Photoelectrochemical Reduction of Carbon Dioxide to Methanol through a Highly Efficient Enzyme Cascade

Article abstract of DOI:10.1002/anie.201611379

Natural photosynthesis is an effective route for the clean and sustainable conversion of CO₂ into high-energy chemicals. Inspired by the natural process, a tandem photoelectrochemical (PEC) cell with an integrated enzyme-cascade (TPIEC) system was designed, which transfers photogenerated electrons to a multienzyme cascade for the biocatalyzed reduction of CO₂ to methanol. A hematite photoanode and a bismuth ferrite photocathode were applied to fabricate the iron oxide based tandem PEC cell for visible-light-assisted regeneration of the nicotinamide cofactor (NADH). The cell utilized water as an electron donor and spontaneously regenerated NADH. To complete the TPIEC system, a superior three-dehydrogenase cascade system was employed in the cathodic part of the PEC cell. Under applied bias, the TPIEC system achieved a high methanol conversion output of 220 μm h^?1, 1280 μmol g^?1 h^?1 using readily available solar energy and water.

Full text of DOI:10.1002/anie.201611379

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201611379
German Edition: DOI: 10.1002/ange.201611379
Biocatalytic Photosynthesis Hot Paper
Photoelectrochemical Reduction of Carbon Dioxide to Methanol
through a Highly Efficient Enzyme Cascade
+
+
Su Keun Kuk , Raushan K Singh , Dong Heon Nam, Ranjitha Singh, Jung-Kul Lee,* and
Chan Beum Park*
Abstract: Natural photosynthesis is an effective route for the
clean and sustainable conversion of CO2 into high-energy
chemicals. Inspired by the natural process, a tandem photo-
electrochemical (PEC) cell with an integrated enzyme-cascade
they suffer from issues of high production cost, poor stability
[5]
under harsh conditions, and O2 sensitivity. In principle,
a cascade involving three different enzymes, namely formate
dehydrogenase (FDH), formaldehyde dehydrogenase
(FaldDH), and alcohol dehydrogenase (ADH), can reduce
(
TPIEC) system was designed, which transfers photogenerated
electrons to a multienzyme cascade for the biocatalyzed
CO to formate, formaldehyde, and methanol in a stepwise
2
reduction of CO to methanol. A hematite photoanode and
manner. However, each of the energetically uphill reductions
requires the supply of a stoichiometric amount of expensive
nicotinamide cofactor (NADH) to achieve catalytic turnover,
and the biocatalytic photochemical systems reported to date
2
a bismuth ferrite photocathode were applied to fabricate the
iron oxide based tandem PEC cell for visible-light-assisted
regeneration of the nicotinamide cofactor (NADH). The cell
utilized water as an electron donor and spontaneously
regenerated NADH. To complete the TPIEC system, a superior
three-dehydrogenase cascade system was employed in the
cathodic part of the PEC cell. Under applied bias, the TPIEC
system achieved a high methanol conversion output of
for the conversion of CO into methanol are limited by their
2
low yields of NADH regeneration.
Here, we report an inorganic/bioorganic hybrid photo-
synthetic platform consisting of an NADH-regenerating
photoelectrochemical (PEC) cell and a multienzyme cascade
(Scheme 1). The tandem PEC cell with an integrated enzyme-
cascade (TPIEC) system mimics the natural photosynthetic
Z-scheme by acquiring photogenerated electrons from the
oxidation of water on illumination with visible light and
transferring them to the enzyme cascade for the biocatalytic
ꢀ1
ꢀ1 ꢀ1
2
20 mm h , 1280 mmolg
h
using readily available solar
energy and water.
P
hotosynthesis occurs in green plants through a highly
efficient, photoinduced energy-transfer cascade consisting of
the Z-scheme and the Calvin cycle. The beauty of this natural
process has inspired many researchers to develop eco-
reduction of CO to methanol. In the photoelectrode-type Z-
2
scheme system, the anodic and cathodic photoelectrodes are
separated into two compartments by an ion-exchange salt
bridge and connected by a conducting wire to prevent product
[1]
friendly, solar reductions of CO2 to hydrocarbon fuels.
Methanol, a useful reduced form of CO2, can be synthesized
[
2]
from CO by the use of different types of photo- or electro-
2
chemical catalysts, such as metal complexes and oxide-based
[3]
semiconductors. These catalysts, however, exhibit critical
problems: for example, metal complexes often suffer from
severe photocorrosion in an aqueous medium when illumi-
[
4]
nated by light, while semiconductors generate low yields
with poor selectivity and require harsh conditions such as high
temperatures and pressures for better output. Recently, redox
biocatalysts have been utilized for the photochemical reduc-
tion of CO . These biocatalysts have several advantages,
2
including the ability to function under mild conditions with
extremely high specificity without side reactions, although
[
+]
[
*] S. K. Kuk, Dr. D. H. Nam, Prof. Dr. C. B. Park
Department of Materials Science and Engineering
Korea Advanced Institute of Science and Technology
3
35 Science Road, Daejeon 305-701 (Republic of Korea)
Scheme 1. Schematic illustration of the solar-assisted production of
E-mail: parkcb@kaist.ac.kr
methanol from CO and water through a three-enzyme (CcFDH,
[
+]
2
Dr. R. K. Singh, R. Singh, Prof. Dr. J. K. Lee
Department of Chemical Engineering, Konkuk University
PcFaldDH, YADH) cascade. Under illumination by visible light, water
is oxidized at the photoanode (Co-Pi/a-Fe O ) and serves as an
2
3
120 Neungdong-ro, Seoul 143-701 (Republic of Korea)
electron donor. Photoexcited electrons are transferred to the photo-
E-mail: jkrhee@konkuk.ac.kr
+
cathode (BiFeO ), followed by reduction of NAD to NADH via
3
+
[
] These authors contributed equally to this work.
