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Starchy biomass-powered enzymatic biofuel cell based on amylases and glucose oxidase multi-immobilized bioanode
Kazuhiro Yamamoto, Takuya Matsumoto, Shota Shimada, Tsutomu Tanaka and Akihiko Kondo
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

The present study reports the design of a novel bioanode to directly utilize starch as a fuel in an enzymatic biofuel cell. The enzymatic fuel cell is based on three enzymes (alpha-amylase, glucoamylase and glucose oxidase). The carbon paste electrode containing these three enzymes and tetrathiafulvalene can both saccharize and oxidize starchy biomass. In cyclic voltammetry, catalytic currents were successfully observed with both glucose and starchy white rice used as a substrate. Finally, a membraneless white rice/O2 biofuel cell was assembled and the electrochemical performance was evaluated. The three enzyme based electrode was used as a bioanode and an immobilized bilirubin oxidase (derived from Myrothecium verrucaria) electrode was used as a biocathode. The biofuel cell delivered an open circuit voltage of 0.522 V and power density of up to 99.0 mW cm2. Our results show that a readily available fuel can be used for enzymatic fuel cells, and will lead to new designs.

Introduction
Biofuel cells are based on bio-functional anodes and cathodes (bioanodes and biocathodes), which convert organic substrates to electrical energy. Bioanodes used in microbial fuel cells are based on microorganisms that can oxidize bioorganic substrates and generate currents [1,2]. In addition, bioanodes used in enzymatic biofuel cells are based on oxidizing enzymes that generate current by oxidizing substrates such as alcohols or carbohydrates [3]. Enzymatic biofuel cells have the attractive property of operating under physiological conditions. By harnessing this property, enzymatic biofuel cells can be developed for implantable applications [4]. In addition, the simple mechanism of bioanodes and the availability of a variety of biological materials for use as substrates render them as attractive potential energy sources. Basically, enzymatic bioanodes are composed of an oxidizing enzyme and a redox mediator. The diversity of oxidizing enzymes enables the use of a variety of fuels. Enzymatic glucose fuel cells have been particularly well-characterized. These bioanodes utilize either glucose oxidase (GOx) or glucose dehydrogenase (GDH) as the oxidizing enzyme
Corresponding author: Tanaka, T. (tanaka@kitty.kobe-u.ac.jp)

[5,6], while alcohol dehydrogenase is utilized in alcohol fuel cells and fructose dehydrogenase is utilized in fructose fuel cells [7,8]. Although several enzymatic monosaccharide (glucose, fructose, and so on.) and alcohol fuel cells have been described in the literature, there has been comparatively little study of bioanodes that can directly oxidize oligosaccharides as fuel sources [9,10]. Several works have shown microbial fuel cells operating with starchy or cellulosic biomass [11,12], but as of yet, only one enzymatic fuel cells as we understand it. This is because there is no enzyme that can directly oxidize long-chain polysaccharides. Starchy or cellulosic biomass is composed of polysaccharides, and thus represents an environmentally friendly fuel for bioconversion processes involving enzymes or microorganisms. Starch is contained in many staple foods (e.g. potatoes, wheat, corn, rice) and consists of a large number of glucose units conjugated with glycosidic bonds. However, while most oxidizing enzymes can utilize monosaccharides, they cannot directly process polysaccharides. Hence, a saccharication process is required to hydrolyze polysaccharides into monosaccharides before use as a biofuel. In the presence of alpha-amylase, starch is hydrolyzed to oligosaccharides or dextrin, both of which are hydrolyzed to glucose by
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glucoamylase. Similarly, in the degradation of cellulosic materials into glucose, several kinds of cellulase are required. Recently, carboxymethyl cellulose (CMC), which is one of the water-soluble cellulosic materials, was utilized as a fuel for enzymatic fuel cells. However, the bioanode was not able to oxidize CMC, and it required a multi-step saccharication process using three types of cellulase to generate electricity from CMC [13]. To simplify this multi-step process, which requires saccharication of polysaccharides and utilization of monosaccharides, biochemical engineers have developed bioconversion processes in which polysaccharides are simultaneously saccharized and fermented (SSF). Bioconversion processes have been developed involving recombinant microorganisms in which alpha-amylase and glucoamylase genes can be induced to enable direct utilization of starchy biomass. These processes have been used to successfully convert starchy biomass into bio-ethanol fuel [14,15]. In this study, a novel bioanode inspired by the above-mentioned SSF process was designed to directly utilize starch as a fuel for an enzymatic fuel cell. The novel bioanode enables simultaneous saccharication and oxidization of starch on the electrode surface. Here, we demonstrate that the enzymatic fuel cell can operate using a suspension of white rice as fuel, with a multi-immobilized bioanode of hydrolases and oxidase. Using multi-immobilization of GOx, alphaamylase and glucoamylase on a carbon paste electrode (CPE), we developed an electrode that can both saccharize and oxidize starchy biomass.

