Conversion of CO2 to formate in an electroenzymatic cell using Candida boidinii formate dehydrogenase

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 9–15

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Conversion of CO2 to formate in an electroenzymatic cell using Candida boidinii formate dehydrogenase Sungrye Kim a , Min Koo Kim b , Sang Hyun Lee b , Sungho Yoon c , Kwang-Deog Jung a,∗ a

Clean Energy Research Centre, Korea Institute of Science and Technology, P.O. Box 131, Cheongryyang, Seoul 136 791, Republic of Korea Department of Microbial Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Department of Bio & Nano Chemistry, College of Natural Science, Kookmin University, 861-1, Jeongneung-dong, Sungbuk-gu, Seoul 137-702, Republic of Korea b c

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 25 October 2013 Received in revised form 3 January 2014 Accepted 7 January 2014 Available online 17 January 2014 Keywords: Candida boidinii CO2 reduction Formate formation NADH regeneration

Electroenzymatic conversion of environmentally detrimental CO2 into useful chemicals using a NADHdependent formate dehydrogenase has been of great interest, but the system remained at a conceptual level because of its complexity. In this study, we found that CO2 was a direct substrate for formate formation with Candida boidinii formate dehydrogenase (CbsFDH), but HCO3 − was not. Enzymatic formate formation with CbsFDH was inhibited at NADH concentrations higher than 0.35 mM. Electrochemical NADH regeneration was performed at a Cu electrode using the [Cp*Rh(bpy)Cl]+ complex, which catalyzed the conversion of NAD+ into the active NADH with almost 100% selectivity. The electroenzymatic reaction for HCO2 H formation was conducted at 1.0 V, 0.25 mM NADH and 0.25 mM Rh complex during electrochemical NADH regeneration. CO2 was reduced into formate by enzymatic catalysis under the NADH regeneration condition, where protons and electrons was continuously supplied into a cathode cell through Nafion® membrane from water splitting at a Pt anode. The interactions of the Cu electrode, Rh mediator, NADH, and CbsFDH were analyzed for the first time. The Rh(III) mediator was hydrolyzed and reduced reversibly into a Rh(I) intermediate (Mred1 ) as well as irreversibly into a Rh(I) hydride intermediate (Mred2 ) at the Cu electrode. Interestingly, the Rh(I) species showed activity toward the direct reduction of CO2 as well as NADH regeneration, although the primary CO2 reduction occurred through CbsFDH at 1.0 V. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Interest in the electrochemical conversion of CO2 using renewable energy resources such as solar, geothermal, and wind power has grown significantly. CO2 can be reduced to CO, HCO2 H, CH3 OH, and CH4 via the transfer of protons and electrons generated through the electrochemical splitting of water as follows. CO2 + 2 H+ + 2 e− → CO

E o = 0.0 Vvs.SHE

CO2 + 2 H+ + 2 e− → HCO2 H

E o = −0.225 Vvs.SHE

CO2 + 6 H+ + 6 e− → CH3 OH

E o = −0.225 Vvs.SHE

CO2 + 8 H+ + 8 e− → CH4

E o = 0.169 Vvs.SHE

∗ Corresponding author. Tel.: +82 2 9585218; fax: +82 2 9585219. E-mail addresses: [email protected], [email protected] (K.-D. Jung). 1381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2014.01.007

Six and eight electrons are required to produce methanol and methane from CO2 , respectively, which require higher energy as compared to CO and HCO2 H production by two-electron transfers. Additionally, HCO2 H synthesis is more attractive than those of methanol and methane, because HCO2 H commands higher prices than CH3 OH and CH4 . Furthermore, HCO2 H can be used as a feedstock for a recently developing fuel cell [1,2]. Commercially, HCO2 H is produced through the hydrolysis of methyl formate [3,4], which, in turn, is prepared via several pathways from methanol [3,5,6]. A direct CO2 hydrogenation has been studied to reduce CO2 into formate. The key problem was that product was metal formate, although TON was very high [7,8]. Recently, a one-step preparation of HCO2 H from water and CO2 by an electrochemical method has attracted much interest. The direct electrochemical reduction of CO2 at metal electrodes has been studied for various electrode systems [9–12]. However, the process requires a reduction potential higher than −1.8 V vs. SHE and is nonspecific, resulting in broad product distributions comprising CO, hydrocarbons, formic acid, and alcohols. Recently, the direct CO2 reduction was performed at low overpotential on Cu electrodes successfully, but still showed the non-selective

S. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 9–15

products based on carbon [13–15]. On the other hand, the oxidation of formate to CO2 occurs with concomitant reduction of NAD+ to NADH catalyzed by formate dehydrogenase (FDH). Interestingly, the reverse reaction, the reduction of CO2 to formate, could also be catalyzed by the enzyme [16–18]. FDH from Pseudomonas oxalaticus was used to reduce CO2 into formate using oxidized methyl viologen as an electron relay [19]. The tungsten-containing FDH from Syntrophobacter fumaroxidans, known to be the most active catalyst for the reaction so far, was also used to reduce CO2 [20]. The NADH-independent FDH enzymes from P. oxalaticus and S. fumaroxidans are highly unstable and inactive in the presence of O2 , limiting their practical application. Thus, the NADH-dependent FDH from Candida boidinii (CbsFDH) has been used to enzymtically regenerate NADH, and was sufficiently stable for commercial use. Herein, we present a previously unavailable whole system analysis of the electrochemical conversion of CO2 employing with a Cu electrode, a Rh mediator, CbsFDH, and NAD+ . 2. Experimental 2.1. CbsFDH(Candida boidinii formate dehydrogenase)-catalyzed CO2 reduction For the kinetic analysis of the CO2 reduction using NaHCO3 , the reaction was carried out in 0.10 M phosphate buffered saline (PBS) solution in a 1.2 mL quartz cuvette cell at 25 ◦ C. The protein content of CbsFDH (C. boidinii formate dehydrogenase) was fixed at 312 ␮g, while the concentrations of NADH and NaHCO3 varied from 0.10 to 0.30 mM and 10 to 50 mM, respectively. The initial rates of the reaction were determined by measuring the concentration change of NADH. The concentration of NADH was measured at 340 nm using a UV–Vis spectrophotometer (Varian Cary 100) equipped with a thermostated cell-holder. For the reduction using CO2 gas, CO2 -saturated 0.10 M PBS solution (1.8 mL), NADH solution (0.20 mM), and CbsFDH solution (0.40 mL) were charged in a 2.4 mL UV cell. The NADH concentration change was monitored in the UV cell at 25 ◦ C by UV spectroscopy. The amount of the enzyme was fixed at 3.7 mg. For comparison, the reduction of CO2 with CbsFDH was conducted with 30 mM NaHCO3 under the same reaction conditions. The 0.10 M PBS solution was prepared with NaH2 PO4 (Kanto Chem.) and Na2 PO4 ·H2 O (Sigma S9390). The NADH (␤-nicotinamide adenine dinucleotide, reduced dipotassium salt, Sigma N4505) and CbsFDH (Sigma F8649) were purchased from Sigma-Aldrich (St. Louis, MO, USA).The protein content of CbsFDH was determined by the Bradford method. Formic acid was analyzed by ion chromatography (DIONEX IC25A) with a conductivity detector using a Dionex IonPacTM AS19 column (4 × 250 mm; Guard column: IonPacTM AG19). Typical ion chromatograms were shown in Fig. S3. 2.2. Electrochemical NADH regeneration An H-shaped cell made from Teflon was used for electrochemical NADH regeneration. Pt foil (2.5 cm × 2.0 cm) was used as the anode and Cu foil (2.5 cm × 2.0 cm) was used as the cathode. The Cu foil was polished with polishing papers (1200 grit-3 M 50022; 4000 grit-3 M 50026) before the experiment. A Nafion® -117 membrane was placed between the anode and cathode cells for proton transfer. The reference Ag/AgCl electrode was placed in the cathode cell. The volume of each half cell was 20 mL. The PBS solution (0.10 M) was placed in the anode cell and the NAD+ (␤-nicotinamide adenine sodium salt, Sigma N0632) in 0.10 M PBS solution was placed in the cathode cell. The [Cp*Rh(bpy)Cl]Cl complex, where Cp* and bpy are pentamethylcyclopentadienyl and bipyridine ligands, respectively, was used as an electron mediator [21,22]. Both

5 4

1/ (min/mM)

10

3 2 1 0 2

4

6 8 1/NADH (mM-1)