+
a rhodium-based mediator (M=[Cp*Rh(bpy)H O] , Cp*=C Me ,
2
5
5
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201611379.
bpy=2,2’-bipyridine). Ultimately, the excited electrons from reduced
NAD are delivered to the three-enzyme cascade for the biocatalytic
synthesis of methanol.
+
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
mixing and side reactions, a feature which is advantageous for
biocatalyst-based” Z-scheme PEC systems because sepa-
rated cells allow biocatalysts to maintain their optimal
“
[
6]
activities. Furthermore, the separation of the anodic and
cathodic reactions (i.e. water oxidation reaction and biocat-
alytic methanol synthesis) can avoid potential damage to
anaerobic enzymes from the oxygen-evolving reaction and
reoxidation of formate (an intermediate product) at the Co-Pi
photoanode. We conducted in situ NADH regeneration using
a Co-Pi/a-Fe O3 photoanode and a ferroelectric BiFeO3
2
photocathode. The photoanode takes up electrons from the
water oxidation, and the photocathode transports the elec-
2
+
trons to an electron mediator M ([CpRh(bpy)(H O)] ) and
2
+
NAD through an internal electric field induced by remnant
polarization to regenerate the enzymatically active 1,4-
NADH. We reduced CO to methanol in the photocathodic
2
compartment, which contains the FDH-FaldDH-ADH
enzyme cascade. In this cascade, FaldDH is a rate-limiting
enzyme because of its low catalytic activity during the
reduction of formate to formaldehyde. To overcome this
limitation, we identified Pseudomonas cepacia genomvar II
FaldDH (PcFaldDH) by a sequence comparison based on the
Basic Local Alignment Search Tool (BLAST), systematic
sequence analysis of 100 formate-assimilating microorgan-
isms (see Table S1), and testing of the formate reduction
capacity of 30 FaldDHs (see Table S2). We demonstrated that
the formate-reducing PcFaldDH shows a high binding affinity
to formate and high formate reduction activity, which
Figure 1. a) Linear sweep voltammetry (LSV) scans of bare hematite
and hematite with deposited Co-Pi under chopped light illumination
using a three-electrode configuration setup (pH 12). b) LSV scans of
a thin film of BiFeO
chopped light illumination (pH 7). c) Cyclic voltammogram (CV)
before and after poling at +8 V or ꢀ8 V under
3
curves of a thin film of BiFeO poled at +8 V in the absence and
3
+
presence of M and NAD at pH 7. d) Illustration of the Co-Pi/a-
Fe O jBiFeO tandem PEC cell for the NADH regeneration system
2
3
3
with visible light. The remnant polarization inside the BiFeO film
3
induced the internal electric field, which promotes charge separation.
significantly enhances the rate of conversion of CO into
2
methanol.
very efficiently. According to the literature, the remnant
polarization should generate an internal electric field that
induces accumulation of surface charge and drives an upward
For efficient photoelectrochemical regeneration of
NADH, we designed an a-Fe O j BiFeO tandem PEC
2
3
3
[
8]
array. a-Fe O , an n-type semiconductor, was chosen as
bending of the energy band of the BiFeO film. Upward
2
3
3
a
photoanode material because its optical band gap
bending of the energy band inhibited recombination of the
photogenerated electron-hole pair and expedited electron
transfer toward the electrolyte. As shown in Figure 1b, the
bending of the energy band derived from poling treatment
(
ca. 2.1 eV) provides respectable photocurrents under irradi-
[
7]
ation with solar light (l > 420 nm). BiFeO , a p-type
3
perovskite semiconductor, is a ferroelectric material that
exhibits a band gap of approximately 2.2 eV as well as
enhanced photocurrent generation and charge transfer from
the electrode to the electrolyte in response to a polarization-
induced internal electric field. These two iron oxide
materials are stable and show suitable alignment of their
band edges within the energy ranges of each electrode, which
can pump electrons up to a higher potential simultaneously
under irradiation with visible light. We prepared a Co-Pi/a-
Fe O photoanode with a band gap of 2.18 eV (see Figures S1
affected the photocurrent of the BiFeO photocathodes: the
3
ꢀ
2
BiFeO films poled at + 8 Vachieved 0.24 mAcm at ꢀ0.4 V
3
(versus Ag/AgCl), which is 1.6 times greater than that without
polarization. In contrast, the photoresponse of the BiFeO3
films was reduced after poling at ꢀ8 V. To explore the
[
8]
photogenerated electron transfer from the BiFeO photo-
3
+
cathode to M and NAD , we constructed cyclic voltammo-
gram (CV) curves of BiFeO electrodes poled at + 8 V under
3
+
a cathodic potential and with M and NAD (Figure 1c).
