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A hybrid CO2 electroreduction system mediated by enzyme-cofactor conjugates coupled with Cu nanoparticle-catalyzed cofactor regeneration
Journal of CO₂ Utilization 34 (2019) 568–575
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Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou
A hybrid CO2 electroreduction system mediated by enzyme-cofactor conjugates coupled with Cu nanoparticle-catalyzed cofactor regeneration Haiyan Songa, Chunling Maa, Pi Liua, Chun Youa,b, Jianping Lina,c, Zhiguang Zhua,b,
T
⁎
a
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West Seventh Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China University of Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing, 100049, China c College of Pharmacy, Nankai University, Nankai District, Tianjin, 300071, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: CO2 electroreduction Hybrid system Cu nanoparticles Enzyme-cofactor conjugates Cofactor regeneration
With the widespread concerns on CO2 capture and utilization, various approaches have been developed to convert CO2 into valuable products. To achieve fast and efficient conversion, a hybrid CO2 electroreduction system was constructed, in which a cofactor-dependent formate dehydrogenase (FDH) was responsible for CO2 enzymatic reduction to formate and Cu nanoparticles (CuNPs) was used for enzyme immobilization and cofactor regeneration. A polyethylene glycol swing arm was further adopted to construct an enzyme-cofactor conjugate and promote the overall reaction. This electron-mediator-free hybrid system containing immobilized FDH-cofactor conjugates and CuNPs achieved a formate production of 8.5 mM and a productivity as high as 11.8 μM mU−1 h−1 with a faraday efficiency of 22.8%, several folds better than systems with unconjugated FDHs alone or with unconjugated FDHs and CuNPs. These results suggest that such hybrid systems may represent a potentially viable alternative for many cofactor-dependent bioelectrochemical applications.
1. Introduction The growing greenhouse effect has aroused widespread concern on carbon dioxide (CO2) reduction and conversion. Various methods have emerged chemically, biologically, electrochemically and photoelectrochemically [1–6]. Among them, the electrochemical reduction of CO2 has gained significant attention because of its modest reaction condition, in connection with renewable energy sources, as well as high reaction rate. However, metal-catalyzed electrochemical methods usually suffer from low reaction selectivity accompanied by mixed byproducts and relying on unsustainable and expensive catalysts [7]. Alternatively, the enzymatic bioelectrocatalysis possessing superior specificity and renewability of the catalyst has been proposed as a promising route recently [8,9]. Formate dehydrogenase (FDH) is a widely-used enzyme reducing CO2 directly to formate, which has been regarded as an important energy carrier and industrial bulk chemical [10]. Compared to metalcontaining FDHs, nicotinamide adenine dinucleotide (NAD+)-dependent FDHs display strong oxygen tolerance and decent catalytic ability and therefore are more frequently used in the bioelectrochemical reduction of CO2 [11,12]. For example, Srikanth et al. used non-immobilized FDHs from Candida boidinii for formate production and
neutral red (NR) for mediated NADH regeneration in a bioelectrochemical system [5]. Later, they introduced carbonic anhydrases to the reaction and immobilized all their enzymes onto electropolymerized NR/Nafion/graphite cathode, observing improved formate production rate and increased stability [13]. Similarly, Zhang et al. optimized the condition of immobilizing CbFDHs in Nafion micelles and obtained a fast formate production rate and a high Faradic efficiency [14]. Additionally, four dehydrogenases and the NAD+ were in situ coencapsulated inside hollow nanofibers to exhibit high conversion activity of CO2 to methanol [15]. More recently, a low-potential redox polymer was prepared to reduce CO2 to formate, yielding an extremely high Faradic efficiency of 99% at a mild applied potential of -0.66 V vs. standard hydrogen electrode [16]. Such studies all relied on electron mediators, either diffusional or immobilized, for electrochemical cofactor regeneration, and the cofactors used were in the free form or simply tethered to the electrode without rational design. Hybrid systems combining chemo- and bio-catalysis are receiving increasing attention, driven by the advantages of high yield, decreased cost and high selectivity [17]. Inspired by nature, a photoelectrochemical (PEC) cell consisting of a photoanode for water oxidation and a biocathode co-immobilized by CbFDH and NADH via polydopamine entrapment was developed and produced formate at a high
⁎ Corresponding author at: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West Seventh Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China. E-mail address: [email protected] (Z. Zhu).
https://doi.org/10.1016/j.jcou.2019.08.007 Received 9 July 2019; Received in revised form 5 August 2019; Accepted 9 August 2019 2212-9820/ © 2019 Elsevier Ltd. All rights reserved.
Journal of CO₂ Utilization 34 (2019) 568–575
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USA). Copper sulfate (CuSO4), citric acid (C6H8O7), sodium acetate (CH3COONa), phenazine methosulfate (PMS), and tetranitroblue tetrazolium chloride (TNBT) were purchased from Sinopharm (Beijing, China). 3-Carboxyphenylboronic Acid (CBA) and 4-(bromomethyl) phenylacetic acid (BPA) were purchased from Innochem (Beijing, China). Amine-polyethylene glycol-carboxylic acid (NH2-PEG-COOH, 3400 kDa) was purchased from Ponsure Biotech (Shanghai, China). NHydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were from J&K Scientific (Beijing, China). Carbon felt (CF) (AvCarb MGL200) was purchased from Fuel Cell Earth (Stoneham, MA, USA). The phosphate buffer solution (PBS, 0.1 M) was the frequently-used buffers in the experiment. All solutions were prepared with the deionized water which was purified with a Millipore-Q purification system (specific resistance > 18.0 MΩ cm).
faradaic efficiency [4]. A biomimicking artificial thylakoid was constructed containing protamine-titania microcapsules and cadmium sulfide quantum dots for the photobiocoupled reduction of CO2 via a single enzyme and multiple enzymes [6]. Similarly, a tandem PEC cell with an integrated enzyme cascade system was constructed for lightassisted cofactor regeneration and enzymatic conversion of CO2 to methanol [18]. In fact, NADH regeneration can be achieved in an electrochemical mode, such as by a cholesterol-modified gold amalgam electrode, a ruthenium-modified glassy carbon (GC) electrode, and a histidine modified silver electrode [19–22]. Recently, Cu nanorods were prepared as the cathode for electrochemical NADH regeneration and CO2 reduction via non-immobilized CbFDH with high yields, and the authors claimed that adding the Rh(III) mediator led to the improved yield of enzymatically active 1,4-NADH [23,24]. It was also reported that Cu foam could be a better electrode material for direct NADH regeneration than Cu foil, and the study showed an optimal 1,4NADH regeneration yield of 80% using Cu foam electrodes [25]. These results suggest that Cu-based nanomaterials may hold the great promise in efficient electrochemical NADH regeneration and can be integrated with biocatalytic systems to increase the enzymatic CO2 reduction rate. Herein, we rationally designed a hybrid CO2 electroreduction system mediated by FDH from Thiobacillus sp. KNK65MA for enzymatic formate production coupled with Cu nanoparticles (CuNPs)-catalyzed NADH regeneration (Scheme 1). Active NADH can be regenerated with a high yield without the need of electron mediators (e.g., Rh(III)). Besides, CuNPs deposited onto carbon felt (CF) electrodes can also serve as a matrix for FDH immobilization through a Cu-S bond formed between the copper and the cysteine residue at the enzyme surface [26]. TsFDH was chosen because of its high CO2 reduction activity reported [11], and its surface Glu262 was mutated to Cys to allow the enzyme immobilization. Moreover, a FDH-NAD+ conjugate crosslinked by a polyethylene glycol (PEG) swing arm was constructed using a previous developed method, in order to immobilize the cofactor in the vicinity of the enzyme [27]. It is expected that the cofactor can swing back and forth between the catalytic site of FDH and the CuNPs with facilitated electron shuttling and increased CO2 reduction rate. Such hybrid systems for CO2 electroreduction to formate may represent a potentially viable alternative for many other bioelectrochemical applications.
2.2. Preparation of CuNPs-electrodeposited electrodes for NADH regeneration A CHI 1000C potentialstat (Shanghai Chenhua Instrument Co., Ltd., Shanghai, China) was used for electrochemical operation. In this study, CuNPs electrodeposited on CF was used for NADH regeneration. Before electrodeposited, the CF surface (1 × 1 cm2) was pretreated using cyclic voltammetry (CV) in 500 mM H2SO4 for 20 cycles from 1.5 V to -1.5 V at a scanning speed of 100 mV s−1. CuNPs were then electrodeposited using CV in various amounts of CuSO4 and 500 mM H2SO4 from -0.2 V to -1.0 V vs Ag/AgCl at 50 mV s−1 for various cycles, resulting in the prepared electrode remarked as CuNPs/CF. The NADH regeneration was carried out at the chosen potential, in a 5 mL threeelectrode system (0.1 M PBS, pH 6.0) with a CuNPs/CF as the working electrode, while a single platinum wire as the counter electrode and a Ag/AgCl as the reference electrode. N2 was continuously injected to remove O2 for 30 min and the reaction was initiated by adding 1 mM NAD+. During the regeneration reaction, 0.1 mL sample was taken from the solution at 30-min intervals and analyzed using a Cary 5000 Agilent spectrophotometer (Santa Clara, CA, USA) at 340 nm for the amount of NADH. The more specific amount of active NADH was measured using 0.1 M PMS as a catalyst and 0.1 M TNBT as a chromogenic reagent. When NAD+ was catalyzed into active NADH, TNBT reduced to a dark blue color (Fig. S1A) that can be quantified using Cary 5000 Agilent spectrophotometer at 654 nm (Fig. S1B). And the standard calibration of active NADH was measured to this method (00.2 mM) (Fig. S1C).
