JP6786885B2 - Biobattery - Google Patents

Description

The present invention relates to a biobattery.

In recent years, the development of biobatteries using the energy conversion system of enzymes and microorganisms has been promoted. A biobattery is a fuel cell that applies an energy conversion system in a living body, and is a power generation device that uses a biocatalyst as an electrode catalyst and couples a redox reaction and an electrode reaction by the biocatalyst to extract electrical energy. A biobattery that utilizes a bioprocess has an advantage that it has mild conditions and high selectivity, and can utilize various substances existing in the environment such as sugar and alcohol as a fuel. Therefore, since it is safe and has a small environmental load, further development as a next-generation power source for small electronic devices such as portable devices and implantable devices is expected.

In the case of a biobattery using an enzyme as an electrode catalyst, electrons and protons are generated by the oxidation reaction of fuel on the negative electrode side, and the extracted electrons are transferred to the electrode base material. Electrons are transported to the positive electrode side via an external circuit, and protons are transported to the positive electrode side via an electrolyte layer or the like, and an oxygen reduction reaction proceeds on the positive electrode side. In recent years, with the aim of improving the performance of biobatteries, research and development have been carried out from the viewpoints of improving stability, optimizing electrode materials, and improving the efficiency of electron transfer between electrodes / enzymes.

For example, in order to realize stable operation of a biobattery for a long period of time, a technique for adjusting the humidity on the positive electrode side within a certain range has been reported (see Patent Document 1). According to the technique of Patent Document 1, by adjusting the relative humidity within a certain range, the performance can be maintained for a long period of time without causing deterioration of the positive electrode, and the durability of the biobattery can be improved.

Further, in order to efficiently extract energy from the fuel, it is required that the enzyme, which is an electrode catalyst, can stably maintain its catalytic activity in a suitable environment on the negative electrode side. For example, a biobattery using an enzyme as an electrode catalyst, which has stable activity in the presence of a compound containing an imidazole ring having excellent buffering capacity and is stable at least in the pH range of 7 to 11 has been reported ( See Patent Document 2). The biobattery of Patent Document 2 uses a compound containing an imidazole ring, which can obtain a sufficient buffering action even when the proton concentration at the electrode increases or decreases, as a buffering solution component in an electrolytic solution or the like. As a result, it is possible to prevent the pH around the enzyme on the electrode from deviating from the optimum pH range of the enzyme, and the enzyme used can function stably in the presence of a compound having an imidazole ring having a high buffering capacity. It is stated that it is possible to efficiently generate electrical energy.

Further, a biobattery stack for efficiently supplying a fuel solution to a negative electrode has been reported (see Patent Document 3). Specifically, the biobattery stack of Patent Document 3 has a fuel manifold in which at least one or more fuel containers are incorporated, an inlet for injecting a fuel solution into the fuel manifold, and an outlet for discharging the fuel solution. It has a fuel supply unit including. The negative electrode is arranged so as to come into contact with the fuel solution in the fuel container, and the fuel solution is supplied into the fuel container of the fuel manifold from an appropriate fuel source. The fuel is consumed during the operation of the biobattery, and the fuel after consumption is configured to be discharged to an appropriate disposal destination.

In addition, the bio is configured to efficiently restore power generation performance by judging the deterioration of battery performance due to fuel consumption, and by adding fuel and equalizing the concentration distribution of the fuel solution through the rotation of the biobattery body. Batteries have also been reported (see, eg, Patent Document 4). Specifically, the biobattery of Patent Document 4 is configured to be provided with a pH detection unit that detects a pH change of the fuel solution by a color change so that the consumption of the fuel solution can be visually detected.

