Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an electrochemical micro-reactor device

Article abstract of DOI:10.1016/j.electacta.2019.135254

This work combines an organic oxidation catalyst, 4-amino-TEMPO (TEMPO-NH₂), and a recombinant enzyme, oxalate decarboxylase (OxDc) to create a hybrid catalytic system able to catalyze complete ethanol electrooxidation. The catalyst system was coupled with a novel small scale electrolysis cell utilizing polycaprolactone (PCL) and carbon composite electrodes for bulk electrolysis. Electrochemical measurements and product detection by nuclear magnetic resonance spectroscopy (NMR) after 12 h of electrolysis have shown for the first time that an organic catalyst and decarboxylase enzyme can oxidize ethanol to carbon dioxide at acidic pH. The success presented here, coupling a hybrid organic catalyst/enzymatic system to simple carbon composites, has potential value for selective alcohol oxidation and is promising for a wide array of applications, including biosensors, environmental monitoring, and biofuel cells.

Full text of DOI:10.1016/j.electacta.2019.135254

Electrochimica Acta xxx (xxxx) xxx
Contents lists available at ScienceDirect
Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Hybrid enzymatic and organic catalyst cascade for enhanced complete
oxidation of ethanol in an electrochemical micro-reactor device
,
Jefferson Honorio Franco a b, Kevin J. Klunder b, Victoria Russell b,
,
, *
Adalgisa R. de Andrade a **, Shelley D. Minteer b
a
~
~
~
Department of Chemistry, Faculty of Philosophy Sciences and Letters at Ribeirao Preto, University of Sao Paulo, 14040-901, Ribeirao Preto, SP, Brazil
b Department of Chemistry, University of Utah, Salt Lake City, UT, 84112, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
This work combines an organic oxidation catalyst, 4-amino-TEMPO (TEMPO-NH2), and a recombinant
enzyme, oxalate decarboxylase (OxDc) to create a hybrid catalytic system able to catalyze complete
ethanol electrooxidation. The catalyst system was coupled with a novel small scale electrolysis cell
utilizing polycaprolactone (PCL) and carbon composite electrodes for bulk electrolysis. Electrochemical
measurements and product detection by nuclear magnetic resonance spectroscopy (NMR) after 12 h of
electrolysis have shown for the first time that an organic catalyst and decarboxylase enzyme can oxidize
ethanol to carbon dioxide at acidic pH. The success presented here, coupling a hybrid organic catalyst/
enzymatic system to simple carbon composites, has potential value for selective alcohol oxidation and is
promising for a wide array of applications, including biosensors, environmental monitoring, and biofuel
cells.
Received 11 October 2019
Received in revised form
3 November 2019
Accepted 8 November 2019
Available online xxx
Keywords:
Enzymatic biofuel cell
Organic catalyst
Oxalate decarboxylase
Electrochemical microfluidic devices
© 2019 Elsevier Ltd. All rights reserved.
1. Introduction
magnitude higher power densities, but can only partially oxidize
the fuel [10].
Ethanol is a popular fuel which is obtained from sugar cane,
corn, and beets, among others sources [1]. High energy density
(8 kW h kg-1), abundance, desirable physicochemical properties,
low cost and low toxicity make ethanol an excellent renewable fuel
choice to generate electricity [2]. Biological fuel cells have been
extensively reported as a efficient and sustainable device to convert
chemical energy to electrical energy [3,4]. Biofuel cells (BFCs) are
alternative energy sources which use enzymes (enzymatic biofuel
cell) or microorganisms (microbial fuel cell) as the electrocatalysts
instead of the traditional noble metal catalysts [4,5].
Microbial biofuel cells have the advantage of possessing long
lifetimes of up to five years [5]. Some microbial fuel cells [6,7] are
capable of completely oxidizing simple sugars to carbon dioxide
[8];however, these have been limited by low current and power
densities owing to slow transport across cellular membranes [9].
On the other hand, enzymatic fuel cells can possess orders of
Enzymes are biocatalysts responsible for oxidizing fuel at the
anode and reducing oxygen at the cathode to generate energy and
convert it to electricity [11]. High stability and high activity at
physiological pH at room temperature, in addition to fuel flexibility
are some of the advantages of the enzymatic fuel cell [12,13].
Enzyme cascades for complete oxidation of ethanol have recently
developed by biofuel cell researchers [14,15]. The advantage of
enzymes is that they exhibit high specificity and turnover rate
[15,16]; however, mimicing the natural metabolism from ethanol
through electrometabolic pathways is enormously difficult [17] due
to problems of stability, and a narrow optimal pH and temperature
range of the enzymes used. The high number of individual enzymes
required to achieve complete ethanol oxidation is also a limiting
factor.
To overcome the limitations of using multiple enzymes, a small
molecule organic catalyst can be employed. TEMPO (2,2,6,6-
tetramethylpiperidin-1-yl) oxyl) is a small organic catalyst that
does not present limited substrate specificity that enzymes face,
due to its ability to oxidize oxygen, nitrogen and sulfur-containing
functional groups [16]. Additionally, TEMPO exhibits favorable
catalysis in biofuel oxidation processes at room temperature and
under mild aqueous conditions [18,19]. However, TEMPO is not able
* Corresponding author.,
** Corresponding author.
E-mail addresses: ardandra@usp.br (A.R. de Andrade), minteer@chem.utah.edu
(S.D. Minteer).
https://doi.org/10.1016/j.electacta.2019.135254
0013-4686/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

2
J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
to break carbon-carbon bonds, making it impossible to achieve
complete substrate oxidation with solely the use of TEMPO [20].
