Communication—CO2 reduction to formate: An electro-enzymatic approach using a formate dehydrogenase from rhodobacter capsulatus

Article abstract of DOI:10.1149/2.0531809jes

CO₂ utilization for producing value-added chemicals has recently emerged as a strategy to mitigate atmospheric CO₂ levels. Given that (i) certain formate dehydrogenases are capable of interconverting CO₂ and formate, and (ii) formate is versatile in various industries, we, herein, aimed to demonstrate FDH-driven formate production from CO₂. Because of its O₂ stability, we selected FDH from Rhodobacter capsulatus (RcFDH) and then constructed a mediated electro-enzymatic system. The mediated electro-enzymatic kinetic parameters (k_red and k_ox) were calculated to optimize the reaction conditions favorable for CO₂ reduction. Finally, a RcFDH-driven electro-enzymatic system successfully produced 6 mM of formate in 5 hours.

Full text of DOI:10.1149/2.0531809jes

H446
Journal of The Electrochemical Society, 165 (9) H446-H448 (2018)
0013-4651/2018/165(9)/H446/3/$37.00 © The Electrochemical Society
Communication—CO2 Reduction to Formate: An
Electro-Enzymatic Approach Using a Formate Dehydrogenase
from Rhodobacter capsulatus
1,z
Eun-Gyu Choi,1 Young Joo Yeon,2 Kyoungseon Min,3 and Yong Hwan Kim
1School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 44919, Korea
2Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung 25457, Korea
3Gwangju Bioenergy R&D Center, Korea Institute of Energy Research, Gwangju 61003, Korea
CO2 utilization for producing value-added chemicals has recently emerged as a strategy to mitigate atmospheric CO2 levels. Given
that (i) certain formate dehydrogenases are capable of interconverting CO2 and formate, and (ii) formate is versatile in various
industries, we, herein, aimed to demonstrate FDH-driven formate production from CO2. Because of its O2 stability, we selected
FDH from Rhodobacter capsulatus (RcFDH) and then constructed a mediated electro-enzymatic system. The mediated electro-
enzymatic kinetic parameters (kred and kox) were calculated to optimize the reaction conditions favorable for CO2 reduction. Finally,
a RcFDH-driven electro-enzymatic system successfully produced 6 mM of formate in 5 hours.
© 2018 The Electrochemical Society. [DOI: 10.1149/2.0531809jes]
Manuscript submitted January 31, 2018; revised manuscript received May 23, 2018. Published June 2, 2018.
The combustion of fossil fuels has accelerated the accumulation
Experimental
of atmospheric CO2 and causes climate change and global warming.
Thus, recent studies have focused on diminishing atmospheric CO2
levels via the capture, storage, and utilization of CO2.1 Among the
strategies, CO2 utilization is emerging for producing useful fuels and
chemicals from CO2 as a cheap, abundant, and renewable feedstock.2
Various fuels and chemicals can be obtained from CO2 (e.g., CO,
methanol, and hydrocarbon), and formate is of primary interest be-
cause of its versatility. Formate has been used not only in various
industrial applications3 but also as a key platform chemical for further
applications such as a carbon source for formatotrophs4 and fuel for
a formate fuel cell.5
Chemical and electrochemical methods have been attempted to
convert CO2 to formate, but practical applications are still limited.
CO2 conversion to formate is usually performed under harsh con-
ditions (i.e., high temperature and pressure),6 and by-products (e.g.,
hydrogen, CO, and ethylene) are frequently produced, resulting in low
selectivity.7 Given that a biocatalyst usually functions under moderate
conditions (i.e., neutral pH, ambient temperature, and atmospheric
pressure) and has higher substrate specificity, the enzymatic CO2 con-
version to formate might be a potential alternative.
Although formate dehydrogenase (FDH, E.C. 1.2.1.2) usually cat-
alyzes formate oxidation to CO2, certain FDHs are capable of re-
versibly interconverting CO2 and formate.8–10 Hirst et al. reported
that the tungsten-containing formate dehydrogenase 1 from Syntro-
phobacter fumaroxidans (SfFDH)8 and the molybdenum-containing
formate dehydrogenase H from Escherichia coli (EcFDH)9 are elec-
troactive and can catalyze CO2 reduction to formate. However, SfFDH
and EcFDH function under strict anaerobic conditions, thereby re-
strictive applications.8,9 Thus, we focused on FDH from Rhodobac-
ter capsulatus (RcFDH), a kind of molybdenum-containing FDH.
Hartmann et al. reported that RcFDH is NAD-linked and cat-
alyzes both CO2 reduction and formate oxidation under aerobic
conditions.10
Herein, we aimed to develop a RcFDH-driven formate production
system utilizing CO2 and thus constructed by a mediated electro-
enzymatic system (Scheme 1). To optimize a mediated electro-
enzymatic system favorable for CO2 reduction rather than for formate
oxidation, we calculated electro-enzymatic kinetic constants (kred and
kox) for various pH and electron mediators based on the limiting
current measured by cyclic voltammetry (CV). Furthermore, we suc-
cessfully achieved measurable formate production from CO2 under
aerobic conditions in our optimized electro-enzymatic system.