Electron transfer from the photocathode to M and NAD was
2
3
[
7b]
+
and S2) by a solution-based method.
conditions (pH 12), the Co-Pi/a-Fe O thin film achieved an
Under alkaline
confirmed by enhancement of the cathodic peak current and
2
3
ꢀ2
[9]
anodic photocurrent density 3.2 times higher (1.7 mAcm ) at
.6 V (versus Ag/AgCl) than that of bare hematite (Fig-
ure 1a).
a potential shift, in agreement with the literature. In the
+
0
presence of both M and NAD , the generation of photo-
current at the cathodic peak increased from ꢀ0.38 to
ꢀ
2
+
A 300-nm-thick polycrystalline BiFeO photocathode was
ꢀ0.6 mAcm as a result of the reduction of NAD by M.
The relationship between the energy band edges of the
photoanode, photocathode, and electron mediator M plays an
3
[
8a]
prepared by a spin-coating method.
This photocathode
exhibits a suitable band gap for the absorption of visible light
2.32 eV, see Figure S3). Polarization-electric (P-E) hysteresis
[
10]
(
important role in the design of tandem PEC cells.
The
loop tests on BiFeO photocathodes showed that the internal
valence band maximum (VBM) and conduction band mini-
3
electric field of spin-coated BiFeO films can be controlled by
mum (CBM) of the BiFeO film were calculated to be 0.82
3
3
poling treatment (see Figure S4). Moreover, the application
of a + 8 Vexternal electric field induced remnant polarization
and ꢀ1.5 eV, respectively (versus Ag/AgCl), according to
analysis by ultraviolet photoelectron spectroscopy (see Fig-
2
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
ure S5). The VBM of BiFeO was higher than the conduction
3
band edge of hematite, and its CBM was lower than the
reduction potential of the electron mediator M (ꢀ0.73 V
+
versus Ag/AgCl) that transfers hydride ions to NAD . These
edge configurations of the energy band indicate that BiFeO3
is a proper photocathode material in the tandem PEC cell for
in situ regeneration of NADH under irradiation by visible
light (Figure 1d). The cathodic current from poled BiFeO3
was lower than the anodic current (see Figure S6), thus
implying that BiFeO was a current-limiting component in the
3
tandem PEC cell. Without applied voltage, a spontaneous
ꢀ2
current of approximately 100 mAcm was predicted from the
overlap onset potential of each photoelectrode under irradi-
ation with visible light.
We assembled the Co-Pi/a-Fe O j BiFeO tandem PEC
2
3
3
NADH regeneration system in a two-electrode configuration
containing Co-Pi/a-Fe O as a working electrode and BiFeO
3
2
3
as a counter electrode under ambient conditions with visible
light. The two-component cell was separated into an oxygen-
evolving reaction (anodic) part and a photobiocatalytic
methanol-formation (cathodic) part. The photoanode was
employed under alkaline conditions (pH 12), thereby induc-
ing the oxidation of water (see Figure S7). Enzymes were
applied with the photocathode at the optimal pH value
Figure 2. a) Photocurrent response in a two-electrode configuration
with Co-Pi/a-Fe O (pH 12) and BiFeO (pH 7) as a function of the
2
3
3
applied voltage under various irradiation conditions (active surface
2
area: 1 cm ). b) Corresponding EABPE of the Co-Pi/a-Fe O jBiFeO
2
3
3
tandem PEC cell. c) Regeneration yield of NADH by the Co-Pi/a-
Fe O jBiFeO tandem PEC cell as a function of the applied voltage.
2
3
3
(
pH 7) and shielded from oxygenic reactions, which could
d) Time profiles of the NADH-regenerating PEC cell under constant
electrical bias (0.8 V) over 8 h with illumination by visible light. Inset:
chronoamperogram of the PEC cell.
damage metal ions (Zn or W) or iron sulfur clusters of enzyme
reaction centers. The difference in the pH values between the
two compartments in the tandem PEC cell causes a chemical
bias in the PEC system (0.059 V per pH unit, 0.295 V), which
drives the electron transfer from the photoanode to the
photocathode. The two separated cells were connected by
a salt bridge to complete the ionic circuit of tandem cell.
Figure 2a shows linear sweep voltammetry scans for each
photoelectrode of the tandem PEC cell under visible light.
Without irradiation of a-Fe O by light, no current passed
at various applied voltages in the presence of the electron
mediator (M) and illumination by light for 2 h (Figure 2c).
We measured the concentrations of NADH through its
absorption at l = 340 nm by using a UV spectrophotometer.
Under the chemical-biased conditions (without applied
voltage), the tandem PEC cell showed a 3% spontaneous
regeneration of NADH through the PEC platform. As the
applied voltage between the two photoelectrodes increased to
1 V, the yield of NADH regeneration increased up to 70.54%
(see Figure S9). At an applied electrical bias of over 0.8 V, the
efficiency of the NADH regeneration increased significantly,
consistent with the EABPE.
