Self-circulation System of Insect Hemolymph for Insect-mountable Biofuel Cell K. Shoji1*, Y. Akiyama1, M. Suzuki2, T. Hoshino1, N. Nakamura2, H. Ohno2 and K. Morishima1, 3 1 Department of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan 2 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan 3 Department of Mechanical Engineering, Osaka University, Osaka, Japan *Presenting Author: kan.shouji@gmail.com Abstract: A self-circulation system is presented for a self-refueling insect-mountable biofuel cell (BFC) using trehalose, the main sugar of insect hemolymph. The maximum power density of 10.5 µW/cm2 was obtained from cockroach hemolymph (CHL) with added trehalase and mutarotase. The electrodes were protected by a dialysis membrane to prevent decrease of open-circuit voltage by adsorption of proteins in the CHL. Furthermore, a melody integrated circuit (IC) was driven by five BFCs connected in series. The self-circulation system was developed using abdominal motion and connected to a cockroach with a tube and a check-valve. Keywords: Biofuel cell, Insect hemolymph, Trehalose, Self-circulation current density of a few mA/cm2 and power density of a few mW/cm2. BFCs using glucose in blood have INTRODUCTION already been reported for a cardiac pacemaker and an Insects are extremely successful animals, living implantable sensor [11]. A maximum power density of almost everywhere on the earth [1]. Insect cells have 50 µW/cm2 was obtained for BFCs from bovine serum been used for recombinant protein production [2]. [12]. Furthermore, high performing micro-robots have been However, while the main sugar in mammal blood developed by mimicking their functions and structures is glucose, the main sugar of insect hemolymph is [3] [4]. In this way, insects have been applied in many trehalose which is a disaccharide composed of two industrial fields. Especially, insect cyborgs which are glucose molecules. A power generation system using robots controlled by electric stimulation of their brain glucose oxidase (GOD) cannot be directly applied to a and neurons are desired for rescue, environmental trehalose BFC. In this study, we propose a trehalose monitoring and working in a radiation environment. BFC system that is applied to the GOD reaction For example, Hozer and Shimoyama [5] analyzed the system after trehalose molecules are decomposed to locomotion reaction of an insect and controlled its glucose molecules by trehalase. Therefore, this walking by electric stimulation of its neurons. Kanzaki trehalose BFC system is able to generate power from et al. [6] developed a sensitive sensor robot using moth both trehalose and glucose. antennas. In studies, Sato et al. [7] and Bozkurt et al. In this study, we made electrochemical [8] succeeded in controlling the flight of insects by measurements using insect hemolymph and evaluated electrical stimulation to their brain and neurons. the performance of the trehalose BFC. Furthermore, Coin batteries which are used for insect cyborgs the CHL self-circulation system using abdominal are too big and heavy for small insects and they restrict motion and the pressure difference in the dorsal vessel their motion. In addition, it is impossible to drive an IC was developed. for a long time without exchanging or recharging the batteries. A battery which is mountable on insects and EXPERIMENTAL has a semi-permanent life-time is desired. However, Preparation of electrodes downsizing, weight saving and longevity of the power The electrode compositions are shown in table 1. source for insect cyborgs has never been examined. Ketjen 600EC, which consists of carbon nano particles, Therefore, we have proposed to transduce the kinetic was used for immobilization on the carbon paper energy of insects into electric energy using a piezo electrodes. The anode was made by successively element directly [9]. dropping solutions of the mediator (ferrocene) and the Fuel cells which transduce chemical energy to enzyme (GOD) which oxidizes glucose onto the base electric energy directly have already come into electrode and drying at room temperature after each. practical use. However, they have many issues Furthermore, poly-L- lysine (PLL) and poly sodium 4including the need for expensive rare metals in their styrenesulfonate (PSS) were successively dropped onto manufacture, the dissipation of generated reaction heat the surface and dried in air to form the insoluble PLLand the finiteness of energy resources. Therefore, PSS complex film (poly ion complex) [13]. The BFCs are of particular interest due to the suitability of cathode surface was modified by dropping BOD a bioprocess based on enzymes and microorganisms solution onto the base electrode. [10]. Glucose BFCs have an electromotive force of about 1.2 V and they have recently achieved the Fig. 1: Power generation mechanism from trehalose. Table 1: Compositions of anode and cathode Anode Cathode Surface Ketjen600 Ketjen600 Mediator Ferrocene Enzyme GOD BOD Polymer PLL-PSS Power generation from insect hemolymph Trehalose which is the main sugar of insect hemolymph is a disaccharide composed of two glucose molecules. A trehalose