Biosensors and Bioelectronics, 2008

Cite this article as : A. Ramanavicius A. Kausaite-Minkstimiene, A.

Ramanaviciene, Enzymatic biofuel cell based on anode and cathode powered by

ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

Full printed journal version is available at:

http://www.sciencedirect.com/science/article/pii/S0956566308003357

RESEARCH ARTICLE

(Advanced authors version)

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

Enzymatic biofuel cell based on anode and cathode powered by

ethanol

Arunas Ramanavicius1∗, Asta Kausaite

1,2, Almira Ramanaviciene

1,2,

1 Center of Nanotechnology and Material Science, Faculty of Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius,

Lithuania

2 Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Moletu pl. 29, 08409 Vilnius,

Lithuania

Abstract

Enzymatic biofuel cell based on enzyme modified anode and cathode electrodes are both powered by ethanol and

operate at ambient temperature is described. The anode of the presented biofuel cell was based on immobilized quino-

hemoprotein-alcohol dehydrogenase (QH-ADH), while the cathode on coimmobilized alcohol oxidase (AOx) and

microperoxidase (MP-8). Two enzymes AOx and MP-8 acted in the consecutive mode and were applied in the design of

the biofuel cell cathode. The ability of QHADH to transfer electrons directly towards the carbon-based electrode and

the ability of MP-8 to accept electrons directly from the same type of electrodes was exploited in this biofuel cell

design. Direct electron transfer (DET) to/from enzymes was the basis for generating an electric potential between the

anode and cathode. Application of immobilized enzymes and the harvesting of the same type of fuel at both electrodes

(cathode and anode) avoided the compartmentization of enzymatic biofuel cell. The maximal open circuit potential of

the biofuel cell was 240 mV.

c 2008 Elsevier B.V. All rights reserved.

Keywords: Enzymatic fuel cell, Fuel cell, Biofuel cell, PQQ, Alcohol dehydrogenase, Alcohol oxidase, Microperoxidase.

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

1. Introduction

The production of electrical power from low-cost

biofuels is an important challenge in the energetics, since

biofuels are renewable, sustainable, offer lower

greenhouse-gas emissions and reduce the demand for

common fuel sources, these are of special interest of

modern-energetics (Pizzariello et al., 2002). As the

majority of organic substrates undergo combustion with

the evolution of energy, the biocatalyzed oxidation of

organic substrates by oxygen at electrode interfaces

provides a means for the conversion of the chemical

energy to an electrical one (Willner et al., 1998a).

Significant concentrations of ethanol and other alcohols

are often present in the various biological substances

(Lim and Wang, 2003). Basic fermentation processes

might be applied to increase concentration of alcohols in

these substances. The chemical energy of abundant

biological substrates including polyhydroxylic

(Arechederra et al., 2007) and monohydroxylic alcohols

(Ikeda and Kano, 2001; Ramanavicius et al., 2005)may

be converted into electrical current by biofuel cells. The

advantage of biofuel cells when compared to standard

fuel cells is that biofuel cells are able to operate at a low

substrate concentration which can be even at the

micromolar level (Pizzariello et al., 2002). In the future

biofuel cells can be used as alternative energy supply

sources for biosensors and for other bio electronic

devices (Willner et al., 2001). These cells may also be

used as power sources of implantable devices (Barton et

al., 2004). The combination of bioelectronics with

nanotechnology allows integration of biofuel cells within

the operating devices, while the nanotechnology offers

novel perspectives for the miniaturization of

bioelectronic devices and the increase of their efficiency

(Willner et al., 2001; Wang et al., 2005). Microorganisms

and/or enzymes are catalysts that are able to convert

chemical energy to electrical energy (Katz et al., 1999),

for this reason they are important subjects of biofuel cells

(Bullen et al., 2006). The most efficient biofuel mediators

based system for re-oxidation of its active site. It was

demonstrated that both types of alcohol harvesting

enzymes could be applied in the design of biofuel cells,

however usually they are applied for designing of biofuel

cell anode. The majority of oxidases and dehydrogenases

require the application of red-ox mediators to establish

direct electron transfer (DET) with electrode

(Habermuller et al., 2000; Malinauskas et al., 2004).