Bioanode preparation
The CPE containing tetrathiafulvalene (TTF) and GOx from Aspergillus niger (GOx, purchased from Nakarai tesuque, Kyoto, Japan), alpha-amylase from S. bovis 148 (AmyA), and glucoamylase from Rhizopus oryzae (GluR, TOYOBO Co., Ltd., Osaka, Japan) (GOx/ AmyA/GluR/CPE) was prepared as follows: Enzymes solutions (containing 75.8 mg GOx, 245 U mg1; 18.2 mg GluR, 37 U mg1; 1; or 15 mg AmyA, 70 000 U mg1) and a solution of TTF (30 mL in a saturated solution of methanol) were mixed with carbon paste (BAS Inc., Tokyo, Japan) (60 mg). The carbon paste mixture was packed into the electrode (diameter 3 mm) and compressed with clean copy paper. Then the electrode surface was coated with an aqueous solution of polyarylamine (PAA) (1.0% (w/v) in distilled water, 10 mL) and glutaraldehyde (GA) (0.5% (w/v) in distilled water, 1 mL) complex lm. Electrodes were allowed to dry for 2 hours at ambient temperature. The CPE containing TTF and GOx (GOx/CPE) was prepared as follows: Enzymes solution (containing 75.8 mg GOx) and a solution of TTF (30 mL in a saturated solution of methanol) were mixed with carbon paste (60 mg). This carbon paste mixture was also packed into the electrode and compressed with clean copy paper, and then the surface was coated with PAA and GA complex lm.

Research Paper

Biocathode preparation
Enzyme solution (containing 45 mg billirubin oxidase (BOx) from Myrothecium verrucaria, 5.6 U mg1, SigmaAldrich Corp., St. Louis, MO, USA) and a solution of 2,20 -azino-bis(3-ethylbenzothazolin-6-sulfonic acid) (ABTS) (30 mL in a saturated solution of water) containing carbon paste (60 mg) were packed into the electrode and compressed with clean copy paper. Then the electrode surface was coated with an aqueous solution of PAA (1.0% (w/v) in distilled water, 10 mL) and GA (0.5% (w/v) in distilled water, 1 mL) complex lm. Electrodes were allowed to dry for 2 hours at ambient temperature.

Materials and methods Construction of expression plasmids

KOD FX polymerase (TOYOBO Co., Ltd., Osaka, Japan) was used for PCR, and the PCR-amplied sequence was veried by DNA sequencing. The expression plasmid for Alpha-amylase (AmyA) derived from Streptococcus bovis 148 was constructed as follows. The gene encoding AmyA was obtained by PCR using a vector from a previous report [14] as a template with the 50 primer (50 -GGG GTA CCG GAT CCG ATA TCG ATG AAC AAG TGT C-30 ) and the 30 primer (50 -CCC AAG CTT GAA TTC TTA TTT TAG CCC ATC TTT ATT AT-30 ). The amplied fragment was subcloned into the KpnI/ EcoRI sites of the pET32b(+) vector (Novagen, San Diego, CA, USA) to yield pET32b(+)-AmyA.