JP-A-2015-228321 Japanese Unexamined Patent Publication No. 2014-182887 Special Table 2010-516017 Japanese Unexamined Patent Publication No. 2013-33630

However, for bio battery of Patent Document 1, the current density refers only to the low output of .mu.A / cm ² level, the case is supplied repeatedly fuel mA / cm ² level or more has not been studied. In the biobatteries of Patent Documents 2 to 4, the fuel is oxidized by the enzyme which is an electrode catalyst on the negative electrode side as the biobattery operates, and the influence of the organic acid generated by the oxidation of the fuel on the enzyme is examined. There is a need to. Since organic acids are generated in large quantities compared to the concentration of the buffer solution component contained in the electrolytic solution, fuel solution, etc., the pH on the negative side shifts to acidic, and as a result, the pH deviates from the optimum range of the enzyme. It is expected that the output of the biobattery will decrease due to the decrease in enzyme activity. However, in any of the conventional techniques, there is room for improvement in dealing with the decrease in the output of the biobattery due to the production of organic acids.

Therefore, it is an object to prevent a decrease in enzyme activity due to an organic acid produced by oxidation of fuel. Another object of the present invention is that the enzyme, which is an electrode catalyst, can stably maintain its catalytic activity in a suitable environment. Another object of the present invention is to provide a biobattery capable of improving power generation efficiency and to provide a biobattery capable of improving power generation duration.

As a result of diligent research to solve the above problems, the present inventors have turned the negative electrode, which produces an organic acid by the oxidation of the fuel after the operation of the biobattery, into an alkaline buffer solution containing a buffer solution component having a certain concentration or higher. It has been found that by impregnation, the pH on the negative electrode side can be returned to the optimum range of the enzyme, the enzyme which is the electrode enzyme can be reactivated, and the power generation efficiency and the power generation duration can be improved. Further, by adding a buffer solution component having a certain concentration or more to the fuel solution to be additionally supplied and adjusting the concentration to alkaline, the enzyme which is an electrode enzyme can be reactivated, and the power generation efficiency and power generation can be increased. We have found that the duration can be improved. Based on these findings, the present inventors have completed the present invention.

That is, in order to solve the above problems, the inventions shown in the following [1] to [8] are provided.

[1] A biobattery including a negative electrode using an enzyme that oxidizes fuel to produce an organic acid as an electrode catalyst.
A pH adjusting unit for adjusting the pH fluctuation in the negative electrode due to the organic acid generated by the oxidation of the fuel by the negative electrode is provided.
The pH adjuster
(I) A fuel containing the fuel and a buffer solution having a concentration of 0.5 times or more that of the fuel and being adjusted to be alkaline to be supplied to the negative electrode to replace the used fuel solution. Solution exchange part and
(Ii) At least one selected from a negative electrode impregnated portion in which the negative electrode is impregnated with a buffer solution having a concentration of 0.5 times or more that of the fuel supplied to the negative electrode and adjusted to be alkaline. Biobattery composed of.

According to the configuration of [1] above, it is possible to prevent the pH on the negative electrode side from becoming acidic due to the organic acid generated when the enzyme which is the electrode catalyst of the negative electrode oxidizes the fuel, and also to prevent the pH on the negative electrode side from becoming acidic. Even if the pH of the negative electrode tends to be acidic, the pH on the negative electrode side can be returned to the optimum range of the enzyme on the negative electrode. The enzyme that is the electrode catalyst of the negative electrode can be maintained in the active state. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, it is possible to provide a biobattery that exhibits excellent battery characteristics with the above configuration. Further, the pH on the negative side can be adjusted simply by replacing the used fuel solution with a fuel solution having an adjusted buffer component concentration and pH, or by impregnating the buffer solution having an adjusted buffer component concentration and pH. It can be performed by a simple mechanism and does not cause the bio-cell to become large and complicated.

[2] The replacement fuel solution supplied to the negative electrode by the fuel solution exchange section contains 0.75 M or more of a buffer solution component.

According to the configuration of the above [2], the pH on the negative electrode side can be effectively adjusted, and the enzyme, which is the electrode catalyst of the negative electrode, can be maintained on the negative electrode side in a state suitable for exerting the activity. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[3] The buffer solution in which the negative electrode is impregnated by the negative electrode impregnated portion contains 1 M or more of a buffer solution component.

According to the configuration of the above [3], the pH on the negative electrode side can be effectively adjusted, and the enzyme, which is the electrode catalyst of the negative electrode, can be maintained on the negative electrode side in a state suitable for exerting the activity. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[4] The alkalinity is in the range of pH 8 to 11.