Among the other types of enzymes, some

pyrroloquinoline quinine (PQQ)-dependent red-ox

enzymes were also employed for the construction of

biofuel cells (Ikeda and Kano, 2003). However the

majority of PQQ-dependent enzymes are unable to

transfer electrons without additional red-ox mediators

(Lapenaite et al., 2006; Habermuller et al., 2000). The

detailed characterization of the interfacial electron

transfer rates is essential in the construction of biofuel

cells, since the enzyme and electronic conductors are

foreign components in respect of one to the other, that

leads to a lack of electric current between them. The

modification of PQQ-dependent enzymes with covalently

attached red-ox mediators can be applied to facilitate

electron transfer from active site of enzyme towards

electrode (Laurinavicius et al., 2004). An alternative to

this is to apply DET-able enzymes (Gorton et al., 1999).

The red-ox enzymes containing heme-c are very

promising in this context (Freire et al., 2003;

Ramanavicius et al., 2006a). Quino-hemoprotein-alcohol

dehydrogenase from Gluconobacter sp. 33 (QH-ADH)

demonstrates DET toward glassy carbon (Ikeda et al.,

1993), other forms of carbon (Razumiene et al., 2002),

gold (Ikeda et al., 1993) and conducting polymer

polypyrrole (Ramanavicius et al., 1999). Another class of

enzymes, heme-c containing peroxidases, are able for

direct electron transfer and also are very promising for

construction biofuel cell cathode (Willner et al., 1998a,b;

Ferapontova and Gorton, 2001). Direct electrochemistry

of microperoxidases with a gold electrode (Willner et al.,

1998a), carbon electrodes (Ruzgas et al., 1996), along

with platinum electrodes modified with carbon nanotubes

(Wang et al., 2005),were investigated and exploited in

the design of biofuel cell cathode. However, DET-based

enzymatic biofuel cell utilizing the same substrate at both

electrodes is still a challenge. The aim of this study was

to design basic, non-compartmentalized, mediator free

biofuel cell based on enzymes exhibiting direct

bioelectrocatalysis and able to convert the chemical

energy of biological substrate – ethanol – at both cathode

and anode. cell designs allow operation’s without

compartments dividing membranes (Chen et al., 2001;

Ramanavicius et al., 2005). It allows applications of

biofuel cells as portable power sources. Biofuel cells

utilize biocatalysts for the conversion of chemical energy

to electrical energy (Katz et al., 1999; Chen et al., 2001;

Mano et al., 2002, 2003). The methodology based on the

application of purified redox enzymes for the targeted

oxidation and reduction of specific fuel and oxidizer

substrates at the electrode supports and the generation of

the electrical current output is used for the development

of biofuel cells (Chen et al., 2001). There are known

several red-ox enzymes able gain electrons from various

alcohols: methanol (Zhang et al., 2006a,b), ethanol

(Ramanavicius et al., 2006a), glycerol (Lapenaite et al.,

2006) and other alcohols (Ivnitski et al., 2006). Alcohol

oxidases (AOxs) utilizes oxygen as natural electron

acceptor thus producing hydrogen peroxide

(Ramanavicius et al., 2006b; Malinauskas et al., 2005).

Alcohol dehydrogenases are using NAD/NADH or

artificial red-ox mediators based system for re-oxidation

of its active site. It was demonstrated that both types of

alcohol harvesting enzymes could be applied in the

design of biofuel cells, however usually they are applied

for designing of biofuel cell anode. The majority of

oxidases and dehydrogenases require the application of

red-ox mediators to establish direct electron transfer

(DET) with electrode (Habermuller et al., 2000;

Malinauskas et al., 2004). Among the other types of

enzymes, some pyrroloquinoline quinine (PQQ)-

dependent red-ox enzymes were also employed for the

construction of biofuel cells (Ikeda and Kano, 2003).