Electrochemical measurements
The electrochemical measurements were performed using a Model 2323 Bi-Potentiostat (BAS Inc.) and a conventional three-electrode cell. The Ag/AgCl electrode and a Pt wire were used as the reference electrode and the counter electrode, respectively. The bioanode described above was used as the substrate of the working electrode. Cyclic voltammogram (CV) analysis was used to characterize the electrocatalytic properties of the bioanodes with respect to glucose or starchy biomass oxidation, and CV curves ranging between 0.2 V and +0.35 V were measured. Open circuit potential analysis was used to measure the electromotive force of cells consisting of a bioanode and a biocathode, and measurement time was 10 s. Linear sweep voltammogram analysis was done to measure the maximum power density of cells. All electrochemical measurements were conducted at ambient temperature and physiological pH.

Expression and purication of enzymes

AmyA was expressed and puried as follows. The pET32b(+)-AmyA plasmid was introduced into Escherichia coli BL21 (DE3). Cells were grown in LB medium to an OD (600 nm) of 0.5 at 378C, then cells were incubated an additional 30 min at 258C. Expression of the protein was induced by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG) to a nal concentration of 0.5 mM. After growth for an additional 24 hours at 258C, cells were harvested by centrifugation. The cell pellets were resuspended in 50 mM phosphate, 150 mM NaCl, pH 8.0, and lysed by sonication. AmyA was puried from the soluble fraction using TALON metal afnity resin (Takara Bio, Inc., Shiga, Japan) according to the manufacturers protocol, and dialyzed against 20 mM phosphate, 150 mM NaCl, pH 8.0. The concentration of puried AmyA was determined using a BCA protein assay kit (Pierce). AmyA hydrolysis activity was assayed by an alpha-amylase assay kit (Kikkoman Corp., Chiba, Japan).
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Preparation of rice crude solution

Polished white rice (kindly provided from Hyogo Prefecture, Japan) was ground to produce our with a particle size of approximately 0.5 mm using a laboratory disintegrator. The our was suspended in phosphate buffer and electrochemical determination was performed.

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Results and discussion Preparation of anodic enzymes

AmyA derived from S. bovis 148 was utilized, which can efciently adsorb and hydrolyze starch [16,17]. AmyA was produced by recombinant E. coli, and puried AmyA retained efcient hydrolysis activity (70 000 U mg1). In addition, glucoamylase from R. oryzae (GluR) was utilized, which is a very efcient hydrolytic enzyme. Starchy biomass has been efciently hydrolyzed to glucose by a combination of AmyA and GluR in previous studies [14,15]. The GOx that we utilized is produced by A. niger. GOx is a avin adenine dinucleotide (FAD)-binding enzyme, and has been used as a glucose sensor and in enzymatic glucose fuel cells [18,19].
FIGURE 1

The design of three enzyme co-immobilized bioanode

The design of the bioanode is illustrated in Fig. 1. Production of the bioanode required the multi-immobilization of the three enzymes (AmyA, GluR and GOx) in a densely packed conguration while retaining the enzymatic activity. TTF was also immobilized on the bioanode to serve as a redox mediator, because oxidized derivatives of TTF had high electrical conductivity. Glassy carbon electrode is often utilized for the preparation of biosensor or bioelectrode of biofuel cells, however, obtaining a sufcient density of enzymes on a surface such as glassy carbon is usually difcult because of surface area constraints. CPEs represent an attractive alternative for multi-immobilization of

Direct production of electrical current from starchy biomass using a GOx/ AmyA/GluR-multi-immobilized bioanode.

enzymes because multiple enzymes can be densely packed by kneading them with a carbon powder [2022]. Parafn modied simple carbon powder was utilized in this study, although a porous carbon powder is more suitable to expand the surface area of the electrode. To reduce the potential for deactivation of enzymes by adsorption to the carbon paste, enzymes were assembled using GA crosslinking of their amino groups, which increased enzymatic stability. The use of GA crosslinking for the

