Electrocatal (2010) 1:8794 DOI 10.

1007/s12678-010-0013-2

An Overview of Enzymatic Biofuel Cells

S. Aquino Neto & J. C. Forti & A. R. De Andrade

Published online: 20 April 2010 # Springer Science+Business Media, LLC 2010

Abstract The continuous increase in the search for alternative energy sources worldwide is pushing the demand for clean and efficient energy production processes. The concept of biofuel cell has been known since 1912 but only in 1964 that the first enzyme-based biofuel cell was described. Since then, this kind of device has been the target of many research teams worldwide. This device is an alternative to the fuel cells employing metal catalysts that is capable of converting chemical energy into electricity, with the advantage of using biological molecules as catalysts. This new technology offers several other advantages over traditional batteries, including the use of renewable and clean catalysts, reaction selectivity, fuel flexibility, and the ability to operate at milder temperature. Recent studies have demonstrated promising characteristics of these devices; however, despite the several advances in this area, some challenges still need to be faced. This manuscript aims to provide the reader with an overview of the state of the art in enzymatic biofuel cells by discussing the latest papers in this field and presenting an outlook for future research in this area. Keywords Biofuel cell . Enzyme immobilization . Bioanode

in the search for alternative energy sources worldwide. In this context, fuel cells have shown great potential among the various alternatives for energy production. Fuel cell technology seeks, through the use of catalysts, the generation of electrons from the oxidation of a fuel, usually H2, methanol, or ethanol among others. Although several studies have presented some promising results, the conventional fuel cells still have to face some challenges in order to find future large-scale commercial application. The main concern to be overcome is the high cost of metal catalysts (mainly Pt), problems with electrode passivation, besides the inefficient oxidation of the by-products from carbonyl fuels. Biofuel cells are a particular class of fuel cells in which enzymes or microorganisms are employed instead of metallic inorganic catalysts for electrode activity. The concept of biofuel cell has been known since the first demonstration in 1912 [1], but only in 1964 the first enzyme-based biofuel cell was described [2]. Since then, this kind of device has been the target of many research teams worldwide [35]. Many groups are currently investigating both microbial and enzymatic biofuel cells. The latter uses an enzyme to catalyze fuel oxidation, such as the oxidation of methanol mediated by NADH [6]:
alcoholdehydrogenase CH3 OH 2NAD CH2 O 2NADH !

Introduction Economic and environmental factors are pushing the demand for clean and efficient energy production processes, which means that there is a continuous increase

1
CH2 O H2 O 2NAD HCOOH 2NADH !
aldehydedehydrogenase

2
HCOOH 2NAD ! CO2 2NADH 3
formatedehydrogenase
diaphorase

S. A. Neto : J. C. Forti : A. R. De Andrade (*) Departamento de Qumica, Faculdade de Filosofia Cincias e Letras de Ribeiro Preto, Universidade de So Paulo, 14040-901 Ribeiro Preto, So Paulo, Brazil e-mail: ardandra@ffclrp.usp.br

3NADH 6 mediatorred 3NAD 6 mediatorox 4 !

6 mediatorox ! 6 mediatorred 6e

anode

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In turn, microbial fuel cells (MFCs) use living cells as catalyst, such as the oxidation of Shewanella putrefaciens with an anode via a membrane-cytochrome [4]: lactate waste membrane cytochrome !
anode

microbial

membrane cytochrome membrane cytochrome e !

7 In recent years, the technology using microbial fuel cells that convert energy from organic compounds to electric energy via catalytic reactions performed by microorganisms has gained considerable attention among researchers worldwide [7]. Bacteria can be used in MFCs to generate electricity while accomplishing the biodegradation of organic matter or waste [8]. Recently, there has been a significant advance in MFC research, and the number of publications in this field has risen [9, 10]. In this paper, the main focus will be given to the enzymatic biofuel cells. A literature search conducted in the Chemical Abstract database using the term biofuel cell (Fig. 1) indicates that the number of publications in this area has increased dramatically over the last 7 years, thus demonstrating the growing interest in this new technology. The exponential interest in biofuel cell allows us to present and discuss the very recent papers which can contribute to the continued development of this subject. This manuscript aims to provide the reader with an overview of the state of the art in enzymatic biofuel cells by discussing the latest papers in this field and presenting an outlook for future research in this area.

