Electroreduction of O2 to water on the wired laccase cathode

Article abstract of DOI:10.1021/jp012488b

Oxygen was electrocatalytically reduced to water at a current density of 5 mA/cm² and at +0.7 V (NHE) in pH 5 citrate buffer at 37.5 ?°C. The electrocatalyst was a composite of laccase cross-linked with a redox polymer on a hydrophilic cloth of 10 ??m diameter carbon fibers. The redox polymer, PVI-Os(tpy)(dme-bpy)^2+/3+, [poly-N-vinyl imidazole with 1/5^th of the imidazoles complexed with [Os(tpy)(dme-bpy)]^2+/3+ (tpy=terpyridine; dme-bpy= 4,4a?2-dimethyl-2,2a?2-bipyridine)], electrically connected ( wired ) the laccase reaction centers to the fibers.

Full text of DOI:10.1021/jp012488b

J. Phys. Chem. B 2001, 105, 11917-11921
11917
Electroreduction of O2 to Water on the “Wired” Laccase Cathode†
Scott Calabrese Barton, Hyug-Han Kim, Gary Binyamin, Yongchao Zhang, and Adam Heller*
Department of Chemical Engineering and the Texas Materials Institute, The UniVersity of Texas,
Austin, Texas 78712
ReceiVed: July 2, 2001; In Final Form: August 27, 2001
2
Oxygen was electrocatalytically reduced to water at a current density of 5 mA/cm and at +0.7 V (NHE) in
pH 5 citrate buffer at 37.5 °C. The electrocatalyst was a composite of laccase cross-linked with a redox
polymer on a hydrophilic cloth of 10 µm diameter carbon fibers. The redox polymer, PVI-Os(tpy)(dme-
bpy)2+/3+
th
2+/3+
, [poly-N-vinyl imidazole with 1/5 of the imidazoles complexed with [Os(tpy)(dme-bpy)]
(
tpy)terpyridine; dme-bpy) 4,4′-dimethyl-2,2′-bipyridine)], electrically connected (“wired”) the laccase
reaction centers to the fibers.
2
3
Introduction
but the current densities were less than 50 µA/cm . Santucci
et al. studied the heterogeneous electron transfer to Rhus laccase
and ascorbate oxidase on gold, showing that trapping of the
enzymes in a cross-linked tributylmethyl phosphonium chloride-
modified polystyrene membrane enhanced the O2 electroreduc-
The electroreduction of O2 to water near neutral pH and at
ambient temperature has been one of the longest-standing and
best-researched problems of electrochemistry. It has been a
missing element of a future biofuel cell with a noncorrosive
electrolyte and operating near ambient temperature. The pres-
ently used platinum group metal-based cathodes of sub-100 °C
H2/O2 fuel cells operate near pH 0 and the silver-activated
carbon cathodes of ambient-temperature zinc-air batteries oper-
ate at pH 14. In contrast, the laccase-based cathode of this paper
operates at 37.5 °C and at pH 5 in a citrate buffer. O2 is
electrocatalytically reduced on the cathode to water at a current
tion below 0.4 V at neutral pH. The currents observed were
1 µA/cm . Trudeau et al. were the first to “wire” laccase,
immobilizing it in a 0.44 V redox potential polymer and forming
a pH 4.7 azide sensor. The sensor operated at room temperature
at a current density of 100 µA/cm when poised below 0.4 V.
Lee et al. reported a series of laccase-based oxygen cathodes,
where the enzyme was in direct electrical contact with pyrolytic
carbon. They observed a current density of 190 µA/cm at +0.2
V at 22 °C and at pH 3.8. Palmore et al. described a fuel cell,
2 4
<
2
5
2
2
2
density of 5 mA/cm and at +0.7 V (NHE).
6
Laccase cathodes on which O2 is electroreduced to water were
introduced by Tarasevich et al. They formed these by directly
adsorbing laccase on Teflon-bound high-surface area carbon-
black particles. Their cathode was stable and operated at pH 5
at a current density of 175 µA/cm when poised at 1.10 V.
its cathode compartment containing a homogeneous laccase
solution and a vitreous carbon electrode poised at 0.4 V,
operating at a current density of 120 µA/cm at pH 4. Thuesen
2
7
1
2
and co-workers reported a current density of 13 µA/cm at 0.36
2
8
V for a monolayer of surface-immobilized laccase at pH 3.3.