However the majority of PQQ-dependent enzymes are

unable to transfer electrons without additional red-ox

mediators (Lapenaite et al., 2006; Habermuller et al.,

2000). The detailed characterization of the interfacial

electron transfer rates is essential in the construction of

biofuel cells, since the enzyme and electronic conductors

are foreign components in respect of one to the other, that

leads to a lack of electric current between them. The

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

modification of PQQ-dependent enzymes with covalently

attached red-ox mediators can be applied to facilitate

electron transfer from active site of enzyme towards

electrode (Laurinavicius et al., 2004). An alternative to

this is to apply DET-able enzymes (Gorton et al., 1999).

The red-ox enzymes containing heme-c are very

promising in this context (Freire et al., 2003;

Ramanavicius et al., 2006a). Quino-hemoprotein-alcohol

dehydrogenase from Gluconobacter sp. 33 (QH-ADH)

demonstrates DET toward glassy carbon (Ikeda et al.,

1993), other forms of carbon (Razumiene et al., 2002),

gold (Ikeda et al., 1993) and conducting polymer

polypyrrolen (Ramanavicius et al., 1999). Another class

of enzymes, heme-c containing peroxidases, are able for

direct electron transfer and also are very promising for

construction biofuel cell cathode (Willner et al., 1998a,b;

Ferapontova and Gorton, 2001). Direct electrochemistry

of microperoxidases with a gold electrode (Willner et al.,

1998a), carbon electrodes (Ruzgas et al., 1996), along

with platinum electrodes modified with carbon nanotubes

(Wang et al., 2005),were investigated and exploited in

the design of biofuel cell cathode. However, DET-based

enzymatic biofuel cell utilizing the same substrate at both

electrodes is still a challenge. The aim of this study was

to design basic, non-compartmentalized, mediator free

biofuel cell based on enzymes exhibiting direct

bioelectrocatalysis and able to convert the chemical

energy of biological substrate – ethanol – at both cathode

and anode.

2. Experimental

2.1 Chemicals

Alcohol dehydrogenase from Gluconobacter sp. 33 (E.C.

1.1.99.8) was isolated and purified at the Institute of

Biochemistry (Vilnius, Lithuania). The enzyme had an

activity of 171 U/ml and 7.6mg/ml concentration of

proteins. Microperoxidase-8 (MP-8) from horse heart 250

U/mg; AOx from Pichia pastoris (E.C.1.1.3.13), 50

U/ml, 25% glutaraldehyde and 96% ethanol were

purchased from Sigma (Berlin, Germany). Carbon rod

electrodes “Ultra F purity” 3mm in diameter obtained

from Ultra Carbon Division of Carbon USA, RAVEN-M

were used. Carbon rod electrodes were sealed into epoxy

to prevent the contact of electrode side surface with the

solution.

2.2 Preparation of electrodes

Graphite electrodes modified with enzymes were

prepared as follows: (i) rods of spectroscopic graphite

were cut, and polished on fine emery paper, followed by

rinsing the electrode surface with ethanol and water.

Electrodes were dried at room temperature before coating

with enzyme; (ii) enzyme solutions were deposited on the

electrode surface and the coated electrodes were kept

for 20 h over the 5% solution of glutaraldehyde at 4 ◦C in

the closed vessel as it was previously described

(Ramanavicius, 2007). In preparing the anode, 6µl of

QH-ADH solution was thoroughly distributed on the

surface of the electrode and kept over glutaraldehyde

solution. In preparing the cathode, 3µl of MP-8 solution

(10mg/ml) was thoroughly distributed over the electrode

and dried for 30 min. Afterwards, 3µl of AOx solution

(10mg/ml) was deposited and the electrode was kept over

glutaraldehyde. Control carbon electrode modified with

MP-8 was prepared by deposition of 3µl ofMP-8 solution

(10mg/ml) and cross-linked by glutaraldehyde using the

same cross-linking protocol.

Fig. 1. Configuration of biofuel cell, using ethanol as a

fuel for cathode and anode.