The Concept of Biofuel Cells The biofuel cell can be understood as a system that directly converts chemical energy into electricity by means of

reactions involving biochemical steps. A biofuel cell is similar to the proton exchange membrane fuel cell, which consists of a cathode and an anode separated or not by a polymeric membrane. Biofuel cells or biological fuel cells are defined as a fuel cell in which the activity of the cell (or part of it) is due to the activity of an enzyme catalyst or even a fuel cell that uses a biocatalyst [3]. Basically, the operation of a fuel cell involves oxidation reactions at the anode side and reduction reactions at the cathode side. As a result of this process, an electron flow by an external circuit provides electrical work. Biofuel cells classified direct are the focus of current research. In this type of device, the biocatalysts are directly involved in the reaction generating electricity. In this biofuel cell, the fuel is enzymatically oxidized at the anode side, producing protons and electrons. At the cathode side, the oxidant (usually O2) reacts with the protons and electrons, generating water. Figure 2 shows a schematic example of a direct biofuel cell. Alternatively to the fuel cells using metal catalysts, the biofuel cell is capable of converting chemical energy into electricity, with the advantage of using biological molecules. This new technology offers several advantages over traditional batteries, including the use of renewable and clean catalysts, the selectivity of the reaction, fuel flexibility, and the ability to operate at milder temperature. Taken together, all these advantages lead to an economically viable process. While traditional fuel cells designated as low temperature operate at approximately 80 C, biofuel cells can operate in the range of 20 to 40 C and at physiological pH. These properties make biofuel cells an interesting alternative especially for devices that need to operate at milder temperatures and conditions. Moreover, the variety of reactions that can be catalyzed by enzymes enables the use of various fuel types. The main differences between conventional fuel cells and biofuel cells are summarized in Table 1. Despite the many advantages provided by the biofuel cells, some factors are crucial for a higher efficiency device. The main critical points in the construction of a biofuel cell are enzyme immobilization on the electrode and the speed of electron transfer from the enzyme active site to the electrode surface. Electrons Transfer A very important characteristic of enzymatic biofuel cells is the electron transfer between the reactive site of the enzyme and the electrode surface. The key to achieving efficient enzymatic biofuel cells is related to the speed of this process, and the best performance will depend on how easily this reaction takes place. Figure 3 shows the two possibilities of arrangement between the enzyme and the electrode surface.

Fig. 1 A literature search carried out in the Chemical Abstract database using the term biofuel cell. Source: SciFinder Scholar

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Fig. 3 Schematic representations of the possible electron transfer processes between enzymes and electrodes. a Direct electron transfer. b Mediated electron transfer Fig. 2 Schematic representation of a direct biofuel cell

The first case refers to the system where direct electron transfer (DET) occurs between the enzyme active site and the electrode. This feature is advantageous because the cells operate at a closer potential of that of the enzyme, thereby reducing interfering reactions and avoiding the use of other reagents in the sequence of enzymatic reactions. The DET is a new approach in biofuel cells and was proposed in late 2006, when researchers developed biocathodes for the reduction of oxygen using laccase and bilirubin oxidase and bioanodes using glucose oxidase [11, 12]. The main problem encountered with this approach was electron exchange with the electrode surface. This is because the redox center of the enzymes is located in the inner regions of the enzyme, in areas of difficult access [13]. In the case of mediated electron transfer (MET), a mediator molecule is used to improve the efficiency of charge transfer to the electrode. The mediators usually involve organic dyes or organometallic complexes, which are dissolved in solution or immobilized on the electrode.
Substrate mediatorox product mediadorred !
enzyme