Tarasevich et al. also reported a current density approaching
Jin et al. electroreduced O2 at a current density of 0.3 µA/cm2
2
1
0 mA/cm at pH 3.5, for an electrode poised at 0.8 V. The
on an electrode poised at -0.1V at pH 5.8, using a homogeneous
laccase solution and cytochrome c as mediator. Tsujimura et
al. recently described a bilirubin oxidase-carbon felt electrode
operating at pH 7 at a current density of 0.89 mA/cm when
2
electrode, made by loading 1-2 mg/cm of a laccase from the
Institute of Biochemistry of the Armenian SSR on 1 cm diameter
carbon substrates with various micro-, meso- and macropore
9
2
2
ratios was, however, difficult to reproduce in absence of
10
poised at 0.22 V.
information on the species from which the laccase was derived
and on the size and the size distribution of the carbon particles.
Papers written in the past decade do not mention the high current
density O2 cathodes, even though some of these were written
by members of the team that reported the high current density
cathodes. Yaropolov et al. studied the bioelectrocatalysis of O2
reduction on laccase-modified graphite and carbon electrodes,
using laccases from Rhus Vernicifera (a tree) and from Coriolus
hirsutus (a fungus). They showed that the tree laccase films
catalyzed O2 electroreduction near neutral pH, but only at
potentials negative of 0.4 V. The fungal laccase films catalyzed
O2 electroreduction at pH 5 at potentials as oxidizing as 0.8 V,
Our initial communication described the operation and the
1
1
design principles of the cathode of this paper. The electro-
catalyst was a composite of carbon cloth, consisting of 10 µm
diameter hydrophilic carbon fibers, coated with the cross-linked
electrostatic adduct of laccase from Coriolus hirsutus (a
polyanion) and an electron-conducting redox polymer (a poly-
cation). The redox polymer electrically connected (“wired”) the
laccase reaction centers to the carbon fibers. The structure of
the 0.78 V redox potential polymer is shown in Figure 1. The
polymer was made of poly-N-vinyl imidazole by coordinating
th
2+/3+
1
/5 of the rings to [Os(tpy)(dme-bpy)]
(tpy ) 2,2′:6′,2′′-
terpyridine; dme-bpy ) 4,4′-dimethyl-2,2′-bipyridine). The
catalytic scheme whereby O2 is electroreduced to water is shown
in Scheme 1. The open-circuit potential of an electrode made
by directly adsorbing C. hirsutus laccase on vitreous carbon is
0.82 V at 1 atm O2 and at pH 5. The laccase oxidizes the Os2
†
Part of the special issue “Howard Reiss Festschrift”.
*
To whom correspondence should be addressed.
All reported potentials are vs the potential of the normal hydrogen
‡
+
electrode (NHE).
1
0.1021/jp012488b CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/12/2001

11918 J. Phys. Chem. B, Vol. 105, No. 47, 2001
Barton et al.
SCHEME 1: The Electrocatalytic Cascade of the
Cathode
Figure 1. Structure of the laccase wiring redox polymer.
centers of the electron and ion-conducting redox polymer to
3+
Os , and the redox polymer is electroreduced at the carbon
surface at a potential near 0.65 V.
As will be seen below, the O2 electroreduction potential and
current density of the laccase cathode depend on the redox
polymer used to “wire” the enzyme, on the weight fractions of
the components of the electrocatalytic composite, on the
temperature and O2 pressure, and on the concentrations of
inhibitors of laccase.
Experimental Section
Chemicals. Laccase (EC 1.10.3.2) from Coriolus hirsutus,
(
(
60 catechol units per mg protein) was received from SynectiQ
Dover, NJ) as a pH 6.5, 3 mg/mL solution in 1:1 glycerol-
Figure 2. SEM (electron backscattering mode) of the composite 350
2
µm thick carbon cloth cathode loaded with 0.78 mg/cm “wired” laccase
sodium phosphate buffer. The solution was concentrated to 50
mg/mL by ultrafiltration and washing with distilled water.