10

12

Fig. 1. Lineweaver Burk plots for FDH-catalyzed NADH oxidation at various sodium bicarbonate concentrations (*: 10 mM, 䊉: 20 mM, : 30 mM, : 40 mM, : 50 mM) in 0.10 M PBS solution.

half cells were purged with Ar gas for 1 h before the reaction and then, a constant potential using a potentiostat (Wonatech WPG100e) was supplied for the regeneration of NADH. The activity of NADH was measured using an enzymatic essay [24]. Unfortunately, the lipoamide dehydrogenase (Sigma L-2002) was no longer available commercially, leading us to use the diaphorase from Clostridium kluyveri (Sigma D55400), which was successful in confirming the generation of active NADH. Linear polarization and cyclic voltammetry experiments of NAD+ and the Rh complex were performed in a conventional three electrode cell. Cu and Pt foils were used as the working and counter electrodes, respectively, and Ag/AgCl was used as the reference electrode. 2.3. Electroenzymatic CO2 reduction The electroenzymatic CO2 reduction was performed in the Hshaped cell. Cu foil (2.5 cm × 2.0 cm) was used as the cathode, as previously described. A 0.10 M PBS solution with CbsFDH, NADH, and the Rh complex was charged into the cathode cell. Then, CO2 was introduced into the cathode cell at a flow rate of 10 mL/min. The CO2 reduction was conducted by applying voltage to the electrodes. Formate was analyzed by an ion chromatography as described above. 3. Results and discussion 3.1. CbsFDH catalysis of CO2 reduction Fig. 1 shows the kinetic data for NADH oxidation with CbsFDH with respect to the concentration of NaHCO3 and NADH. The NADH oxidation catalyzed by FDH was carried out at 25 ◦ C in 0.10 M sodium phosphate buffer (pH 7.0) in a reaction volume of 1.2 mL. According to the ternary complex mechanism proposed by Cleland [23], kinetic parameters were determined.

v=

Vmax [A][B] KiA KB + KB [A] + KA [B] + [A][B]

(1)

The values of the kinetic constants were obtained using the linear regression method by assigning NADH as A and carbon dioxide as B. KA and KB are the Michaelis constants for substrates A and B, respectively, in the presence of saturating concentrations of the other substrate. KiA is an inhibition constant for A. Each kinetic constant is shown in Table 1. The kcat /KB value for the reduction of the HCO3 − anion was estimated to be 0.33 × 10−3 (mM s)−1 . On the other hand, the kcat /KB value for the formate oxidation was 0.153 (mM s)−1 , indicating that the formate formation from CO2

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11

Table 1 Kinetic parameters for NADH oxidation with NaHCO3 in 0.1 M PBS solution at 25 ◦ C. Kinetic parameters

KA (mM)

KiA (mM)

KB (mM)

kcat (s−1 )

kcat /KB (mM s)−1

Values

0.696

0.328

27.3

0.0091

0.33 × 10−3

A and B represent NADH and NaHCO3 , respectively. Table 2 NADH conversion and HCOOH yield at 0.25 mM NADH, 3.7 mg CbsFDH, and 0.1 M PBS solution in 2.4 mL cell. Substrate

pH

CO2 CO2 CO2 NaHCO3 (30 mM)

6 7 8 6

Reaction at 30 min

Reaction at 90 min

NADH Conv. (%)

HCOOH yield (%)

HCOOH production rate (umol/min/mg)

NADH Conv. (%)

HCOOH yield (%)

HCOOH production rate (␮mol/min/mg)

