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Technical Brief

An Integrated Bio-Anode Using Yeast Extract for a High-Temperature Glucose Fuel Cell OPEN ACCESS

[+] Author and Article Information
Koichi Kasahara

Department of Chemical and Environmental Engineering,
Gunma University,
1-5-1, Tenjincho,
Kiryu 376-8515, Gunma, Japan
e-mail: t12801406@gunma-u.ac.jp

Hirokazu Ishitobi

Division of Environmental Engineering Science,
Gunma University,
1-5-1, Tenjincho,
Kiryu 376-8515, Gunma, Japan
e-mail: ishitobi@gunma-u.ac.jp

Shota Yamamori

Department of Chemical and Environmental Engineering,
Gunma University,
1-5-1, Tenjincho,
Kiryu 376-8515, Gunma, Japan
e-mail: t15803052@gunma-u.ac.jp

Nobuyoshi Nakagawa

Division of Environmental Engineering Science,
Gunma University,
1-5-1, Tenjincho,
Kiryu 376-8515, Gunma, Japan
e-mail: nob.nakagawa@gunma-u.ac.jp

1Corresponding author.

Manuscript received February 22, 2016; final manuscript received June 16, 2016; published online July 19, 2016. Assoc. Editor: San Ping Jiang.

J. Electrochem. En. Conv. Stor. 13(1), 014501 (Jul 19, 2016) (4 pages) Paper No: JEECS-16-1029; doi: 10.1115/1.4033970 History: Received February 22, 2016; Revised June 16, 2016

By modifying the carbon electrode with a yeast extract (YE) using a support material (SM), a complete bio-anode was established without adding any extrinsic enzymes and mediators in a glucose–air fuel cell. The yeast extract was mixed into a paste with carbon black and an SM, i.e., glutaraldehyde (GA), TritonX-100, polyethyleneglycol, chitosan, or agarose. Chitosan was the best support, producing lower overpotentials and a good stability. Optimization of the paste composition and its loading were carried out for the bio-anode of a glucose–air fuel cell. The fuel cell generated a power of 33 μW cm−2 at 333 K with an aqueous glucose solution without adding any extrinsic enzymes and mediators. It showed about 70% of the initial power output at a stable condition. The bio-anode is expected to be used for energy recovery from hot wastewater-containing glucose.

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Biofuel cells have received much attention as a power source from biodegradable compounds, as the fuel with a simple system structure operated under moderate conditions. They are categorized based on the type of biocatalyst, i.e., microbial fuel cells that use living microorganisms [1], organelle biofuel cells with orangelike mitochondria [2,3], and enzymatic fuel cells that apply an isolated specific enzyme [4,5]. The enzymatic bio-electrodes need to have a set of specific enzymes and pathways for the charge transfer between the reaction sites and the current corrector. They usually perform at higher current densities compared to that of the other types due to lack of cellular walls and/or organelle membranes which control the mass and charge transports during the electrochemical redox processes. One of the issues of the enzymatic electrodes is the relatively short lifetime of the catalytic activity. On the other hand, microbial and organelle systems have a complete set of enzymes and pathways to achieve metabolism; they have a mediator or a special pathway like a nanowire [6,7] for the charge transport. If the set of enzymes and pathways in the microbes and/or organs can be properly modified on the electrode, it would be expected to form a complete bio-anode in which the enzyme and mediator are comodified with an accelerated mass and charge transport.