2. Experimental section 2.1. Materials
2.3. Preparation of FDH Nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide reduced (NADH), sodium formate, sodium bicarbonate, and acetamide were purchased from Sigma-Aldrich (St. Louis, MO,
Plasmids pET20b-ts-fdh was constructed using Simple Cloning [28]. The FDH gene was PCR-amplified from the genomic DNA of T. KNK65MA using a pair of primers (forward: 5′-TAACT TTAAG AAGGA GATAT ACATA TGGCG AAAAT ACTTT GCGTT CTCT-3′; reverse: 5′GTGCT CGAGT GCGGC CGCAA GCTTG CCGGC CTTCT TGAAC TTCGC G-3′). The vector backbone of pET20b was also PCR-amplified using a pair of primers (forward: 5′- CGTGA TGGAG TAACT TGGTA TGACC TCGAG CACCA CCACC ACCAC CACT-3′; reverse: 5′-ATCGT CGAAA CGGTA TTTGT CATAT GTATA TCTCC TTCTT AAAGT TAAA-3′). The PCR products were purified using the Zymo Research DNA Clean & Concentrator Kit (Irvine, CA). The DNA inserter and vector backbone were assembled using prolonged overlap extension PCR, and subsequently, the amplified DNA multimer was directly transformed into E. coli TOP10 cells, yielding the desired plasmid. The Glu262Cys mutation was performed using a site-directed mutagenesis kit from New England Biolabs (Ipswich, MA, USA). The recombinant FDH was produced in the E. coli BL21 (DE3) strain harboring the plasmid and grown in LuriaBertani medium with 100 μM ampicillin at 37 °C, using a T&J-Atype 5L × 2 Parallel Bioreactor (T&J Bio-engineering Co.,LTD, Shanghai, China). The his-tagged FDH protein was purified by HisPur Ni-NTA Resin columns from Thermo Fisher Scientific (Waltham, MA, USA). The
Scheme 1. Schematic illustration of the hybrid CO2 electroreduction system. 569
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Methanol was estimated using a colorimetric assay. First, methanol was catalyzed to formaldehyde by alcohol oxidase (1 U mL−1) from Pichia Poastoris with 10 μM flavin adenine dinucleotide as cofactor. Then the sample was poured into a centrifuge tube to 500 μL with water. And 50 μL solution (50 g ammonium acetate, 6 mL glacial acetic acid and 0.5 mL acetylacetone were dissolved in 100 mL H2O) was added into the centrifuge tube and heated in water at 60 ℃ for 15 min. Finally, the sample was detected at a spectrum of 414 nm. Ethanol was estimated through HPLC with a Bio-rad 87H chromatographic column at 60 ℃. The mobile phase was 5 mM H2SO4 and the flow velocity was 0.6 mL min−1. The faraday efficiency (FE) for formate production was calculated as follows:
activity assay of FDH was measured at various pH values of 5.0, 6.0 and 7.0 in 0.1 M PBS, 50 mM NaHCO3, 0.2 mM NADH at 25 °C. One unit of reduction activity was defined as the amount of enzyme required to consume 1 μmol NADH per minute under standard conditions. 2.4. Preparation of FDH-NAD+ conjugates To construct FDH-NAD+ conjugates, the NAD+-CBA-PEG-COOH was prepared first, using a method developed by our group previously [29]. Then, 100 mmol EDC and 100 mmol NHS were added to activate the carboxyl group of the resultant NAD+-CBA-PEG-COOH (10 mmol), and 100 μmol FDH was added to the above mixture for 2 h to finally form FDH-NAD+ conjugates. The amount of NAD+ in the conjugate was determined by measuring the absorbance change at 340 nm where NAD+ acted as a cofactor was turned into NADH by the catalysis of FDH with 50 mM formate as the substrate. On the other side, the concentration of FDH was determined by the Bradford assay using bovine serum albumin (BSA) as a standard. Therefore, the relative molecular ratio of NAD+ moiety and enzyme (FDH) in FDH-NAD+ conjugates can be calculated. The surface Met1 of FDH was identified according to its crystal structure (PDB: 3WR5), which maybe applied in the conjugate formation.
FE=
2nformate t ∫0 I dt
Where nformate is the mole of formate generated, 2 is the number of electrons transferred from one molecule CO2 to formate. I is the reduction current intensity (A) and t is the reaction time (s) obtained from (Fig. S3). 3. Results and discussion
2.5. Construction and characterization of enzyme-immobilized bioelectrodes
First of all, an electrochemical NADH regeneration system catalyzed by CuNPs was developed and optimized. A three-dimensional porous CF electrode with a large surface area of ∼100 m2 g−1 was used as the substrate electrode in order to maximize the catalyst loading. Cyclic voltammetry (CV) from −0.2 V to −1.0 V vs Ag/AgCl at 50 mV s−1 was applied in 1 mM CuSO4 to electrodeposit CuNPs onto CF electrodes and the potential applied for NADH regeneration was set as −1.2 V. Fig. 1A shows that 30 or 40 CV cycles in the process of CuNPs electrodeposition result in a NADH regeneration yield of ∼40% from CuNPs/CF electrodes constructed, higher than that with 20 cycles. Therefore, the condition of 30 cycles was used for the preparation of CuNPs/CF electrodes in the following study. The concentration of CuSO4 was optimized next. As revealed from Fig. 1B, the NADH regeneration yield achieves a maximum of 92.1% at 2 mM CuSO4, larger than 41.2% obtained at 1 mM CuSO4. Yet, the yield reduces to 65.2% with the concentration of CuSO4 increasing to 3 mM, which may be caused by the diminished Cu active sites from overfull CuNPs deposited on the CF electrode. Moreover, the potential applied for electrochemical NADH regeneration was also evaluated (Fig. 1C). The yield achieves to more than 90% when using a potential of −1.0 V or −1.2 V. Instead, it drops to only 45% at a reduced potential of −0.8 V. Notably, nonselective electrochemical reduction of NAD+ may occur depending on the reduction potential and the catalyst, and the formation of enzymatically inactive NADH dimer can decrease the regeneration yield of enzymatically active 1,4-NADH [19]. As a result, we further measured the amount of 1, 4-NADH using a method involving phenazine methosulfate (PMS) catalyzing 1, 4-NADH to NAD+ and tetranitroblue tetrazolium chloride (TNBT) reduced to a dark blue color that can be quantified at 654 nm (Fig. S1) [30]. The yield of 1, 4-NADH achieves ∼82.3% at the optimal condition of 30 CV circles, 2 mM CuSO4, and -1.0 V applied potential (Fig. 1D). In comparison, a Cu nanorods-electrodeposited GC electrode exhibited an active NADH regeneration yield of 67% and a Cu foam electrode could achieve an optimal yield of 80% [23,25]. These results suggest that CuNPs/CF electrodes were successfully constructed with the highest efficiency for the regeneration of enzymatically active 1, 4-NADH without the necessity of adding electron mediators. Next, FDHs were adsorbed onto the CuNPs/CF electrodes by forming Cu-S bonds between a CuNP and a thiol group at the FDH surface, which was introduced by mutating Glu264 to Cys. The fabricated bioelectrodes were characterized by electrochemical impedance spectroscopy (EIS) which can probe the features of the surface-modified
Different amounts of FDH-NAD+ conjugates or FDH were dropped onto CuNPs/CF and incubated at 25 °C overnight. Cu-S bonds were formed between the enzyme and the CuNPs and the unbound enzymes were cleaned by 0.1 M PBS (pH 6.0). The amount of enzymes adsorbed on the electrode was quantified by the difference between the amount of the enzyme to be dispensed and the amount of enzyme shedding, which were all detected by Bradford. The bioelectrode was designated as FDH-NAD+/CuNPs/CF or FDH/CuNPs/CF. In addition, the CuNPs/ CF and CF electrodes were prepared for the control experiments. The electrochemical impedance spectroscopy was performed at open circuit potential over an AC frequency range of 100 kHz to 1 Hz, with a sinusoidal perturbation of 5 mV, in order to obtain the resistance on bioelectrodes constructed. Scanning electron microscopy (SEM) was carried out by using a JSM-6701 field emission SEM instrument (JEOL, Akishima, Japan) at an accelerating voltage of 10 kV with a Phoenix energy-dispersive X-ray analyzer (NS7, Thermo, USA). Raman spectra was carried out by using a LabRAM HR Evolution spectra instrument (HORIBA Jobin Yvon, France). 2.6. Formate production by the hybrid CO2 electroreduction system The formate production by bioelectrochemical CO2 reduction was performed in a bioelectrochemical system. A 5 mL single-chamber reactor consisting of an enzyme-immobilized electrode as a working electrode, a single platinum wire as the counter electrode, and a Ag/ AgCl as the reference electrode was used. The electrolyte contained several mM NADH and CO2 gas in 0.1 M PBS. The CO2 was reduced by FDH to formate, meanwhile the NADH molecule was converted into NAD+. Then, NAD+ acquired electrons from electrode, in turn, to regenerate NADH under catalysis of CuNPs. Formate was analyzed according to Kuk’s method with a little modification [18]. Briefly, 100 μL of sample containing formate was mixed with 5 mM NaHCO3, 0.2 mL of solution A, 10 μL of solution B, 0.7 mL of 100% acetic anhydride, and incubated at 50 °C for 0.5 h with occasional rapid mixing. A red color could thereby be developed and quantified at 515 nm. Solution A was prepared by dissolving 0.5 g of citric acid and 10 g of acetamide in 100 mL of isopropanol; solution B was prepared by dissolving 30 g of sodium acetate in 100 mL of water. Sodium formate dissolved in 0.1 M PBS (pH 6.0) was used for standard calibration (0–10 mM) (Fig. S2A). 570
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Fig. 1. The effect of CV cycles (A) and Cu ion concentration (B) in CuNPs/CF electrode preparation on the yield of NADH regeneration. The effect of applied reduction potential on the yield of NADH regeneration using the CuNPs/ CF electrode (C) and the yield of 1, 4-NADH at the optimal condition (30 CV cycles, 2 mM CuSO4, and -1.0 V applied potential) with time (D).