According to the configuration of the above [4], the pH of the fuel solution supplied to the negative electrode by the fuel solution exchange part and the buffer solution impregnated with the negative electrode by the negative electrode impregnated part is in the range of 8 to 11. By doing so, the pH on the negative electrode side can be effectively adjusted, and the negative electrode side can be maintained in a state suitable for exerting the activity without inactivating the enzyme which is the electrode catalyst of the negative electrode. .. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[5] The buffer solution component contains an imidazole compound.

According to the configuration of the above [5], since the imidazole compound used as the buffer solution component has an excellent buffering action, it can effectively buffer the pH fluctuation due to the organic acid generated on the negative electrode side, and in particular, a high-concentration fuel. The pH on the negative electrode side can be effectively adjusted even at the time of high output using the above, and the negative electrode side can be maintained in a state suitable for exerting the activity of the enzyme which is the electrode catalyst of the negative electrode. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[6] The buffer capacity of the buffer solution component is adjusted by a strong basic compound.

According to the configuration of [6] above, the presence of the strong basic compound in addition to the buffer component improves the buffering capacity on the alkaline side, so that the pH fluctuation due to the organic acid generated on the negative electrode side is effective. Can be buffered. In particular, even at high output using a high concentration fuel, the pH on the negative electrode side can be effectively adjusted, and the negative electrode side is maintained in a state suitable for exerting the activity of the enzyme that is the electrode catalyst of the negative electrode. can do. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[7] The pH adjusting unit is configured to operate repeatedly when the fuel solution is replaced.

According to the configuration of [7] above, by repeatedly operating the pH adjusting unit when exchanging the fuel solution, the pH fluctuation due to the organic acid generated on the negative electrode side can be continuously buffered, and the negative electrode side is used as the negative electrode catalyst. It can be maintained in a state suitable for exerting the activity of a certain enzyme for a long period of time. As a result, stable and sustainable operation of the biobattery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and in particular, durability can be improved. Therefore, with the above configuration, it is possible to provide a biobattery that exhibits more excellent battery characteristics.

[8] The enzyme is nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, or pyrroloquinoline quinone-dependent dehydrogenase.

According to the configuration of [8] above, stable and continuous operation of a biobattery using nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, or pyrroloquinoline quinone-dependent dehydrogenase as an electrode catalyst of the negative electrode. Can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and in particular, durability can be improved. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, or pyrroloquinoline quinone-dependent dehydrogenase is an enzyme that is widely used in the negative electrode of biobattery, such as catalyzing the oxidation reaction of sugars. Biobattery availability is improved.