2.3. Electrochemical measurements

All electrochemical measurements were performed

by potentiostat–galvanostat PGSTAT 30 using

specialized software GPES3 v3,2 Echochemie/Autolab

(Utrech, Netherlands). Two-electrode circuit was applied

for registration of electrical potential between electrodes.

Some potentiometric measurements were performed at

open circuit; during some other experiments the resistors

were switched into the external circuit (Fig. 1) to imitate

circuit-load on biofuel cell.

3. Results and discussion

Current developments in nanotechnology enables serious

thoughts about nano-devices and even nano-robots, but in

many cases such systems require miniature power

sources (Chen et al., 2001) otherwise such devices will

be very limited in function and application. Major idea of

the work presented here was to apply environmentally

friendly and/or biodegradable materials. This concept

does not allow us to apply any soluble red-ox mediators

or hazardous red-ox polymers. Direct electron transfer

exhibiting hemoproteins (Gorton et al., 1999) were

selected for this study to avoid application of any

additional environmentally and/or biologically hazardous

red-ox materials. Enzymes MP-8 and QH-ADH were

selected for the design of biofuel cell as the catalysts.

These enzymes are able to transduce chemical energy

into electrical one and to transfer electrical current

directly to the carbon electrodes. Carbon is selected as a

matrix-material, because various carbon forms are very

often applied in various nanotechnological applications

(Malinauskas et al., 2005). Moreover carbon is especially

suitable for application in bioelectronic devices since it

supports sufficient electron transfer due to proper enzyme

orientation (Shleev et al., 2005). The QH-ADH is

comprised of three subunits (Fre’bortova et al., 1998).

The ability of QH-ADH to transfer electrons directly

towards the electrodes was employed for generation of

anodic current in amperometric biosensors

(Ramanavicius et al., 1999) and later this process was

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

applied in biofuel cell harvesting ethanol and glucose as

biofuels (Ramanavicius et al., 2005). The anode of the

constructed enzymatic fuel cell was based on

immobilized QH-ADH, which oxidized ethanol as the

anode fuel. The cooperative action of the enzyme-

integrated prosthetic groups pyrroloquinoline quinone

and hemes-c were assumed to allow direct electron-

transfer from the enzyme’s catalytic site to the carbon

electrode. The electrical contact between QH-ADH and

carbon electrode was efficient and the electrons extracted

from ethanol (1) were directly transferred to carbon

surface (Fig. 1, anode reaction). This phenomenon has

been exploited in anodes of other biofuel cells

(Ramanavicius et al., 2005; Treu et al., 2006). This

enzyme becomes attractive because of the possible

improvements when applying other carbon-based

nanomaterials, including carbon nanotubes (Treu et al., in

press).

Fig. 2. Open circuit potentials of: (1) QH-ADH functionalized

anode as a function of ethanol concentration; (2)MP-8/AOx functionalized cathode as a function of ethanol concentration;

(3) MP-8/AOx functionalized cathode as function of hydrogen

peroxide concentration; (4) QH-ADH functionalized anode and MP-8/AOx functionalized cathode-based biofuel cell as a

function of ethanol concentration. Investigations performed in

50mM Na acetate solution, pH 6.0, with 100mM KCl at open circuit.

The open circuit potential of here studied QH-ADH-

based anode was approximately −125mV (Fig. 2, curve

1). Anode reaction:

Ethanol QH−-→ADHEthanal+2H+ + 2e− (1)

The cathode reaction is also very important during the

creation of biofuel cells since it can significantly increase

electric potential and current of biofuel cell. Various

strategies were applied to maximize the performance of

cathodes in biofuel cells and the application of laccases

seems the most popular for this purpose (Barton et al.,

2001; Tarasevich et al., 1979; Tayhas et al., 1999), it was

demonstrated that some carbon-based nanomaterials may

significantly improve the current of laccase-based biofuel

cell cathode (Gallaway et al., 2008) and negative

influence of some halide ions could be eliminated by

proper orientation of laccases (Vaz-Dominguez et al.,

2008). Advantageous properties of laccases including

major DET aspects were in details reviewed (Shleev et

al., 2005), however some alternative ways including

application of heme-c-dependent enzymes could be also

applied to design cathodes of biofuel cells (Bullen et al.,

2006). In this respect, previously we have demonstrated

that glucose oxidase might be principally applied in the

design of biofuel cell cathode if combined with

microperoxidase-8 (Ramanavicius et al., 2005).