Fig. 2. Electrochemical impedance (A) of CF electrode (red line); CuNPs/CF (black line); FDH/CuNPs/CF (blue line); Raman spectra (B) of CF (black), FDH (blue), CuNPs/CF (red) and FDH/CuNPs/CF (green).
characterize the morphology of the bioelectrode surface. A three-dimensional wire with smooth surface was observed for bare CF electrodes (Fig. 3A). Meantime, X-Ray Microanalysis (EDS-Mapping) of CF showed that only carbon element (red) existed in the bare CF (Fig. 3B and C). After electrodeposited with CuNPs, their surface became rough and contained numerous spherical and uniform particles with an approximate diameter of 20–30 nm (Fig. 3D). The obvious copper element (blue) indicated the effective CuNPs modification (Fig. 3E–G). Upon the immobilization of FDH, the electrode presented a disordered porous, filamentous and reticulated structure (Fig. 3H) and appeared about 9% nitrogen element (yellow) representing FDH (Fig. 3I–L), illustrating the successful construction of FDH/CuNPs/CF electrodes. After rinse (Fig. 3M), the nitrogen element reduced to 7%, and reflected that most FDH was immobilized on the electrode by Cu-S bond (Fig. 3N–Q). It should be noted that the size of CuNPs obtained in this study was smaller than that reported elsewhere (i.e., 94–282 nm), possibly because of different carbon electrodes employed. [23] Using CF rather than GC as the substrate electrode can allow better distribution of nanoparticles due to the large surface area of the former and might lead to enhanced catalytic performance. For the proof-of-concept of CO2 electroduction and formate generation, a 5 mL 3-electrode bioelectrochemical reactor was constructed
electrodes. Fig. 2A shows a Nyquist plot of different electrodes in 5 mM K3Fe(CN)6/K4Fe(CN)6 redox probe in 0.1 M KCl. The bare CF (red line) displays a small resistance, while the resistance of CuNPs/CF electrodes (black line) is even smaller, demonstrating that CuNPs are excellent electrically conducting materials and able to promote the electron transfer. Upon the immobilization of FDH (blue line), an insulating protein layer was formed at the electrode surface, evident from the appearance of a significantly increased semicircular portion of the resulting spectrum. This can be attributed to the block of the redox probe Fe(CN)63−/4− from reaching the electrode by immobilized FDHs and inhibited interfacial electron transfer, indicating a successful immobilization of enzymes. Raman spectra of different modified electrodes was also presented (Fig. 2B). The peak at ∼1328 cm-1 and 1600 cm-1 showed D and G bands featuring the characteristic for carbon felt (black). The peak at ∼1126 cm-1 was caused by the CeC and CeN telescopic vibration of the protein. In addition, the peak at ∼1445 cm-1 belonged to FDH (blue). [31] The 525 cm-1 and 625 cm-1 ascribed to copper (red), illustrating the CuNPs modification. [32] When FDH modified on CuNPs/CF, another three peaks at 300 cm-1, 400 cm-1, and 448 cm-1, belonged to Cu-S(Cys) bond [33] appeared, indicating the formation of Cu-S bonds and the successful immobilization of FDH. In addition, scanning electron microscope (SEM) was further used to 571
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Fig. 3. Scanning electron microscope of CF electrode (A–C); CuNPs/CF (D–G); FDH/CuNPs/CF (H–L) and FDH/CuNPs/CF after rinse (M–Q). X-Ray Microanalysis (EDS-Mapping) showing (B, E, I, N): C element; (F, J, O): Cu element; (K, P): N element, and (C, G, L, Q): percentage of element.
Fig. 4. Formate production by various electrodes (A) and the effect of applied FDH amount (B), reaction pH (C), and NADH concentration (D) on formate production.
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further promote the CO2 electroreduction, as the NAD+ generated by enzymatic CO2 reduction can be in situ regenerated by CuNPs on the electrode surface, thus accelerating the overall reaction rate. To compare the formate production between the systems consisting of a FDHPEG(n)-NAD+ conjugate/CuNPs/CF electrode and a FDH/CuNPs/CF electrode with free NAD+, an optimized amount of FDH or the conjugates was immobilized onto the electrode as mentioned above, corresponding to an overall amount of 0.24 mM of NAD+ in the system (Fig. 5A). As expect, the FDH-PEG(2000 or 3400)-NAD+/CuNPs/CF electrode produced ∼1.7 mM formate after 6 h without additional free NAD+, far greater than that from the FDH-casted electrode with the same concentration of NAD+ supplemented in solution. A control of deactivated FDH-PEG(2000)-NAD+/CuNPs/CF electrode yielded no formate at all. Besides, when NAD+ was directly attached to the surface of FDH without a PEG swing arm (the case of using a FDH-PEG(0)NAD+/CuNPs/CF electrode), no formate was produced. This can be explained as the position of NAD+ crosslinked on the surface of FDH is far from its active center. According to its crystal structure (Fig. S6), the direct distance is ∼5 nm between the active center and Met1 of FDH or ∼3 nm between the active center and Glu262, and therefore both PEG (2000) and PEG (3400) with the length longer than 10 nm can easily swing back and forth to shuttle NAD+/NADH between the active center and the electrode. When there is no PEG swing arm between FDH and NAD+, the shuttle and regeneration of NAD+/NADH is impossible. Meantime, we also observed that the activity of FDH-NAD+ decreased ∼15%, kcat decreased ∼27%, but km increased ∼58% compared to that of unconjugated FDH (Tables S1 & S2), possibly due to the influence from enzyme modification. In spite of the slightly reduced enzyme activity, the conjugate significantly improves in formate production by increasing the local relative cofactor concentration around the FDH and CuNPs, thus allowing fast cofactor regeneration. When the optimal concentration of overall 3 mM NAD+ was applied (with additional free NAD+ in solution), the formate production from the FDH-NAD+/ CuNPs/CF electrode further increased to ∼8.5 mM after 4 h with a faraday efficiency of 22.8%, while only 5 mM of formate was produced from the FDH/CuNPs/CF electrode. As illustrated in the Fig. S5, the formation of FDH-NAD+ conjugate further reduces the cofactor traveling distance between the enzyme and the CuNPs and therefore speeds up the overall CO2 electroreduction reaction. Compared with other works (Table 1), this study under the help of the enzyme-cofactor conjugate using a PEG swing arm exhibits a high molar productivity of formate based on the enzyme unit loading as high as 11.8 μM mU−1 h−1, much higher than other FDH-mediated CO2 electroreduction systems and slightly lower than that with the addition of carbonic anhydrase (CA). The product titer at the stable state was 8.5 mM, comparable with those reported elsewhere. After 4 h, the reaction may come to a state where the CO2 reduction and formate oxidation reach an equilibrium. To further increase the titer, efforts should be made towards increasing the CO2 reduction activity of FDH and
containing 1 mM NADH and CO2 gas bubbled in 0.1 M PBS buffer (pH 6.0) (Fig. S4). Formate was analyzed by a chromogenic reaction adopted elsewhere and the standard curve was drawn (Fig. S2) [18]. At a potential of −1.0 V vs Ag/AgCl, the system consisting a FDH/CuNPs/ CF was able to generate a maximal formate amount of 3.2 mM after 4 h, representing a specific formate productivity of 1.3 × 10−3 μmol min-1 mg-1 (Fig. 4A). These rates are comparable with that of a free CbFDH/ Cu/Rh complex-mediated CO2 reduction system [24]. As the control, those with a CF electrode, a CuNPs/CF electrode, or a deactivated FDH/ CuNPs/CF electrode could barely produce any formate due to the absence of active FDHs. If without enzyme immobilization, the formate generation from a system containing the equal amount of free FDH and a CuNPs/CF electrode only reached ∼1.5 mM. This is expected as the immobilization of FDH shortens the distance of diffusional-controlled cofactor-mediated electron transfer between the enzyme and the CuNPs (Fig. S5). We then optimized the conditions to improve the formate production from CO2 electroreduction via the FDH/CuNPs/CF electrodes. Various loadings of FDHs (based on enzyme unit) were applied onto the CuNPs/CF electrodes, and then the unbound enzymes were rinsed away and detected by Bradford to find out the optimal immobilized FDH amount. Fig. 4B reveals that the immobilized FDH amount