RcFDH was expressed as previously described,10 and purified as
the following the steps. The cell pellet was suspended in Bugbuster
Master Mix (Merck) solution, and then cell debris was removed by
centrifugation. Ni-NTA agarose (QIAGEN) was added to the cell
lysate, and the mixture was incubated in ice. The mixture was loaded
into the column and the resin was washed with washing buffer (50
mM NaHPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). The bound
RcFDH was eluted with elution buffer (50 mM NaHPO4, 300 mM
NaCl, 250 mM imidazole, pH 8.0). The elution buffer was replaced
with storage buffer (75 mM potassium phosphate, 10 mM KNO3,
pH 7.5) by Amicon Ultra centrifugal filters (50 kDa, Merck). The for-
mate oxidation activity of RcFDH was measured with 2 ml of 100 mM
Tris-HCl buffer (pH 9.0) containing 6 mM sodium formate and 2 mM
NAD+ at 25◦C. The activity was calculated by the increase in the ab-
sorbance of NADH at 340 nm with a UV-spectrophotometer (εNADH
:
6,220 M−1cm−1). All CV measurements were carried out at a 10 mV/s
scan rate in 10 ml of argon-saturated 100 mM potassium phosphate
buffer (KPB) at 30◦C, containing 100 mM potassium bicarbonate and
potassium formate for bicarbonate reduction and formate oxidation,
respectively. 44 μM of alizarin red S (ARS), anthraquinone-2-sulfonic
acid (AQ2S), benzyl viologen (BV), and methyl viologen (MV) were
tested to determine the most effective electron mediator for RcFDH-
driven CO2 reduction. In the following equations, (i) kcat, red, kcat, ox
and ilim, red, ilim, ox are the catalytic constants and the limiting cur-
rents for bicarbonate reduction and formate oxidation, respectively,
(ii) KM, red, KM, ox and CM, red, CM, red are the Michaelis-Menten con-
stants and concentrations for the reduced and oxidized electron me-
−
−
diators, respectively, (iii) nM, nHCO , and nHCOO are the numbers of
3
electrons in the mediator, HCO3−, and HCOO−, respectively, and (iv)
F, A, and DM are the Faraday constant, the electrode surface area,
and diffusion constants of the mediator, respectively.11
2
i
lim, red
(
)
kcat, red
F AC
M, ox
kred
≡
=
−
KM, red
nHCO DMCE
3
2
)
i
lim, ox
(
kcat, ox
F AC
M, red
kox
≡
=
−
KM, ox
nHCOO DMCE
The mediated electro-enzymatic CO2 reduction was performed in
an electrochemical reactor, in which the anodic and cathodic compart-
ments were separated by a proton exchange membrane (Nafion115,
DuPont). The anodic compartment was equipped with Pt wire and
filled with 1 mM H2SO4. A glassy carbon plate (2.0 cm × 1.5 cm) and
an Ag/AgCl electrode were placed in the cathodic compartment that
contained 10 ml of 200 mM KPB, 10 mM KNO3, 10 mM MV, and
zE-mail: metalkim@unist.ac.kr
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Journal of The Electrochemical Society, 165 (9) H446-H448 (2018)
H447
Figure 1. Cyclic voltammograms of 44 μM MV without RcFDH (dashed line), and with RcFDH (solid line) at a glassy carbon electrode in the 100 mM KPB at
30◦C (scan rate: 10 mV/s). a) 100 mM potassium bicarbonate reduction at pH 7.0. b) 100 mM potassium formate oxidation at pH 7.0.
65 U of RcFDH. CO2 gas (99.999%) was purged into the cathodic
compartment at a flow rate of 1.0 ml/s. Formate was quantified by
a HPLC (Agilent 1200 series) equipped with an Aminex HPX 87-H
column (300 × 7.8 mm, Bio-Rad) and a refractive index detector.2
MV. Thus, we selected MV as the most suitable electron mediator for
the electro-enzymatic CO2 reduction. Sakai et al. previously reported
that a correlation between formal potential of the mediators and logk
values in the MeFDH-driven electro-enzymatic reaction.11 In that pa-
per, logkred was linearly decreased with the formal potential of the
Results and Discussion
As previously reported, we successfully obtained a heterologously
expressed recombinant RcFDH in Escherichia coli and purified it un-
der aerobic condition (Fig. S1). In order to calculate the amount of
catalytically active RcFDH, reduction spectra were recorded as shown
in Fig. S2, thereby indicating that the purified recombinant RcFDH
was fully active. When using sodium formate as the substrate, kcat
and KM of the purified recombinant RcFDH were determined to be
7,440.4 min−1 and 237.5 μM, respectively (Fig. S3). Fig. 1 indi-
cates that the reduction and oxidation peak potential of MV were
determined at −0.68 V (vs. Ag/AgCl, dashed line in Fig. 1a) and
−0.64 V (vs. Ag/AgCl, dashed line in Fig. 1b), respectively. The lim-
iting current plateau of the mediated electro-enzymatic bicarbonate
reduction and formate oxidation were observed by adding RcFDH at
potentials below of −0.7 V (vs. Ag/AgCl, solid line in Fig. 1a) and
above of −0.6 V (vs. Ag/AgCl, solid line in Fig. 1b), respectively.