Despite their individual limitations, we considered the possibility
of combining the advantages of a organic catalyst with enzymatic
oxidation in order to obtain the benefits of each catalytic motif
while minimizing the demands of complex electrooxidative cas-
cades via hybrid systems [21,22].
miniaturization of an electrolysis cell to accommodate a small
volume (z1 mL) which is beneficial for initial fundamental studies
of reaction products by nuclear magnetic resonance spectroscopy
(NMR).
2. Experimental
Recent work reported a hybrid catalyst cascade using the
organic catalyst TEMPO in the complete oxidation of fuels, such as
glycerol [16,23] and ethanol [22]. Franco et al. employed TEMPO in
order to oxidizes ethanol to acetic acid through 2 steps. Next, the
combination of TEMPO and oxalate oxidase (OxOx) transformed
acetic acid to formic acid, and finally CO2. However, the TEMPO
electrocatalyst operates best at higher pH (7e10) while the OxOx
enzyme demonstrates activity only at mildly acidic pH. To address
this issue, recent work evaluated the organic catalyst 4-amino-
TEMPO, TEMPO-NH2, and confirmed it was capable of operating
under acidic conditions that are necessary for enhancing complete
oxidation of fuels from hybrid systems [16].
However, the low specific activity of OxOx activity and the
limited substrate range of OxOx substrates hindered the hybrid
system from achieving excellent results [22,23]. To improve this
cascade with OxOx, Minteer et al. demonstrated the use of a hybrid
catalytic system combining TEMPO-NH2 (an organic oxidation
catalyst) with oxalate decarboxylase (OxDc) from Bacillus subtilis,
another recombinant enzyme, for the complete electrocatalytic
oxidation of glycerol [23]. OxDc and can be expressed easily with
E. coli, while OxOx requires a eukaryote system [24,25]. Addition-
ally, OxDc provides advantages in terms of cost, specific activity,
while OxOx requires a eukaryote system [24,25].
In this work, we employed a hybrid system combining the
organic oxidation catalyst, 4-amino-TEMPO (TEMPO-NH2), and an
enzyme, oxalate decarboxylase (OxDc) to complete electrochemical
oxidation of ethanol to CO2, while collecting up to 12 electrons per
ethanol molecule (Scheme 1). The device used to perform the full
oxidation was a bulk electrolysis micro-reactor which was an
adaptation from a previous report [26].
The new electrolysis cell design is composed of simple graphite
and thermoplastic materials used for commercial electrolysis/fuel
flow cells. The unique fabrication method allows for
2.1. Chemicals
4-amino-TEMPO (free radical), ethanol, acetaldehyde, acetic
acid, and formic acid were purchased from Sigma Aldrich, while
ethanol (2e13C, 99%) was purchased from Cambridge Isotope
Laboratories, and these reagents were used as received without
additional purification. 150 mM citric acid-phosphate buffers
(pH ¼ 5.2) and 200 mM phosphate buffered saline (pH ¼ 5.7). Ox-
alate decarboxylase (OxDc) was also expressed and purified in the
lab (vide infra) and stored in ꢀ80 ꢁC until use. Water purified using a
Millipore Milli-Q system was used to prepare all solutions. All
chemicals were used as they were received without further
purification.
2.2. Expression and purification of the enzyme oxalate
decarboxylase (OxDc) from Bacillus subtilis
Oxalate decarboxylase (OxDc) expression and purification was
adapted from a previously reported method [23]. The plasmid pET-
9c-OxDc was used to transform the strain E. coli BL21(DE3). A
starter culture was grown overnight at 37 ꢁC in LB broth in the
presence of 100 m
g mLꢀ1 kanamycin and chloramphenicol, and then
was used to inoculate 6 L of LB broth. The inoculated culture was
grown at 37 ꢁC and 220 rpm until an OD600nm value of 0.5 was
reached. The cells were heatshocked at 42 ꢁC for 15 min and then
induced for expression by adding 1 mM IPTG and 5 mM MnCl2. The
induced cells were incubated for 4 h at 30 ꢁC and shaken at
220 rpm. The cells were then collected by centrifugation followed
by resuspension in 50 mM Tris-HCl buffer (pH 7.0); these were then
disrupted using a microfluidizer. After centrifugation, the soluble
fraction of the cell lysate was applied to a Q-Sepharose Fast Flow
column (1 ꢂ 25 cm) that had been equilibrated with 50 mM Tris-
HCl (pH 7.0). Elution was performed using a 400-mL linear
gradient from 0 to 1 M NaCl. The fractions that contained purified
OxDc were combined and concentrated by ultracentrifugation in an
Amicon centrifugal filter. The OxDc solution was then run through
an FPLC desalting column (HiPrep desalting column, 15 mL, GE
Healthcare) and stored at ꢀ80 ꢁC. A protein concentration of
55.4 mg mLꢀ1 was measured with Pierce™ BCA protein assay kit
(Life Technologies™, Carlbad, CA).