increases with the initial applied FDH amount. Meanwhile, the formate production achieves to the maximum at an immobilized FDH amount of 180 mU. Therefore, this loading was chosen for the following experiments. Besides, pH and cofactor concentrations were also well-known to play significant roles in FDH-mediated enzymatic reactions. [11] Fig. 4C demonstrates that the formate production at pH 6 is slightly higher than that at pH 5, and much better than that at pH 7, which are in accordance with the CO2 reduction activities of FDH at different pHs (Table S1). When increasing NADH concentration from 1 mM to 4 mM, the maximal formate yield is also increased (Fig. 4D). Compared with the above-mentioned condition using 1 mM of NADH, the optimal 3 mM NADH registered a ∼70% increase in the formate generation. Finally, we constructed an enzyme-cofactor conjugate-based electrode to further increase the performance of our hybrid CO2 electroreduction system. It has been widely reported that co-immobilization of cofactors and redox enzymes allows cofactors in the vicinity of enzymes and can promote many cofactor-dependent reactions [34,35]. Recently, we have constructed a dehydrogenase-NAD+ conjugate and achieved a distinct enhancement in the electricity generation rate of a biofuel cell, although without enzyme immobilization [27]. By using the similar approach, a FDH-NAD+ conjugate was constructed for this present study. Based on the known structure of TsFDH (PDB: 3WR5, Fig. S6), the surface amino acid with a free amine group was identified at the residue Met1, thus indicating that each FDH may only link with one NAD+. Experimentally, we proved that the ratio of the NAD+ moiety to the FDH was measured to be 1:1. Besides, PEG was adopted to bridge NAD+ and FDH as a flexible swing arm. The designed conjugate would
Fig. 5. Formate production by various electrodes with or without enzyme-cofactor conjugation in presence of 0.24 mM additional NAD+ (A) or 3 mM overall NAD+ (B). 573
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Table 1 Molar productivity of formate based on the enzyme unit loading in this work and other reported references. FDH form
NADH (mM)
NADH regeneration by
Product titer at stable state (mM)
Molar productivity (μM∙mU−1∙h−1)
Faraday efficiency
References
Free Immobilized and free CA Immobilized Free Free Immobilized
4 4 1 0.25 1 3
Graphite + neutral red Graphite + neutral red Graphite + poly neutral red Cu foil + Rh(III) Cu nanorod + Rh(III) CuNPs
8.9 13.5 2.8 2.5 2.9 8.5
4.7 15.3 4.2 3.3 9.6 11.8
9.84% 8.35% 77.08% – – 22.80%
[5] [13] [36] [24] [37] In this work
Development Program of China (2018YFA0901300), the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (ZDRW-ZS-2016-3S), the National Natural Science Foundation of China (21878324) and the CAS Pioneer Hundred Talent Program (Type C, reference # 2016-081).
removing or consuming the formate by other means to drive the reduction reaction. The faraday efficiency of 22.8% presented in this work was decent. Even so, it was not high enough due to by-products generated during the electrocatalysis. As others demonstrate, a high potential applied can increase the reaction rate at a certain point while it leads to a low faraday efficiency [5,13]. Hydrogen evolution is one of the most common reaction in the CO2 electroreduction. In our hybrid, a potential of -1.0 V was applied, resulting in a mitigated hydrogen evolution reaction as reported elsewhere [14]. Meantime, insufficient NADH regeneration may lead to the generation of NAD2 that would lower the overall reaction yield. Additionally, CO, methane, and ethylene may be produced during the CO2 electroreduction [38,39], especially under a high voltage (e.g., -2 V). In this work, a trace amount of methanol (lower than 10 μM, roughly estimated by a colorimetric assay, Fig. S7A) and a significant amount of ethanol (greater than 1 mM, estimated using a HPLC, Fig. S7B) were discovered because of the catalysis of CO2 by CuNPs. We suspected that most methanol was evaporated as gas although we did not detect it using a gas phase chromatography. This observation was also reported in the literature where CO2 was electrochemically reduced to alcohols including methanol and ethanol in a filter-press electrochemical cell using Cu/Bi metal-organic framework [38,40]. Although those by-products mainly came from the catalysis of copper oxide, we cannot exclude that there was no Cu(I) or Cu(II) during our preparation of CuNPs. In short, to further improve the faraday efficiency of our system, a thorough analysis of all possible byproducts and a careful preparation of the abiotic catalyst should be performed.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jcou.2019.08.007. References [1] J. Schneider, H.F. Jia, J.T. Muckerman, E. Fujita, Thermodynamics and kinetics of CO2, CO, and H+ binding to the metal centre of CO2 reduction catalysts, Chem. Soc. Rev. 41 (2012) 2036–2051. [2] N. Yang, S.R. Waldvogel, X. Jiang, Electrochemistry of carbon dioxide on carbon electrodes, ACS Appl. Mater. Interfaces 8 (2016) 28357–28371. [3] S. Bajracharya, S. Srikanth, G. Mohanakrishna, R. Zacharia, D.P. Strik, D. Pant, Biotransformation of carbon dioxide in bioelectrochemical systems: state of the art and future prospects, J. Power Sources 356 (2017) 256–273. [4] S.Y. Lee, S.Y. Lim, D. Seo, J.Y. Lee, T.D. Chung, Light-driven highly selective conversion of CO2 to formate by electrosynthesized enzyme/cofactor thin film electrode, Adv. Energy Mater. 6 (2016) 1502207. [5] S. Srikanth, M. Maesen, X. Dominguez-Benetton, K. Vanbroekhoven, D. Pant, Enzymatic electrosynthesis of formate through CO2 sequestration/reduction in a bioelectrochemical system (BES), Bioresour. Technol. 165 (2014) 350–354. [6] S. Zhang, J. Shi, Y. Sun, Y. Wu, Y. Zhang, Z. Cai, Y. Chen, C. You, P. Han, Z. Jiang, Artificial thylakoid for the coordinated photoenzymatic reduction of carbon dioxide, ACS Catal. 9 (2019) 3913–3925. [7] R.J. Lim, M.S. Xie, M.A. Sk, J.M. Lee, A. Fisher, X. Wang, K.H. Lim, A review on the electrochemical reduction of CO2 in fuel cells, metal electrodes and molecular catalysts, Catal. Today 233 (2014) 169–180. [8] R.K. Singh, R. Singh, D. Sivakumar, S. Kondaveeti, T. Kim, J.L. Li, B.H. Sung, B.K. Cho, D.R. Kim, S.C. Kim, V.C. Kalia, Y.H.P.J. Zhang, H.M. Zhao, Y.C. Kang, J.K. Lee, Insights into cell-free conversion of CO2 to chemicals by a multienzyme cascade reaction, ACS Catal. 8 (2018) 11085–11093. [9] R. Cai, R.D. Milton, S. Abdellaoui, T. Park, J. Patel, B. Alkotaini, S.D. Minteer, Electroenzymatic C-C bond formation from CO2, J. Am. Chem. Soc. 140 (2018) 5041–5044. [10] K. Sakai, Y. Kitazumi, O. Shirai, K. Takagi, K. Kano, High-power formate/dioxygen biofuel cell based on mediated Electron transfer type bioelectrocatalysis, ACS Catal. 7 (2017) 5668–5673. [11] H. Choe, J.C. Joo, D.H. Cho, M.H. Kim, S.H. Lee, K.D. Jung, Y.H. Kim, Efficient CO2reducing activity of NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA for formate production from CO2 gas, PLoS One 9 (2014) e103111. [12] J. Shi, Y. Jiang, Z. Jiang, X. Wang, X. Wang, S. Zhang, P. Han, C. Yang, Enzymatic conversion of carbon dioxide, Chem. Soc. Rev. 44 (2015) 5981–6000. [13] S. Srikanth, Y. Alvarez-Gallego, K. Vanbroekhoven, D. Pant, Enzymatic electrosynthesis of formic acid through carbon dioxide reduction in a bioelectrochemical system: effect of immobilization and carbonic anhydrase addition, Chemphyschem 18 (2017) 3174–3181. [14] L.J. Zhang, J.Y. Liu, J. Ong, S.F.Y. Li, Specific and sustainable bioelectro-reduction of carbon dioxide to formate on a novel enzymatic cathode, Chemosphere 162 (2016) 228–234. [15] X.Y. Ji, Z.G. Su, P. Wang, G.H. Ma, S.P. Zhang, Tethering of nicotinamide adenine dinucleotide inside hollow nanofibers for high-yield synthesis of methanol from carbon dioxide catalyzed by coencapsulated multienzymes, ACS Nano 9 (2015) 4600–4610. [16] M.W. Yuan, S. Sahin, R. Cai, S. Abdellaoui, D.P. Hickey, S.D. Minteer, R.D. Milton, Creating a low-potential redox polymer for efficient electroenzymatic CO2 reduction, Angew. Chem. Int. Ed. 57 (2018) 6582–6586. [17] F. Rudroff, M.D. Mihovilovic, H. Groger, R. Snajdrova, H. Iding, U.T. Bornscheuer, Opportunities and challenges for combining chemo- and biocatalysis, Nat. Catal. 1 (2018) 306-306. [18] S.K. Kuk, R.K. Singh, D.H. Nam, R. Singh, J.K. Lee, C.B. Park, Photoelectrochemical reduction of carbon dioxide to methanol through a highly efficient enzyme cascade,