substrate or product. Also, mediators make the anode structure complicated and sometimes are expensive and toxic to the biological arrangement. Immobilization and Enzyme Stability For better stability to be achieved, it is crucial to ensure that the enzyme is placed in a suitable environment, so that it can resist sudden changes in temperature, pH, and solution composition, which often distort or inactivate enzymes. The choice of immobilization process during the anchoring of enzymes on the electrode surface is of great importance because it directly affects the lifetime of the immobilized enzyme. It is usually necessary to immobilize the enzyme in a membrane on the electrode surface, and it is desirable to obtain a chemically and mechanically stable tie layer without formation of a capacitive region. The ion-exchange membrane Nafion has been frequently used as an ionic conductor in fuel cells; however, the use of non-modified layer in biofuel cells has not been successful due to its highly acidic environment, which decreases enzyme lifetime and activity. Recent studies have shown that the treatment of Nafion membranes with tetrabutylammonium salts results in an ideal environment for immobilization of dehydrogenase enzymes on the electrode surface. This modification neutralizes the Nafion acidity and increases its pore size, thereby promoting enzyme fixation and enhancing its stability. The biofuel cell lifetime achieved

8 9

Mediatorred mediatorox e !

cathode

A disadvantage of the mediated process (MET) is the possible interference of side reactions, once this arrangement may also facilitate electron transfer from redox reactions that occur in parallel with the reaction between the enzyme and the
Table 1 Main features of conventional fuel cells and biofuel cells Catalyst pH Temperature Efficiency (%) Fuels

Traditional fuel cell Noble metals Highly acid or basic pH Above 80 C 40 to 60 H2, methanol, etc.

Biofuel cell Microorganisms or enzymes pH between 7.0 and 9.0 22 to 25 C 40 Carbohydrate, methanol, ethanol

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with this immobilization technique can increase considerably, as reported elsewhere [14]. Another immobilization method is the use of redox polymers, where a polymer with high redox potential (e.g., 0.58 to 0.79 V vs. SHE) is employed at the cathode, and another polymer with a low redox potential (0.02 to 0.32 V vs. SHE) is used at the anode side [15]. One report described that enzymes are usually mixed with these polymers, which often contains an osmium redox center. A miniature biofuel cell with glucose oxidase and bilirubin oxidase immobilized on a redox polymer has shown a lifetime of 20 days at 37 C [15]. Enzyme immobilization can also be accomplished by encapsulating the enzymes inside a solgel matrix [16]. One example is the glucose/O2 biofuel cell developed by immobilization of enzymes inside a silica matrix solgel, also incorporating carbon nanotubes within the matrix, to increase the driving electronics and the electrode active area. Chitosan and modified Nafion have also been proposed for immobilization of dehydrogenase enzymes through the formation of a micellar membrane [17].

A Brief History and Obtained Results Biofuel Cell Development The development of biofuel cells started in the 1960s, being the majority of the studies focused on the glucose/O2 biofuel cell. Most studies were interested in the electrocatalysis of immobilized enzymes, focusing on fundamental studies of enzyme electron transfer or/and catalytic mechanism. In 1964, Yahiro et al. reported the first enzyme biofuel cell [2]; glucose and glucose oxidase were placed in the anode solution, and the cathode (Pt) was open to air. The cell gave an open circuit potential (OCP) in the range of 625 to 750 mV; however, the obtained current density was very low, only 30 nA cm2 at 330 mV. This low current density was probably due to the absence of any mediator molecule. The development of enzymatic biofuel cells was revived in the 1980s, when some papers focused on alcohol dehydrogenase for methanol oxidation. In 1981, a biofuel cell based on methanol was reported by Plotkin et al. [18], who used a bacterial methanol dehydrogenase in solution as the anode catalyst and phenazine ethosulfate as mediator. An OCP of 300 mV was achieved, with a maximum current density of 30 A cm2. In the 1990s, Palmore et al. [6] demonstrated a breakthrough research involving an enzymatic cascade employing alcohol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase for methanol complete oxidation. In 1998, Willner and Katz reported a membraneless fuel cell with immobilized enzymes as catalysts at both