2.3. Enzyme activity assays
OxDc activity in solution was evaluated by indirect enzymatic
UVeVis spectrophotometric assay using a 96-well plate and the
Synergy HTX Multi-Mode Reader (BioTek). OxDc (60
in 100 L mixture containing different concentrations of oxalate
(from 0 to 50 mM) in 150 mM phosphate buffer (pH 4.0). After
mg) was added
m
5 min, the reactions were quenched with 140 mL of 0.2 M K2HPO4
followed by the addition of 5 mM NADþ. The levels of produced
formic acid were established in a coupled assay with the addition of
formate dehydrogenase and the absorbance was recorded at
340 nm (ε ¼ 6220 Mꢀ1 cmꢀ1). The formate concentration was
quantified by the Michaelis-Menten equation containing Km and
Vm of the formate dehydrogenase obtained in the same condition of
this essay. The obtained oxalate decarboxylase extract was deter-
mined to have a specific activity of 11.74 U/mg (mmol of substrate
converted per minute per milligram of enzyme).
Scheme 1. Electrocatalytic oxidation cascade of ethanol by TEMPO-NH2/OxDc hybrid
system. The blue line represents the oxidation mediated by TEMPO-NH2, while
decarboxylation reaction catalyzed by OxDc is represented by red lines.
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
3
2.4. Electrochemical measurements
2.5. Ethanol oxidation cascade and CO2 detection
A CH Instruments 660 E Electrochemical Workstation combined
with a standard three-electrode cell was used for electrochemical
experiments. Cyclic voltammetry (CV) experiments were per-
formed using a 3 mm glassy carbon (GC) as a working electrode, a
saturated calomel (SCE) reference electrode, and a Pt mesh counter
electrode. CV experiments were performed at a scan rate of
Bulk electrolysis experiments were performed using a SCE
reference electrode and a custom micro-reactor with integrated
working and counter electrode (Fig. S1). The fabrication of the
microfluidic cell and the polycaprolactone (PCL) carbon composite
electrode was described by Klunder et al. [26]. The 12-h ethanol
oxidation cascade was performed by bulk electrolysis Eap ¼ 0.8 V
(vs. SCE) using a small cell containing 30 mM 13C2-ethanol, 50 mM
TEMPO-NH2, in presence of 20 U mLꢀ1 OxDc with 200 mM phos-
phate buffered saline (pH ¼ 5.7), at 25 ꢁC. The oxidation cascade
was run using a potential of 0.8 V (vs. SCE) for 12 h.
Carbon dioxide (CO2) after bulk electrolysis was detected via
NMR. 13C NMR analysis was performed on a 400 MHz NMR. The
NaOH was used to capture 13C-enriched CO2 through the formation
of Na2CO3 þ H2O, which was detected by 13C NMR (D2O). Samples
were analyzed using D2O at 25 ꢁC. All of the analyses were carried
out in triplicate. Commercial standards were used to identify the
products formed in the reaction.
10 mV sꢀ1
, a step potential of 0.001 V, a potential range of
0.00e1.0 V (vs. SCE), and 25 ꢁC unless otherwise noted.
Unless otherwise stated, all amperometric titrations were car-
ried out at a fixed oxidative potential 0.8 V vs. SCE using constant
potential amperometry in a 150-mM citric acid-phosphate buffer
(pH ¼ 5.2) with consecutive additions of substrate.
Power and current density measurements were conducted in a
two-compartment cell separated by a Nafion® membrane. The
cathode counterpart in direct contact with air is separated from the
aqueous anodic chamber using a gas diffusion membrane (ELAT)
composed of 20% platinum on Vulcan XC-72 which is hot-pressed
in a Nafion® 212 membrane. The cell compartment volume of
10 mL was filled with 150 mM citric acid-phosphate buffer
(pH ¼ 5.2) containing 100 mM ethanol. The open-circuit potential
of the cell was monitored for 1 h until it stabilized. Linear polari-
zation was performed starting from OCP to 0 V at 1 mV sꢀ1. The
obtained data was used to calculate power density. All results
presented are based on triplicate samples. Uncertainties corre-
spond to one standard deviation.
3. Results and discussion
3.1. Bioelectro-oxidation of ethanol experiments at hybrid
bioanodes
In order to evaluate the electrochemical properties of the hybrid
system TEMPO-NH2/OxDc, ethanol electro-oxidation was investi-
gated. Fig. 1 shows the cyclic voltammetric (CV) profile of the
Fig. 1. Catalytic cyclic voltammetric (CV) curves of 5 mM TEMPO-NH2 in the presence of 20 U mLꢀ1 OxDc (red line). CVs were performed in the absence of any substrate (red line)
and in the presence (black line) of 100 mM ethanol (A), acetaldehyde (B), acetic acid (C) and formic acid (D). Supporting electrolyte: 150-mM citric acid-phosphate buffer (pH ¼ 5.2),
potential range of 0.00e1.0 V (vs. SCE),
article.)
n
¼ 10 mVsꢀ1, and 25 ꢁC. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

4
J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
TEMPO-NH2/OxDc electrode. The black line represents the vol-
tammetric response in the absence of substrate (only hybrid system
in buffer) and the red line represents in the presence of 50 mM of
ethanol (A), acetaldehyde (B), acetic acid (C), and formic acid (D).
Fig. S2 presents the CV for the bare electrode in the presence of
buffer (dashed line) and with TEMPO-NH2 (black line).
First, the electrocatalytic activity of hybrid system was tested
against ethanol, acetaldehyde, acetic acid and formic acid (Fig. 1).
The maximum current density generated a substantial increase in
the presence of ethanol (0.56 mA cmꢀ2), acetaldehyde
(0.34 mA cmꢀ2), acetic acid (0.42 mA cmꢀ2), and formic acid
(0.51 mA cmꢀ2) at 0.8 V vs. SCE, which confirmed that TEMPO-NH2/
OxDc has the capability to readily oxidize several key intermediates
in the multi-step oxidation of ethanol.