5 16 15 9

5 12 12 2

2.7E − 04 6.5E − 04 6.5E − 04 1.1E − 04

11 28 27 12

10 23 24 5

1.8E − 04 4.1E − 04 4.3E − 04 0.9E − 04

was unfavorable with CbsFDH. This clearly suggested that NADH and CO2 should be maintained at high concentrations to produce formate. NaHCO3 was used to generate the CO2 substrate during the kinetic analysis. However, the mechanistic observation with CbsFDH suggested that CO2 was binding to the active sites of the enzyme rather than HCO3 − [24], indicating that only neutral CO2 was a direct substrate for the formation of formate. In that case, as HCO3 − cannot be a direct substrate, CO2 from the equilibrium between HCO3 − and H2 CO3 can be used for the formate formation. To investigate this, the initial NADH oxidation rates with NaHCO3 and CO2 were compared. Fig. 2 shows the specific activity for the NADH oxidation with respect to NADH concentration in 0.10 M PBS at pH 7.0. Both saturated CO2 and 30 mM NaHCO3 solution in the phosphate buffer were used to supply CO2 . The concentration of saturated CO2 was estimated to be 33 mM at a CO2 partial pressure of 1 atm [25]. Therefore, 30 mM NaHCO3 was used for comparison with the CO2 saturated solution, because the CO2 solubility in PBS was slightly lower than that in water. The CO2 saturated solution in 0.10 M PBS at pH 7.0 exhibited a pH 6.5. The specific activity for the NADH oxidation with gaseous CO2 was lower than the one using NaHCO3 . It may originate from the pH being lower than 7.0. Table 2 shows that the conversion of NADH at pH 6.0 was much lower than that at pH 7.0. The specific activity for the NADH oxidation with CO2 increased with an increase in NADH concentration, but began to decrease slightly at 0.35 mM NADH. The dependency of the specific activity on NADH concentration with NaHCO3 was similar to that with CO2 .

Specific activity ( mol/min/mg)

4.E-03 4.E-03 3.E-03 3.E-03 2.E-03 2.E-03 1.E-03 5.E-04 0.E+00

0

0.2 0.4 0.6 NADH concentration (mM)

0.8

Fig. 2. Specific activities of NADH oxidation in () saturated CO2 and () 30 mM NaHCO3 in 0.10 M PBS solution.

The low activity of CbsFDH for NADH oxidation at high concentration suggested that CbsFDH could be inhibited by the NADH cofactor as NADH accumulated. These experimental results supported the previous reports of the inhibition of NAD+ and NADH on FDH [26,27]. To confirm formate formation, reactions employing CO2 and NaHCO3 with 0.25 mM NADH were analyzed by ion chromatography. Table 2 shows the conversion of NADH and product yields of HCO2 H at 30 and 90 min in 0.25 mM NADH and 0.10 M PBS solution. The initial specific activity with the NaHCO3 substrate for the NADH oxidation was higher than that with CO2 , but the conversion of NADH with NaHCO3 was similar to that with CO2 after 90 min. It is interesting to note that the observed yield of HCO2 H with the NaHCO3 substrate was much lower than the consumed NADH, while the yield of formate with the CO2 substrate was almost equivalent to the amount of converted NADH. This indicates that HCO3 − is not a direct substrate for the formation of formic acid with CbsFDH but CO2 , supporting the suggestion of Popov and Lamzin [28]. The activities of CbsFDH at pH 7.0 and 8.0 were higher than that at pH 6.0; the conversion of NADH with CbsFDH was almost identical to the consumed formate at pH 6.0. From the kinetic point of view, the rate at lower pH would be higher than that at higher pH, but the experimental result was the opposite. It seems to be related to the intrinsic characteristics of CbsFDH. The production rates at 30 min were calculated to be 2.7 × 10−4 and 6.5 × 10−4 ␮mol/min/mg at pH 6.0 and pH 7.0, respectively. 3.2. Regeneration of NADH at Cu metal electrode Fig. 3a shows the linear polarization curve of Cu electrode in the 1.0 mM NAD+ and 0.10 M PBS. The peak at about 1.2 V is due to the reduction of phosphate ions, as shown in the linear polarization curve in PBS solution only [29,30]. The distinctive reduction peak of NAD+ with the Cu electrode was not observed in PBS solution. It was reported that the hydrogen evolution reaction (HER) is predominant at metal electrodes [31]. The high current density in the experimental ranges was due to the HER. Therefore, the absence of the NAD+ reduction peak did not only result from the irreversible adsorption of NAD+ , but also from the low reduction rate of NAD+ as compared to the HER. Nonetheless, the addition of NAD+ increased the cathodic current density. The current density difference in 0.10 M PBS with and without NAD+ on the Cu electrode was also plotted with respect to the potential vs. Ag/AgCl in Fig. 3b. The reduction potential of NAD+ was determined from the difference of the current densities of the linear polarization curves with and without NAD+ , assuming that the presence of NAD+ has no influence on the HER, which occurs at 1.1 V. The reduction potential

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Fig. 4. Cyclic voltammograms recorded using a Cu electrode in 0.10 M PBS solution containing the 0.25 mM Rh complex (solid line: no NAD+ , dotted line: 0.25 mM NAD+ , double dot-dashed line: 0.5 mM NAD+ , single dot-dashed line: 1.0 mM NAD+ , dashed line; 2.0 mM NAD+ ).