2
3
through the tandem PEC cell, while further irradiation of the
BiFeO photocathode increased the photocurrent. This obser-
3
vation indicates that the PEC reaction driven by the photo-
anode was further enhanced by electron excitation at the
electric-poled BiFeO3 photocathode. In the case of the
illumination of both electrodes by light, a spontaneous
We further investigated the performance and stability of
the NADH-regenerating tandem PEC cell under continuous
illumination of visible light at an external bias of 0.8 V. A
ꢀ2
photocurrent of 38.3 mAcm was generated with an open
circuit voltage of 909.2 mV (see Figure S8). This photocurrent
was lower than the predicted value and attributed to ohmic
loss in the ionic pathway of the circuit through the salt bridge
ꢀ
2
photocurrent of approximately 0.48 mAcm was observed
with good stability during the 8 h reaction, and resulted in an
80% yield for NADH regeneration with 90% of the initial
current density retained during the reaction (Figure 2d,
Figure S10). To confirm that the photocurrent of the inte-
grated PEC cell originated from the oxidation of water on the
a-Fe O photoanode, we measured the amount of evolved O
[10b,11]
and electrolyte.
We estimated the efficiency of the NADH-regenerating
Co-Pi/a-Fe O j BiFeO tandem PEC cell in the two-electrode
2
3
3
platform by calculating the external applied bias photon to
current efficiency (EABPE). In the NADH regeneration
system, we defined EABPE according to the following
2
3
2
on the photoanodic part at an external bias of 0.8 V by using
a calibrated fluorescence-based oxygen sensor. From Fig-
ure S11 and Figure 2d, the corresponding rates for product
generation and faradic efficiencies were estimated to be
ꢀ
1
equation: EABPE =j j jꢀ (V ꢀV ꢀVbias) ꢀ Ptotal
Note
th
chem
.
that Vth is a theoretical voltage difference between the
electrolysis potential of water and the reduction potential of
M (1.76 V), and Vchem is a chemical bias between the two
chambers (0.295 V). The best EABPE (0.24%) was obtained
at an applied electrical bias of 0.73 V for the NADH-
regenerating tandem PEC cell (Figure 2b). We applied the
ꢀ1
ꢀ1
4.08 mmolh , 94.1% for O evolution, and 7.26 mmolh ,
2
88.4% for NADH regeneration. We attribute the reasons why
the faradaic efficiencies were less than 100% to hole
[
12]
consumption on the photoanode,
electron losses arising
+
tandem PEC cell to the regeneration of NADH from NAD
from photodegradation of the BiFeO3 photocathode, and
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
multiple electron-transfer steps in the cathodic compartment.
The faradaic efficiencies of approximately 90% and a nearly
:1 ratio of the rates of NADH regeneration and O evolution
2
2
indicate that most of the photogenerated electrons generated
from the oxidation of water in the photoanode were used for
the regeneration of NADH in the photocathode. These results
suggest that the Co-Pi/a-Fe O j BiFeO -integrated PEC cell
2
3
3
is a highly efficient solar-to-NADH regeneration system that
utilizes earth-abundant iron oxide based materials.
To construct the TPIEC system based on the Co-Pi/a-
Fe O j BiFeO -integrated PEC cell, we designed a novel
2
3
3
enzymatic reaction platform involving a cascade of three
dehydrogenases (i.e. FDH, FaldDH, and ADH). PcFaldDH,
which encodes a polypeptide of 399 amino acids, was cloned,
overexpressed, and purified (see Figure S12 in the Supporting
Information). The protein yield of PcFaldDH was estimated
to be 41 mg per liter of culture. PcFaldDH belongs to the zinc-
containing, medium-chain alcohol dehydrogenase family; it
contains two zinc ions per subunit, one catalytic and one
structural (Figure 3a). The catalytic zinc center is coordinated
by three residues (C47, H68, and D170; Figure 3b). Under
optimum conditions, the apparent formate to formaldehyde
ꢀ
1
reduction activity (k ) of purified PcFaldDH was 0.653 s
cat
(
Table S3, Figure S13). In contrast, PpFaldDH, which is
a commercial FaldDH, showed no detectable reduction
activity. To the best of our knowledge, PcFaldDH is the first
FaldDH enzyme with evident activity for the reduction of
formate. To further investigate the ability of PcFaldDH to
reduce formate, we performed bioelectrochemical analysis as
Figure 3. a) Homology model of PcFaldDH showing the catalytic
residues (green sticks) and NADH. b) Active site pocket of PcFaldDH
highlighting the catalytic residues (green color). The NADH and the
catalytic Zn center in the active site pocket of the PcFaldDH are
indicated. c) Comparison of the linear sweep voltammetry curves of
bare carbon felt (black), PpFaldDH (green), and carbon felt with
adsorbed PcFaldDH with (red) and without substrate (blue) at pH 7.
d) Methanol production as a function of time in multienzymatic
systems. The initial NADH concentration was 5 mm. e) Photoelectro-
chemical production of methanol by TPIEC under various applied
[
13]
previously described. The linear sweep voltammetry curve
of a porous carbon felt electrode with adsorbed PcFaldDH
(
Figure 3c) showed a huge formate reduction peak of
ꢀ2
ꢀ
1.37 mAcm
compared
to
with
PpFaldDH
ꢀ
2
(
ꢀ0.16 mAcm ). This result shows that PcFaldDH possesses
+
potentials with illumination by visible light. The initial NAD concen-
high catalytic activity for formate reduction which can
improve the rate-determining step in the enzyme taking
part in the methanol cascade. To elucidate the interaction
between PcFaldDH and ligands (i.e. formate and NADH), we
performed isothermal titration calorimetry (ITC; see Fig-
tration was 5 mm. f) Methanol production of the TPIEC system as
a function of time in various multienzymatic systems with an external
voltage of 0.8 V.
ure S14 and Table S4). The binding constants (K values) of
production of methanol, respectively. This finding indicates
d
formate and NADH to PcFaldDH were 6.20 and 0.67 mm,
that PcFaldDH plays a crucial role in the reduction of CO to
2
respectively. These results strongly suggest that PcFaldDH is
methanol.
a suitable biocatalyst that can improve the rate of CO to
Prior to applying the enzyme cascade system to the Co-Pi/
a-Fe O j BiFeO tandem PEC system, we tested each enzyme
2
methanol conversion in a multienzyme cascade system.