Additionally, it was observed that TEMPO-NH2 alone is not able
to react with acetic acid (Fig. S3). The use of the OxDc enzyme is
required to complete this step of the cascade reaction. This finding
is confirmed by Franco et al. who describe the oxidation of alcohols
and aldehydes using TEMPO-mediated electrocatalysis [15,22]. The
oxidative current densities by cyclic voltammetry (CV) for the
system containing only OxDc did not show catalytic activity to-
wards ethanol, acetaldehyde and formic acid (Fig. S4). These results
indicate that OxDc does not catalyze the oxidation of these sub-
strates. We demonstrated the use of recombinant OxDc with acetic
acid (Fig. S4) and confirmed the capacity of OxDc enzyme to cata-
lyze the cleavage of the carbon-carbon bond in acetic acid to pro-
duce formic acid. Thus, we conclude that the observed increase in
anodic current density is the result of enzyme activity rather than
the activity of the TEMPO-NH2 catalyst.
significantly increased the anode catalytic activity, and was able to
collect more electrons from ethanol.
An oxidation potential of 0.8 V (vs. SCE) which is 50 mV above
the peak oxidation potential was used in chronoamperometric
studies that were undertaken with the prepared hybrid system.
Concentrations of ethanol, acetaldehyde, acetic acid, and formic
acid in the electrolyte solution were gradually increased. Repre-
sentative amperometric titration response curves for sequential
additions of ethanol, acetaldehyde, acetic acid, and formic acid (A-
D, respectively) are shown in Fig. 2.
The titration curves generated maximum current densities of
0.88 mA cmꢀ2 for acetaldehyde, 1.02 mA cmꢀ2 for acetic acid, and
1.13 mA cmꢀ2 for formic acid. The highest catalytic current density
of 1.45 mA cmꢀ2 was observed in the presence of ethanol for con-
centrations ranging between 0 and 140 mM. The high catalytic
activity of TEMPO-NH2 toward primary alcohols is demonstrated by
these results.
In addition, the amperometric responses of the hybrid system
with all substrates showed the current increased linearly with
substrate concentration until it reached a steady state, indicating
that the system has reached saturation.
Fig. S5 depicts a representative chronoamperometric assay of a
TEMPO-NH2 prepared bioanode in buffer containing acetic acid, it
is observed that the organic catalyst TEMPO-NH2 alone cannot
perform acetic acid oxidation. In contrast, Fig. S6 shows the same
assay performed in the presence of OxDc at 0.8 V (vs. SCE) with each
of the four substrates/intermediates; only acetic acid demonstrates
a catalytic response while ethanol, acetaldehyde, and formic acid
do not.
The CV data clearly indicated that the hybrid system
Successive additions of different ethanol concentrations,
Fig. 2. Chronoamperometric assay performed with TEMPO-NH2/OxDc with sequential addition of 10 mM ethanol, acetaldehyde, acetic acid, and formic acid (A-D, respectively).
Eap ¼ 0.8 V vs. SCE. 150-mM citric acid-phosphate buffer (pH ¼ 5.2) was used as the supporting electrolyte.
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
5
acetaldehyde and formic acid to the enzymatic system did not
cause an increase in the current output. However, consecutive ad-
ditions of acetic acid lead to consecutive increases in Jmax with an
observed current density maximum of 0.85 mA cmꢀ2; these results
demonstrate the ability of the OxDc biocatalyst to cleave the CeC
bond of acetic acid. Therefore, a noticeable increase occurs in cat-
alytic current densities of the hybrid system in the presence of all
substrates when compared to the systems containing only TEMPO
or enzyme, confirming that their simultaneous presence has a
synergistic interaction to oxidize these substrates and complete the
total oxidation of ethanol.
Since current is a direct measurement of reaction rate then
based on cyclic voltammetry and chronoamperometry data, the
rate limiting oxidation step can be investigated. It can be seen from
Fig. S4 that the enzyme modified electrode had the lowest current
density (0.9 mA cmꢀ2 at 0.8 V vs. SCE) in comparison to the other
substrates. Direct electron transfer (DET) from the enzyme is
limited by enzyme orientation to the electrode surface and ability
to shuttle electrons to the enzyme active site. Overall, it was the
main thrust of this work to demonstrate the complete oxidation of
ethanol. In order to increase cell efficiencies, enzyme activity
should be enhanced. Common methods to enhance active are to
synergistically pair the enzyme with specialized polymers which
enhance DET or increase the surface area of the electrode with
porous nanocarbon [20].
Fig. 1B shows that the oxidation of acetaldehyde had the second
lowest current density by cyclic voltammetry. Conversion of alde-
hyde to carboxylic acid moieties have been reported to have vary-
ing kinetics based on substrate structure, with simple aliphatic
aldehydes having slow turnover numbers at low pH [27]. Perhaps a
combination of low pH and the fact that the oxidation product,
acetic acid, is a determining factor for the diminished current with
acetaldehyde relative to the other substrates. Based on the current
density shown in Figs. 1, 2, and S4, it is concluded that the sub-
strates with more favorable oxidation kinetics under acidic condi-
tions and the unique catalytic system are formic
acid > acetaldehyde > acetic acid.
Fig. 3. (A) Pieces of the micro-reactor (B) Picture of PCL/graphite system used for
electrolysis.