of NAD+ on the Cu electrode in 0.10 M PBS was relatively high as compared with the redox potential of NAD+ /NADH (−0.54 V vs. Ag/AgCl at pH 7). The estimated NAD+ reduction potential of about 1.1 V was in good agreement with that at other modified Au, Hg and Ru electrodes [32]. It was proposed that the reduction potential of NAD+ was not dependent on electrode materials, but on the orientation of the nicotinamide moiety on the electrode surface. It was reported that the formation of an enzymatically inactive NAD-dimer species at the metal electrode can be suppressed if an electron mediator is used [33–35]. Here, after several mediators were tested and compared, the most active complex, [Cp*Rh(bpy)Cl]Cl, was selected. Fig. 4 shows the cyclic voltammograms of NAD+ in 0.25 mM Rh complex and 0.10 M PBS solution. The reduction of the Rh(III) cation into the Rh(I) ion is shown in the cyclic voltammogram. The current density increased with the addition of NAD+ up to 0.5 mM NAD+ , and then decreased with the further NAD+ addition. It should be noted that an anodic current was observed at concentrations of NAD+ higher than 0.5 mM. The observed reduction behavior of NAD+ in the presence of the Rh mediator was similar to the result reported at an Au electrode [35]. The Rh(III) species was reduced into the Rh(I) species (Mred1 ) by accepting electrons from the electrode, and the protonation of Mred1 irreversibly generated a second intermediate, the Rh hydride complex (Mred2 ). The appearance of an anodic current in the cyclic voltammogram at a high NAD+ concentration was attributed to the depletion of protons in the diffusion layer, because Mred2 formation was prevented. The protons were consumed to generate NADH from NAD+ and were depleted at high concentrations of NAD+ . Then, Mred1 (reversible Rh(I)) was re-oxidized to Rh(III) under the proton-depleted conditions,

1

NADH concentration (mM)

Fig. 3. (a) Linear polarization curves of Cu electrode and (b) differential polarization curve in 0.10 M PBS solution (----: without NAD+ , –: with 1.0 mM NAD+ ).

because the irreversible formation of the Rh hydride complex did not occur, showing the anodic peak. Fig. 5 shows the effect of the Rh(III) mediator concentration on NADH regeneration, which was conducted at 1.0 V vs. Ag/AgCl in 1.0 mM NAD+ and 0.10 M PBS solution. The initial NADH regeneration rate increased with an increase in the concentration of the Rh mediator. However, the resultant NADH was converted back to NAD+ at high Rh mediator concentration, showing a maximum concentration of generated NADH with respect to time. The Rh mediator can donate or accept electrons to NAD+ or from NADH, respectively. It was reported that the hydride exchange of NADH and Rh mediator was reversible [36–38]. Therefore, in the presence of a high concentration of Rh mediator, the concentration of the Rh(III) mediator can be high enough to oxidize NADH to NAD+ . As mentioned before, proton depletion can also explain these results. It was shown that metal complex converted into M-H in the presence of NADH so fast under no proton donor [a8]. NADH can be oxidized to NAD+ in the presence of the Rh(III) species under that condition, as described in Fig. 4. Fig. 6 shows the NADH regeneration rate with respect to the potential vs. Ag/AgCl in 0.10 M PBS buffer solution at pH 6.0 at the Cu electrode with the Rh mediator. The NADH regeneration rates were highest at about 1.0 V at the Cu electrode with the Rh mediator. The concentration of NADH at the potential higher than 1.0 V decreased slightly. The NADH concentration decrease at the high potential

0.8 0.6 0.4 0.2 0 0

30

60

90

120

150

180

210

NADH regeneration time (min) Fig. 5. Dependence of Rh complex concentration on NADH regeneration using a Cu electrode in 0.10 M PBS solution containing 1.0 mM NAD+ at pH 6 and 1.0 V (䊉: no Rh complex, : 0.25 mM Rh complex, : 0.50 mM Rh complex, : 1.00 mM Rh complex): The potential was referenced to Ag/AgCl.