the anode and cathode [19]. A pyrroloquinoline monolayer and a microperoxidase-11-modified Au electrode were used as anode and cathode, respectively. The device generated a power of 8 W cm2 and an open circuit voltage of 320 mV. In 1999, Palmore and Kim described dioxygen reduction using the enzyme laccase as catalyst and employing 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) as mediator [20]. In 2001, a very tiny enzyme-based device was developed for microsized implanted medical devices. Chen et al. [21] demonstrated a glucose biofuel cell made of two 7-mdiameter electrocatalyst-coated carbon fiber electrodes placed in 1-mm grooves machined into a polycarbonate support. The cell gave a power output of 600 nW at 37 C, which is enough to power small silicon-based microelectronics. The anode coating was glucose oxidase covalently bound to a reducing potential copolymer based on osmium complexes, and the cathode coating was based on laccase [21]. Another biofuel cell scope discussed over the last few years has been genetic improvement of enzymes before the immobilization step. In 2002, Halliwell et al. [22] showed the use of L-lactate dehydrogenase mutants and their suitability as potential anodic biocatalysts for biofuel cells. The enzymes were genetically modified for later immobilization on poly(aniline)-poly(vinyl sulfonate). The authors demonstrated that the enzymes can be readily immobilized while maintaining their specific activities toward the substrate and NAD+. The so-called PANi-enzyme electrodes revealed good potential for biosensor and biofuel cell applications [22]. An electroswitchable and tunable biofuel cell based on glucose and cytochrome oxidase at the anode and cathode sides, respectively, was described by Katz and Willner in 2003 [23]. This system was assembled on the hybrid Cu+2/Cu0poly(acrylic acid) matrix. The employed switching process allowed for reversible activation and deactivation of the cell, which works as a power source or as a self-powered glucose sensor. This paper was the first to describe an example of an electrochemically switchable and tunable biofuel cell for future use in implantable devices in physiological fluids, such as pacemakers and insulin pumps [23]. So far, the focus of this discussion has been mainly placed on alcohol/sugar fuels, but hydrogen has long been the standard fuel due to its low reduction potential and easy supply as gas, also called the transportable fuel of the future. Some interesting efforts toward the use of hydrogenases to replace currently Pt have also been published. Studies on hydrogenases and laccase/bilirubin oxidase are being conducted without redox mediators [24]. Some good results concerning enzyme activity have been reported; however, most hydrogenases are inactivated even by traces of O2, which makes them difficult to use in particular situations [25].

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In 2004, Heller focused on improvements in the performance of cathodes for the O2 reduction reaction [15]. The development of these cells occurred by using mainly laccase and bilirubin oxidase embedded into a conducting hydrogel. These biofuel cells presented a lifetime ranging from 8 h, in the case of the enzyme in solution, to about 710 days for the immobilized enzymes. Papers reporting lifetime extension were followed by several papers showing an increase in the power density for small electronic devices [15]. Many review contributions regarding biological fuel cell have been presented in the literature, standing out was a contribution from Barton et al. covering papers until 2004 that fully reviewed different aspects concerning biological fuel cells such as immobilization, applications, and differences between biofuel cell and biosensors, and mainly, a careful presentation of diffusional mediators for both NADH oxidation and O2 reduction was the main focus of the paper [26]. In addition, Bullen et al. in 2006 critically reviewed the papers published in the period from 1994 to 2005 quite well; the review focuses mainly on parameters such as power density, OCV, and cell conditions [3]. More recently, some researchers have also been interested in developing biofuel cells based on ethanol/O2. Although many advances have been made in this area, one of the major obstacles to the preparation of an efficient biofuel cell is bioanode stability and lifetime. Recently, many important studies aiming at biofuel cells based on ethanol have been published; some papers have already indicated that this new technology can lead to the production of batteries that allow for the operation of electronic devices like cell phones [14, 27, 28]. In 2008, Sokic-Lazic and Minteer described the complete oxidation of ethanol to carbonic gas by mimicking the citric acid cycle, which is the main metabolic pathway employed by living cells to convert carbon fuels to CO2 [29]. Dehydrogenase enzymes were immobilized in cascades at a carbon electrode, and an increase in the current density was observed according to the amount of the enzyme. Another biochemical cycle that has been mimicked is the Krebss cycle, by employing bioanodes with dehydrogenases in cascade for the complete oxidation of pyruvate [30]. Finally, in 2009, Sony Corporation presented a glucosefueled enzyme fuel cell for powering small consumer electronics [31]. In this cell, glucose dehydrogenase and bilirubin oxidase, both in solution with electron mediators and separated by a membrane, are used as the anode and cathode catalysts, respectively. Biofuel cells are a very developable subject, so many research teams worldwide are currently investigating several points regarding bioanode preparation and enzyme immobilization, also looking for higher power output values and longer biofuel cell lifetime. In 2009, the main focus of the papers was the immobilization step; once a good anchoring condition is obtained, both power output and bioanode