3.2. Bulk electrolysis and product identification by NMR
The long-term electrolysis of ethanol was performed at 0.8 V vs.
SCE for 12 h and the products were then characterized using C13-
NMR to detect the possible products formed. We chose to
develop a small volume (z1 mL) electrolysis cell (Fig. 3) specifically
for NMR detection, which was constructed out of poly-methyl
methacrylate (PMMA) with a polycaprolactone/graphite compos-
ite electrode. Polycaprolatone (PCL) is used in medical devices and
is biologically compatible [28]. A traditional CO2 laser was used to
cut out the individual pieces, and the PCL/graphite composite was
then hot molded into the templates [26]. The composition of the
PCL/graphite composite is analogous to that of bipolar plates which
are used in commercial fuel cells [29].
It is important to emphasize that the optimal ratio between 20
U mLꢀ1 OxDc (55.4 mg mLꢀ1), and 5 mM 4-Amino-TEMPO
(8.6 mg mLꢀ1) concentration in the reactor was 6.4, ie the enzyme
concentration is 6.4 times higher than the organic catalyst con-
centration. The organic catalyst has a much higher catalytic activity
than the enzyme, acting on the oxidation of OH group-containing
compounds, while the enzyme is more specific and only acts on a
specific substrate. Then, to balance the activities of both catalysts, 6
times more enzyme was added to the solution.
The profile for ethanol bioelectro-oxidation after bulk electrol-
ysis (12 h) employing the PCL/graphite micro-reactor are described
in Fig. 4. The Fig. 4 indicated that the catalytic activity of the hybrid
system (red line) showed that the amount of current density
Fig. 4. Current versus time measurement for 30 mM 13C2-ethanol during long-term
electrolysis employing the system containing only TEMPO-NH2 (blue line) and in the
presence of a hybrid system TEMPO-NH2/OxDc (red line). The control sample (black
line) was performed, in the absence of TEMPO-NH2 and OxDc, in the presence of buffer
only. Eap ¼ 0.8 V (vs. SCE). Supporting electrolyte: 200 mM phosphate buffered saline
(pH ¼ 5.7). (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

6
J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
generated during the 12 h of electrolysis was two times higher
compared to the system containing only TEMPO-NH2 (blue line).
Therefore, the hybrid system showed that the TEMPO-NH2 and
OxDc act together to enhance the amount of ethanol oxidized. The
catalytic activity generated by the control sample - black line
(absence of TEMPO-NH2 and OxDc) was much lower when
compared to the current density value of the system in the pres-
ence of organic catalyst and/or enzyme, suggesting that both are
active in the ethanol oxidation.
solution, and carbon-labeled substrates, intermediates, and prod-
ucts were observed using 13C NMR. Additionally, all samples
analyzed have no peaks related to the ethanol oxidation at t ¼ 0 h.
Fig. 5A shows that the enzyme only system had no peaks
Acetic acid electrolysis was conducted with TEMPO-NH2
(Fig. S7) and OxDc (Fig. S8). As expected, no current density value
was observed for the TEMPO-NH2 electrode after electrolysis for
12 h with acetic acid, which indicated that organic catalyst is not
able to cleave carbon-carbon bonds, acting only in oxidation of
primary alcohol. We performed a long-term electrolysis for the
enzymatic system (Fig. S8) in the presence of acetic acid (green
line) and showed an outstanding electrocatalytic activity toward
acetic acid electrooxidation, confirming the action of the oxalate
decarboxylase to break the carbon-carbon bond from acetic acid.
Finally, the work here utilized PCL as a plastic binder material.
While PCL has excellent biocompatibility, it is unclear if any
leaching or degradation of the material may have an effect on the
cell performance/stability. The choice of polymer binder as well as
purity of the carbon should be a consideration when constructing
devices for long-term electrolysis (days-years).
NMR experiments were performed to identify the products
formed after 12 h ethanol oxidation by bulk electrolysis. To
demonstrate the ability of TEMPO-NH2 with OxDc to completely
oxidize ethanol, 13C-labeled ethanol was used as a substrate; the
produced 13CO2 was converted to Na123CO3 using a 0.1 M NaOH
Fig. 6. Ethanol/O2 biofuel cell power density curves obtained for different systems
employed: hybrid system TEMPO-NH2/OxDc (black line), only with organic catalyst
TEMPO-NH2 (dashed line) and only with OxDc enzyme (red line) before bulk elec-
trolysis (12 h). Supporting electrolyte: 150-mM citric acid-phosphate buffer (pH ¼ 5.2),
and 100 mM ethanol. (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)
Fig. 5. 13C NMR spectrum of OxDc system (A), and TEMPO-NH2 system (B) from 13C2-labeled ethanol after 12 h of electrolysis. (C) 13C NMR spectrum of NaOH exposed to ambient
conditions for 12 h as a control, and (D) the product of the complete oxidation of 13C2-labeled ethanol by TEMPO-NH2 and OxDc, 13CO2, trapped in the form of Na123CO3 by reaction
with NaOH.
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
7
Table 1
Evaluation of power density values obtained for all systems analyzed before and after 12 h of electrolysis and 30 days of storage in a refrigerator. Conditions used: 100 mM
ethanol; 150 mM pH 5.2 citric acid-phosphate buffer; 12 h of total electrolysis time; Eap of 0.8 V (vs. SCE).