molecule can be decomposed into two glucose molecules by Tre. Therefore, the conventional GOD reaction system can be applied after decomposition of trehalose. In addition, higher output voltage is expected since two glucose molecules are obtained for each trehalose molecule. The power generation mechanism of the trehalose BFC is shown in Fig. 1. First, trehalose molecules in insect hemolymph are hydrolyzed to α-glucose molecules by trehalase. Second, α-glucose molecules are transformed to β-glucose molecules by mutarotase. Then, electric power is generated from β-glucose molecules by the GOD reaction system. The β-glucose is oxidized by GOD immobilized on an anode and electrons are produced. The electrons are transferred to an anode by a mediator. Oxygen is reduced by bilirubin oxidase (BOD) immobilized on a cathode and the electrochemical conversion of hydrogen and oxygen into water occurs. As a result, electricity is generated. Madagascar cockroaches which were administered trehalose-rich feed (Pro Jelly, KB Farm) were used in this study. The CHL was collected with fat bodies and purified by centrifugation. Trehalase and mutarotase were added to CHL and this solution was kept for a day at room temperature. Then the solution was dialyzed by a cellulose tube in order to prevent the adsorption of proteins in CHL. Then the electrodes were put into the solution and power density was measured by an electrochemical measurement system (SI 1280B, Solartron). The cathode was between both the air and the solution. Fig. 2: Schematic image of the insect-mountable BFC with a circulation system. Fabrication of insect hemolymph BFC (IHBFC) Carbon paper electrodes of 2.5 cm longth and 2.0 cm width which were mountable on the cockroach were prepared. The IH-BFC consisted of the anode, cathode and chamber and they were glued in place by grease. Then we demonstrated the IH-BFC could drive a melody IC (UM66T-68L, Bowin Electronic Co.) and produce music. Self-circulation of CHL It is necessary supply trehalose to the reaction part continuously in order to generate electricity semipermanently. The trehalose concentration in CHL is about 100 mM [14] and the volume of CHL is about 20 % of the body weight [15]. One cockroach includes about 140 µmol trehalose. Trehalose is synthesized from fat bodies and the Americana cockroach is able to synthesize more than 2 mg of trehalose per day [16]. When trehalose in CHL is pumped to a reaction part, it is able to create a semi-permanent power generation system. In this study, we attempted to circulate CHL through the IH-BFC by the abdominal motion and dorsal vessel peristaltic movement leading to a selfcharging IH-BFC. A schematic illustration of the insect-mountable BFC is drawn in Fig. 2. The connector was halved and the two halves were glued onto the exoskeleton by epoxy (Araldite). Then a 2 mm in diameter holes were made through the exoskeleton in the center of the half connectors. A silicone tube (3 mm inside diameter) was filled with cockroach saline that included micro fluorescent beads (20 µm diameter) and the tube was connected to the cockroach (Fig. 3). Furthermore, a check-valve which was a 100 µm thick PDMS (polydimethylsiloxane) membrane was fastened inside the tube. The displacement of the beads in the tube was observed by fluorescent microscopy. Fig. 5: Photograph of the IH-BFC consisting of the chamber, anode and cathode. Fig. 3: Photograph of a Madagascar Cockroach with connectors, tube and check-valve. Top inset: Cross section view of check-valve. Bottom inset: Cross section view of connector. Fig. 6: Displacement of the beads in the tube. The velocity was about 129.2 µm/s. Fig. 4: (a) Time course of open-circuit voltages with and without a dialysis membrane. (b) Performance of the BFC using an air diffusion cathode with CHL and added trehalase and mutarotase. The open-circuit voltage and the maximum current density were about 225 mV and 120 µA/cm2, respectively. RESULTS AND DISCUSSION Power generation from CHL A maximum power density of 10.5 µW/cm2 was obtained from CHL with added trehalase and mutarotase (Fig. 4 (b)). In the IH-BFC, trehalose and glucose in CHL were used for fuel. Trehalose in CHL was hydrolyzed to α-glucose by trehalase. Then, αglucose was transformed to β-glucose by mutarotase. The β-glucose was oxidized by GOD immobilized on the anode and electrons were produced. The electrons were transferred to the anode by ferrocene. Oxygen in the air was reduced by the air diffusion cathode. Furthermore, the electrodes were protected from proteins in CHL by a dialysis membrane. As a result, the open-circuit voltage was stabilized at 300 mV for 30 min (Fig. 4(a)). Because the dialysis membrane we used had a molecular weight cut off of 12,000-16,000, proteins in CHL were cut off but fat bodies in CHL were passed through the dialysis membrane. Higher electric power may be possible by selecting enzymes and mediators which exhibit better reaction characteristics and by increasing the surface area through making nano structure electrodes using Carbon-MEMS (C-MEMS) technology and oriented carbon nano tubes on the electrodes. Fabrication of the