FIGURE 2

Cyclic voltammograms of the GOx/AmyA/GluR/CPE (a) or GOx/CPE (b) in 0.1 M, pH 7.2, phosphate buffer (blue line) containing 50 mM glucose (green line) or raw starch (weight equivalent to 50 mM glucose: red line). (c) Cyclic voltammograms of the BOx/CPE in N2 (blue line) or O2 (red line) saturated 0.1 M, pH 7.2, phosphate buffer. Data were collected at 10 mV s1.
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assembly and stabilization of enzymes has been reported elsewhere [23,24]. Another signicant concern when using CPE is the potential for exsorption of the enzymes and/or mediator from the electrode surface. To prevent exsorption, CPE can be covered with a thin membrane such as a nitrocellulose lm or a poly-ion complex (PIC) membrane [25]. In this study, we covered the CPE with a PAA membrane, which can be easily produced by gelatication of PAA with the addition of GA.

Cyclic voltammetry of multi-functional bioanode

The electrochemical performance of the bioanode was assessed using CV analysis. The analysis was conducted in a 0.1 M phosphate buffer (pH 7.2). GOx/AmyA/GluR/CPE was tested for direct oxidation of raw starchy biomass. Hyogo Kinuhikari white rice was chosen as a model of starchy biomass. The results of CV analysis of simultaneous saccharication and oxidation of white rice and glucose using the GOx/AmyA/GluR/CPE are shown in Fig. 2a. Catalytic currents were observed with both glucose and white rice used as a substrate. However, with a GOx-only immobilized bioanode, GOx/CPE, no catalytic current was observed in the presence of white rice (Fig. 2b). These results indicated that simultaneous saccharication and oxidation of white rice requires co-immobilization of GOx along with AmyA and GluR. Although anodic currents were observed in the presence of white rice with the GOx/AmyA/ GluR/CPE, this current was lower than that obtained with glucose, although equivalent initial amounts of glucose and raw starch were used. Polished white rice is also composed of water, protein, lipid and ash. The usable raw starch component is 80%, and therefore we expected that the anodic current obtained with white rice would be 80% of that obtained with glucose. Actually, the obtained anodic current from white rice was approximately 70% of that obtained with glucose. Thus, GOx/AmyA/GluR/CPE efciently operated even with white rice suspended in crude solution, showing that a solid solute reaction (degradation of starchy biomass to glucose) and a solutesolute reaction (oxidation of glucose) occurred consecutively. Incidentally, the extra addition of AmyA or GluR in the bioanode solution did not result in more efcient simultaneous saccharication and oxidation of white rice (data not shown). This result indicated that the addition of oxidase and hydrolase contained in the bioanode was suitable for simultaneous saccharication and oxidation. Even more remarkable, with GOx/CPE in addition to AmyA and GluR in the solution, a poor catalytic current was observed in the presence of white rice (data not shown). This result clearly indicated that multi-immobilization of GOx, AmyA and GluR was an effective approach for simultaneous saccharication and oxidation of starchy biomass.
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FIGURE 3

The assembly of a membrane-less white rice/O2 biofuel cell.

FIGURE 4

The power density curve of the white rice/O2 biofuel cell obtained by LSV in 0.1 M, pH 7.2, phosphate buffer containing 50 mM white rice (weight equivalent to 50 mM glucose). Data were collected at 10 mV s1.