Power density
W cmꢀ2
Before Electrolysis
Power density
W cmꢀ2
After Electrolysis
Power density (
After 15 days
m
W cmꢀ2
)
Power density
W cmꢀ2
After 30 days
Power density
stability compared
after 30 days (%)
(m
)
(
m
)
(m
)
Enzymatic (OxDc) system stability
TEMPO-NH2 system stability
TEMPO-NH2/OxDc
49
59
78
2
3
5
27
32 28
69
1
21
29
63
1
2
5
15
25
60
1
2
4
30
43
77
2
3
6
4
Hybrid system stability
indicating oxidation products after reaction with ethanol. In the
system containing TEMPO-NH2 only (Fig. 5B), a peak at 30.1 ppm
and 200.5 ppm was attributed to acetaldehyde. The acetic acid was
detected at 20.7 ppm and 177.2 ppm. These results obtained by
NMR demonstrated the action of TEMPO-NH2 to oxidize the first
two catabolic steps, which involve 4 eꢀ in the formation of acetic
acid (Scheme 1).
Control experiments were also performed with NaOH in the
presence of phosphate buffered saline (pH ¼ 5.7), which confirmed
that NaOH does not oxidize the buffer, i.e., no product was formed
(Fig. 5C). Finally, a significant peak at 168 ppm (Fig. 5D) indicates
that the hybrid system was able to produce 13CO2 from the cascade
oxidation of 13C-ethanol. The formation of acetaldehyde was
observed, but not acetic acid. Probably, the acetic acid formed by
the action of the TEMPO-NH2 system was converted to CO2 since
OxDc acts on and cleaves the carbon-carbon bond in acetic acid.
A reaction scheme for the complete oxidation is proposed in
Scheme 1 were TEMPO-NH2 catalyzes the first two oxidation steps
of ethanol to acetic acid (collecting up to 4 eꢀ), followed by OxDc-
catalyzed decarboxylation of acetic acid by cleavage of a carbon-
carbon bond to generate formate. Thus, due to extreme potential
or TEMPO-NH2, the formate is oxidized easily to CO2, enabling the
collection of an additional 2 eꢀ.
system when compared with the others systems is explained by
NMR data, which showed that this configuration was able to uptake
the maximum energy possible from ethanol, e.g., 12 electrons
producing CO2.
Comparison of the power density yielded by the hybrid system
with the system containing organic catalyst or enzymes only,
showed significant improvement in bioanode performance. The
increase in the number of electrons (12 electrons) exchanged in the
complete oxidation of ethanol is likely the reason for the increased
performance of the organic catalyst/enzyme system [14,30].
Notably, the developed system herein is in accordance with the
literature and possesses a high enough energy density to be suit-
able for applications in small portable devices [31].
The evaluation and comparison of power density values ob-
tained during storage in a refrigerator for 30 days and after bulk
electrolysis for all systems analyzed, as shown in Table 1. The hybrid
system presented a good stability after 12 h of electrolysis and 30
days of storage, with 89 and 77% power retention, respectively. In
contrast, the stability of the individual TEMPO-NH2 and enzymatic
systems after 12 h electrolysis is much lower compared with the
hybrid system. Indeed, TEMPO-NH2 (25
2 m
W cmꢀ2) and OxDc
(15
1
m
W cmꢀ2) system showed low stability after 30 days of
storage, indicating 30 and 43% loss in the power density, respec-
tively. Thus, the hybrid bioanode enables high stability when the
organic catalyst and enzyme are combined in the system.
3.3. Power density tests
4. Conclusions
Following the cyclic voltammetry and the chronoamperometry
assays, polarization tests for the systems investigated were per-
formed. Fig. 6 displays representative power density curves of
complete biofuel cells with freshly prepared bioanodes containing
TEMPO-NH2/OxDc (black line), only with organic catalyst TEMPO-
NH2 (dashed line) and only with OxDc enzyme (red line) before
bulk electrolysis (12 h). The goal of the power density tests is to
verify and compare the reproducibility before electrolysis and the
stability after bulk electrolysis of the each system analyzed. A
In this investigation, TEMPO-NH2 organic catalyst and OxDc
enzyme were shown to synergystically promote a compete oxida-
tion of ethanol. The hybrid catalytic design enhances the overall
steps in the cascade operating at an optimum pH for both the
organic catalyst and the enzyme.To validate the reaction sequence,
a novel micro-reactor device enabled low-cost product analysis and
allows for high analyte sensitivity.Overall, this work demonstrated
that bi-cascade of TEMPO-NH2 and OxDc can be combined in an
electrochemical cell to synergistically catalyze the oxidation of
substrates that make up the steps of the ethanol oxidation cascade
and enable enhanced electrochemical oxidation of ethanol to CO2.
maximum current density of 147
8 m
A cmꢀ2, power density of
49
2
m
W cmꢀ2, and open circuit potential (OCP) of 0.394 V (vs.
SCE) are obtained for bioanodes which were prepared with only the
enzyme. Those prepared with only TEMPO-NH2 demonstrated
316 10
m
A cmꢀ2, 59
3 m
W cmꢀ2, and 0.440 V for the maximum
current density, maximum power density, and OCP, respectively.
Finally, those with both TEMPO-NH2 and OxDc demonstrated a
maximum current density, maximum power density, and OCP of
Declaration of competing interest
The authors declare no competing financial interest.
353 15
m
A cmꢀ2 and 78
5 m
W cmꢀ2, and 0.468 V, respectively.
Thus, the higher the power density value of a system, more energy
must be generated and consequently more electrons will be
collected from the ethanol molecule.