4. Conclusions In summary, our strategy of combining enzymatic reduction and metal-catalyzed cofactor regeneration system have demonstrated the merits of high product selectivity and fast cofactor regeneration. This study under the help of the enzyme-cofactor conjugate using a PEG swing arm, exhibits a molar productivity of formate based on the enzyme unit loading as high as 11.8 μM mU−1 h−1 with a faraday efficiency 22.8%, among one of the best results reported. In addition, CuNPs electrodeposited on carbon felt not only serve as a good immobilization substrate, but also are highly efficient for NADH regeneration without the addition of Rh(III). Our results suggest that Cubased nanomaterials may become a promising catalyst and material in many cofactor regeneration-involved bioelectrocatalytic processes. We anticipate that the strategy of constructing this hybrid CO2 electroreduction system will be of interest to others in the field of bioelectrocatalysis. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was supported by the National Key Research and 574
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from NADP(+) to NAD(+), Sci. Rep. 6 (2016) 32644. [31] R.D. Ming-Qian, L.U. Shun-Hua W, Detection of recombinant protein expression of formate dehydrogenase in single living Escherichia coli cell by laser tweezers Raman spectroscopy, Chin. J. Anal. Chem. 40 (2012) 1845–1851. [32] H.Y.H. Chan, C.G. Takoudis, M.J. Weaver, Oxide film formation and oxygen adsorption on copper in aqueous media As probed by surface-enhanced raman spectroscopy, J. Phys. Chem. B 103 (1999) 357–365. [33] H.Y.Colin R. Andrew, Joan Selverstone Valentine, B. Goran Karlsson, Nicklas Bonander, Gertievan Pouderoyen, Gerard W. Canters, Thomas M. Loehr, Joann Sanders-Loehr, Raman spectroscopy as an Indicator of Cu-S bond length in type 1 and type 2 copper cysteinate proteins, J. Am. Chem. Soc. (1994) 11489–11498. [34] S. Velasco-Lozano, A.I. Benitez-Mateos, F. Lopez-Gallego, Co-immobilized phosphorylated cofactors and enzymes as self-sufficient heterogeneous biocatalysts for chemical processes, Angew. Chem. Int. Ed. 56 (2017) 771–775. [35] J.L. Fu, Y.R. Yang, A. Johnson-Buck, M.H. Liu, Y. Liu, N.G. Walter, N.W. Woodbury, H. Yan, Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm, Nat. Nanotechnol. 9 (2014) 531–536. [36] L. Zhang, J. Liu, J. Ong, S.F. Li, Specific and sustainable bioelectro-reduction of carbon dioxide to formate on a novel enzymatic cathode, Chemosphere 162 (2016) 228–234. [37] S.-H. Kim, G.-Y. Chung, S.-H. Kim, G. Vinothkumar, S.-H. Yoon, K.-D. Jung, Electrochemical NADH regeneration and electroenzymatic CO 2 reduction on Cu nanorods/glassy carbon electrode prepared by cyclic deposition, Electrochim. Acta 210 (2016) 837–845. [38] J. Albo, M. Perfecto-Irigaray, G. Beobide, A. Irabien, Cu/Bi metal-organic framework-based systems for an enhanced electrochemical transformation of CO2 to alcohols, J. Co2 Util. 33 (2019) 157–165. [39] V. Merino-Garcia, J. Albo, A. Irabien, Productivity and selectivity of Gas-PhaseCO2 electroreduction to methane at copper nanoparticle based electrodes, Energy Technol-Ger 5 (2017) 922–928. [40] J. Albo, G. Beobide, P. Castaño, A. Irabien, Methanol electrosynthesis from CO 2 at Cu 2 O/ZnO prompted by pyridine-based aqueous solutions, J. Co2 Util. 18 (2017) 164–172.
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Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou
A hybrid CO2 electroreduction system mediated by enzyme-cofactor conjugates coupled with Cu nanoparticle-catalyzed cofactor regeneration Haiyan Songa, Chunling Maa, Pi Liua, Chun Youa,b, Jianping Lina,c, Zhiguang Zhua,b,
T
⁎
a
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West Seventh Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China University of Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing, 100049, China c College of Pharmacy, Nankai University, Nankai District, Tianjin, 300071, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: CO2 electroreduction Hybrid system Cu nanoparticles Enzyme-cofactor conjugates Cofactor regeneration
With the widespread concerns on CO2 capture and utilization, various approaches have been developed to convert CO2 into valuable products. To achieve fast and efficient conversion, a hybrid CO2 electroreduction system was constructed, in which a cofactor-dependent formate dehydrogenase (FDH) was responsible for CO2 enzymatic reduction to formate and Cu nanoparticles (CuNPs) was used for enzyme immobilization and cofactor regeneration. A polyethylene glycol swing arm was further adopted to construct an enzyme-cofactor conjugate and promote the overall reaction. This electron-mediator-free hybrid system containing immobilized FDH-cofactor conjugates and CuNPs achieved a formate production of 8.5 mM and a productivity as high as 11.8 μM mU−1 h−1 with a faraday efficiency of 22.8%, several folds better than systems with unconjugated FDHs alone or with unconjugated FDHs and CuNPs. These results suggest that such hybrid systems may represent a potentially viable alternative for many cofactor-dependent bioelectrochemical applications.
1. Introduction The growing greenhouse effect has aroused widespread concern on carbon dioxide (CO2) reduction and conversion. Various methods have emerged chemically, biologically, electrochemically and photoelectrochemically [1–6]. Among them, the electrochemical reduction of CO2 has gained significant attention because of its modest reaction condition, in connection with renewable energy sources, as well as high reaction rate. However, metal-catalyzed electrochemical methods usually suffer from low reaction selectivity accompanied by mixed byproducts and relying on unsustainable and expensive catalysts [7]. Alternatively, the enzymatic bioelectrocatalysis possessing superior specificity and renewability of the catalyst has been proposed as a promising route recently [8,9]. Formate dehydrogenase (FDH) is a widely-used enzyme reducing CO2 directly to formate, which has been regarded as an important energy carrier and industrial bulk chemical [10]. Compared to metalcontaining FDHs, nicotinamide adenine dinucleotide (NAD+)-dependent FDHs display strong oxygen tolerance and decent catalytic ability and therefore are more frequently used in the bioelectrochemical reduction of CO2 [11,12]. For example, Srikanth et al. used non-immobilized FDHs from Candida boidinii for formate production and
neutral red (NR) for mediated NADH regeneration in a bioelectrochemical system [5]. Later, they introduced carbonic anhydrases to the reaction and immobilized all their enzymes onto electropolymerized NR/Nafion/graphite cathode, observing improved formate production rate and increased stability [13]. Similarly, Zhang et al. optimized the condition of immobilizing CbFDHs in Nafion micelles and obtained a fast formate production rate and a high Faradic efficiency [14]. Additionally, four dehydrogenases and the NAD+ were in situ coencapsulated inside hollow nanofibers to exhibit high conversion activity of CO2 to methanol [15]. More recently, a low-potential redox polymer was prepared to reduce CO2 to formate, yielding an extremely high Faradic efficiency of 99% at a mild applied potential of -0.66 V vs. standard hydrogen electrode [16]. Such studies all relied on electron mediators, either diffusional or immobilized, for electrochemical cofactor regeneration, and the cofactors used were in the free form or simply tethered to the electrode without rational design. Hybrid systems combining chemo- and bio-catalysis are receiving increasing attention, driven by the advantages of high yield, decreased cost and high selectivity [17]. Inspired by nature, a photoelectrochemical (PEC) cell consisting of a photoanode for water oxidation and a biocathode co-immobilized by CbFDH and NADH via polydopamine entrapment was developed and produced formate at a high
⁎ Corresponding author at: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West Seventh Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China. E-mail address: [email protected] (Z. Zhu).
https://doi.org/10.1016/j.jcou.2019.08.007 Received 9 July 2019; Received in revised form 5 August 2019; Accepted 9 August 2019 2212-9820/ © 2019 Elsevier Ltd. All rights reserved.