Hydrogen peroxide, which is produced by oxidase, is a

strong oxidizer. However electrochemical reduction of

hydrogen peroxide at the carbon electrode occurs with

very high over-potential, which may be reduced if the

carbon surface is modified by peroxidase activity

exhibiting enzymes (Ruzgas et al., 1996). From the

variety of peroxidases we have selected the smallest one,

MP-8, because this enzyme is very stable what is

determined by basic structure of this microperoxidases.

Although, this biocatalyst was expected to have the most

efficient carbon surface covering due to lowest molecular

weight (Adams, 1991). The influence of an efficient

surface covering was demonstrated by investigation of

several carbon electrode-covering strategies. The layered

design of the cathode based on two separated layers of

MP-8 and AOx was selected for this study, because the

investigations showed that in the case when pure MP-8

layer was deposited as first layer on these carbon

electrodes, then higher potential and higher current

density of the cathode are achieved in comparison with

the cathode based on mixed MP-8 and AOx. This type of

a layered design guaranties a higher surface-density of

MP-8, which is directly deposited on the electrode.

Hydrogen peroxide was formed in the reaction catalyzed

by AOx. Then hydrogen peroxide was used as a substrate

for MP-8, and electrons from this reaction were

transferred from the electrode to the active site of MP-8,

which was acting as a reducer of hydrogen peroxide (Fig.

1, cathode interface reaction). Due to the consecutive

action of AOx and MP-8 immobilized on the cathode,

chemical energy of ethanol oxidation by dissolved

oxygen was converted into electrical current. Cathode

interface reactions:

Ethanol + O2 + H2O−AO→xEthanal+H2O2 (2)

H2O2 + 2H+M−→P-82H2O − 2e− (3)

The potential generated by MP-8/AOx cathode in the

presence of different ethanol concentrations was

examined (Fig. 2, curve 2). The slope of the second curve

(Fig. 2, curve 2) seems much lower if compared with that

achieved by QH-ADH-based anode (Fig. 2, curve 1).

This effect might be based on diffusion of ethanol and

hydrogen peroxide, which was formed during the action

of AOx. From the potential values of MP-8/AOx

modified electrode at 2mM and higher hydrogen

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

peroxide concentrations (Fig. 2, curve 3), the theoretical

limit of the MP-8 modified electrode potential is

estimated to be 150mV. The maximal voltage generated

by MP-8/AOx modified cathode was close to that

generated by biofuel cell cathode based on MP-11

modified gold cathode (Willner et al., 1998a). The

cathode potential exceeded 125mV if 5mM and higher

ethanol concentrations were added into the cell (Fig. 2,

curve 1). The maximal open circuit potential of the

complete biofuel cell was 240mVat ethanol

concentrations of 20mMand higher (Fig. 2, curve 4).

Experimental results indicates, that the open circuit

potential in this type of measurements is dependent on

substrate concentration because of some limitations in

measurement: (i) the “open circuit” is not ideal, despite

of very high resistance of measuring device (300M_), the

resistance of circuit cathode/solution/anode was just

1±0.2M_, because of conductivity of used buffer

solution, for this reason the measured potential at least

partially depends on enzymatic reaction rate, which is

highly influenced by concentration of substrate—ethanol.

Fig. 3. The pH dependence of open circuit potential for: (1) MP-

8 functionalized cathode in presence 20mMof hydrogen peroxide; (2)QH-ADH functionalized anode in presence 20mM

of ethanol; (3) MP-8/AOx functionalized cathode in presence

20mM of ethanol and (4) biofuel cell based on MP-8/AOx functionalized cathode and QH-ADH functionalized anode in

the presence 20mMof ethanol. Investigations performed at an

open circuit in 50mMof sodium acetate with accordingly adjusted pH, 100mM of potassium chloride.