Carbon cloth (Toray TGPH-030, Japan) was received as a
sample from E-TEK (Somerset, NJ). Ultrapure O2 and air
(45 wt % laccase, 49 wt % redox polymer, 6 wt % PEGDGE) The
lighter areas are coated with more heavy atoms (osmium).
an aqueous 7.6 mg/mL redox polymer solution were mixed with
(
Praxair), sodium chloride and sodium hydroxide (Aldrich,
Milwaukee, WI), and poly(ethylene glycol) diglycidyl ether
PEGDGE, Polysciences Inc., Warrington, PA) were used as
2
µL of 200 mM citrate buffer (pH 5), 2 µL of a 35 mg/mL
laccase solution, and 2 µL of 5 mg/mL PEGDGE. An 8 µL
aliquot of this mixture was pipetted onto the mounted hydro-
philic carbon cloth, which was promptly wetted and penetrated
by the solution. The composite mounted disks were cured for
(
received.
Polymer Synthesis. 2,2′:6′,2′′-terpyridine (tpy) was com-
1
2
plexed to (NH4)OsCl6 to produce Os(tpy)Cl3 as described
>12 h in air at room temperature.
except that reactants, at 1:1 molar ratio, were dissolved in
ethylene glycol and refluxed under argon for 1 h. The Os(tpy)-
Cl3 produced was precipitated by pouring the reaction mixture
into ethyl ether (yield 90%). The complex was then reacted with
Instrumentation and Cell. The measurements were per-
formed using a CH-Instruments Electrochemical Detector
Model #CHI832 and the data were collected by a dedicated
computer. The electrodes were rotated using a Pine Instruments
Rotator. The water-jacketed electrochemical cell had a Pt wire
or vitreous carbon counter electrode and an Ag/AgCl reference
electrode. It contained 100 mL of citrate buffer (200 mM, pH
,4′-dimethyl-2,2′-bipyridine (dme-bpy)1 at a 1:1 molar ratio
3
4
to form [Os(tpy)(dme-bpy)Cl]Cl2, which was also precipitated
by pouring the reaction solution into an excess of ethyl ether
14
(yield 85%). The redox polymer was prepared by complexing
5
) and was maintained at 37.5 °C. To maintain a fixed volume
the reduced complex, [Os(tpy)(dme-bpy)Cl]Cl, with poly(N-
vinylimidazole) in a 1:4 molar ratio and was purified as
described.
Electrode Preparation. The composite electrodes were
assembled on 3 mm diameter vitreous carbon rotating disk
electrodes (RDE) mounted in Teflon sleeves. These disks were
sanded with ultrafine sand paper, polished with alumina slurry
of electrolytic solution in the cell the admitted gases were
presaturated with water by passage through a bubbler, which
contained a solution similar to that in the cell.
1
4
Results
(
1 µm) and sonicated for 5 min in deionized water. The polishing
Composite Electrodes. The distribution of osmium in the
hydrogel-carbon cloth composite after its dehydration in the
evacuated chamber of an electron microscope is shown in
electron micrograph of Figure 2. In obtaining the picture, the
electron microscope was operated the electron backscattering
mode. The figure shows large, solvent accessible pores between
the 10 µm fibers. Because the backscattering of electrons
increases with atomic mass, regions of high osmium concentra-
tion appear as bright patches. The presence of the osmium-
containing polymer is particularly pronounced in patches
step was repeated until no voltammetric features beyond water
oxidation were observed in a 50 mV/s scan in phosphate buffer,
pH 7 in the range between 0.2 and 1 V. The 350 µm thick carbon
cloth had a nominal 78% void fraction and consisted of 10 µm
diameter fibers. The cloth was cut into 4 mm diameter disks,
which were cemented, using conductive carbon paint (SPI, West
Chester, PA), to the surface of the vitreous carbon RDE. The
rod-bound cloth was made hydrophilic by exposure to a 1 Torr
O2 plasma for 5 min.15 For the base-case electrode, 10 µL of

Electroreduction of O2 to Water
J. Phys. Chem. B, Vol. 105, No. 47, 2001 11919
Figure 4. Dependence of the current density of O
.62 V (NHE) on the square root of the angular velocity of the rotating
electrode. Conditions as in Figure 3b.