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USA). Copper sulfate (CuSO4), citric acid (C6H8O7), sodium acetate (CH3COONa), phenazine methosulfate (PMS), and tetranitroblue tetrazolium chloride (TNBT) were purchased from Sinopharm (Beijing, China). 3-Carboxyphenylboronic Acid (CBA) and 4-(bromomethyl) phenylacetic acid (BPA) were purchased from Innochem (Beijing, China). Amine-polyethylene glycol-carboxylic acid (NH2-PEG-COOH, 3400 kDa) was purchased from Ponsure Biotech (Shanghai, China). NHydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were from J&K Scientific (Beijing, China). Carbon felt (CF) (AvCarb MGL200) was purchased from Fuel Cell Earth (Stoneham, MA, USA). The phosphate buffer solution (PBS, 0.1 M) was the frequently-used buffers in the experiment. All solutions were prepared with the deionized water which was purified with a Millipore-Q purification system (specific resistance > 18.0 MΩ cm).
faradaic efficiency [4]. A biomimicking artificial thylakoid was constructed containing protamine-titania microcapsules and cadmium sulfide quantum dots for the photobiocoupled reduction of CO2 via a single enzyme and multiple enzymes [6]. Similarly, a tandem PEC cell with an integrated enzyme cascade system was constructed for lightassisted cofactor regeneration and enzymatic conversion of CO2 to methanol [18]. In fact, NADH regeneration can be achieved in an electrochemical mode, such as by a cholesterol-modified gold amalgam electrode, a ruthenium-modified glassy carbon (GC) electrode, and a histidine modified silver electrode [19–22]. Recently, Cu nanorods were prepared as the cathode for electrochemical NADH regeneration and CO2 reduction via non-immobilized CbFDH with high yields, and the authors claimed that adding the Rh(III) mediator led to the improved yield of enzymatically active 1,4-NADH [23,24]. It was also reported that Cu foam could be a better electrode material for direct NADH regeneration than Cu foil, and the study showed an optimal 1,4NADH regeneration yield of 80% using Cu foam electrodes [25]. These results suggest that Cu-based nanomaterials may hold the great promise in efficient electrochemical NADH regeneration and can be integrated with biocatalytic systems to increase the enzymatic CO2 reduction rate. Herein, we rationally designed a hybrid CO2 electroreduction system mediated by FDH from Thiobacillus sp. KNK65MA for enzymatic formate production coupled with Cu nanoparticles (CuNPs)-catalyzed NADH regeneration (Scheme 1). Active NADH can be regenerated with a high yield without the need of electron mediators (e.g., Rh(III)). Besides, CuNPs deposited onto carbon felt (CF) electrodes can also serve as a matrix for FDH immobilization through a Cu-S bond formed between the copper and the cysteine residue at the enzyme surface [26]. TsFDH was chosen because of its high CO2 reduction activity reported [11], and its surface Glu262 was mutated to Cys to allow the enzyme immobilization. Moreover, a FDH-NAD+ conjugate crosslinked by a polyethylene glycol (PEG) swing arm was constructed using a previous developed method, in order to immobilize the cofactor in the vicinity of the enzyme [27]. It is expected that the cofactor can swing back and forth between the catalytic site of FDH and the CuNPs with facilitated electron shuttling and increased CO2 reduction rate. Such hybrid systems for CO2 electroreduction to formate may represent a potentially viable alternative for many other bioelectrochemical applications.
2.2. Preparation of CuNPs-electrodeposited electrodes for NADH regeneration A CHI 1000C potentialstat (Shanghai Chenhua Instrument Co., Ltd., Shanghai, China) was used for electrochemical operation. In this study, CuNPs electrodeposited on CF was used for NADH regeneration. Before electrodeposited, the CF surface (1 × 1 cm2) was pretreated using cyclic voltammetry (CV) in 500 mM H2SO4 for 20 cycles from 1.5 V to -1.5 V at a scanning speed of 100 mV s−1. CuNPs were then electrodeposited using CV in various amounts of CuSO4 and 500 mM H2SO4 from -0.2 V to -1.0 V vs Ag/AgCl at 50 mV s−1 for various cycles, resulting in the prepared electrode remarked as CuNPs/CF. The NADH regeneration was carried out at the chosen potential, in a 5 mL threeelectrode system (0.1 M PBS, pH 6.0) with a CuNPs/CF as the working electrode, while a single platinum wire as the counter electrode and a Ag/AgCl as the reference electrode. N2 was continuously injected to remove O2 for 30 min and the reaction was initiated by adding 1 mM NAD+. During the regeneration reaction, 0.1 mL sample was taken from the solution at 30-min intervals and analyzed using a Cary 5000 Agilent spectrophotometer (Santa Clara, CA, USA) at 340 nm for the amount of NADH. The more specific amount of active NADH was measured using 0.1 M PMS as a catalyst and 0.1 M TNBT as a chromogenic reagent. When NAD+ was catalyzed into active NADH, TNBT reduced to a dark blue color (Fig. S1A) that can be quantified using Cary 5000 Agilent spectrophotometer at 654 nm (Fig. S1B). And the standard calibration of active NADH was measured to this method (00.2 mM) (Fig. S1C).
2. Experimental section 2.1. Materials
2.3. Preparation of FDH Nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide reduced (NADH), sodium formate, sodium bicarbonate, and acetamide were purchased from Sigma-Aldrich (St. Louis, MO,
Plasmids pET20b-ts-fdh was constructed using Simple Cloning [28]. The FDH gene was PCR-amplified from the genomic DNA of T. KNK65MA using a pair of primers (forward: 5′-TAACT TTAAG AAGGA GATAT ACATA TGGCG AAAAT ACTTT GCGTT CTCT-3′; reverse: 5′GTGCT CGAGT GCGGC CGCAA GCTTG CCGGC CTTCT TGAAC TTCGC G-3′). The vector backbone of pET20b was also PCR-amplified using a pair of primers (forward: 5′- CGTGA TGGAG TAACT TGGTA TGACC TCGAG CACCA CCACC ACCAC CACT-3′; reverse: 5′-ATCGT CGAAA CGGTA TTTGT CATAT GTATA TCTCC TTCTT AAAGT TAAA-3′). The PCR products were purified using the Zymo Research DNA Clean & Concentrator Kit (Irvine, CA). The DNA inserter and vector backbone were assembled using prolonged overlap extension PCR, and subsequently, the amplified DNA multimer was directly transformed into E. coli TOP10 cells, yielding the desired plasmid. The Glu262Cys mutation was performed using a site-directed mutagenesis kit from New England Biolabs (Ipswich, MA, USA). The recombinant FDH was produced in the E. coli BL21 (DE3) strain harboring the plasmid and grown in LuriaBertani medium with 100 μM ampicillin at 37 °C, using a T&J-Atype 5L × 2 Parallel Bioreactor (T&J Bio-engineering Co.,LTD, Shanghai, China). The his-tagged FDH protein was purified by HisPur Ni-NTA Resin columns from Thermo Fisher Scientific (Waltham, MA, USA). The
Scheme 1. Schematic illustration of the hybrid CO2 electroreduction system. 569
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Methanol was estimated using a colorimetric assay. First, methanol was catalyzed to formaldehyde by alcohol oxidase (1 U mL−1) from Pichia Poastoris with 10 μM flavin adenine dinucleotide as cofactor. Then the sample was poured into a centrifuge tube to 500 μL with water. And 50 μL solution (50 g ammonium acetate, 6 mL glacial acetic acid and 0.5 mL acetylacetone were dissolved in 100 mL H2O) was added into the centrifuge tube and heated in water at 60 ℃ for 15 min. Finally, the sample was detected at a spectrum of 414 nm. Ethanol was estimated through HPLC with a Bio-rad 87H chromatographic column at 60 ℃. The mobile phase was 5 mM H2SO4 and the flow velocity was 0.6 mL min−1. The faraday efficiency (FE) for formate production was calculated as follows:
activity assay of FDH was measured at various pH values of 5.0, 6.0 and 7.0 in 0.1 M PBS, 50 mM NaHCO3, 0.2 mM NADH at 25 °C. One unit of reduction activity was defined as the amount of enzyme required to consume 1 μmol NADH per minute under standard conditions. 2.4. Preparation of FDH-NAD+ conjugates To construct FDH-NAD+ conjugates, the NAD+-CBA-PEG-COOH was prepared first, using a method developed by our group previously [29]. Then, 100 mmol EDC and 100 mmol NHS were added to activate the carboxyl group of the resultant NAD+-CBA-PEG-COOH (10 mmol), and 100 μmol FDH was added to the above mixture for 2 h to finally form FDH-NAD+ conjugates. The amount of NAD+ in the conjugate was determined by measuring the absorbance change at 340 nm where NAD+ acted as a cofactor was turned into NADH by the catalysis of FDH with 50 mM formate as the substrate. On the other side, the concentration of FDH was determined by the Bradford assay using bovine serum albumin (BSA) as a standard. Therefore, the relative molecular ratio of NAD+ moiety and enzyme (FDH) in FDH-NAD+ conjugates can be calculated. The surface Met1 of FDH was identified according to its crystal structure (PDB: 3WR5), which maybe applied in the conjugate formation.