lifetime are enhanced. In 2009, Kuwahara et al. [32] proposed a bioanode prepared with a conducting polymer film electrochemically copolymerized with 3-methylthiophene and thiophene-3-acetic acid. With these polymers, the authors claimed that many functional groups can be introduced into the main chain by selection of appropriate monomers that facilitate access to the enzyme binding sites. In this same year, Wu et al. have demonstrated a membraneless fructose/air biofuel by using D-fructose dehydrogenase and bilirubin oxidase and employing a cellulosemultiwalled carbon nanotube matrix [33]. This kind of matrix provided a promising environment for enzyme anchoring since relative high power density and lifetime were achieved. Once the biofuel cells based on dehydrogenase enzymes require the use of mediators, mediatorless biofuel cells are currently the target of some research teams. Miyake et al. [34] demonstrated the use of glucose dehydrogenase and its cofactor on ketjenblack electrode with density power values enough for some medical sensors. Polyaniline is one of the often used conducting polymers, and bioanode composite films made of polyaniline and carboxydextrangold hybrid nanomaterials were reported by Lee et al. [35, 36]. The high electrocatalytic performance makes such material a good candidate for enzyme immobilization. Some good results using pyrroloquinoline onto gold electrodes for lactate dehydrogenase immobilization and an electrochemical study using ultra-small silicon nanoparticles for glucose oxidation were also described [37, 38]. Power Density Besides the immobilization step and the biofuel cell lifetime, another important point for the good efficiency of a biofuel cell is the power density, which is the relation between the power generated in the electrode and the area or volume of the cell. Contrary to the traditional fuel cells or metal-catalyzed fuel cells, the enzymatic fuel cells generally result in low power density. The typical power output values are in the range of micro- to milliwatts, much lower if compared with the kilowatt output values of traditional metal catalyst fuel cells. In situations where higher power density is required, this value can be increased by using a series of biofuel cells, for instance. For this reason, the development of biofuel cells aims at high power output values, and literature discussions are also based on this parameter. In 2008, Habrioux et al. built a prototype of a glucose biofuel cell on a cylindrical shape [39]. In this cell, the cathode was coated with laccase using ABTS [2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] as mediator on the external surface of the cylinder, for O2 reduction. The anode side was coated with glucose oxidase (10 mM) using HQs (8-hydroxyquinoline-5-sulfonic acid)

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as mediator on the inner cylinder surface. The enzymes and their mediators were immobilized on electrodes by means of a polypyrrole film, and the experiments were conducted at pH = 7.4 and 37 C. These biofuel cells led to a power density of 42 W cm2 at 0.32 V. In 2003, Kim et al. reported the preparation of a microbiofuel cell using glucose oxidase [40]. The results under physiological conditions (pH = 7.4, NaCl 0.14 M, 37.5 C, 15 mM glucose) revealed a power density of 50 W cm2 at 0.5 V. In 2007, Gao et al. also developed a biofuel cell glucose/O2 based on the use of the same enzymes with different mediators [41]. The obtained power density was 53.9 W cm2; the OCP and the current density were 0.53 V and 28 A cm2, respectively. Lim et al. studied the fabrication and characterization of a biofuel cell glucose/O2, with a generated power density of 0.12 mW cm2 at 0.24 V. The OCP was 0.48 V at room temperature [16]. Topcagic and Minter [27] described a biofuel cell based on alcohol dehydrogenase for oxidation of ethanol at the anode and bilirubin oxidase for O2 reduction at the cathode, in the midst of phosphate buffer pH = 7.15. As the oxidation and reduction were governed by selective enzymatic catalysis, i.e., anode and cathode modified with enzymes, it was necessary to use a membrane for separation. Results showed a 30-day lifetime, power density of 0.46 mW cm2, and OCP in the range of 0.680.83 V. A multi-enzymatic biofuel cell was developed by Palmore et al. [6] for methanol oxidation. The enzymes alcohol, aldehyde, and formate dehydrogenases were used with NADH as mediator. A graphite and a Pt cathode
Table 2 Summary of enzymatic biofuel cells latest papers Immobilization methodology Substrate Enzymatic system