2
electroreduction at
0
Figure 3. (a) Cyclic voltammograms (50 mV/s sweep rate, 500 rpm)
of the vitreous carbon and the composite carbon cloth electrodes under
argon. (b) Potential-dependence of the O
density at 1 mV/s sweep rate at 1 atm O pressure. Loading as in Figure
, 0.2 M citrate buffer, pH 5, 37 °C. Current densities were based on
2
electroreduction current
2
2
2
the projected area of the 4-mm carbon cloth disk (0.13 cm ).
clinging to the binder that glues the carbon fibers of the cloth.
Figure 3a shows cyclic voltammograms of a plain vitreous
carbon electrode and of the carbon-cloth composite electrode
under argon at 50 mV/s sweep rate. The only voltammetric
waves observed were those associated with the electrooxidation/
reduction of PVI[Os(tpy)(dme-bpy)]2+/3+, centered at 0.77 V.
The waves are about an order of magnitude larger for the carbon-
cloth composite electrode than for the polished vitreous carbon
electrode, suggesting an order of magnitude difference in the
electrochemically accessible, redox-polymer coated surface area.
The polarization curves of the carbon cloth composite and
the polished vitreous carbon cathodes, both under 1 atm O2
rotated at 500 and 4000 rpm in 0.2 M pH 5 citrate buffer at
2
Figure 5. Dependence of the O electroreduction current density at
0.62 V (NHE) on the laccase loading. Total loading was fixed at 0.78
2
mg/cm and PEGDGE constituted 6 wt % of the total mass. Conditions
as in Figure 3b.
repeatability error. Observed current densities at 0.62 V were
found to vary directly with both the loading of the wired laccase,
2
for loadings between 0.2 and 1.3 mg/cm , and the thickness of
the carbon cloth for thicknesses of 90 µm to 350 µm (data not
shown).
3
7.5 °C, are seen in Figure 3b. In the absence of laccase O2 is
2
not electroreduced on either electrode. At 0.5 mg/cm laccase
loading O2 is electroreduced when the electrodes are poised at
When the pH was raised above its optimal value of pH 5 the
current density dropped and at pH 7 the electrode was inactive
(Figure 6). Chloride also inhibited the electroreduction: The
potentials negative of 0.85 V, the potential where the electrore-
duction of PVI[Os(tpy)(dme-bpy)]3+ becomes significant (Figure
-
2
current density declined by 60% when the Cl concentration
3
a). The current density, based on the 0.13 cm carbon cloth
2
was raised from nil to 0.1 M. Under ∼0.1 N/m shear stress,
disk, increases nearly linearly with decreasing electrode potential
until the mass-transport defined plateau is reached. The observed
linearity suggests that the current density is limited by ohmic
resistance. The O2 mass transport defined plateau of the current
density is a function of the O2 partial pressure, the temperature
and the angular velocity of the rotating electrode below about
exerted by rotation at 1000 rpm, the carbon-cloth composite
cathodes were much more stable than the electrodes made by
coating the polished vitreous carbon electrodes with films of
wired laccase (Figure 7a). The O2 electroreduction current of
the composite electrodes dropped to 1/3rd of its initial value in
2
+/3+
3
3
4.7 days and their voltammetric Os
electrooxidation/
1
0 rpm (Figure 4). Above 10 rpm, the plateau is defined by
reduction peaks declined by 42% ( 5% (Figure 7b). The loss
of current was at least three times faster in the noncomposite
vitreous carbon electrodes. Although the half-lives of the shear-
stressed composite electrodes were of ∼3 days, the half-lives
the kinetics of the O2 electroreduction and increases with
temperature. The current density reaches a remarkably high
2
value of 10 mA/cm at 1 atm O2, 37 °C, and 4000 rpm. In a
quasi-stagnant, air-saturated solution the current density at 37
16
2
of the noncomposite electrodes were only of ∼1 day.