Acknowledgements
The authors are grateful for the financial support from Brazilian
research funding agencies FAPESP (2017/20431e7) and Coor-
The data obtained from the biofuel cell test showed the same
behavior observed by CV and chronoamperometry results, con-
firming once again the enhanced performance of the hybrid bio-
anode against the systems with only TEMPO-NH2 or OxDc.
Futhermore, the highest power density attributed to the hybrid
~
denaçao de Aperfeiçoamento de Pessoal de Nível Superior Brasil
(CAPES) Finance Code 001, as well as the Army Research Office
MURI (W911NF-14-1-0263).
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254

8
J.H. Franco et al. / Electrochimica Acta xxx (xxxx) xxx
ja5098379.
Appendix A. Supplementary data
[17] S. Aquino Neto, S.D. Minteer, A.R. de Andrade, Developing ethanol bioanodes
using hydrophobically modified linear polyethylenimine hydrogel for
a
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.electacta.2019.135254.
immobilizing an enzyme cascade, J. Electroanal. Chem. (2017), https://doi.org/
10.1016/j.jelechem.2017.09.001.
ꢀ
[18] A. Dijksman, A. Marino-Gonzalez, A. Mairata
i Payeras, I.W.C.E. Arends,
R.A. Sheldon, Efficient and selective aerobic oxidation of alcohols into alde-
hydes and ketones using ruthenium/TEMPO as the catalytic system, J. Am.
Chem. Soc. 123 (2001) 6826e6833, https://doi.org/10.1021/ja0103804.
[19] L. De Luca, G. Giacomelli, S. Masala, A. Porcheddu, Trichloroisocyanuric/
TEMPO oxidation of alcohols under mild conditions: a close investigation,
J. Org. Chem. 68 (2003) 4999e5001, https://doi.org/10.1021/jo034276b.
[20] D.P. Hickey, R.D. Milton, D. Chen, M.S. Sigman, S.D. Minteer, TEMPO-modified
linear poly(ethylenimine) for immobilization-enhanced electrocatalytic
oxidation of alcohols, ACS Catal. 5 (2015) 5519e5524, https://doi.org/10.1021/
acscatal.5b01668.
[21] F.C. Macazo, D.P. Hickey, S. Abdellaoui, M.S. Sigman, S.D. Minteer, Polymer-
immobilized, hybrid multi-catalyst architecture for enhanced electrochemical
oxidation of glycerol, Chem. Commun. 53 (2017) 10310e10313, https://
doi.org/10.1039/C7CC05724E.
[22] J.H. Franco, P.Z. de Almeida, S. Abdellaoui, D.P. Hickey, P. Ciancaglini, M. de
Lourdes, T.M. Polizeli, S.D. Minteer, A.R. de Andrade, Bioinspired architecture
References
[1] Y. Yan, I. Baravik, R. Tel-Vered, I. Willner, An ethanol/O2 biofuel cell based on
an electropolymerized bilirubin oxidase/Pt nanoparticle bioelectrocatalytic
O2-reduction cathode, Adv. Mater. 21 (2009) 4275e4279, https://doi.org/
10.1002/adma.200900206.
[2] N.L. Akers, C.M. Moore, S.D. Minteer, Development of alcohol/O2 biofuel cells
using salt-extracted tetrabutylammonium bromide/Nafion membranes to
immobilize dehydrogenase enzymes, Electrochim. Acta 50 (2005) 2521e2525,
https://doi.org/10.1016/j.electacta.2004.10.080.
[3] O. Ellabban, H. Abu-Rub, F. Blaabjerg, Renewable energy resources: current
status, future prospects and their enabling technology, Renew. Sustain. Energy
Rev. 39 (2014) 748e764, https://doi.org/10.1016/j.rser.2014.07.113.
[4] S. Aquino Neto, A.R. De Andrade, New energy sources: the enzymatic biofuel
cell, J. Braz. Chem. Soc. 24 (2013) 1891e1912, https://doi.org/10.5935/0103-
5053.20130261.
of
a hybrid bifunctional enzymatic/organic electrocatalyst for complete
[5] M. Rasmussen, S. Abdellaoui, S.D. Minteer, Enzymatic biofuel cells: 30 years of
critical advancements, Biosens. Bioelectron. 76 (2016) 91e102, https://
doi.org/10.1016/j.bios.2015.06.029.
ethanol oxidation, Bioelectrochemistry 130 (2019) 107331, https://doi.org/
10.1016/j.bioelechem.2019.107331.
[23] S. Abdellaoui, D.P. Hickey, A.R. Stephens, S.D. Minteer, Recombinant oxalate
decarboxylase: enhancement of a hybrid catalytic cascade for the complete
electro-oxidation of glycerol, Chem. Commun. 51 (2015) 14330e14333,
https://doi.org/10.1039/C5CC06131H.
[24] P. Moussatche, A. Angerhofer, W. Imaram, E. Hoffer, K. Uberto, C. Brooks,
C. Bruce, D. Sledge, N.G.J. Richards, E.W. Moomaw, Characterization of Cer-
iporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris,
Arch. Biochem. Biophys. 509 (2011) 100e107, https://doi.org/10.1016/
j.abb.2011.02.022.
[25] M.M. Whittaker, J.W. Whittaker, Expression of recombinant galactose oxidase
by Pichia pastoris, Protein Expr. Purif. 20 (2000) 105e111, https://doi.org/
10.1006/prep.2000.1287.