YE is the common name of processed yeast products made by extracting the cell internals after removing the cell walls. It is usually used as a food additive, flavoring, or as a nutrient for bacterial culture media. YE is commercially available in a dried powder form, being inexpensive and not hazardous. Previously, one of the authors reported [8] that YE shows catalytic activity for glucose oxidation and/or dehydrogenation, and also it includes an internal mediator. The key component as the catalyst must be glucose oxidase and/or glucose dehydrogenase, while it may be denatured, and that as the mediator would be the riboflavin [8,9]. The glucose–air fuel cell with an anolyte solution, which dissolves glucose and YE, generated power without adding any extrinsic mediators. However, the power density was low, but it was significantly enhanced by the addition of methylene blue [8]. Interestingly, the power output was maximized at an elevated temperature as high as 333 K [8] similar to that of the mitochondria biofuel cell [3]. This would be due to the effect of denatured enzyme [10]. Such a high temperature is advantageous for directly utilizing hot wastewater from a factory without cooling and keeping the anode solution from a proliferation of microorganisms. However, the addition of YE and methylene bule to the wastewater should be avoided in a practical application for the energy recovery because another water treatment to remove YE and mediator is required. Hence, immobilization of YE on the electrode and fuel cell power generation without adding any extrinsic enzymes and mediators is necessary. However, an immobilization method for YE has been little reported so far, although there is a variety of immobilization methods for enzymes and mediators on the electrode [11].

In this study, modification of the electrode with YE was evaluated in order to demonstrate a complete bio-anode using YE. A method to mix YE into a carbon paste with SMs [1216] is used because it is an easy and effective method. The YE was mixed into a carbon paste with carbon black and an SM; then, the paste was deposited on a carbon cloth. The composition of the paste and its loading were optimized in order to maximize the electrode performance, and the optimized bio-anode was used in a glucose–air fuel cell. The power generation performance of the glucose–air fuel cell with the bio-anode was evaluated, and a stability test by the replacement of the anode solution was also conducted.

Carbon Paste With YE and SM.

For the modification with YE, a method to mix YE into a carbon paste with an SM was applied. The YE was a dry powder from Nihon Pharmaceutical Co., Ltd, (Tokyo, Japan). It was mixed with carbon black (CB, Ketjen Black EC, Lion Co., Ltd., Tokyo, Japan), which increased the actual electrode surface, and with one of the five SMs, i.e., polyethylene glycol (PEG, 20,000, Wako Co., Ltd., Tokyo, Japan), GA (GA, 25% aqueous solution, Wako Co., Ltd.), TritonX-100 (Molecular Biology Grade, Wako Co., Ltd.), chitosan (100, Wako Co., Ltd.), or agarose (Agarose S, Wako Co., Ltd.), which are commonly used for the immobilization of enzymes in previous studies [4,5,11].

CB, YE, and the SM were weighed in a certain ratio and then mixed using a certain amount of distilled water. The mixture was stirred for 30 min and then ultrasonicated for 30 min to disperse the CB particles in the mixture. Only in the case of chitosan, a 0.5 wt. % acetic acid aqueous solution was used instead of the distilled water to dissolve the chitosan into the solution. The mixture was uniformly dropped on the carbon cloth (CC, ECC-CC1-060, Toyo Technica Co., Ltd., Tokyo, Japan), 30 mm × 30 mm, by repeating the dropping and drying under ambient conditions. Finally, the carbon cloth with the mixture layer was dried in an oven at 373 K for 1 hr. The carbon cloth was then cut into a square, 25 mm × 25 mm, and used as a bio-anode.

A Glucose–Air Fuel Cell and Electrochemical Measurements.

A membrane electrode assembly (MEA) was prepared by hot-pressing the bio-anode to a membrane of Nafion 117 (Du Pont, Co., Ltd., Tokyo, Japan), 50 mm × 50 mm, as the electrolyte and a ready-made Pt(1 mg cm−2)/C electrode (Electro Chem, Inc., Boston, MA), 25 mm × 25 mm, as the cathode at 410 K and 4.02 MPa for 3 min. The MEA was placed in a holder, which has a chamber with 90 ml for the anode solution, while its cathode was exposed to air. Its detailed structure was the same as that described in a previous report [8].

After adding a glucose solution, 20 gl−1 glucose and 50 mM phosphate buffer (pH = 6.8) of 70 ml to the chamber, the electrode potentials at open circuit became constant, and the relationship between the current density and the electrode potentials was measured. The electrochemical measurements were conducted using a Galvano/Potentiostat (HA-151B, Hokutodenko Co., Ltd., Tokyo, Japan) with a Hg/Hg2SO4 reference electrode dipped in the anode solution.