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13

1.4

Formate (mM)

1.2 1 0.8 0.6 0.4 0.2 0 0

may result from the HER; the HER can occur at the Cu electrode, as reported. Then, the concentration of available protons for the formation of NADH would be limited by the fast HER. The optimized potential for the regeneration of NADH was equivalent to the NAD+ reduction potential as shown in Fig. 3. Fig. 6 also shows the pH effect on NADH regeneration in 0.10 M PBS buffer solution at the Cu electrode with the Rh mediator. The regeneration rate of NADH was higher at lower pH. Similarly to the effect of the potential, it can be concluded that the regeneration rates were proportional to the concentration of protons. Table 3 summarized NADH yields and active NADH selectivity at 180 min reaction time in Fig. 5 and Fig. 6. 3.3. Formation of formic acid from CO2 in bioelectrochemical reaction with CbsFDH An H-shaped cell was used for the bioelectrochemical reaction with CbsFDH. A Cu electrode was immersed in CO2 saturated PBS solution in a cathode cell after an Ar purge to remove oxygen, while a Pt coil was placed to generate protons from water in the PBS solution in the anode cell. CO2 (10 mL/min) was introduced into the cathode cell for the formation of HCO2 H. Fig. 7 shows the effects of the potential vs. Ag/AgCl on the formate formation with 0.25 mM NADH and CbsFDH in 0.10 M PBS solution at pH 6.0 during the regeneration of NADH. It should be noted that the formate formation rate at 1.0 V increased with respect to the reaction time after 150 min. The formate production rates at 0.80, 0.90 and 1.0 V at 30 min were estimated to be 2.5 × 10−4 , 2.4 × 10−4 and 3.2 × 10−4 ␮mol/min/mg, respectively. The formate formation rates in the H-shaped cell at 30 min were similar to the enzymatic Table 3 NADH yield and Active NADH selectivity on the different NADH regeneration condition at 180 min reaction in 1.0 mM NAD+ and 0.1 M PBS solution. Rh complex (mM)

pH

Potential vs Ag/AgCl (V)

NADH yield (%)

Active NADH selectivity (%)

0.25 0.25 0.25 0.25 0.25 0.25 0.5 1.0 0.0 0.0 0.0

6 6 6 6 7 8 6 6 6 6 6

−0.7 −0.8 −1.0 −1.2 −1.0 −1.0 −1.0 −1.0 −0.8 −1.0 −1.2

68.5 78.2 96.5 79.4 56.0 12.1 85.2 56.9 5.3 69.7 47.6

92.8 91.1 94.6 100.0 100 93.8 100.0 92.2 31.1 67.4 24.1

120 180 240 Reaction Time (min)

300

360

Fig. 7. Potential effect on formate formation in an electroenzymatic cell using a Cu electrode in 0.10 M PBS solution containing 0.25 mM NADH, 30 mg CbsFDH and 0.25 mM Rh complex at pH 6.0 (䊉: 0.80 V, : 0.90 V, : 1.0 V).

formation rate of formate at 30 min as described in Table 2. The formate production rates at 330 min were 1.2 × 10−4 , 1.8 × 10−4 and 2.1 × 10−3 ␮mol/min/mg at 0.80, 0.90, and 1.0 V, respectively. In particular, the formate production rate at 0.80 V decreased with the reaction time, similar to the result of the enzymatic reaction (Fig. S1), which was due to the low rate of NADH regeneration. On the other hand, the rate of formate production in the electroenymatic reaction at 1.0 V increased slightly with the reaction time. The observed high production rate at 1.0 V may partially originate from the high regeneration rate of NADH. The other reason may be a high proton transfer rate, generated from water splitting in the anodic cell. The proton transfer rates are proportional to the pH difference between the anode and cathode cell. During the reaction, the pH value in cathode cell was maintained at about 6.2 irrespective of the potential, while the pH value in the anode cell steadily decreased with reaction time, reaching pH 6.0, 5.5, and 4.4 at 0.80, 0.90, and 1.0 V after 330 min, respectively. The dramatic change in pH indicates that many protons are supplied to the cathode cell for CO2 reduction, resulting in the high formate formation rate. It should be noted that the observed behavior of the pH difference between the cathode and anode cells for CO2 reduction is similar to that between chloroplast stroma and the thylakoid lumen in natural photosynthesis. Fig. 8 shows the combined effects of the amount of CbsFDH enzyme, the capability of the Cu electrode, and the Rh complex on 1.8 1.6 1.4 Formate (mM)

Fig. 6. pH and potential dependence on NADH regeneration using a Cu electrode in 0.10 M PBS solution containing 1.0 mM NADH and 0.25 mM Rh complex (䊉: 1.0 V at pH 8.0, : 1.0 V at pH 7.0, : 1.0 V at pH 6.0, : 0.7 V at pH 6.0, : 0.80 V at pH 6.0, : 1.2 V at pH 6.0): The potential was referenced to Ag/AgCl.