Based on the mediated electro-enzymatic kinetic parameters intro-
duced by Sakai et al.,11 we calculated the kinetic parameters of the
RcFDH-driven CO2 reduction (kred ≡ kcat, red/KM, red) and the formate
oxidation (kox ≡ kcat, ox/KM, ox).
We tested MV, BV, ARS, and AQ2S to determine the most ef-
fective electron mediator for RcFDH-driven CO2 reduction. Fig. 2a
shows that MV was more efficient for CO2 reduction than other me-
diators at pH 7.0, and the parameters kred was larger than kox for
Figure 2. Logarithmic values of kinetic constants between RcFDH and elec-
tron mediators. a) Effect of mediators on the kinetic constants; MV, BV, ARS,
and AQ2S. b) Effect of pH on the kinetic constants; pH range of 6.5–8.0. kred
:
Scheme 1. Schematic illustration of a mediated electro-enzymatic system for
RcFDH-driven formate production from CO2.
kinetic constant of bicarbonate (CO2) reduction; left y-axis ( ), kox: kinetic
constants of formate oxidation; left y-axis ( ), and kred/kox; right y-axis ( ).
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H448
Journal of The Electrochemical Society, 165 (9) H446-H448 (2018)
In an abiotic system as a negative control, no formate production was
observed (× in Fig. 3), even though sufficient CO2 and reduced MV
were provided in the reaction solution. To the best of our knowledge,
this is the first report of measurable FDH-driven formate production
from CO2.
Summary
Herein, we constructed a mediated electro-enzymatic system for
producing formate from CO2 as a sustainable feedstock. Based on
the electro-enzymatic kinetic constants (kred and kox), the system was
optimized with MV at pH 6.5. Finally, RcFDH-driven CO2 reduction
resulted in 6 mM of formate in 5 hours under aerobic condition.
Consequently, the results discussed herein not only might open up the
next door to produce the versatile chemical formate from CO2 as a
cheap, abundant, and renewable feedstock but also might contribute
to mitigate the atmospheric CO2 level as a climate change mitigation
strategy.
Acknowledgments
Figure 3. Formate production from CO2 in the mediated electro-enzymatic
system (200 mM KPB containing 10 mM KNO3, pH 7.0). 10 mM of MV was
used as the electron mediator.
This research was supported by a grant from the Korea CCS
Research & Development Center (KCRC) (2014M1A8A1049296)
and C1 Gas Refinery Research & Development Center (CGRC)
(2015M3D3A1A01064919).
mediator, and it was also diminished with the reduction peak potential
of the mediator. However, we did not observe a relationship between
logkred and the reduction peak potential (the reduction and oxidation
peak potentials of each mediator are summarized in Table S1), proba-
bly because of structural differences (e.g., Mo-containing for RcFDH
and W-containing for MeFDH) that might lead to mediator preference
under different circumstances.12–14
ORCID
Yong Hwan Kim https://orcid.org/0000-0002-5947-4169
We investigated the effect of pH on the mediated electro-enzymatic
system. The RcFDH-driven CO2 reduction was more predominant at
an acidic pH, whereas formate oxidation was favorable at a basic
pH (Fig. 2b). This phenomenon might be due to concentrated protons
(H+), which are inevitably required to convert CO2 to formate, thereby
favoring an acidic pH. On the other hand, formate oxidation to CO2
releases protons and easily occurs at a basic pH. Accordingly, the ratio
of kred to kox (ꢀ in Fig. 2b) implied that pH 6.5 was the most suitable
for CO2 reduction in our mediated electro-enzymatic system.
Nevertheless, RcFDH had low stability at an acidic pH, especially
at pH 6.0, retaining 81% of the initial activity after 7 hours (Fig. S4a)
and thus RcFDH-driven CO2 reduction reaction was performed at pH
7.0. In addition, nitrate enhanced the stability of RcFDH (Fig. S4b),
with 10 mM of KNO3 employed as the stabilizing additive in the
mediated electro-enzymatic system.15
Finally, RcFDH-driven formate production from CO2 was per-
formed under optimized conditions with MV as the electron mediator
at pH 7.0. When using 65 U of RcFDH, we successfully achieved
6 mM of formate production in 5 hours. The current density decreased
from initially 0.65 mA/cm2 to finally 0.55 mA/cm2, consistent with
the decline of formate production rate in Fig. 3, because probably
the activity of RcFDH decreased under electrochemical conditions.
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