The fuel cell was operated in an oven in which the temperature and humidity were controlled. The temperature was set at 308 K, 323 K, or 333 K, and the relative humidity of air was set at 70% in all the experiments. The current density was calculated on the basis of the projected area, 25 mm × 25 mm, of the electrode.

Figure 1 shows the anode potentials of the bio-anode prepared with different SMs at 308 K. The bio-anode was prepared using CB, YE, and the SM in the weight ratio of CB:YE:SM as 1:2:2, and the CB loading was 4.4 mg cm−2. It is noted that every electrode worked as an anode, at 50 μA cm−2 under the overpotential within 150 mV, without adding any extrinsic mediators. This means co-immobilization of the enzyme, and the mediator was established in all the cases. It is also noted that the onset potential was quite different from the SM. In the previous study [8], the onset potential decreased with the increase of the YE concentration of the anolyte solution [8] and was explained by the redox potential of mediator. The potential is a function of the concentrations of the oxidized and reduced forms of mediator. The difference in the onset potential among the different SMs would reflect different densities of electronic connection between the immobilized mediators and the current corrector on the electrode. In case of GA, the onset potential was the lowest showing an advantage for the fuel cell. On the other hand, in case of PEG, the onset potential was the highest. This suggests that the SM PEG may strongly interrupt the connection in the catalyst layer. However, GA showed the higher overpotentials at the higher current densities over 20 μA cm−2 than that of the others. The overpotential at the higher current density would be caused by the mass transport resistance of the reactants between the immobilized mediators and the bulk solution through the catalyst layer. GA, TritonX-100, and PEG resulted in the higher overpotentials suggesting the high mass transport resistance through the catalyst layer. Chitosan and agarose showed the relatively low overpotentials that would be due to their layer structures for an enhanced transport of glucose through the layer. In the cases of GA and TritonX-100, after a few hours of operation, the anode solution becomes muddy due to the disconnection of the carbon particles from the deposited layer suggesting that these materials were not sufficient as a binder for this purpose. Although agarose showed a better performance in this experiment, it is not appropriate for high-temperature operation around 333 K as its gelling temperature is around 311 K. Hence, we chose chitosan as a proper SM for the bio-anode, and, hereafter, the experiment was conducted using chitosan as the SM.

Figure 2 shows the performance of the bio-anode with different chitosan contents by keeping the CB and YE loadings at 4.4 mg cm−2 and 8.8 mg cm−2, respectively. The weight percent of chitosan is based on the total amounts of CB, YE, and chitosan.

It is noted that the anode overpotential decreased with the increasing chitosan content up to approximately 33 wt. % and then increased at a higher content. Large amount of chitosan content would obstruct the electronic connection between the CB particles and also mass transport of the reactants through the layer of the mixture, which then increased the overpotential. It was found that about 20–30 wt. % is appropriate for the mixture.

Figure 3 shows the effect of the loading of the mixture at the optimum composition using chitosan. As the loading increased up to around 12 mg-CB cm−2, both the onset potential and the overpotential decreased. The decreased electrode potential would be due to the increased density of the electronic connection between the immobilized mediator and the current collector due to the increased coverage of the carbon cloth with the increasing loading. The higher loading of 16.7 mg-CB cm−2 showed a performance similar to that of the 12 mg-CB cm−2 suggesting that the loading of 12 mg-CB cm−2 is the optimum. Since the thickness of the bio-anode, the mixture layer, increased as the loading increased, there must be an effective thickness that is limited by the electron conduction and mass transport through the layer.

Figure 4 shows the power generation performance of the glucose–air fuel cell with the bio-anode, prepared at the optimum conditions, operated at different temperatures 308 K, 323 K, and 333 K. As shown in Fig. 4(a), the anode potential decreased with the increase in the temperature. Also, the cathode potentials improved due to the temperature increase. The power output then reached 33 μW cm−2 at 333 K. This was an equivalent output compared to that obtained from the previous glucose–air fuel cell using an anolyte solution with YE (10 g l−1), glucose (10 g l−1), and methylene blue (5 mM) [8]. The equivalent power output was successfully demonstrated using the modified anode with YE, without adding YE, and external mediator into the anode solution. It was found that the relatively high cathode overpotential governed the loss of power. A higher power output can then be expected by improving the cathode performance.