The assembly of a membrane-less white rice/O2 biofuel cell

To evaluate the electrochemical performance as an enzymatic biofuel cell operating with raw starch biomass, linear sweep voltammetry (LSV) was used. GOx/AmyA/GluR/CPE was used for a bioanode, and bilirubin oxidase derived from M. verrucaria (BOx) and ABTS immobilized CPE (BOx/CPE) was used for a biocathode [26,27]. ABTS is commonly used as a substrate for peroxidase, laccase and bilirubin oxidase, and serves as an electron donor. The BOx/CPE performance was assessed by CV in a 0.1 M phosphate buffer (pH 7.2). The result of CV analysis of O2 oxidation is shown in Fig. 2c. Catalytic current was observed only in the O2 saturated condition,
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indicating that the BOx/CPE was suitable as a biocathode of our biofuel cell. To assemble a membrane-less white rice/O2 biofuel cell, these two electrodes were immersed in O2 saturated 0.1 M phosphate buffer (pH 7.2) containing 50 mM white rice (weight equivalent to 50 mM glucose) (shown in Fig. 3). Fig. 4 shows the current and the power density of the biofuel cell as a function of the operating cell voltage (IV and PV curve) at room temperature. As shown in Fig. 4, open-circuit voltage (Voc) and the short-circuit current of the biofuel cell are 0.522 V and 0.469 mA cm2, respectively. The maximum power density of the biofuel cell was 99.0 mW cm2 at 0.211 V. Although our designed bioanode saccharized and oxidized starch biomass simultaneously on the electrode surface, the value of the power density was almost the same order as those reported in the literature such as for a gold nanoparticle modied Au electrode, which can operate without redox mediator [28], and one in which maltodextrin was used as a power source [10]. In summary, we developed a novel enzymatic biofuel cell that operates with starchy biomass. The biofuel cell was based on an AmyA, GluR and GOx multi-immobilized bioanode that can

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directly utilize starch. The biofuel cell delivered an open circuit voltage of 0.522 V and power density of up to 99.0 mW cm2. Our results which show that a readily available fuel can be used for enzymatic fuel cells will promote their study as logic gate systems such as typical Boolean logic operations which operate with concerted catalysis of multi-enzyme reaction [2931] and will lead to new designs.

Acknowledgements
This work was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows, by Grant-in-Aid for Scientic Research(B) and by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.

