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).
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