It is a figure which shows the measurement result of the redox potential of the negative electrode internal composition produced in the reference example 1 which verified the negative electrode internal composition of a biobattery. It is a graph which shows the result of the reference example 2 which verified the optimum pH of the enzyme used for a positive electrode and a negative electrode (PQQGDH-mPMS system), and shows the pH dependence example of the activity of a positive electrode enzyme and a negative electrode enzyme. It is a schematic diagram of the biobattery produced in Reference Example 3 in which the influence of the pH environment on the biobattery output was verified (PQQGDH-mPMS system). It is a graph which shows the result of the comparative example 1 which performed the verification-1 (PQQGDH-mPMS system) of the voltage holding time by exchanging a fuel solution. It is a graph which shows the result of the comparative example 2 which performed the verification-2 (NADGDH-ACNQ system) of the voltage holding time by exchanging a fuel solution. It is a graph which shows the result of the comparative example 3 which performed the verification 3 (NADGDH-ACNQ system) of the voltage holding time by exchanging a fuel solution. It is a schematic diagram which shows the electrode exchange method of the biobattery produced in Reference Example 4 which verified from the electrode of the battery capacity decrease factor (NADGDH-ACNQ system). It is a graph which shows the result of the reference example 4 which verified the influence from the electrode of the battery capacity decrease factor (NADGDH-ACNQ system). It is a graph which shows the result of the reference example 5 which performed the verification (NADGDH-ACNQ system) from the internal component of the negative electrode of the battery capacity decrease factor. It is a graph which shows the result of the reference example 6 which performed the verification-1 (NADGDH-ACNQ system) from the organic acid generated by the fuel consumption of the battery capacity decrease factor. It is a graph which shows the result of the reference example 7 which performed the verification-2 (NADGDH-ACNQ system) of the influence from the organic acid generated by the fuel consumption of the battery capacity decrease factor. It is a schematic diagram which shows the impregnation method of the component | component in the negative electrode in Example 1 which verified the influence of the buffer solution impregnation for pH adjustment on a negative electrode (NADGDH-ACNQ system). It is a graph which shows the result of Example 1 which verified the influence of the buffer solution impregnation for pH adjustment on the negative electrode (NADGDH-ACNQ system). It is a graph which shows the result of Example 2 which verified the influence of the buffer solution concentration in the fuel solution added at the time of fuel solution exchange (NADGDH-ACNQ system). It is a graph which shows the result of Example 3 which verified the kind (NADGDH-ACNQ system) of the buffer solution in the fuel solution added at the time of fuel solution exchange. It is a graph which shows the result of Example 3 which verified the kind (NADGDH-ACNQ system) of the buffer solution in the fuel solution added at the time of fuel solution exchange. It is a graph which shows the result of Example 4 which performed the influence of the high concentration buffer solution (NADGDH-ACNQ system) in the fuel solution added at the time of fuel solution exchange and its pH. It is a graph which shows the result of Example 5 which verified the influence of the buffer solution concentration in the fuel solution added at the time of fuel solution exchange (NADGDH-ACNQ system). It is a graph which shows the result of Example 6 which verified the influence of the buffer solution concentration and pH (PQQGDH-mPMS system) added at the time of exchanging a fuel solution. It is a graph which shows the result of the comparative example 4 which verified the behavior at the time of a low-concentration fuel solution (NADGDH-ACNQ system). It is a graph which shows the result of Example 8 which verified the influence of the pH of the buffer solution (NADGDH-ACNQ system) at the time of impregnating the negative electrode with the buffer solution. The graph which shows the result of Example 9 which verified the influence of the fuel concentration and the buffer solution concentration in the fuel solution added at the time of fuel solution exchange (NADGDH-ACNQ system).

Hereinafter, the invention of the present application will be described in detail.

(Biobattery of the present invention)
In the biobattery of the present invention, the positive electrode and the negative electrode are arranged so as to face each other with a diaphragm interposed therebetween, and the positive electrode and the negative electrode are connected by an external circuit. The biobattery of the present invention is provided with a pH adjusting unit and is configured to be able to adjust the pH inside the negative electrode.

The negative electrode of the biobattery of the present invention is provided with an enzyme that oxidizes fuel to produce an organic acid as an electrode catalyst, and by the catalytic action of the enzyme, a reaction of extracting biological energy from the fuel (substrate) and converting it into electrical energy is performed. It is configured as an enzyme electrode.

An enzyme is an enzyme that oxidizes a fuel to produce an organic acid, and produces a product having a higher acidity than the fuel used as a substrate. Here, the production of the organic acid includes not only the case where the organic acid is produced by the direct catalytic action of the enzyme, but also the case where the organic acid is produced by the subsequent non-enzymatic action on the product produced by the direct catalytic action of the enzyme.

As the enzyme, only one type can be used alone, or a combination of two or more types of enzymes can be used. When constructing a multi-step reaction system by combining a plurality of types of enzymes, it is sufficient that an organic acid is produced at any of the steps, and preferably an enzyme in which the final product of the multi-step reaction is an organic acid. Combinations can be used.

The origin of the enzyme is not particularly limited as long as it has the above-mentioned properties. Therefore, it may be of natural origin purified by isolation and purification technology of an appropriate protein from a naturally occurring organism, and it may be produced as a recombinant by a genetic engineering method or chemically synthesized. There may be something. Furthermore, a commercially available product can also be used.