To build a methanol-producing, multienzymatic cascade
system, we adopted FDH from Clostridium carboxidivor-
2
3
3
with the NADH-regenerating PEC platform and confirmed
that each biocatalytic redox reaction was induced by the
generated NADH in the PEC system (see Figures S21—S23).
Thereafter, we fabricated the TPIEC system by coupling the
enzyme cascade with the NADH-regenerating PEC platform
for the production of methanol. Solar-assisted, CO2 to
methanol conversion was performed for 6 h in the TPIEC
system and analyzed by gas chromatography and NMR
[14]
ans P7T (CcFDH)
(see Figures S15 and S12) and the
commercial enzyme YADH, along with PcFaldDH. The
protein yield of CcFDH was estimated to be 27 mg per liter
of culture. CcFDH displayed better catalytic activity and
binding constants than the commercial enzyme CbFDH for
the reduction of CO (see Figures S14 and S17). In collabo-
2
[15]
ration with PcFalDH, CcFDH converted CO sequentially
spectroscopy (Figure 3e, Figure S24).
Until the external
2
into formate and then into formaldehyde (see Figures S18–
S20). Compared to other multienzyme cascades, the CcFDH-
PcFaldDH-YADH cascade system showed superior activity
applied voltage reached 0.8 V, only a small amount of
methanol was detected; however, when more than 0.8 V
was applied, photobiocatalytically produced methanol was
obtained at a concentration of 1.31 mm with a faradaic
efficiency of 31.9%. We attribute this significant increment to
for the CO to methanol conversion (Figure 3d). Individual
2
replacement of CcFDH and PcFaldDH in the cascade
reaction resulted in a 3.4- and 5.6-fold increase in the
+
the NADH/NAD ratio, which was dependent on the applied
4
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
[
16]
voltage (see Figure S25). According to the literature, the
Table 1: Comparison of the efficiencies for the CO to methanol
2
conversion in various photocatalytic systems.
+
NADH/NAD ratio significantly affects the catalytic per-
formances of NADH-dependent enzymes. As the reactions
catalyzed by these enzymes are reversible, the NADH/NAD
[
a]
[b]
Category
Photocatalyst
Electron
donor
Cavg
Ravg
+
ratio is critical to the production of methanol in the
biocatalytic PEC system. Figure S25 and Figure 2c show
that the NADH/NAD ratio increased substantially when the
this study
photoenzymatic
BiFeO3
CCG-IP
Eosin Y
H O
TEOA
TEOA
220.0 1279.8
2
[5b]
7.5
6.0
46.3
186.4
+
[5c]
[
1f]
electrochemical bias reached 0.8 V and was only slightly
enhanced after 0.8 V, consistent with the NADH generation
profile. These results indicate an inefficiency in energy
transfer at the high potential, which is in agreement with
the significant decrease in EABPE above 0.8 V (Figure 2b).
Furthermore, high voltages can induce the formation of by-
photochemical, non-
enzymatic
Bi
2
WO
6
H
2
O
107.1 75
586.7
Au-Cu/graphene/ H O
–
2
[
21a]
Cu O
2
[
21b]
NiO/InNbO4
H O
31.5 1.58
98 56
2
carbon QD/
H O
2
[21c]
Cu O
2
[
21d]
Bi S /CdS
H O
122.6 88
44.9 44.9
118.5 118.5
2
3
2
[17]
[21e]
products in PEC systems. The production of methanol by
the TPIEC system in Figure 3e showed a similar profile as the
CdS/Bi
RuO2/
2
S
3
/TiO
2
H
2
O
H2
[
21f]
+
x y z k
Cu Ag In Zn S
NiO/InTaO4
m
NADH/NAD ratio versus applied voltage (see Figure S25),
[
21g]
H O
2
–
11.1
and the optimal electrochemical bias for the biocatalytic
synthesis of methanol through photoelectrochemical NADH
regeneration was approximately 0.8 V. Hence, we performed
subsequent experiments with a voltage of 0.8 V. We con-
ducted several negative control experiments for each compo-
nent of the TPIEC system (see Figure S26). According to our
results, methanol was not synthesized at all in the absence of
[a] Average rate of methanol formation per reaction volume, in
ꢀ
1 [20b,c]
mmh
.
[b] Average rate of methanol formation per photocatalyst
ꢀ
1
ꢀ1 [21g]
weight, in mmolg
h
.
TEOA=triethanolamine.
with visible light under ambient conditions based on photo-
enzymatic,
and photochemical pathways.