FE=
2nformate t ∫0 I dt
Where nformate is the mole of formate generated, 2 is the number of electrons transferred from one molecule CO2 to formate. I is the reduction current intensity (A) and t is the reaction time (s) obtained from (Fig. S3). 3. Results and discussion
2.5. Construction and characterization of enzyme-immobilized bioelectrodes
First of all, an electrochemical NADH regeneration system catalyzed by CuNPs was developed and optimized. A three-dimensional porous CF electrode with a large surface area of ∼100 m2 g−1 was used as the substrate electrode in order to maximize the catalyst loading. Cyclic voltammetry (CV) from −0.2 V to −1.0 V vs Ag/AgCl at 50 mV s−1 was applied in 1 mM CuSO4 to electrodeposit CuNPs onto CF electrodes and the potential applied for NADH regeneration was set as −1.2 V. Fig. 1A shows that 30 or 40 CV cycles in the process of CuNPs electrodeposition result in a NADH regeneration yield of ∼40% from CuNPs/CF electrodes constructed, higher than that with 20 cycles. Therefore, the condition of 30 cycles was used for the preparation of CuNPs/CF electrodes in the following study. The concentration of CuSO4 was optimized next. As revealed from Fig. 1B, the NADH regeneration yield achieves a maximum of 92.1% at 2 mM CuSO4, larger than 41.2% obtained at 1 mM CuSO4. Yet, the yield reduces to 65.2% with the concentration of CuSO4 increasing to 3 mM, which may be caused by the diminished Cu active sites from overfull CuNPs deposited on the CF electrode. Moreover, the potential applied for electrochemical NADH regeneration was also evaluated (Fig. 1C). The yield achieves to more than 90% when using a potential of −1.0 V or −1.2 V. Instead, it drops to only 45% at a reduced potential of −0.8 V. Notably, nonselective electrochemical reduction of NAD+ may occur depending on the reduction potential and the catalyst, and the formation of enzymatically inactive NADH dimer can decrease the regeneration yield of enzymatically active 1,4-NADH [19]. As a result, we further measured the amount of 1, 4-NADH using a method involving phenazine methosulfate (PMS) catalyzing 1, 4-NADH to NAD+ and tetranitroblue tetrazolium chloride (TNBT) reduced to a dark blue color that can be quantified at 654 nm (Fig. S1) [30]. The yield of 1, 4-NADH achieves ∼82.3% at the optimal condition of 30 CV circles, 2 mM CuSO4, and -1.0 V applied potential (Fig. 1D). In comparison, a Cu nanorods-electrodeposited GC electrode exhibited an active NADH regeneration yield of 67% and a Cu foam electrode could achieve an optimal yield of 80% [23,25]. These results suggest that CuNPs/CF electrodes were successfully constructed with the highest efficiency for the regeneration of enzymatically active 1, 4-NADH without the necessity of adding electron mediators. Next, FDHs were adsorbed onto the CuNPs/CF electrodes by forming Cu-S bonds between a CuNP and a thiol group at the FDH surface, which was introduced by mutating Glu264 to Cys. The fabricated bioelectrodes were characterized by electrochemical impedance spectroscopy (EIS) which can probe the features of the surface-modified
Different amounts of FDH-NAD+ conjugates or FDH were dropped onto CuNPs/CF and incubated at 25 °C overnight. Cu-S bonds were formed between the enzyme and the CuNPs and the unbound enzymes were cleaned by 0.1 M PBS (pH 6.0). The amount of enzymes adsorbed on the electrode was quantified by the difference between the amount of the enzyme to be dispensed and the amount of enzyme shedding, which were all detected by Bradford. The bioelectrode was designated as FDH-NAD+/CuNPs/CF or FDH/CuNPs/CF. In addition, the CuNPs/ CF and CF electrodes were prepared for the control experiments. The electrochemical impedance spectroscopy was performed at open circuit potential over an AC frequency range of 100 kHz to 1 Hz, with a sinusoidal perturbation of 5 mV, in order to obtain the resistance on bioelectrodes constructed. Scanning electron microscopy (SEM) was carried out by using a JSM-6701 field emission SEM instrument (JEOL, Akishima, Japan) at an accelerating voltage of 10 kV with a Phoenix energy-dispersive X-ray analyzer (NS7, Thermo, USA). Raman spectra was carried out by using a LabRAM HR Evolution spectra instrument (HORIBA Jobin Yvon, France). 2.6. Formate production by the hybrid CO2 electroreduction system The formate production by bioelectrochemical CO2 reduction was performed in a bioelectrochemical system. A 5 mL single-chamber reactor consisting of an enzyme-immobilized electrode as a working electrode, a single platinum wire as the counter electrode, and a Ag/ AgCl as the reference electrode was used. The electrolyte contained several mM NADH and CO2 gas in 0.1 M PBS. The CO2 was reduced by FDH to formate, meanwhile the NADH molecule was converted into NAD+. Then, NAD+ acquired electrons from electrode, in turn, to regenerate NADH under catalysis of CuNPs. Formate was analyzed according to Kuk’s method with a little modification [18]. Briefly, 100 μL of sample containing formate was mixed with 5 mM NaHCO3, 0.2 mL of solution A, 10 μL of solution B, 0.7 mL of 100% acetic anhydride, and incubated at 50 °C for 0.5 h with occasional rapid mixing. A red color could thereby be developed and quantified at 515 nm. Solution A was prepared by dissolving 0.5 g of citric acid and 10 g of acetamide in 100 mL of isopropanol; solution B was prepared by dissolving 30 g of sodium acetate in 100 mL of water. Sodium formate dissolved in 0.1 M PBS (pH 6.0) was used for standard calibration (0–10 mM) (Fig. S2A). 570
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Fig. 1. The effect of CV cycles (A) and Cu ion concentration (B) in CuNPs/CF electrode preparation on the yield of NADH regeneration. The effect of applied reduction potential on the yield of NADH regeneration using the CuNPs/ CF electrode (C) and the yield of 1, 4-NADH at the optimal condition (30 CV cycles, 2 mM CuSO4, and -1.0 V applied potential) with time (D).
Fig. 2. Electrochemical impedance (A) of CF electrode (red line); CuNPs/CF (black line); FDH/CuNPs/CF (blue line); Raman spectra (B) of CF (black), FDH (blue), CuNPs/CF (red) and FDH/CuNPs/CF (green).
characterize the morphology of the bioelectrode surface. A three-dimensional wire with smooth surface was observed for bare CF electrodes (Fig. 3A). Meantime, X-Ray Microanalysis (EDS-Mapping) of CF showed that only carbon element (red) existed in the bare CF (Fig. 3B and C). After electrodeposited with CuNPs, their surface became rough and contained numerous spherical and uniform particles with an approximate diameter of 20–30 nm (Fig. 3D). The obvious copper element (blue) indicated the effective CuNPs modification (Fig. 3E–G). Upon the immobilization of FDH, the electrode presented a disordered porous, filamentous and reticulated structure (Fig. 3H) and appeared about 9% nitrogen element (yellow) representing FDH (Fig. 3I–L), illustrating the successful construction of FDH/CuNPs/CF electrodes. After rinse (Fig. 3M), the nitrogen element reduced to 7%, and reflected that most FDH was immobilized on the electrode by Cu-S bond (Fig. 3N–Q). It should be noted that the size of CuNPs obtained in this study was smaller than that reported elsewhere (i.e., 94–282 nm), possibly because of different carbon electrodes employed. [23] Using CF rather than GC as the substrate electrode can allow better distribution of nanoparticles due to the large surface area of the former and might lead to enhanced catalytic performance. For the proof-of-concept of CO2 electroduction and formate generation, a 5 mL 3-electrode bioelectrochemical reactor was constructed
electrodes. Fig. 2A shows a Nyquist plot of different electrodes in 5 mM K3Fe(CN)6/K4Fe(CN)6 redox probe in 0.1 M KCl. The bare CF (red line) displays a small resistance, while the resistance of CuNPs/CF electrodes (black line) is even smaller, demonstrating that CuNPs are excellent electrically conducting materials and able to promote the electron transfer. Upon the immobilization of FDH (blue line), an insulating protein layer was formed at the electrode surface, evident from the appearance of a significantly increased semicircular portion of the resulting spectrum. This can be attributed to the block of the redox probe Fe(CN)63−/4− from reaching the electrode by immobilized FDHs and inhibited interfacial electron transfer, indicating a successful immobilization of enzymes. Raman spectra of different modified electrodes was also presented (Fig. 2B). The peak at ∼1328 cm-1 and 1600 cm-1 showed D and G bands featuring the characteristic for carbon felt (black). The peak at ∼1126 cm-1 was caused by the CeC and CeN telescopic vibration of the protein. In addition, the peak at ∼1445 cm-1 belonged to FDH (blue). [31] The 525 cm-1 and 625 cm-1 ascribed to copper (red), illustrating the CuNPs modification. [32] When FDH modified on CuNPs/CF, another three peaks at 300 cm-1, 400 cm-1, and 448 cm-1, belonged to Cu-S(Cys) bond [33] appeared, indicating the formation of Cu-S bonds and the successful immobilization of FDH. In addition, scanning electron microscope (SEM) was further used to 571
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Fig. 3. Scanning electron microscope of CF electrode (A–C); CuNPs/CF (D–G); FDH/CuNPs/CF (H–L) and FDH/CuNPs/CF after rinse (M–Q). X-Ray Microanalysis (EDS-Mapping) showing (B, E, I, N): C element; (F, J, O): Cu element; (K, P): N element, and (C, G, L, Q): percentage of element.
Fig. 4. Formate production by various electrodes (A) and the effect of applied FDH amount (B), reaction pH (C), and NADH concentration (D) on formate production.