60

1.2 1.0 0.8 0.6 0.4 0.2 0.0

0

60

120 180 240 Reaction time (min)

300

360

Fig. 8. Electroenzymatic CO2 conversion to formate using a Cu electrode at 1.0 V in 0.10 M PBS solution at pH 6.0 (: buffer only, : 0.25 mM Rh complex, : 0.25 mM Rh complex/0.25 mM NADH, 䊉: 0.25 mM Rh complex/0.25 mM NADH/20 mg CbsFDH, : 0.25 mM Rh complex/0.25 mM NADH/30 mg CbsFDH, : 0.25 mM Rh complex/0.25 mM NADH/45 mg CbsFDH).

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4. Conclusion

Scheme 1. Schematic diagram of electroenzymatic CO2 reduction using CbsFDH.

formate formation. The reaction was conducted at 1.0 V in 0.10 M PBS buffer solution. The Cu metal electrode has negligible capability to produce formate; the formate concentration with a Cu electrode only was 0.02 mM after 330 min. The concentration effects of the Rh complex for CO2 reduction was shown in Fig. S2. The formate concentrations with 0.5 mM and 0.75 mM Rh complex were 1.1 mM and 2.5 mM after 330 min, respectively. From these experiments, Rh complex has a capability to reduce CO2 into formate as well as to transfer hydride to NAD+ . The Rh complex with NADH yielded 0.43 mM formate after 330 min. Formate yield of the Rh complex was slightly increased in the presence of NADH. The conversion of NAD+ to NADH consumes two electrons and one proton. Therefore, NADH can supply electrons and protons to the Rh complex as well as accept electrons from the Rh complex, indicating that hydride transfer between NADH and the Rh complex can be reversible. The experimental results indicated that NADH reversibly supplied electrons and protons to the Rh complex. It was clear that formate formation increased with an increase in the amount of CbsFDH, indicating that the main CO2 reduction was due to the enzymatic reaction. CbsFDH requires active NADH for formate formation, and the NADH concentration is thus a limiting factor in the enzymatic reaction. Furthermore, high NADH concentration inhibits the reduction of CO2 by CbsFDH, indicating the existence of an optimized NADH concentration (0.25 mM NADH under our experimental conditions) for formate formation. The highest enzymatic NADH conversion was 28% (max. 0.08 mM HCO2 H) at pH 8.0 as shown in Table 2 and Fig. S-1. Therefore, an NADH regeneration system was required to increase the yield of HCO2 H. As tested, the Cu electrode for NADH regeneration was best among Au, Ag and In as shown in Table S1, but the selectivity for the generation of active NADH at the Cu electrode was below 70%. It is worth noting that the Rh mediator not only increased the active NADH selectivity to ∼100%, but also the yield of NADH; the Cu foil showed negligible activity for formate formation under our experimental conditions. The Rh complex showed significant CO2 reduction capability at 1.0 V, as shown in Fig. 8, indicating that the Rh complex can play an interactive role as a CO2 reduction catalyst as well as an electron mediator for NADH regeneration. From these observations, a scheme for CO2 reduction in this electroenzymatic system (Cu/Rh complex/NADH/CbsFDH) was depicted in Scheme 1. Water is split into 2H+ , 1/2O2 , and electrons and the generated protons are transferred through the Nafion® membrane. The [Cp*Rh(bpy)Cl]Cl mediator is readily hydrolyzed in water to give [Cp*Rh(bpy)H2 O]2+ (Rh(III)), which is reduced to [Cp*Rh(bpy)] (Mred1 /Rh(I)) by electron transfer from the Cu cathode. Mred1 is further protonated to [Cp*Rh(bpy)H]+ (Mred2 , Rh hydride complex) [28]. Both NAD+ and CO2 can be reduced to NADH and HCOO− by the Rh hydride complex. CO2 is also converted into HCOO− by CbsFDH in the presence of NADH.