References
der U, Keller J, Freguia S, et al. Microbial [1] Logan BE, Hamelers B, Rozendal R, Schro fuel cells: methodology and technology. Environmental Science and Technology 2006;40:518192. [2] Chaudhuri SK, Lovley DR. Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nature Biotechnology 2003;21:122932. [3] Willner I, Yan Y-M, Willner B, Tel-Vered R. Integrated enzyme-based biofuel cells a review. Fuel Cells 2009;9:724. [4] Barton SC, Gallaway J, Atanassov P. Enzymatic biofuel cells for implantable and microscale devices. Chemical Reviews 2004;104:486786. [5] Chen C, Wang L, Tan Y, Qin C, Xie F, Fu Y, et al. High-performance amperometric biosensors and biofuel cell based on chitosan-strengthened cast thin lms of chemically synthesized catecholamine polymers with glucose oxidase effectively entrapped. Biosensors and Bioelectronics 2011;26:23116. [6] Tan Y, Deng W, Ge B, Xie Q, Huang J, Yao S, et al. Biofuel cell and phenolic biosensor based on acid-resistant laccase-glutaraldehyde functionalized chitosan-multiwalled carbon nanotubes nanocomposite lm. Biosensors and Bioelectronics 2009;24:222531. [7] Deng L, Shang L, Wen D, Zhai J, Dong S. A membraneless biofuel cell powered by ethanol and alcoholic beverage. Biosensors and Bioelectronics 2010;26:703. [8] Wu X, Zhao F, Varcoe JR, Thumser AE, Avignone-Rossa C, Slade RC, et al. A onecompartment fructose/air biological fuel cell based on direct electron transfer. Biosensors and Bioelectronics 2009;25:32631. [9] Rasmussen M, Ritzmann RE, Lee I, Pollack AJ, Scherson D. An implantable biofuel cell for a live insect. Journal of the American Chemical Society 2012;134:145860. [10] Zhu Z, Wang Y, Minteer SD, Zhang YP. Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway. Journal of Power Sources 2011; 196:75059. [11] Ren Z, Ward TE, Regan JM. Electricity production from cellulose in a microbial fuel cell using a dened binary culture. Environmental Science and Technology 2007;41:47816. der U, Scholz F. Exploiting complex carbohydrates for microbial [12] Niessen J, Schro electricity generation a bacterial fuel cell operating on starch. Electrochemistry Communications 2004;6:9558. [13] Cheng H, Qian Q, Wang X, Yu P, Mao L. Electricity generation from carboxymethyl cellulose biomass: a new application of enzymatic biofuel cells. Electochimica Acta 2012;82:2037. [14] Yamada R, Bito Y, Adachi T, Tanaka T, Oginoa C, Fukuda H, et al. Efcient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated a-amylase and glucoamylase genes. Enzyme and Microbial Technology 2009;44:3449. [15] Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A. Novel strategy for yeast construction using delta-integration and cell fusion to efciently produce ethanol from raw starch. Applied Microbiology and Biotechnology 2010;85: 14918. [16] Satoh E, Niimura Y, Uchimura T, Kozaki M, Komagata K. Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli. Applied and Environment Microbiology 1993;59:366973. [17] Lin SC, Liu WT, Liu SH, Chou WI, Hsiung BK, Lin IP, et al. Role of the linker region in the expression of Rhizopus oryzae glucoamylase. BMC Biochemistry 2007;8:9. [18] Mano N, Mao F, Shin W, Chen T, Heller A. A miniature biofuel cell operating at 0.78 V. Chemical Communications 2003;5189. [19] Wong CM, Wong KH, Chen XD. Glucose oxidase: natural occurrence, function, properties and industrial applications. Applied Microbiology and Biotechnology 2008;78:92738. [20] Yang X, Tian G, Jiang N, Su B. Immobilization technology: a sustainable solution for biofuel cell design. Energy & Environmental Science 2012;5: 554063. [21] Liu S, Ju H. Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modied carbon paste electrode. Biosensors and Bioelectronics 2003;19:17783. [22] Wang J, Musameh M, Mo JW. Acid stability of carbon paste enzyme electrodes. Analytical Chemistry 2006;78:70447. [23] Kowal R, Parsons RG. Stabilization of proteins immobilized on Sepharose from leakage by glutaraldehyde crosslinking. Analytical Biochemistry 1980;102:726. [24] Yu CM, Yen MJ, Chen LC. A bioanode based on MWCNT/protein-assisted coimmobilization of glucose oxidase and 2,5-dihydroxybenzaldehyde for glucose fuel cells. Biosensors and Bioelectronics 2010;25:251521. [25] Komaba S, Mitsuhashi T, Shiraishi S. Optimization of enzyme anode and cathode with polyion complex for the application to biofuel cells. Electochemistry 2008;76:61924. [26] Ivnitski D, Artyushkova K, Atanassov P. Surface characterization and direct electrochemistry of redox copper centers of bilirubin oxidase from fungi Myrothecium verrucaria. Bioelectrochemistry 2008;74:10110. [27] Szczupak A, Kol-Kalman D, Alfonta L. A hybrid biocathode: surface display of O2-reducing enzymes for microbial fuel cell applications. Chemical Communications 2012;48:4951. [28] Wang X, Falk M, Ortiz R, Matsumura H, Bobacka J, Ludwig R, et al. Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modied electrodes. Biosensors and Bioelectronics 2012;31:21925. [29] Zhou M, Zheng X, Wang J, Dong S. A self-powered and reusable biocomputing security keypad lock system based on biofuel cells. Chemistry A European Journal 2010;16:771924. mek J, Tam TK, Strack G, Bocharova V, Pita M, Katz E, et al. Self-powered [30] Hala biomolecular keypad lock security system based on a biofuel cell. Chemical Communications 2010;46:24057. [31] Strack G, Pita M, Ornatska M, Katz E. Boolean logic gates that use enzymes as input signals. Chembiochem 2008;9:12606.

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