used as a cathode and a platinum wire (thickness 0. 5 mm, length 150 mm) was employed as an anode. Electric potential charged to NR-cathode was precisely adjusted to -2 V.
section_name: Electrode

section_content: The cyclic voltammetry was employed in order to analyze electrochemical redox reactions between electrode and intact cell of C. acetobutylicum. The cyclic voltammetry was conducted using a voltammetric potentiostat (BAS model CV50W, USA) linked to a data acquisition system. Aglassy carbon electrode (5mm diameter, Electrosynthesis, USA), a platinum wire, and an Ag/AgCl electrode (redox potential, +0. 2 V vs. NHE, Electrosynthesis, USA) were utilized as a working electrode, counter-electrode, and reference electrode, respectively. The reactant was composed of 25 mM Tris-HCl buffer (pH 7. 5) containing 5 mM NaCl and 100 μM NR. C. acetobutylicum that was anaerobically cultivated in the modified M9 medium for 48 hr under H 2 atmosphere was anaerobically centrifuged at 1500 × g and 4˚C for 60 min. 

The precipitated bacterial cells were suspended in 0. 05 volume of the oxygen-free reaction mixture, in which NR was spontaneously immobilized in bacterial cells. Prior to and during the cyclic voltammetry measurement, argon (99. 999%) was sparged into headspace of reaction beaker in order to protect contamination of the reaction mixture by oxygen. The scanning rate was 25 mV•s -1 over a range of 0 to -1200 mV. During cyclic voltammetry for NR dissolved in reactant or immobilized in C. acetobutylicum, the variations of upper voltammetric peaks (an indicator for electron transfer from electrode to bacterial cells through NR) and lower voltammetric peaks (an indicator for electron transfer from bacterial cells to electrode through NR) by addition of CO 2 were recorded.
section_name: Analysis of Electrochemical Reaction of C. acetobutylicum

section_content: Bacterial metabolites were analyzed using a Gas Chromatography/Mass Spectrometry (Clarus 600 series + TurboMatrix HSS Trap, PerkinElmer, USA) equipped with Elite-FFAP column (ID 0. 25 μm, OD 0. 32 μm, length 30 m) and electron ionization system. Bacterial culture was centrifugation at 10,000  g and 4˚C for 30 min and filtered with membrane filter (pore 0. 22 μm), and then directly injected into the GC/MS injector. Con-centration of metabolites was determined based on peak area of standard compounds and chemical species was determined based on mass profile database.
section_name: Analysis of Metabolites

section_content: C. acetobutylicum was cultivated in the electrochemical bioreactor using the defined medium under strict anaerobic CO 2 atmosphere and in the complex medium under strict anaerobic N 2 atmosphere for 5 days. Total RNA was isolated and purified from harvested bacterial cells using a RNA purification kit (Total RNA, spin-column format, Oligotex mRNA mini kit, Qiagen Korea, Seoul). Microarray analysis of mRNA was conducted at Genomictree (Daejeon, Korea) using the systems, kits, DNA chips, and analysis software offered by Agillent Technologies (Korea branch, Seoul) via a turnkey-based analyzing order. The significantly expressed genes that are concerned with CO 2 fixation and energy metabolism were selectively analyzed to compare the relationship between metabolic pathway related with CO 2 fixation and cultivation conditions.
section_name: Microarray of mRNA