+
[5]
[19]
[20]
the enzymes, Rh complex M, or NAD , which indicates
electroenzymatic,
photoelectrochemical,
[
21]
methanol was generated only through the three-enzyme
cascade reactions. We noted that a small amount of formate
In summary, we have demonstrated a PEC synergistic
biocatalyzed conversion of CO2 into methanol by the
integration of an NADH-regenerating tandem PEC cell and
a multienzyme cascade. The design of the iron oxide based,
NADH-regenerating Co-Pi/a-Fe O j BiFeO tandem PEC
(
0.15 mm) was formed with M in the absence of enzymes and
+
NAD after incubation for 6 h, thus indicating that CO can
2
be partially reduced to formate by M, which was used for the
generation of formate and coupled with a subsequent enzy-
2
3
3
[15b,18]
matic reaction in recent studies.
However, the amount of
cell was based on proper alignment of the band edges of
two photoelectrodes and the internal electric field of
formate (2.03 mm) produced with CcFDH in the NADH-
regenerating PEC system was less than 10% (see Figure S21).
In addition, we carried out a control experiment of the TPIEC
system without CcFDH. According to the result (see Fig-
ure S27), only small amounts of methanol (0.03 mm) and
formate (0.11 mm) were synthesized relative to the full
TPIEC system (Figures 3 f and S26). These limited conver-
sions indicate that most of the methanol was generated in the
TPIEC system through the consecutive enzymatic reactions.
We investigated TPIEC systems with different multi-
enzymatic combinations over 6 h (Figure 3 f). When
PcFaldDH was not involved in the multienzymatic cascade,
the TPIEC systems failed to produce methanol. In the
PcFaldDH-containing TPIEC systems, the replacement of
CbFDH by CcFDH resulted in a 6.5-fold increase in the
methanol concentration. Thus, the improved reduction of
formate by PcFaldDH and the slow oxidation of formate by
a BiFeO photocathode, which induced electron transfer to
M and NAD . The tandem PEC cell utilized water as an
electron donor and generated NADH from NAD without
electrical bias. The yield of NADH regeneration increased up
to 80% at an applied bias of 0.8 V. For the efficient
3
+
+
biocatalytic conversion of CO into methanol, the superior
2
formate-reducing ability of PcFaldDH was identified and
exploited for the first time in a three-dehydrogenase cascade
system. The combination of the NADH-regenerating PEC
system and the CcFDH-PcFaldDH-YADH cascade system
resulted in high efficiencies for methanol formation compared
with other systems mediated by visible light. Overall, this two-
system-integration approach is a promising strategy for the
conversion of CO2 into valuable chemicals with highly
selectivity from inexpensive materials by using a PEC
system and using readily available solar energy and water.
CcFDH led to the rapid and efficient reduction of CO in the
2
photobiocatalytic reaction system. We estimated the efficien-
cies for the conversion of CO into methanol in the TPIEC Acknowledgements
2
system on the basis of the average rate of methanol formation
ꢀ
1
per reaction volume (C , mm h ) and photocatalyst weight
This study was supported by the National Research Founda-
tion (Grant nos.: NRF-2015R1A3A2066191 and
avg
ꢀ1
ꢀ1
(
Ravg, mmolgcat h ). The calculated C and Ravg values for
avg
the Co-Pi/a-Fe O j BiFeO tandem PEC cell integrated with
2013M3A6A8073184) and the Samsung Research Funding
Center of Samsung Electronics (Grant no.: SRFC-TA1403-
01), Republic of Korea.
2
3
3
the CcFDH-PcFaldDH-YADH enzyme cascade were
ꢀ1
ꢀ1 ꢀ1
2
20 mm h and 1280 mmolgcat h , respectively (Table 1).
These values are high compared to other systems reported
to date for the conversion of CO into methanol by irradiation
2
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Mater. 2010, 22, 1763; d) A. M. Glass, D. V. D. Linde, D. H.
Auston, T. J. Negran, J. Electron. Mater. 1975, 4, 915 – 943.
9] S. H. Lee, G. M. Ryu, D. H. Nam, J. H. Kim, C. B. Park,
ChemSusChem 2014, 7, 3007 – 3011.
Conflict of interest
[
The authors declare no conflict of interest.
[
10] a) A. J. Nozik, Appl. Phys. Lett. 1976, 29, 150 – 153; b) Y. H. Lai,
D. W. Palm, E. Reisner, Adv. Energy Mater. 2015, 5, 1501668.
Keywords: biocatalysis · CO reduction · enzymes ·
2
methanol production · photosynthesis
[11] J. Newman, J. Electrochem. Soc. 2013, 160, F309 – F311.
12] a) Y. Li, W. Zhang, J. F. Niu, Y. S. Chen, ACS Nano 2012, 6,
164 – 5173; b) X. J. Shi, Y. Choi, K. Zhang, J. Kwon, D. Y. Kim,
J. K. Lee, S. H. Oh, J. K. Kim, J. H. Park, Nat. Commun. 2014, 5,
775.
[
5
4
[
1] a) N. S. Lewis, D. G. Nocera, Proc. Natl. Acad. Sci. USA 2006,
03, 15729 – 15735; b) E. E. Barton, D. M. Rampulla, A. B.
[13] a) S. L. Li, S. Freguia, S. M. Liu, S. S. Cheng, S. Tsujimura, O.
Shirai, K. Kano, Biosens. Bioelectron. 2010, 25, 2651 – 2656;
b) A. Bassegoda, C. Madden, D. W. Wakerley, E. Reisner, J.