A known method can be used when producing by a genetic engineering method. Specifically, a desired enzyme is obtained from genomic DNA derived from an organism, cDNA synthesized by a reverse transcription reaction from total RNA, or the like by a hybridization method using DNA prepared based on the base sequence of a desired enzyme gene as a probe. A nucleic acid molecule encoding the above can be prepared. The probe used here is an oligonucleotide containing a sequence complementary to the desired enzyme, and can be prepared by a conventional method. For example, in addition to the chemical synthesis method, a restriction enzyme fragment or the like can be used when the target nucleic acid has already been obtained.

Similarly, by PCR using a primer prepared based on the base sequence of a desired enzyme gene, a nucleic acid molecule encoding the desired enzyme can be prepared using genomic DNA or cDNA derived from an organism as a template. The primer used when utilizing PCR is an oligonucleotide containing a sequence complementary to the nucleic acid sequence encoding the desired enzyme, and can be prepared by a conventional method in the same manner as the above probe. When a probe or primer is prepared based on a chemical synthesis method, the probe or primer is designed based on the sequence information of the target nucleic acid prior to the synthesis.

Here, complementary means that the probe or primer and the target nucleic acid molecule can specifically bind to each other according to the base pairing rule to form a stable double-stranded structure. Here, not only perfect complementarity, but only some nucleobases follow the base pairing rule, as long as the probe or primer and the target nucleobase are sufficient to form a stable duplex structure with each other. Even partial complementarity that fits together is acceptable. The length of the probe or primer is determined depending on the sequence information of the target nucleic acid such as GC content and many factors such as the reaction temperature and the hybridization reaction conditions such as the salt concentration in the reaction solution.

Furthermore, a nucleic acid molecule encoding a desired enzyme can be chemically synthesized by using a DNA synthesis method such as a conventional phosphoramidite method.

Then, using the obtained nucleic acid molecule, a desired enzyme can be produced by a gene recombination technique known to those skilled in the art.

Specifically, a nucleic acid molecule encoding a desired enzyme is inserted into an appropriate expression vector, and this is introduced into a host to prepare a transformant. Here, the usable vector is not particularly limited as long as it can incorporate foreign DNA and can autonomously replicate in a host cell. Then, the vector may be integrated so that the foreign gene can express its function, and may contain other known base sequences necessary for function expression. For example, a promoter sequence, a leader sequence, a signal sequence, a ribosome binding sequence, and the like can be mentioned. Further, a marking sequence or the like capable of imparting phenotypic selection in the host can also be included.

For insertion of a foreign gene into a vector, for example, a method is used in which a nucleic acid molecule encoding a desired enzyme is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multicloning site of an appropriate vector for ligation. Can, but is not limited to.

The host cell for producing the transformant is not particularly limited as long as it is a host cell capable of efficiently expressing a foreign gene. Prokaryotic cells can be preferably used, and especially Escherichia coli can be used. In addition, Bacillus subtilis, Bacillus bacterium, Pseudomonas bacterium and the like can also be used. Furthermore, it is possible to utilize eukaryotic cells without being limited to prokaryotes. As the transformation method, a known method such as a calcium chloride method, an electroporation method, a liposome fermentation method, or a microinjection method can be used.

The resulting transformant is then cultured in a suitable nutrient medium under conditions that allow the expression of the introduced nucleic acid molecule to produce the desired enzyme. The culture can be carried out according to a conventional method, and the culture conditions may be selected in consideration of the nutritional and physiological properties of the host cell. The medium used is not particularly limited as long as it contains nutrients that can be assimilated by the host cell and can efficiently express the protein in the transformant. Further, the culture form is not particularly limited, but a liquid medium can be preferably used from the viewpoint of mass culture.

The selection of host cells carrying the desired recombinant vector can be performed, for example, by the presence or absence of expression of the marking sequence. For example, when a drug resistance gene is used as a marking sequence, it can be carried out by culturing in a drug-containing medium corresponding to the drug resistance gene.

In order to isolate and purify the desired enzyme from the culture of the transformant, a conventional protein isolation and purification method can be used. For purification, a method similar to a general protein isolation and purification method may be applied from the culture of the transformant according to the fraction in which the desired enzyme is present. Specifically, when the desired enzyme is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by a step such as centrifugation or filtration to obtain a culture supernatant. Subsequently, the culture supernatant can be isolated and purified by appropriately selecting a known protein purification method.