anode were used in buffer solution, pH = 7.5. This biofuel cell produced 0.68 mW cm2 at 0.49 V (OCP = 0.8 V and i = 2.6 A cm2). On the developing of ethanol-based biofuel cells, some promising features, especially in relation to the power density values, have been obtained. Akers and coworkers studied the oxidation of ethanol to acetate in two steps using a carbon anode with polymerized modified biocatalysts of alcohol and aldehyde dehydrogenases and NADH as mediator [14]. The Pt cathode was used in buffer pH = 7.15. The biofuel ethanol/ O2-based cell presented a power density of 1.16 mW cm2 in a simple enzymatic system (alcohol dehydrogenase, OCP = 0.82 V, i= 5.2 A cm2) and 2.04 mW cm2 in a dual enzymatic system (alcohol and aldehyde dehydrogenase, OCP = 0.82 V, i = 5.2 A cm2). For the methanol/O2based biofuel cell, a power density of 1.55 mW cm2 (OCP = 0.71 V, i = 5 A cm2) was achieved. We have recently demonstrated a bioanode for ethanol biofuel cell based on alcohol dehydrogenase using PAMAM dendrimers as matrix for enzyme immobilization on carbon cloth platform [42]. The device provided a power density of 0.28 mW cm2 at 0.3 V and an OCP of 0.72 V. The prepared bioanode provided a lifetime of 90 days based on power density measurements. Numerous efforts have been devoted to improving the generated power density; the use of multiple layers of enzymes is an example. The very recent studies have shown a significant increase in the power density values compared to the ones obtained in studies published 10 years ago. The latest papers report values in the range of 0.1 mW to power output enough for real-life applications (100 mW), high

Power output Reference (mWcm2) 0.46 0.12 0.035 0.0015 1.01 0.042 0.93 1.45 0.15 0.12 0.052 0.14 0.0037 0.28 Topcagic and Minteer [27] Lim et al. [16] Klotzbach et al. [17] Ramanavicius et al. [28] Sokic-Lazic and Minteer [29] Habrioux et al. [39] Sokic-Lazic and Minteer [30] Sakai et al. [31] Kuwahara et al. [32] Wu et al. [33] Miyake et al. [34] Lee et al. [3537] Choi et al. [38] Forti et al. [42]

Modified Nafion membrane Solgel silica matrix/carbon nanotubes Modified chitosan and Nafion membranes Glutaraldehyde Modified Nafion membrane Polypyrrole film Modified Nafion membrane Polyacrylic acid sodium salt Conducting polymer copolymerized with 3-methylthiophene Cellulosemultiwall carbon nanotubes matrix Ketjenblack electrode Pyrroloquinoline onto gold electrodes Ultra-small silicon nanoparticles PAMAM dendrimers

Ethanol/O2 Glucose/O2 Glucose Ethanol Ethanol Glucose/O2 Pyruvate Glucose/O2 Glucose/O2 Fructose/O2 Glucose Lactate Glucose Ethanol

Alcohol dehydrogenase/bilirubin oxidase Glucose oxidase and bilirubin oxidase Glucose dehydrogenase Quino-hemoprotein-alcohol dehydrogenase Dehydrogenase enzymes in cascade Glucose dehydrogenase/laccase Dehydrogenase enzymes in cascade Glucose dehydrogenase/Bilirubin oxidase Glucose oxidase and bilirubin oxidase
D-fructose

dehydrogenase/bilirubin oxidase Glucose dehydrogenase Lactate dehydrogenase Glucose dehydrogenase Alcohol dehydrogenase