section_name: Results

section_content: Cyclic voltammetry is a useful technique to measure electrochemical coupling redox reaction of electron mediator immobilized in bacterial cells. In the cyclic voltammetry without bacterial cells, the electrochemical redox reaction of NR was measured to be -0. 52 V (vs. Ag/AgCl), which is very similar to the experimental value -0. 525 V (vs. Ag/AgCl) measured in standard condition. Both the upper and lower voltammetric peaks were not altered by addition of CO 2 as expected; in contrast, the upper voltammetric peak generated by NR immobilized in C. acetobutylicum was shifted upward from 2. 4 to 2. 9 μA and rightward from -0. 52 to -0. 55 V by addition of CO 2 as shown in Figure 2. Increase of 0. 4 μA of current indicates that electrons are transferred from electrode to bacterial cells coupled to redox reaction of NR. Increase of -0. 3 V of redox potential is a clue that electrons are transferred from electrode to NR by lower electrode (working electrode) potential than intrinsic redox potential of NR. Relatively higher electron-driving force (electrode potential) may be required for electrons to move through the electric resistance generated between electrode and NR immobilized in bacterial membrane. Meanwhile, other upper voltammetric peak (bold arrow mark) located at -0. 9 V (vs. Ag/AgCl) was also shifted upward from 2. 7 to 3. 0 μA and rightward from -0. 9 V to -0. 93 V by addition of CO 2. It seems possible that electrons are transferred from the electrode via one of the electron carriers located in the bacterial membrane, allowing for current and potential increase by addition of CO 2. CO 2 could act as an electron acceptor to induce biochemical oxidation of NADH that may be electrochemically regenerated, by which electrons may be transferred from electrode to bacterial cells via the coupling redox reaction of NR and NAD +.
section_name: Electrochemical Redox Reaction in Coupling with CO 2

section_content: C. acetobutylicum did not grow and didn't produce metabolites in the defined medium under N 2 atmosphere (DM-N 2 ) but grew and produced acetate, propionate, and butyrate under H 2 atmosphere (DM-H 2 ), as shown in Table 1. The metabolites detected in the chromatography for culture fluid of C. acetobutylicum cultivated in the DM-N 2 may have originated from the metabolites contained in the inoculum. The electrochemical reducing power generated from NR-cathode (reduced NR) is converted to the biochemical reducing power (NADH), which can be presumed on the basis of the growth and metabolite production of C. acetobutylicum cultivated in the DM-ER. C. acetobutylicum cultivated with electrochemical reducing power produced more acetate than butyrate in DM-ER but more butyrate than acetate in CM-ER. These are more clues that biochemical reducing power (NADH) may be regenerated by electrochemical reducing power generated from -2 V of NR-cathode and the high balance of NADH/NAD + may induce metabolic conversion of CO 2 to metabolites in coupling with free energy synthesis. required because some metabolic intermediates derived from amino acids (yeast extract) may be produced by bacterial cells cultivated in the DM-H 2 and DM-ER condition.
section_name: Growth and Metabolite Production of C. acetobutylicum

section_name: Quantitative and Qualitative Verification of Metabolites

section_content: Significant genes (higher than twice the signal intensity) commonly expressed in C. acetobutylicum that was electrochemically cultivated in the defined medium under CO 2 -atmosphere and in the complex medium under N 2 -atmosphere numbered in 318. All of the fundamental genes related to CO 2 -fixation were not detected; however, the genes of encoding the enzymes catalyzing acetyl-CoA synthesis from CO 2, ATP synthesis, and butyrate production were quantitatively analyzed and compared as shown in Table 2. The genes encoding the enzymes catalyzing acetyl-CoA generation from CO 2 and ATP synthesis in pathway from acetyl-CoA to acetate were more expressed but those catalyzing NADH regeneration coupled to oxidation of substrates (metabolic intermediates) and butyric acid production were less expressed in C. acetobuylicum cultivated in DM-ER condition than CM-N 2 condition (Table 1 ).
section_name: Analysis of Genes Induced by Electrochemical Reducing Power