When the desired enzyme is produced in the host cell, the host cell is recovered by a step such as centrifugation or filtration of the culture. Subsequently, the host cells are disrupted by an enzymatic disruption method or a physical disruption method such as ultrasonic treatment, freeze-thaw, or osmotic shock. After crushing, the solubilized fraction is collected by steps such as centrifugation and filtration. The obtained solubilized fraction can be isolated and purified by treating it in the same manner as in the case where it can be produced extracellularly as described above.

In addition, an enzyme having a known amino acid sequence can also be produced by a chemical synthesis technique. For example, it can be prepared by synthesizing all or a part of the amino acid sequence of a desired enzyme using a peptide synthesizer and reconstructing the obtained polypeptide under appropriate conditions.

Furthermore, the enzyme is not limited to the above-mentioned refined products, but is in the form of organisms such as microorganisms, organelles and cells as long as it has catalytic activity that can extract the chemical energy of the fuel (substrate) and convert it into electrical energy. There may be. In addition, crude products from these organisms may be used.

The enzyme is not particularly limited in the presence or absence of coenzyme and cofactor requirement, but is preferably nicotinamide adenine dinucleotide (hereinafter, may be referred to as "NAD" or "NAD ⁺ ") or nicotin. It is an adenine dinucleotide phosphate (sometimes referred to as "NADP" or "NADP ⁺ ") -dependent enzyme, or a pyrroloquinoline quinone (hereinafter sometimes referred to as "PQQ") -dependent enzyme. When combining multiple enzymes, it is preferable that at least one enzyme is a NAD-dependent enzyme, a NADP-dependent enzyme, or a PQQ-dependent enzyme, and all the enzymes are NAD-dependent enzymes, NADP-dependent enzymes, or PQQ. It may be a dependent enzyme. Here, the NAD-dependent enzyme and the NADP-dependent enzyme are enzymes that require the coenzyme NAD or NADP to exert the catalytic activity of the enzyme, and catalyz the reaction of oxidizing the fuel (substrate) and reducing NAD or NADP. .. A PQQ-dependent enzyme is an enzyme that requires the coenzyme PQQ to exert its catalytic activity.

As the enzyme, an enzyme that catalyzes a redox reaction can be preferably used, and a dehydrogenase that catalyzes a dehydrogenation reaction can be particularly preferably used. Examples thereof include enzymes that oxidize sugars to produce organic acids such as aldonic acid and aldaric acid. Although not limited to these, glucose dehydrogenase, galactose dehydrogenase, mannose dehydrogenase, glyceraldehyde dehydrogenase, arabinose dehydrogenase, xylose dehydrogenase and the like can be used. Further, aldehyde dehydrogenase, acetaldehyde dehydrogenase and the like can be mentioned. Glucose dehydrogenase is preferred, particularly preferably NAD-dependent glucose dehydrogenase, which can be purchased from Amano Enzyme as GLUCDH “Amano” 2, and PQQ-dependent from the Acinetobacter calcoaceticus NBRC12552 strain. It is a glucose dehydrogenase, which oxidizes glucose to produce gluconolactone, and gluconolactone is hydrolyzed non-enzymatically to produce gluconic acid, which is an organic acid.

In the negative electrode of the biobattery of the present invention, the enzyme is immobilized on an appropriate electrode base material. Here, the enzyme is preferably immobilized at a concentration of 1-100 mg / ml.

The electrode base material is not particularly limited as long as it is a conductive base material that can be connected to an external circuit and can transmit electrons. Carbon materials such as carbon cloth, carbon paper, graphite, and glassy carbon, metals or alloys such as aluminum, copper, gold, platinum, silver, nickel, and palladium, SnO ₂ , In ₂ O ₃ , WO ₃ , TiO _2, etc. Examples thereof include conductive oxides, but the present invention is not limited to these. A conductive base material of a conventionally known material can be used.