In order to study the stability of the modification and the cell performance, measurement repetitions of the current–voltage curve was conducted by replacing the anode solution with a fresh one. After the replacement, the solution was stored for overnight with stirring before the measurement. The maximum obtained power output was reduced to 81.0%, 70.9%, 66.8%, and 69.3% of the initial one for the second, third, fourth, and fifth repetitions, respectively. The drop of the power output from the first to the third would be caused by the leaching of YE weakly immobilized, although change in color of the solution was not observed during the experiment. However, after the third repetition, the power output reached a constant that is about 70% of the initial one suggesting that the modification with YE was stable.

The YE-modified bio-anode is expected to be used for energy recovery from hot wastewater-containing glucose discharged at a sugar factory.

A YE-modified bio-anode was fabricated with CB and different SMs. Chitosan showed the best anode performance with the five different SMs, although each modification succeeded as a bio-anode without adding any extrinsic enzymes and mediators. The glucose–air fuel cell with the optimized bio-anode generated 33 μW cm−2 at 333 K. The fuel cell showed a stable power output that was 70% of the initial after the third replacement of the solution suggesting the stability of the modification.

This research was supported by the JSPS KAKENHI Grant No. 24656479. The authors deeply thank this foundation.

Logan, B. E. , Hamelers, B. , Rozendal, R. , Shroder, U. , Keller, J. , Freguia, S. , Aelterman, P. , Verstraete, W. , and Rabaey, K. , 2006, “ Microbial Fuel Cells: Methodology and Technology,” Environ. Sci. Technol., 40(17), pp. 5181–5192. [CrossRef] [PubMed]
Arechederra, R. L. , and Mintter, S. D. , 2008, “ Organelle-Based Biofuel Cells: Immobilized Mitochondria on Carbon Paper Electrodes,” Electrochim. Acta, 53(23), pp. 6698–6703. [CrossRef]
Arechederra, R. L. , Boehm, K. , and Minteer, S. D. , 2009, “ Mitochondria Bioelectrocatalysis for Biofuel Cell Applications,” Electrochim. Acta, 54(28), pp. 7268–7273. [CrossRef]
Osman, M. H. , Shah, A. A. , and Walsh, F. C. , 2011, “ Recent Progress and Continuing Challenges in Bio-Fuel Cells—Part I: Enzymatic Cells,” Biosens. Bioelectron., 26(7), pp. 3087–3102. [CrossRef] [PubMed]
Leech, D. , Kavanagh, P. , and Schuhmann, W. , 2012, “ Enzymatic Fuel Cells: Recent Progress,” Electrochim. Acta, 84(1), pp. 223–234. [CrossRef]