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enough for many electronic devices [30]. Table 2 summarizes the latest papers main features in enzymatic biofuel.

been investigated by using different fuels, such as methanol, ethanol, hydrogen, and glucose. Micro- and nanofluidic devices have been recently reviewed by Kjeang et al. [47].
Acknowledgments Financial support from FAPESP, CAPES, and CNPq is gratefully acknowledged. Aquino Neto also acknowledges the DS-CAPES fellowship.

Applications, Future, and Challenges Many are the possibilities of biofuel cells use; nowadays, there is a growing interest in enzyme-based biofuel cells as an alternative of renewable and sustainable power supply, with great potential for portable devices. The current research also has future in vivo application in drug delivery as a major target, i.e., the use of biofuel cells in implantable devices where the fuel can be continuously replaced (e.g., glucose in pacemaker devices). Recent studies have revealed promising characteristics of these devices; however, despite the several advances in this area, some challenges still need to be faced. Crucial factors for their future large-scale application, such as enzyme lifetime, generation of higher power densities, overcoming the difficulties in the electron transfer between enzymes and the electrodes, and the development/improvement of enzyme anchoring techniques (immobilization), are important objects of research. In terms of maximizing power density, threedimensional enzymatic biofuel cells appear as an advantageous alternative, since this kind of cells should exhibit multidimensional and multidirectional pore structures. Multidimensionality provides both small pores to support enzyme stabilization and high loading densities. The chitosan polymer described recently is a material that promises control over both the degree of dimensionality and directionality [30, 43]; nevertheless, this approach remains relatively unexplored. The development of miniature biofuel cell has been another focus of recent papers. One of the best advantages of miniaturized cells is the high surface to volume ratio (very important in electrochemical reactions), but it adds up that there are still some technical challenges to be faced in their development [44, 45]. Another device that has been the subject of studies is the microfluidic fuel cell, which can be defined as a cell where the reaction sites and electrode structure are confined in a microfluidic channel, making the separation between cathode and anode unnecessary. In other words, this cell operates without a physical barrier, such as a membrane. This specific area was firstly developed by Moore et al. [46], who showed a microchip-based bioanode with alcohol dehydrogenase enzyme immobilized on a tetrabutylammonium bromide-treated membrane. The microfluidic bioanode generated an open-circuit potential of 0.34 V and a maximum current density of 53 A cm2. These results indicated that the process is probably limited by the diffusion rate of NADH within the membrane. Since its invention in 2002, this field has been the target of many researchers, and it is currently being commercially developed by a US company (INI Power Systems, Morrisville, NC). This kind of device has