section_content: Electron transfer from electrode to bacterial cells can be porarily function in proportion to physiological activity generated by the simultaneous contact of an electron mediator with both electrode and bacterial cells. Contact of an electrode with the electron mediator (NR) immobilized in bacterial cell or contact of bacterial cells with the electron mediator immobilized in an electrode is a unique way to induce electron transfer between bacterial cells and electrode [17] [18] [19]. Patterns of cyclic voltammetry of NR immobilized in bacterial cells were identical to those of NR dissolved in reactant in the condition uncoupled to the external redox reaction, because the redox reaction is proportional to the concentration of NR contacted stably with the electrode (Figure 2 ). Some NRs immobilized in C. acetobutylicum are electrochemically reduced and biochemically oxidized coupled to NADH regeneration [6]. This electrochemical and biochemical coupling redox reaction of NR and NAD + may be continuously repeated in this condition with both electron donor and acceptor. The electrode may be an electron donor and CO 2 may be an electron acceptor in the cyclic voltammetry for the modified C. acetobutylicum with NR, considering that addition of CO 2 induced electron transfer from electrode to bacterial cells via NR immobilized in bacterial cells or other bacterial electron carrier (upper voltammetric peaks in Figure 2 ) can be quantitatively analyzed based on the increase of current (electron number) and variation of redox potential (electron-driving force). The NR immobilized in bacterial cells can tem- In the electrochemical bioreactor equipped with the NR-catho rial cells can't be quantitatively analyzed because the number of electrons (current) transferred from power supply to NR-cathode is not proportional to the metabolic reduction reaction catalyzed by bacterial cells, and -2 V of electrode potential charged to the bioreactor was stronger than the redox potential of NR. This can generate electron-driving force to transfer electrons via NR immobilized in electrode to bacterial cells but may induce electrochemical reduction of medium ingredients and other organic compounds. The -2 V of electrode potential may be too strong to induce the electrochemical reduction of NR (-0. 325 V vs. NHE) but is required to induce electron transfer from electrode to bacterial cells through the electron barrier (cytoplasmic membrane) because electric resistance may be generated by reactor membrane between anolyte and catholyte, connecting error between NR and bacterial cells, and structural mismatch between NR and NAD + [20]. 

It is unquestionable that the NR immobilized in graphite felt electrode mediated electron trans de to bacterial cells and catalyzed regeneration of biochemical reducing power on the basis of acetic and butyric acid production from CO 2 and production of more butyric acid than acetic acid from glucose. This result is very similar to that obtained from C. acetobutylicum culture using H 2 and CO 2 as an electron donor and acceptor. Metabolic production of acetic acid is coupled to NADH regeneration but that of butyric acid is acid than acetic acid by C. acetobutylicum cultivated in CM-ER and CM-H 2 is another clue that H 2 and electrochemically reduced NR may be an additional reducing power to increase ratio of NADH/NAD + [21]. 

Metabolic conversion of CO 2 to metabolites is coupled to oxidation of biochemical reducing power regenerated by the electrochemically reduced NR or H 2 ; however, the metabolic pathways related to the autotrophic C ADH oxi obuylic 2 n can be assumed only by the metabolites produced by C. acetobutylicum cultivated in different media and under different conditions. Microarray analysis of mRNA is effective to analyze variations of metabolic pathway and free energy production related to autotrophic CO 2 fixation or heterotrophic growth. Practically, the specific genes related to the CO 2 fixation and energy metabolism expressed in C. acetobutylicum electrochemically or conventionally cultivated are a clue that the metabolic conversion of CO 2 to metabolites in coupling with the free energy production and redox reaction of reducing power may be generated by the electrochemical reducing power. Theoretically, -2 V of electricity charged to the NRcathode located in culture medium may induce H 2 generation by electrolysis of H 2 O. However, H 2 was not detected in the electrochemical bioreactor even by precision analysis. Accordingly, the NR-cathode may directly transfer electrons from electrode to intact cells of C. acetobutylicum and induce catalyzing of NADH regeneration in metabolism of C. acetobutylicum.
section_name: Discussion

section_content: The electrochemical redox reaction of NR, the catalytic function of NR for NADH regeneration, and t
section_name: Conclusion

section_name: Acknowledgements

section_content: This work was supported by the New e En-Evaluation and Planning (KETEP governmental Ministry of Knowledge Economy ( 2012-T1001100334 ).
section_name: Acknowledgements