Reguera, G. , Nevin, K. P. , and Nicoll, J. S. , 2006, “ Biofilm and Nanowire Production Leads to Increased Current in Geobacter Sulfurreducens Fuel Cells,” Appl. Environ. Microbiol., 72(11), pp. 7345–7348. [CrossRef] [PubMed]
Zhao, Y. , Watanabe, K. , and Nakamura, R. , 2010, “ Three-Dimensional Conductive Nanowire Networks for Maximizing Anode Performance in Microbial Fuel Cells,” Chem. Europ. J., 16(17), pp. 4982–4985. [CrossRef]
Sayed, E. T. , Saito, Y. , Tsujiguchi, T. , and Nakagawa, N. , 2012, “ Calytic Activity of Yeast Extract in Biofuel Cell,” J. Biosci. Bioeng., 144(5), pp. 521–525. [CrossRef]
Kamitaka, Y. , Tsujimura, S. , Setoyama, N. , Kajino, T. , and Kano, K. , 2007, “ Fructose/Dioxygen Biofuel Cell Based on Direct Electron Transfer-Type Bioelectrocatalysis,” Phys. Chem. Chem. Phys., 9(15), pp. 1793–1801. [CrossRef] [PubMed]
Yamazaki, T. , Tsugawa, W. , and Sode, K. , 1999, “ Subunit Analyses of a Novel Thermostable Glucose Dehydrogenase Showing Different Temperature Properties According to Its Quaternary Structure,” Appl. Biochem. Biotechnol., 77–79(1), pp. 325–335. [CrossRef]
Datta, S. , Christena, L. R. , and Rajaram, Y. R. S. , 2013, “ Enzyme Immobilization: An Overview on Techniques and Support Materials,” Biotechnology, 3(1), pp. 1–9.
Pizzariello, A. , Stred'ansky, M. , and Miertuš, S. , 2002, “ A Glucose/Hydrogen Peroxide Biofuel Cell That Uses Oxidase and Peroxidase as Catalysts by Composite Bulk-Modified Bioelectrodes Based on a Solid Binding Matrix,” Bioelectrochemistry, 56(1–2), pp. 99–105. [CrossRef] [PubMed]
Tasca, F. , Gorton, L. , Harreither, W. , Haltrich, D. , Ludwig, R. , and Nöll, G. , 2008, “ Direct Electron Transfer at Cellobiose Dehydrogenase Modified Anodes for Biofuel Cell,” J. Phys. Chem. C, 112(26), pp. 9956–9961. [CrossRef]
Ikeda, T. , Hamada, H. , and Senda, M. , 1986, “ Electrocatalytic Oxidation of Glucose at a Glucose Oxidase-Immobilized Benzoquinone-Mixed Carbon Paste Electrode,” Agric. Biol. Chem., 50(4), pp. 883–890.
Gregg, B. A. , and Heller, A. , 1991, “ Redox Polymer Films Containing Enzymes—1: A Redox-Conducting Epoxy Cement: Synthesis, Characterization, and Electrocatalytic Oxidation of Hydroquinone,” J. Phys. Chem., 95(15), pp. 5970–5975. [CrossRef]
Kandimalla, V . B. , Tripathi, V . S. , and Ju, H. X. , 2006, “ Immobilization of Biomolecules in Sol–Gels: Biological and Analytical Applications,” Crit. Rev. Anal. Chem., 36(2), pp. 73–106. [CrossRef]
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References