Hirst, J. Am. Chem. Soc. 2015, 137, 4592 – 4592.
[14] A. Alissandratos, H. K. Kim, H. Matthews, J. E. Hennessy, A.
Philbrook, C. J. Easton, Appl. Environ. Microbiol. 2013, 79, 741 –
744.
[15] a) X. Y. Ji, Z. G. Su, P. Wang, G. H. Ma, S. P. Zhang, ACS Nano
2015, 9, 4600 – 4610; b) K. Ma, O. Yehezkeli, E. Park, J. N. Cha,
ACS Catal. 2016, 6, 6982 – 6986; c) J. M. Saveant, C. Tard, J. Am.
Chem. Soc. 2016, 138, 1017 – 1021.
[16] a) C. Zhang, K. Ma, X. H. Xing, Int. J. Hydrogen Energy 2009,
34, 1226 – 1232; b) J. Wang, Y. M. Kim, H. S. Rhee, M. W. Lee,
J. M. Park, Bioresour. Technol. 2013, 135, 199 – 206; c) C. Zhang,
F. X. Lv, X. H. Xing, Bioresour. Technol. 2011, 102, 8344 – 8349.
[17] a) J. L. Qiao, Y. Y. Liu, F. Hong, J. J. Zhang, Chem. Soc. Rev.
2014, 43, 631 – 675; b) A. S. Agarwal, Y. Zhai, D. Hill, N. Sridhar,
ChemSusChem 2011, 4, 1301 – 1310.
1
Bocarsly, J. Am. Chem. Soc. 2008, 130, 6342; c) S. H. Lee, J. H.
Kim, C. B. Park, Chem. Eur. J. 2013, 19, 4392 – 4406; d) J. H.
Kim, D. H. Nam, C. B. Park, Curr. Opin. Biotechnol. 2014, 28, 1 –
9
; e) D. Kim, K. K. Sakimoto, D. C. Hong, P. D. Yang, Angew.
Chem. Int. Ed. 2015, 54, 3259 – 3266; Angew. Chem. 2015, 127,
309 – 3316; f) L. Liang, F. Lei, S. Gao, Y. Sun, X. Jiao, J. Wu, S.
3
Qamar, Y. Xie, Angew. Chem. Int. Ed. 2015, 54, 13971 – 13974;
Angew. Chem. 2015, 127, 14177 – 14180; g) M. D. Porosoff, B. H.
Yan, J. G. G. Chen, Energy Environ. Sci. 2016, 9, 62 – 73; h) E. J.
Son, J. W. Ko, S. K. Kuk, H. Choe, S. Lee, J. H. Kim, D. H. Nam,
G. M. Ryu, Y. H. Kim, C. B. Park, Chem. Commun. 2016, 52,
9723 – 9726.
[
2] a) S. Kuwabata, K. Nishida, R. Tsuda, H. Inoue, H. Yoneyama, J.
Electrochem. Soc. 1994, 141, 1498 – 1503; b) J. Graciani, K.
Mudiyanselage, F. Xu, A. E. Baber, J. Evans, S. D. Senanayake,
D. J. Stacchiola, P. Liu, J. Hrbek, J. F. Sanz, J. A. Rodriguez,
Science 2014, 345, 546 – 550.
[18] a) O. Yehezkeli, N. M. Bedford, E. Park, K. Ma, J. N. Cha,
ChemSusChem 2016, 9, 3188 – 3195; b) M. B. Chambers, X.
Wang, N. Elgrishi, C. H. Hendon, A. Walsh, J. Bonnefoy, J.
Canivet, E. A. Quadrelli, D. Farrusseng, C. Mellot-Draznieks,
M. Fontecave, ChemSusChem 2015, 8, 603 – 608.
[
3] a) S. N. Habisreutinger, L. Schmidt-Mende, J. K. Stolarczyk,
Angew. Chem. Int. Ed. 2013, 52, 7372 – 7408; Angew. Chem.
2013, 125, 7516 – 7557; b) J. L. White, M. F. Baruch, J. E. Pander,
Y. Hu, I. C. Fortmeyer, J. E. Park, T. Zhang, K. Liao, J. Gu, Y.
Yan, T. W. Shaw, E. Abelev, A. B. Bocarsly, Chem. Rev. 2015,
[19] a) P. K. Addo, R. L. Arechederra, A. Waheed, J. D. Shoemaker,
W. S. Sly, S. D. Minteer, Electrochem. Solid-State Lett. 2011, 14,
E9 – E13; b) S. Schlager, L. M. Dumitru, M. Haberbauer, A.
Fuchsbauer, H. Neugebauer, D. Hiemetsberger, A. Wagner, E.
Portenkirchner, N. S. Sariciftci, ChemSusChem 2016, 9, 631 –
635.
[20] a) J. L. Yuan, P. C. Wang, C. J. Hao, G. C. Yu, Electrochim. Acta
2016, 193, 1 – 6; b) Z. X. Yang, J. F. Xu, C. X. Wu, H. Jing, P. Q.
Li, H. Z. Yin, Appl. Catal. B 2014, 156, 249 – 256; c) P. Q. Li, J. F.
Xu, H. Jing, C. X. Wu, H. Peng, J. Lu, H. Z. Yin, Appl. Catal. B
2014, 156, 134 – 140; d) J. L. Yuan, C. J. Hao, Sol. Energy Mater.