CbsFDH, an NADH-dependent FDH, is known to be stable, whereas the NADH-independent FDH enzymes from P. oxalaticus and S. fumaroxidans are highly unstable and inactive in the presence of O2 , limiting their practical application. CbsFDH requires NADH, protons and electrons to convert CO2 into formate, which can be supplied by the electrochemical system of the Cu electrode which showed the best activity for NADH regeneration. However, [Cp*Rh(bpy)Cl]Cl mediator was required to achieve ∼100% active NADH. Therefore, an electroenzymatic cell employing a Cu electrode with [Cp*Rh(bpy)Cl]Cl mediator was used to convert CO2 into HCO2 H using CbsFDH in 0.10 M PBS solution. At a high concentration of NADH (more than 0.30 mM NADH), CO2 reduction with CbsFDH was inhibited, indicating that NADH in an optimized concentration (0.20–0.40 mM NADH) should be regenerated continuously to convert CO2 in a realistic preparative way. The Rh complex was hydrolyzed and reduced reversibly into (Mred1 ) and irreversibly into (Mred2 ) Rh(I) species. The reversibility of the Rh complex required optimization of the NADH concentration under a given reaction condition. Interestingly, the Rh(I) species demonstrated activity for CO2 reduction as well as NADH regeneration. From these observations, we concluded that the rate of formate formation catalyzed by CbsFDH can be increased only within the optimized reaction conditions of electrochemical NADH regeneration. Therefore, a robust new FDH, operating in high NADH and enzyme concentrations, should be developed through bioengineering for an electroenzymatic method to convert CO2 into HCO2 H on a preparative scale. Acknowledgments The authors acknowledge financial support for this work from a Korea CCS R&D Center (KCRC) grant and a Converging Research Center Program (2011K000660) by the Ministry of Education and Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molcatb. 2014.01.007. References [1] N.M. Aslam, M.S. Masdar, S.K. Kamarudin, W.R.W. Daud, APCBEE Pocedia 3 (2012) 33–39. [2] X. Yu, P.G. Pickup, J. Power Source 182 (2008) 124–132. [3] W. Reutemann, H. Kieczka, Ullmann’s Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim, 2002. [4] T. Schaub, R.A. Paciello, Angew. Chem. Int. Ed. 50 (2011) 7278–7282. [5] J.S. Lee, J.C. Kim, Y.G. Kim, Appl. Catal. 57 (1990) 1–30. [6] L. Chen, J. Zhang, P. Ning, Y. Chen, W. Wu, J. Nat. Gas Chem. 13 (2004) 225–230. [7] P.G. Jessep, F. Joo, C. Tai, Coord. Chem. Rev. 248 (2004) 2425–2442. [8] Y.N. Himeda, N.O. Komatsuzaki, H. Sugihara, K. Kasuga, Organometallics 26 (2007) 702–712. [9] K.W. Frese, B.P. Sullivan, K. Krist, H.E. Guard, Electrochemical and Electrocatalytic Reactions of Carbon Dioxide, Elsevier, Amsterdam, 1993, pp. 145. [10] M. Gattrell, N. Gupta, A. Co, Energy Convers. Manage. 48 (2007) 1255–1265. [11] H. De Jesus-Cardona, C. del Mora, C.R. Cabrera, J. Electroanal. Chem. 513 (2001) 45–51. [12] Y. Hori, in: W. Vielstich, H.A. Gasteiger, A. Lamm (Eds.), Handbook of Fuel CellsFundamentals, Technology and Applications, 2, Wiley, Chichester, UK, 2003, pp. 720–733. [13] K.P. Kuhl, E.R. Cave, D.A. Abram, T.F. Jaramillo, Energy Envion. Sci. 5 (2012) 7050–7059. [14] C.W. Li, M.W. Kanan, J Am. Chem. Soc. 134 (2012) 7231–7234. [15] R. Kortlever, K.H. Tan, Y. Kwon, M.T.M. Koper, J. Solid State Electrochem. 17 (2013) 1843–1849. [16] V.I. Tishkov, V.O. Popov, Biochemistry 69 (2004) 1252–1267. [17] S. Kuwabata, R. Tsuda, K. Nishida, H. Yoneyama, Chem. Lett. (1993) 1631–1634. [18] S. Kuwabata, R. Tsuda, H. Yoneyama, J Am. Chem. Soc. 116 (1994) 5437–5443.

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