IH-BFC The IH-BFC is shown in Fig. 5. The melody IC was driven by five IH-BFCs connected in series. The melody IC was driven at 1.5 V and the current consumption was 30 µA. The open-circuit voltage of the IH-BFC was about 300 mV and the maximum power was about 50 µW. To downsize the chamber of the IH-BFC, a power source which does not restrict the insect motion should be developed. Self-circulation of CHL Displacements of beads in the tube by abdominal deformation and the dorsal vessel peristaltic movement were confirmed. The displacement of the beads was shown in Fig. 6. The bead displacements were measured directly from a fluorescent microscopic video image by image analysis (DIPP-Motion Pro 2D, DITECT). As a result, the velocity of 129.2 µm/s was obtained and the average velocity was 64.6 µm/s. Therefore, the flow rate was calculated as about 457 nl/s. The maximum current consumption of the IH-BFC was about 600 µA and 1.55 nmol trehalose was consumed per second. Because the trehalose concentration of the CHL was 100 mM, the CHL of 15.5 nl would have to be circulated through the IHBFC per second at a minimum. In this study, we obtained a flow rate about 30 times the lowest value required. Therefore, by downsizing the connector and building the IH-BFC in the self-circulation system, it is possible to fabricate the insect-mountable BFC. CONCLUSION We generated power using electrochemical reactions with trehalose included in CHL and we obtained a maximum power density of 10.5 µW/cm2 from CHL. The electrodes were protected from the adsorption of proteins by using a dialysis membrane and the open-circuit voltage was stabilized at 300 mV for more than 30 min. The self-circulation system of CHL actuated by the abdominal motion and the pressure difference of the dorsal vessel was developed by connecting a tube with a built in check-valve to a cockroach. The flow rate of 457 nl/s was obtained. Furthermore, we expect an onsite semi-permanent power source for insect cyborgs will be possible by combining the IH-BFC and the self-circulation system. ACKNOWLEDMENTS This work was partly supported by the Industrial Research Program of NEDO and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (Nos. 21676002, 21225007, 23111705). REFERENCES [1] P. Gullan and P. Cranston, The Insects, 3rd edition, Blackwell, 2005. [2] B. Maiorella, D. Inlow, A. Shauger, and D. Harano, “Large-Scale Insect Cell-Culture for Recombinant Protein Production,” Nat Biotech, Vol. 6, pp. 1406-1410, 1988. [3] R. Wood, “The First Takeoff of a Biologically Inspired At-Scale Robotic Insect,” IEEE Transactions on Robotics, Vol. 24, pp. 341-347, 2008. [4] K. Akimoto, S. Watanabe, and M. Yano, “An insect robot controlled by the emergence of gait patterns,” Artificial Life and Robotics, Vol. 3, pp. 102-105, 1999. [5] R. Holzer, I. Shimoyama, “Locomotion control of a bio-robotic system via electric stimulation,” Intelligent Robots and Systems, pp. 1514-1519, 1997. [6] R. Kanzaki, “Behavioral and neural basis of instinctive behavior in insects: Odor-source searching strategies without memory and learning,” Robotics and Autonomous Systems, vol. 18, pp. 33-43, 1996. [7] H. Sato, C. Berry, B. Casey, G. Lavella, Ying Yao, J. VandenBrooks, and M. Maharbiz, “A cyborg beetle: Insect flight control through an implantable, tetherless microsystem,” Micro Electro Mechanical Systems, 2008. MEMS 2008. IEEE 21st International Conference, 2008, pp. 164-167. [8] A. Bozkurt, R. Gilmour, D. Stern, and A. Lal, “MEMS based bioelectronic neuromuscular interfaces for insect cyborg flight control,” Micro Electro Mechanical Systems, 2008. MEMS 2008. IEEE 21st International Conference, 2008, pp. 160-163. [9] T. Honna, K. Murata, T. Hoshino, H. Sato, N. Nakamura, H. Ohno, K. Morishima: “Bio Hybrid Power Generator using an Insect,” ROBOMEC 2009, 1A2-M08, 2009. [10]I. Taniguchi, “Biofuel Cells Using Au Nanoparticle Immobilized Carbon Electrodes in Alkaline Solutions,” ECS Meeting Abstracts, Vol. 702, p. 328, 2007. [11]P. Cinquin, C. Gondran, F. Giroud, S. Mazabrard, A. Pellissier, F. Boucher, J. Alcaraz, K. Gorgy, F. Lenouvel, S. Mathé, P. Porcu, and S. Cosnier, “A Glucose BioFuel Cell Implanted in Rats,” PLoS ONE, Vol. 5, p. e10476, 2010. [12]M. Nishizawa, “Innovative Batteries Enhanced Power Supply for the Next Stage,” pp.73-82, NTS, 2006. (in Japanese) [13]S. Komaba, T. Mitsuhashi, and S. Shiraishi, “Optimization of Enzyme Anode and Cathode with Polyion Complex for the Application to Biofuel Cells,” Electrochemistry, Vol. 76, pp. 619–624, 2008. [14]K. Shoji et al., “Biofuel cells with trehalose leading to an insect-implanted power source,” in Solid-State Sensors, Actuators and Microsystems Conference, pp. 2762-2765, 2011. [15]X. Xian, “Changes in hemolymph volume, blood sugar titer, fat body and content of glycogen in fat body of smoky brown cockroach (Periplaneta fuliginosa S.) during fast,” Med. Entomol. Zool., Vol. 47, pp.31-36, 1996. [16]Y. Kono et al. “In vitro trehalose synthesis in American cockroach fat body measured by NMR spectroscopy using 13C-sugars and a trehalase inhibitor, validoxylamine A,” Med. Entomol. Zool., Vol. 49, pp. 321-330, 1998.