Logan, B. E. , Hamelers, B. , Rozendal, R. , Shroder, U. , Keller, J. , Freguia, S. , Aelterman, P. , Verstraete, W. , and Rabaey, K. , 2006, “ Microbial Fuel Cells: Methodology and Technology,” Environ. Sci. Technol., 40(17), pp. 5181–5192. [CrossRef] [PubMed]
Arechederra, R. L. , and Mintter, S. D. , 2008, “ Organelle-Based Biofuel Cells: Immobilized Mitochondria on Carbon Paper Electrodes,” Electrochim. Acta, 53(23), pp. 6698–6703. [CrossRef]
Arechederra, R. L. , Boehm, K. , and Minteer, S. D. , 2009, “ Mitochondria Bioelectrocatalysis for Biofuel Cell Applications,” Electrochim. Acta, 54(28), pp. 7268–7273. [CrossRef]
Osman, M. H. , Shah, A. A. , and Walsh, F. C. , 2011, “ Recent Progress and Continuing Challenges in Bio-Fuel Cells—Part I: Enzymatic Cells,” Biosens. Bioelectron., 26(7), pp. 3087–3102. [CrossRef] [PubMed]
Leech, D. , Kavanagh, P. , and Schuhmann, W. , 2012, “ Enzymatic Fuel Cells: Recent Progress,” Electrochim. Acta, 84(1), pp. 223–234. [CrossRef]
Reguera, G. , Nevin, K. P. , and Nicoll, J. S. , 2006, “ Biofilm and Nanowire Production Leads to Increased Current in Geobacter Sulfurreducens Fuel Cells,” Appl. Environ. Microbiol., 72(11), pp. 7345–7348. [CrossRef] [PubMed]
Zhao, Y. , Watanabe, K. , and Nakamura, R. , 2010, “ Three-Dimensional Conductive Nanowire Networks for Maximizing Anode Performance in Microbial Fuel Cells,” Chem. Europ. J., 16(17), pp. 4982–4985. [CrossRef]
Sayed, E. T. , Saito, Y. , Tsujiguchi, T. , and Nakagawa, N. , 2012, “ Calytic Activity of Yeast Extract in Biofuel Cell,” J. Biosci. Bioeng., 144(5), pp. 521–525. [CrossRef]
Kamitaka, Y. , Tsujimura, S. , Setoyama, N. , Kajino, T. , and Kano, K. , 2007, “ Fructose/Dioxygen Biofuel Cell Based on Direct Electron Transfer-Type Bioelectrocatalysis,” Phys. Chem. Chem. Phys., 9(15), pp. 1793–1801. [CrossRef] [PubMed]
Yamazaki, T. , Tsugawa, W. , and Sode, K. , 1999, “ Subunit Analyses of a Novel Thermostable Glucose Dehydrogenase Showing Different Temperature Properties According to Its Quaternary Structure,” Appl. Biochem. Biotechnol., 77–79(1), pp. 325–335. [CrossRef]
Datta, S. , Christena, L. R. , and Rajaram, Y. R. S. , 2013, “ Enzyme Immobilization: An Overview on Techniques and Support Materials,” Biotechnology, 3(1), pp. 1–9.
Pizzariello, A. , Stred'ansky, M. , and Miertuš, S. , 2002, “ A Glucose/Hydrogen Peroxide Biofuel Cell That Uses Oxidase and Peroxidase as Catalysts by Composite Bulk-Modified Bioelectrodes Based on a Solid Binding Matrix,” Bioelectrochemistry, 56(1–2), pp. 99–105. [CrossRef] [PubMed]
Tasca, F. , Gorton, L. , Harreither, W. , Haltrich, D. , Ludwig, R. , and Nöll, G. , 2008, “ Direct Electron Transfer at Cellobiose Dehydrogenase Modified Anodes for Biofuel Cell,” J. Phys. Chem. C, 112(26), pp. 9956–9961. [CrossRef]
Ikeda, T. , Hamada, H. , and Senda, M. , 1986, “ Electrocatalytic Oxidation of Glucose at a Glucose Oxidase-Immobilized Benzoquinone-Mixed Carbon Paste Electrode,” Agric. Biol. Chem., 50(4), pp. 883–890.
Gregg, B. A. , and Heller, A. , 1991, “ Redox Polymer Films Containing Enzymes—1: A Redox-Conducting Epoxy Cement: Synthesis, Characterization, and Electrocatalytic Oxidation of Hydroquinone,” J. Phys. Chem., 95(15), pp. 5970–5975. [CrossRef]
Kandimalla, V . B. , Tripathi, V . S. , and Ju, H. X. , 2006, “ Immobilization of Biomolecules in Sol–Gels: Biological and Analytical Applications,” Crit. Rev. Anal. Chem., 36(2), pp. 73–106. [CrossRef]

Figures

Grahic Jump Location
Fig. 1

Anode performance of the YE-modified electrode with different SMs at 308 K (mixture: CB:YE:SM = 1:2:2; CB loading: 4.4 mg cm−2; and anode solution: 20 g l−1 glucose and 50 mM phosphate buffer)

Grahic Jump Location
Fig. 2

Effect of chitosan content in the mixture for the YE-modified electrode on the anode performance at 308 K (CB and YE loadings were 4.4 mg cm−2 and 8.8 mg cm−2, respectively; and anode solution: 20 g l−1 glucose and 50 mM phosphate buffer)

Grahic Jump Location
Fig. 3

Effect of the loading of the YE mixture on the anode performance of the glucose electrode at 308 K (mixture: CB:YE:chitosan = 1:2:1.5; and anode solution: 20 g l−1 glucose and 50 mM phosphate buffer)

Grahic Jump Location
Fig. 4

Power generation performance of the glucose–air fuel cell with the YE-modified bio-anode: (a) electrode potentials and (b) cell voltage and power density (anode solution: 20 g l−1 glucose and 50 mM phosphate buffer)

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