°
C is lower by 2 orders of magnitude, about 100 µA/cm .
The optimal enzyme mass fraction was ∼40% when the
Discussion
PEGDGE binder fraction was 6 wt % for both the carbon cloth
composite and for the polished vitreous carbon electrodes
The results show that at pH 5 and at 37 °C and under
(
Figure 5). Although a more rigorous analysis would reveal day-
conditions of high convection oxygen is electroreduced to water
at 0.62 V at a current density exceeding 5 mA/cm on the
2
to-day variations in enzyme activity, this effect was within the

11920 J. Phys. Chem. B, Vol. 105, No. 47, 2001
Barton et al.
at a linear velocity of 10 cm/s, the 0.24 cm cell would produce
3
1
.2 mW. It would generate in one week the amount of energy
stored in the highest energy density lithium battery of equal
volume. A cathode operating in a major blood vessel of an
animal would require a wired laccase electrode that would not
loose its activity when the pH is raised from 5 to 7 and when
-
Cl is present at its physiological concentration. A recent study
suggests that the activity of laccase from Pleurotus ostreatus
declines only moderately upon increasing the pH from 5 to 7.17
The observed optimal enzyme mass fraction was ∼40% for
both the carbon cloth and the vitreous carbon electrodes (Figure
5
). The constancy of the ratio suggests that the thicknesses of
the electroactive zones were similar for both structures, and that
the increased current density with the cloth derives solely from
an increase in active surface area when enzyme kinetics and
Figure 6. Dependence of the current density of the noncomposite
polished and catalyst coated) vitreous carbon electrodes on pH and
1
8
(
electron transport are not re-balanced.
on the chloride ion concentration. 0.62 V (NHE), 900 rpm. Conditions
as in Figure 3b.
The wired laccase cathode was reasonably stable even when
the electrode was continuously stressed by shearing at 0.1 N/m2
through rotation at 1000 rpm (Figure 7a). The major cause of
loss was the stripping of the electrocatalytic film, suggested by
the fact that not only the current density but also the voltam-
metric peaks declined (Figure 7b). If enzyme deactivation were
the dominant cause of loss, only the O2 electroreduction current,
2
+/3+
not the voltammetric peaks of Os
electrooxidation/reduc-
tion, would have declined. After 4.7 days of continuous oper-
ation, 66% of the O2 electroreduction current and 42% of the
2
+/3+
electroactive Os
centers were lost. This suggests that en-
dz ya ym s e. degradation caused only ∼24% of the current loss in 5
Conclusions
O2 was electroreduced to water at pH 5 on a rotating (1000
rpm) carbon-cloth composite “wired” laccase (45 wt % laccase,
2
4
9 wt % redox polymer) cathode at 5 mA/cm current density
at 0.62 V in O2-saturated pH 5 citrate buffer at 37 °C. In
stagnant, air-saturated, citrate buffer the current density was
2
∼
100 µA/cm . The half-life of the cathode was ∼3 days when
it was sheared at a stress comparable to that of fast-flowing
blood in a major blood vessel. The next necessary step toward
a fuel cell implanted in a major blood would be the extension
of the cathode’s operating pH from pH 5 to the physiological
pH 7.2-7.4 range and its operation in the presence of chloride
at its physiological concentration.
Figure 7. Operational stability of the “wired” laccase electrode. (a.)
Time-dependence of the current density of carbon-cloth composite and
of the vitreous carbon noncomposite electrodes poised at 0.62 V (NHE).
Acknowledgment. DARPA/ONR and The Welch Founda-
tion supported this work. The authors thank Jason Cole and
Keith A. Friedman for their advice and assistance.
(b.) The change in the cyclic voltammograms of the carbon cloth
composite electrode after 4.6 days of operation. Scan rate 50 mV/s.
The composition, loading and conditions were as in Figure 3b.
References and Notes
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(
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2
2
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(
(
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(
1
(

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(
(
(
6
(