The MP-8 modified electrode may be effectively applied

in a broad pH diapason: between pH 3 and pH 7 (Fig. 3,

curve 1). However, the QH-ADH modified anode (Fig. 3,

curve 2) and layered MP-8/AOx modified cathode (Fig.

3, curve 3) are much more dependent on pH level. The

result of this is a “sharp” dependence of electric potential

on pH (Fig. 3, curve 4). On the other hand the developed

biofuel cell is able to operate in the physiological pH

range. It is important for possible application of biofuel

cell in vivo (Tsujimura et al., 2001). To enable better

operation of biofuel cell in vivo, some enzymes might be

exchanged into ones harvesting glucose (Malinauskas et

al., 2004) and/or glycerol (Lapenaite et al., 2006). There

are many reasons why pH is affecting open circuit

potentials, e.g.: (i) there are significant differences in

thermodynamics of enzymatic red-ox reactions and in

thermodynamics intrinsic/extrinsic electron transfer

reactions at different pH; (ii) significant differences in

enzymatic activity, because conformation of enzyme is

pH dependent and protonated/deprotonated groups might

be crucial in catalytic site of enzyme; (iii) extrinsic

electron transfer might be dependent on enzyme

conformation, etc. The stability of biofuel cell was

examined at working state in presence of 2mM of ethanol

at the loading resistance of 1M_ as

Fig. 4. Open circuit potentials for: (1) MP-8/AOx functionalized

cathode and (2) QH-ADH functionalized anode as a function of

glucose concentration. Investigations performed at an open circuit in 50mMof sodium acetate, pH6.0, containing 100mM of

potassium chloride and 75mM of ethanol.

Fig. 5. Current–voltage behavior of: (1) complete biofuel cell

based on both MP- 8/AOx cathode and QH-ADH anode; (2) QH-ADH anode vs. unmodified carbon electrode; (3) MP-

8/AOx cathode vs. unmodified carbon electrode. All

measurements were performed in 25mM of ethanol.

a function of time in the presence of ethanol. The half-

life of the biofuel cell was found to be 26 h. Significant

improvement in stability of biofuel cell was detected if it

is compared with the results published (Willner et al.,

1998a) where the cathode was based on MP-11 while the

anode on GOx “wired” by PQQ derivatives assembled on

the gold electrode. The “increased” stability of the

biofuel cell might be based on application of unmodified

enzymes when compared with enzymes treated under

harsh conditions due to immobilization via self-

assembled monolayers or enzyme “wiring” techniques

applied in other studies (Willner et al., 1998a). On the

other hand, the structure of MP-8 is very basic, which

makes this enzyme more stable if comparing it with the

higher molecular weight enzyme—MP-11 used in

previous studies. However in this study used AOx was

found to be relatively unstable; for this reason AOx

negatively influenced stability of biofuel cell cathode.

The least expensive alcohol sources, for example natural

fermentation products, typically contain a lot of various

sugars. In order to study performance of biofuel cells in

an environment containing sugars the effectiveness of

MP-8/AOx (Fig. 4, curve 1) and QH-ADH (Fig. 4, curve

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

2) cathode in presence of glucose was tested. The results

demonstrate that higher concentrations of glucose (over

200mM) decrease generated potential by 7–15% (Fig. 4).

This effect may be based on significant increase in

viscosity. High viscosity of solution can cause a negative

influence in the diffusion of substrate and reaction

products as it was demonstrated in other studies of

biofuel cells (Arechederra et al., 2007).

Fig. 6. Biofuel cell power vs. registered current in presence of

25mM of ethanol.

Current–voltage behavior of biofuel cell represents how

voltage of complete biofuel cell (Fig. 5, curve 1) and

anode and cathode separately (Fig. 5, curves 2 and 3)

depends on load of circuit. The power density vs. current

of the cell (Fig. 6) illustrates that maximal power for

completed biofuel cell was 1.5_W/cm2. Here described

biofuel cell anodes and cathodes might be used in

separated biofuel cells, e.g. the QH-ADH-based anode of

the biofuel cell might be combined with any other type of

biocathodes, e.g. laccase based, that are also very

promising in this for application in biofuel cells including

microfluidic biofuel cells (Lim and Palmore, 2007).