[6] H. Moon, I.S. Chang, B.H. Kim, Continuous electricity production from artificial
wastewater using a mediator-less microbial fuel cell, Bioresour. Technol. 97
(2006) 621e627, https://doi.org/10.1016/j.biortech.2005.03.027.
[7] B.H. Kim, I.S. Chang, G. Cheol Gil, H.S. Park, H.J. Kim, Novel BOD (biological
oxygen demand) sensor using mediator-less microbial fuel cell, Biotechnol.
Lett. 25 (2003) 541e545, https://doi.org/10.1023/A:1022891231369.
[8] D.R. Bond, D.R. Lovley, Evidence for involvement of an electron shuttle in
electricity generation by Geothrix fermentans, Appl. Environ. Microbiol. 71
(2005) 2186e2189, https://doi.org/10.1128/AEM.71.4.2186-2189.2005.
[9] G.T.R. Palmore, G.M. Whitesides, Microbial and enzymatic biofuel cells, in:
Enzymatic Conversion of Biomass for Fuels Production, American Chemical
Society, 1994, pp. 14e271, https://doi.org/10.1021/bk-1994-0566.ch014.
[10] S.C. Barton, J. Gallaway, P. Atanassov, Enzymatic biofuel cells for implantable
and microscale devices, Chem. Rev. 104 (2004) 4867e4886, https://doi.org/
10.1021/cr020719k.
[26] K.J. Klunder, K.M. Clark, C. McCord, K.E. Berg, S.D. Minteer, C.S. Henry, Poly-
caprolactone-enabled sealing and carbon composite electrode integration into
electrochemical microfluidics, Lab Chip 19 (2019) 2589e2597, https://doi.org/
10.1039/C9LC00417C.
[11] R.A. Bullen, T.C. Arnot, J.B. Lakeman, F.C. Walsh, Biofuel cells and their
development, Biosens. Bioelectron. 21 (2006) 2015e2045, https://doi.org/
10.1016/j.bios.2006.01.030.
[12] F. Gouranlou, H. Ghourchian, Enhancement of ethanoleoxygen biofuel cell
output using a CNT based nano-composite as bioanode, Biosens. Bioelectron.
78 (2016) 337e343, https://doi.org/10.1016/j.bios.2015.11.064.
[13] A.K. Sarma, P. Vatsyayan, P. Goswami, S.D. Minteer, Recent advances in ma-
terial science for developing enzyme electrodes, Biosens. Bioelectron. 24
(2009) 2313e2322, https://doi.org/10.1016/j.bios.2008.09.026.
[14] J.H. Franco, S.D. Minteer, A.R. de Andrade, Product analysis of operating an
ethanol/O 2 biofuel cell shows the Synergy between enzymes within an
enzymatic cascade, J. Electrochem. Soc. 165 (2018) H575eH579, https://
doi.org/10.1149/2.0931809jes.
[15] J.H. Franco, S.A. Neto, D.P. Hickey, S.D. Minteer, A.R. de Andrade, Hybrid
catalyst cascade architecture enhancement for complete ethanol electro-
chemical oxidation, Biosens. Bioelectron. 121 (2018) 281e286, https://
doi.org/10.1016/j.bios.2018.09.011.
[16] D.P. Hickey, M.S. McCammant, F. Giroud, M.S. Sigman, S.D. Minteer, Hybrid
enzymatic and organic electrocatalytic cascade for the complete oxidation of
glycerol, J. Am. Chem. Soc. 136 (2014) 15917e15920, https://doi.org/10.1021/
[27] M. Rafiee, Z.M. Konz, M.D. Graaf, H.F. Koolman, S.S. Stahl, Electrochemical
oxidation of alcohols and aldehydes to carboxylic acids catalyzed by 4-
acetamido-TEMPO: an alternative to “anelli” and “pinnick” oxidations, ACS
Catal. 8 (2018) 6738e6744, https://doi.org/10.1021/acscatal.8b01640.
[28] M.A. Woodruff, D.W. Hutmacher, The return of
a forgotten polymer-
dpolycaprolactone in the 21st century, Prog. Polym. Sci. 35 (2010)
1217e1256, https://doi.org/10.1016/j.progpolymsci.2010.04.002.
[29] A. Hermann, T. Chaudhuri, P. Spagnol, Bipolar plates for PEM fuel cells: a
review, Int. J. Hydrogen Energy 30 (2005) 1297e1302, https://doi.org/
10.1016/j.ijhydene.2005.04.016.
[30] S. Aquino Neto, J.C. Forti, V. Zucolotto, P. Ciancaglini, A.R. de Andrade,
Development of nanostructured bioanodes containing dendrimers and de-
hydrogenases enzymes for application in ethanol biofuel cells, Biosens. Bio-
electron. 26 (2011) 2922e2926, https://doi.org/10.1016/j.bios.2010.11.038.
[31] S. Aquino Neto, E.L. Suda, S. Xu, M.T. Meredith, A.R. De Andrade, S.D. Minteer,
Direct electron transfer-based bioanodes for ethanol biofuel cells using PQQ-
dependent alcohol and aldehyde dehydrogenases, Electrochim. Acta 87
(2013) 323e329, https://doi.org/10.1016/j.electacta.2012.09.052.
Please cite this article as: J.H. Franco et al., Hybrid enzymatic and organic catalyst cascade for enhanced complete oxidation of ethanol in an
electrochemical micro-reactor device, Electrochimica Acta, https://doi.org/10.1016/j.electacta.2019.135254