Sol. Cells 2013, 108, 170 – 174; e) K. Rajeshwar, N. R. de Tacconi,
G. Ghadimkhani, W. Chanmanee, C. Janaky, ChemPhysChem
2013, 14, 2251 – 2259.
[21] a) J. G. Hou, H. J. Cheng, O. Takeda, H. Zhu, Angew. Chem. Int.
Ed. 2015, 54, 8480 – 8484; Angew. Chem. 2015, 127, 8600 – 8604;
b) D. S. Lee, H. J. Chen, Y. W. Chen, J. Phys. Chem. Solids 2012,
73, 661 – 669; c) H. T. Li, X. Y. Zhang, D. R. MacFarlane, Adv.
Energy Mater. 2015, 5, 1401077; d) X. Li, J. T. Chen, H. L. Li, J. T.
Li, Y. T. Xu, Y. J. Liu, J. R. Zhou, J. Nat. Gas Chem. 2011, 20,
413 – 417; e) X. Li, H. L. Liu, D. L. Luo, J. T. Li, Y. Huang, H. L.
Li, Y. P. Fang, Y. H. Xu, L. Zhu, Chem. Eng. J. 2012, 180, 151 –
158; f) J. Y. Liu, B. Garg, Y. C. Ling, Green Chem. 2011, 13,
2029 – 2031; g) Z. Y. Wang, H. C. Chou, J. C. S. Wu, D. P. Tsai, G.
Mul, Appl. Catal. A 2010, 380, 172 – 177.
1
15, 12888 – 12935.
4] a) T. E. Rosser, C. D. Windle, E. Reisner, Angew. Chem. Int. Ed.
016, 55, 7388 – 7392; Angew. Chem. 2016, 128, 7514 – 7518;
[
[
2
b) H. Takeda, H. Koizumi, K. Okamoto, O. Ishitani, Chem.
Commun. 2014, 50, 1491 – 1493.
5] a) D. H. Nam, S. K. Kuk, H. Choe, S. Lee, J. W. Ko, E. J. Son,
E. G. Choi, Y. H. Kim, C. B. Park, Green Chem. 2016, 18, 5989 –
5993; b) R. K. Yadav, G. H. Oh, N. J. Park, A. Kumar, K. J. Kong,
J. O. Baeg, J. Am. Chem. Soc. 2014, 136, 16728 – 16731; c) X. Ji,
Z. Su, P. Wang, G. Ma, S. Zhang, Small 2016, 12, 4753 – 4762.
6] a) E. M. Nichols, J. J. Gallagher, C. Liu, Y. Su, J. Resasco, Y. Yu,
Y. Sun, P. Yang, M. C. Chang, C. J. Chang, Proc. Natl. Acad. Sci.
USA 2015, 112, 11461 – 11466; b) C. Liu, J. J. Gallagher, K. K.
Sakimoto, E. M. Nichols, C. J. Chang, M. C. Chang, P. Yang,
Nano Lett. 2015, 15, 3634 – 3639; c) T. Kothe, N. Plumere, A.
Badura, M. M. Nowaczyk, D. A. Guschin, M. Rogner, W.
Schuhmann, Angew. Chem. Int. Ed. 2013, 52, 14233 – 14236;
Angew. Chem. 2013, 125, 14483 – 14486; d) P. Zhou, J. G. Yu, M.
Jaroniec, Adv. Mater. 2014, 26, 4920 – 4935.
[
[
7] a) K. Sivula, F. Le Formal, M. Gratzel, ChemSusChem 2011, 4,
432 – 449; b) J. Y. Kim, G. Magesh, D. H. Youn, J. W. Jang, J.
Kubota, K. Domen, J. S. Lee, Sci. Rep. 2013, 3, 2681; c) D. H.
Nam, G. M. Ryu, S. K. Kuk, D. S. Choi, E. J. Son, C. B. Park,
Appl. Catal. B 2016, 198, 311 – 317.
[
8] a) D. Cao, Z. Wang, Nasori, L. Wen, Y. Mi, Y. Lei, Angew. Chem.
Int. Ed. 2014, 53, 11027 – 11031; Angew. Chem. 2014, 126, 11207 –
11211; b) W. Ji, K. Yao, Y. F. Lim, Y. C. Liang, A. Suwardi, Appl.
Manuscript received: November 21, 2016
Phys. Lett. 2013, 103, 062901; c) W. Ji, K. Yao, Y. C. Liang, Adv.
Final Article published: && &&, &&&&
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biocatalytic Photosynthesis
In synergy: A tandem photoelectrochem-
ical (PEC) cell with an integrated enzyme
cascade has been developed to transfer
photogenerated electrons to a multi-
enzyme cascade for the biocatalyzed
S. K. Kuk, R. K. Singh, D. H. Nam,
R. Singh, J. K. Lee,*
C. B. Park*
&&& — &&&
reduction of CO to methanol in high
2
Photoelectrochemical Reduction of
Carbon Dioxide to Methanol through
a Highly Efficient Enzyme Cascade
yield. The approach makes use of water
as an electron donor, a hematite photo-
anode and a bismuth ferrite photo-
cathode for the regeneration of NADH
with visible light, as well as a three-
dehydrogenase cascade system.
Angew. Chem. Int. Ed. 2017, 56, 1 – 7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!