References
1. Potter MC (1912) Biol Sci 84:260 2. Yahiro AT, Lee SM, Kimble DO (1964) Biochim Biophys Acta 25:88 3. Bullen RA, Arnot TC, Lakeman JB, Walsh FC (2006) Biosens Bioelectron 21:2015 4. Jungbae K, Hongfei J, Wang P (2006) Biotechnol Adv 24:296 5. Minteer SD, Liaw BY, Cooney MJ (2007) Curr Op Biotech 18:228 6. Palmore GTR, Bertschy H, Bergens SH, Whitesides GM (1998) J Electroanal Chem 443:155 7. Moon H, Chang IS, Kim BH (2006) Bioresource Technol 97:621 8. Logan BE, Murano C, Scott K, Gray ND, Head IM (2005) Water Res 39:942 9. Du Z, Li H, Gu T (2007) Biotech Adv 25:464 10. Logan BE, Regan JM (2006) Environ Sci Technol 40:5172 11. Zheng W, Li Q, Su L, Ya Y, Zhang J, Mao L (2006) Electroanal 18:587 12. Ivnitski D, Branch B, Atanassov P, Apblett C (2006) Electrochem Commun 8:1204 13. Dan W, Ying L, Guocheng Y, Shaojun D (2007) Electrochim Acta 52:5312 14. Akers NL, Moore CM, Minteer SD (2005) Electrochim Acta 50:2521 15. Heller A (2004) Phys Chem Chem Phys 6:209 16. Lim J, Malati P, Bonet F, Dunn B (2007) J Electrochem Soc 154:140 17. Klotzbach TL, Watt M, Ansari Y, Minteer SD (2008) J Membrane Sci 311:81 18. Plotkin EV, Higgins IJ, Hill HAO (1981) Biotechnol Lett 3:187 19. Willner I, Katz GAE (1998) Bioelectrochem Bioenerg 44:209 20. Palmore GTR, Kim H (1999) J Electroanal Chem 464:110 21. Chen T, Barton SC, Binyamin G, Gao Z, Zhang Y, Kim H, Heller A (2001) J Am Chem Soc 123:8630 22. Halliwell CM, Simon E, Toh C, Cass AEG, Bartlett PN (2002) Bioelectrochem 55:21 23. Katz E, Willner I (2003) J Am Chem Soc 125:6803 24. Vincent KA, Cracknell JA, Clark JR, Ludwig M, Lenz O, Friedrich B, Armstrong FA (2006) Chem Commun 48:5033 25. Karyakin AA, Morozov SV, Karyakina EE, Zorin NA, Perelygin VV, Cosnier S (2005) Biochem Soc Trans 33:73 26. Barton SC, Galllaway J, Atanassov P (2004) Chem Rev 104:4867 27. Topcagic S, Minteer SD (2006) Electrochim Acta 51:2168 28. Ramanavicius A, Kausaite A, Ramanaviciene A (2008) Biosens Bioelectron 24:761 29. Sokic-Lazic D, Minteer SD (2008) Biosens Bioelectron 24:939 30. Sokic-Lazic D, Minteer SD (2009) Electrochem Solid-State Lett 12:26 31. Sakai H, Nakagawa T, Tokita Y, Hatazawa T, Ikeda T, Tsujimura S, Kano K (2009) Energy Environ Sci 2:133 32. Kuwahara T, Homma T, Kondo M, Shimomura M (2009) Synth Met 159:1859 33. Wu X, Zhao F, Varcoe JR, Thumser AE, Avignone-Rossac C, Slade RCT (2009) Biosens Bioelectron 25:326 34. Miyake T, Oike M, Yoshino S, Yatagawa Y, Haneda K, Kaji H, Nishizawa M (2009) Chem Phys Lett 480:123 35. Lee S, Choi B, Tsutsumi A (2009) J Chem Eng Japan 42:596 36. Lee S, Choi B, Tsutsumi A (2009) Biotechnol Lett 31:851 37. Lee JY, Shin HY, Lee JH, Song YS, Kang SW, Park C, Kim JB, Kim SW (2009) J Mol Catalysis B: Enzym 59:274

94 38. Choi Y, Wang G, Nayfeh MH, Yau S (2009) Biosens Bioelectron 24:3103 39. Habrioux A, Merle G, Servat K, Kokoh KB, Innocent C, Cretin M, Tingry S (2008) J Electroanal Chem 622:97 40. Kim H-H, Mano N, Zhang Y, Heller A (2003) J Electrochem Soc 150:209 41. Gao F, Yan Y, Su L, Wang L, Mao L (2007) Electrochem Commun 9:989

Electrocatal (2010) 1:8794 42. Forti JC, Aquino Neto S, Zucolotto V, Ciancaglini P, De Andrade AR (2010) Biosens Bioelectron (in press) 43. Tan YM, Deng WF, Ge B, Xie QJ, Huang JH, Yao SZ (2009) Biosens Bioelectron 24:2225 44. Mano N, Mao F, Heller A (2002) J Am Chem Soc 124:12962 45. Mano N, Heller A (2003) J Electrochem Soc 150:1136 46. Moore CM, Minteer SD, Martin RS (2005) Lab Chip 5:218 47. Kjeang E, Djilali N, Sintona D (2009) J Power Sources 186:353