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further promote the CO2 electroreduction, as the NAD+ generated by enzymatic CO2 reduction can be in situ regenerated by CuNPs on the electrode surface, thus accelerating the overall reaction rate. To compare the formate production between the systems consisting of a FDHPEG(n)-NAD+ conjugate/CuNPs/CF electrode and a FDH/CuNPs/CF electrode with free NAD+, an optimized amount of FDH or the conjugates was immobilized onto the electrode as mentioned above, corresponding to an overall amount of 0.24 mM of NAD+ in the system (Fig. 5A). As expect, the FDH-PEG(2000 or 3400)-NAD+/CuNPs/CF electrode produced ∼1.7 mM formate after 6 h without additional free NAD+, far greater than that from the FDH-casted electrode with the same concentration of NAD+ supplemented in solution. A control of deactivated FDH-PEG(2000)-NAD+/CuNPs/CF electrode yielded no formate at all. Besides, when NAD+ was directly attached to the surface of FDH without a PEG swing arm (the case of using a FDH-PEG(0)NAD+/CuNPs/CF electrode), no formate was produced. This can be explained as the position of NAD+ crosslinked on the surface of FDH is far from its active center. According to its crystal structure (Fig. S6), the direct distance is ∼5 nm between the active center and Met1 of FDH or ∼3 nm between the active center and Glu262, and therefore both PEG (2000) and PEG (3400) with the length longer than 10 nm can easily swing back and forth to shuttle NAD+/NADH between the active center and the electrode. When there is no PEG swing arm between FDH and NAD+, the shuttle and regeneration of NAD+/NADH is impossible. Meantime, we also observed that the activity of FDH-NAD+ decreased ∼15%, kcat decreased ∼27%, but km increased ∼58% compared to that of unconjugated FDH (Tables S1 & S2), possibly due to the influence from enzyme modification. In spite of the slightly reduced enzyme activity, the conjugate significantly improves in formate production by increasing the local relative cofactor concentration around the FDH and CuNPs, thus allowing fast cofactor regeneration. When the optimal concentration of overall 3 mM NAD+ was applied (with additional free NAD+ in solution), the formate production from the FDH-NAD+/ CuNPs/CF electrode further increased to ∼8.5 mM after 4 h with a faraday efficiency of 22.8%, while only 5 mM of formate was produced from the FDH/CuNPs/CF electrode. As illustrated in the Fig. S5, the formation of FDH-NAD+ conjugate further reduces the cofactor traveling distance between the enzyme and the CuNPs and therefore speeds up the overall CO2 electroreduction reaction. Compared with other works (Table 1), this study under the help of the enzyme-cofactor conjugate using a PEG swing arm exhibits a high molar productivity of formate based on the enzyme unit loading as high as 11.8 μM mU−1 h−1, much higher than other FDH-mediated CO2 electroreduction systems and slightly lower than that with the addition of carbonic anhydrase (CA). The product titer at the stable state was 8.5 mM, comparable with those reported elsewhere. After 4 h, the reaction may come to a state where the CO2 reduction and formate oxidation reach an equilibrium. To further increase the titer, efforts should be made towards increasing the CO2 reduction activity of FDH and
containing 1 mM NADH and CO2 gas bubbled in 0.1 M PBS buffer (pH 6.0) (Fig. S4). Formate was analyzed by a chromogenic reaction adopted elsewhere and the standard curve was drawn (Fig. S2) [18]. At a potential of −1.0 V vs Ag/AgCl, the system consisting a FDH/CuNPs/ CF was able to generate a maximal formate amount of 3.2 mM after 4 h, representing a specific formate productivity of 1.3 × 10−3 μmol min-1 mg-1 (Fig. 4A). These rates are comparable with that of a free CbFDH/ Cu/Rh complex-mediated CO2 reduction system [24]. As the control, those with a CF electrode, a CuNPs/CF electrode, or a deactivated FDH/ CuNPs/CF electrode could barely produce any formate due to the absence of active FDHs. If without enzyme immobilization, the formate generation from a system containing the equal amount of free FDH and a CuNPs/CF electrode only reached ∼1.5 mM. This is expected as the immobilization of FDH shortens the distance of diffusional-controlled cofactor-mediated electron transfer between the enzyme and the CuNPs (Fig. S5). We then optimized the conditions to improve the formate production from CO2 electroreduction via the FDH/CuNPs/CF electrodes. Various loadings of FDHs (based on enzyme unit) were applied onto the CuNPs/CF electrodes, and then the unbound enzymes were rinsed away and detected by Bradford to find out the optimal immobilized FDH amount. Fig. 4B reveals that the immobilized FDH amount increases with the initial applied FDH amount. Meanwhile, the formate production achieves to the maximum at an immobilized FDH amount of 180 mU. Therefore, this loading was chosen for the following experiments. Besides, pH and cofactor concentrations were also well-known to play significant roles in FDH-mediated enzymatic reactions. [11] Fig. 4C demonstrates that the formate production at pH 6 is slightly higher than that at pH 5, and much better than that at pH 7, which are in accordance with the CO2 reduction activities of FDH at different pHs (Table S1). When increasing NADH concentration from 1 mM to 4 mM, the maximal formate yield is also increased (Fig. 4D). Compared with the above-mentioned condition using 1 mM of NADH, the optimal 3 mM NADH registered a ∼70% increase in the formate generation. Finally, we constructed an enzyme-cofactor conjugate-based electrode to further increase the performance of our hybrid CO2 electroreduction system. It has been widely reported that co-immobilization of cofactors and redox enzymes allows cofactors in the vicinity of enzymes and can promote many cofactor-dependent reactions [34,35]. Recently, we have constructed a dehydrogenase-NAD+ conjugate and achieved a distinct enhancement in the electricity generation rate of a biofuel cell, although without enzyme immobilization [27]. By using the similar approach, a FDH-NAD+ conjugate was constructed for this present study. Based on the known structure of TsFDH (PDB: 3WR5, Fig. S6), the surface amino acid with a free amine group was identified at the residue Met1, thus indicating that each FDH may only link with one NAD+. Experimentally, we proved that the ratio of the NAD+ moiety to the FDH was measured to be 1:1. Besides, PEG was adopted to bridge NAD+ and FDH as a flexible swing arm. The designed conjugate would
Fig. 5. Formate production by various electrodes with or without enzyme-cofactor conjugation in presence of 0.24 mM additional NAD+ (A) or 3 mM overall NAD+ (B). 573
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Table 1 Molar productivity of formate based on the enzyme unit loading in this work and other reported references. FDH form
NADH (mM)
NADH regeneration by
Product titer at stable state (mM)
Molar productivity (μM∙mU−1∙h−1)
Faraday efficiency
References
Free Immobilized and free CA Immobilized Free Free Immobilized
4 4 1 0.25 1 3
Graphite + neutral red Graphite + neutral red Graphite + poly neutral red Cu foil + Rh(III) Cu nanorod + Rh(III) CuNPs
8.9 13.5 2.8 2.5 2.9 8.5
4.7 15.3 4.2 3.3 9.6 11.8
9.84% 8.35% 77.08% – – 22.80%
[5] [13] [36] [24] [37] In this work
Development Program of China (2018YFA0901300), the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (ZDRW-ZS-2016-3S), the National Natural Science Foundation of China (21878324) and the CAS Pioneer Hundred Talent Program (Type C, reference # 2016-081).
removing or consuming the formate by other means to drive the reduction reaction. The faraday efficiency of 22.8% presented in this work was decent. Even so, it was not high enough due to by-products generated during the electrocatalysis. As others demonstrate, a high potential applied can increase the reaction rate at a certain point while it leads to a low faraday efficiency [5,13]. Hydrogen evolution is one of the most common reaction in the CO2 electroreduction. In our hybrid, a potential of -1.0 V was applied, resulting in a mitigated hydrogen evolution reaction as reported elsewhere [14]. Meantime, insufficient NADH regeneration may lead to the generation of NAD2 that would lower the overall reaction yield. Additionally, CO, methane, and ethylene may be produced during the CO2 electroreduction [38,39], especially under a high voltage (e.g., -2 V). In this work, a trace amount of methanol (lower than 10 μM, roughly estimated by a colorimetric assay, Fig. S7A) and a significant amount of ethanol (greater than 1 mM, estimated using a HPLC, Fig. S7B) were discovered because of the catalysis of CO2 by CuNPs. We suspected that most methanol was evaporated as gas although we did not detect it using a gas phase chromatography. This observation was also reported in the literature where CO2 was electrochemically reduced to alcohols including methanol and ethanol in a filter-press electrochemical cell using Cu/Bi metal-organic framework [38,40]. Although those by-products mainly came from the catalysis of copper oxide, we cannot exclude that there was no Cu(I) or Cu(II) during our preparation of CuNPs. In short, to further improve the faraday efficiency of our system, a thorough analysis of all possible byproducts and a careful preparation of the abiotic catalyst should be performed.
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4. Conclusions In summary, our strategy of combining enzymatic reduction and metal-catalyzed cofactor regeneration system have demonstrated the merits of high product selectivity and fast cofactor regeneration. This study under the help of the enzyme-cofactor conjugate using a PEG swing arm, exhibits a molar productivity of formate based on the enzyme unit loading as high as 11.8 μM mU−1 h−1 with a faraday efficiency 22.8%, among one of the best results reported. In addition, CuNPs electrodeposited on carbon felt not only serve as a good immobilization substrate, but also are highly efficient for NADH regeneration without the addition of Rh(III). Our results suggest that Cubased nanomaterials may become a promising catalyst and material in many cofactor regeneration-involved bioelectrocatalytic processes. We anticipate that the strategy of constructing this hybrid CO2 electroreduction system will be of interest to others in the field of bioelectrocatalysis. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was supported by the National Key Research and 574
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