4. Conclusions and future developments

An enzymatic biofuel cell based on QH-ADH and

MP-8/AOx was designed. The cell was operating at

ambient conditions in absence of any red-ox mediators.

Substrates of QH-ADH and AOx are aliphatic alcohols

that are often found in high concentrations in fermenting

biological samples. The development of this type of

biofuel cell does not require any sophisticated

procedures; basic enzyme-immobilization method is

applied for the construction of this cell. The biofuel cell

is able to produce an electrical current in the presence of

very common biological substances containing alcohols.

The biofuel cell is non-compartmentalized; this design

allows the application of this biofuel cell directly in a

vessel containing alcohol solution (e.g. barrel of wine

fermentation). The application of DET-able enzymes

ensures that the current generated by such biofuel cells

do not depend on leakage of an red-ox mediator from the

modified electrode. Despite the relatively low electrical

potential (240mV) there are some assumptions that this

type of biofuel cell may be exploited in self-powered

bioelectronical devices, because current developments in

microelectronics, nanoelectronics and molecular

electronics allows to reduce requested potentials for new

electronic devices. Both enzymes used in the present

study exhibit higher red-ox potentials as it was reported

here, e.g. the QH-ADH is able to generate +190mV vs.

Ag/AgCl in presence of a soluble red-ox mediator

(Ramanavicius et al., 2006a) and MP-8 −150mV vs.

Ag/AgCl. This enables the search for more efficient way

to apply these enzymes. In this respect, the application of

carbon nanomaterials including carbon nanotubes seems

especially promising since even glucose oxidase from

Aspergillus niger was reported being able for direct

electron transfer promoted by carbon nanotubes (Cai and

Chen, 2004). Recent works demonstrates that carbon

nanotubes can be employed in the design of enzymatic

fuel cells (Ivnitski et al., 2006; Treu et al., in press).

However the stability of the biofuel cell was not steady

enough for long-term operation, but in this respect the

future investigations might be encouraged by such facts

that significant extension in life time of biofuel cell

function can be achieved if genetically engineered FAD-

based (Zhu et al., 2006) and/or PQQ-based enzymes

(Yuhashi et al., 2005) will be applied for this purpose. In

a continuance of the operational period of biofuel cells

the living Gluconobacter cells containing membrane

bound QH-ADH and/or others PQQ–heme-dependent

enzymes present in Gluconobacter might be adopted.

Previous investigations showed that almost the same

current density of QH-ADH modified electrodes might

be produced in the presence of the same concentrations

of ethanol, propanol or butanol (Laurinavicius et al.,

2002), and such versatility of QH-ADH makes proposed

biofuel cell especially attractive for the application in

mixture of alcohols. Other PQQ-dependent enzymes that

are present in Gluconobacter (e.g. glycerol

dehydrogenase (Arechederra et al., 2007) and lactate

dehydrogenase (Treu and Minteer, in press) might be

combined in this type of biofuel cells in order to enhance

the variety of combusted fuels. For extension of current

density of here presented biofuel cell some strategies: (i)

of proper orientation of enzymes could be applied, as it

was reported in case of application laccase in recent

designs of biofuel cells (Vaz-Dominguez et al., 2008);

(ii) multilayered 3DOM gold based enzyme/(gold

nanoparticles) structures (Deng et al., 2008).

Acknowledgements We are grateful to Dr. R. Meskys, Dr. I.

Bachmatova and Dr. L. Marcinkeviciene for purification and

preparation of QH-ADH. This work was partially financially

supported by Agency for International Science and Technology

Development Programmes in Lithuania COST program action

D40.

A. Ramanavicius A. Kausaite-Minkstimiene, A. Ramanaviciene, Enzymatic biofuel cell based on anode and cathode

powered by ethanol. Biosensors and Bioelectronics 24 (2008), 761–766.

DOI:org/10.1016/j.bios.2008.06.048

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