Further, this may be configured with a single layer or a laminated structure of two or more types. Further, in order to improve the conductivity, commercially available carbon black such as Ketjen black or conductive carbon fine particles such as activated carbon powder may be applied to the base material. At that time, a binder such as PVDF may be used. The size and shape of the electrode base material are not particularly limited, and can be appropriately adjusted according to the purpose of use. It can be configured as a microelectrode whose electrode area is reduced to the order of micrometers.

A substance necessary for the enzyme to exert its catalytic activity may be immobilized on the electrode base material together with the enzyme. For example, in the case of an enzyme that requires a coenzyme or the like to exert the catalytic activity of the enzyme, a substance required for activation can be immobilized together with the enzyme on the electrode substrate. On the other hand, when only the enzyme is immobilized on the electrode substrate, a member for supplying the substance necessary for activation to the enzyme immobilized on the electrode substrate may be provided. As for the enzyme that requires a cofactor, the enzyme to be immobilized may be in the apo form or the holo form, but it must be configured so that it can exert its activity in the electrode reaction. Therefore, when it is held on the electrode base material as the apo form, the enzyme in the apo form is changed to the active holo form by providing a member for supplying a cofactor to the enzyme immobilized on the electrode base material. It is necessary to provide a member for conversion. Further, an electron mediator that mediates electron transfer between the catalytic reaction and the electrode may be immobilized on the electrode base material together with an enzyme.

For example, in the case of a NAD-dependent enzyme and a NADP-dependent enzyme, NAD or NADP, which is a coenzyme required for exerting the catalytic activity of the enzyme, may be immobilized on the electrode substrate together with the enzyme. Here, NAD or NADP is reduced with the oxidation of the fuel of the NAD-dependent enzyme or NADP-dependent enzyme to produce the reduced form of NADH or NADPH. Therefore, this NADH or NADPH is oxidized to return to the oxidized form NAD or NADP, and electrons are transferred to the NADH oxidase or NADPH oxidase such as diaholase that mediates the transfer of electrons to and from the electron mediator, and the electrode substrate. The electronic mediator to be used may be fixed. In the case of a PQQ-dependent enzyme, PQQ, which is a coenzyme required for exerting the catalytic activity of the enzyme, and an electron mediator that transfers electrons to the electrode substrate may be immobilized.

The electronic mediator is not particularly limited as long as it has the above properties. Examples thereof include ferrocene, ferricyanide, quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferredoxins and derivatives thereof, and the most suitable substance is selected according to the type of electrode catalyst. You can select it. Preferably, in the case of a NAD-dependent enzyme and a NADP-dependent enzyme, 2-amino-3-chloro-1,4-naphthoquinone (hereinafter referred to as "ACNQ") can be used. In the case of a PQQ-dependent enzyme, 1-methoxy-5-phenazinemethsulfate (hereinafter abbreviated as "mPMS") can be used.

It is preferable that the positive electrode of the biobattery of the present invention can carry out an oxygen reduction reaction. For the positive electrode, it is preferable to select an enzyme such as oxidase that undergoes an oxygen reduction reaction. The positive electrode may be configured as a biological electrode containing a biocatalyst in at least a part on a suitable electrode base material, a metal electrode such as platinum utilizing a catalytic reaction of a metal, or the like. Here, the biocatalyst is a substance having a substance conversion ability existing in the living body. In addition to natural substances derived from living organisms, it also includes artificial substances that imitate it. For example, enzymes, microorganisms, organelles and cells are included. When a positive electrode is formed as a biological electrode, it is preferable to use an enzyme electrode in which an enzyme is immobilized on an appropriate electrode base material. As the electrode base material, the same conductive base material as that described for the negative electrode can be used.

As the enzyme, oxidoreductases such as bilirubin oxidase, pyruvate oxidase, laccase, and ascorbic acid oxidase are preferably used, and the enzymes can be used alone or in combination of two or more. Here, bilirubin oxidase is a multicopper oxidase having a copper ion as an active center, and is an enzyme that catalyzes the oxidation reaction from bilirubin to biliverdin. It is an enzyme with high utility value as an electrode catalyst for the positive electrode of a biobattery because it has the property of catalyzing the reaction of electron reduction of molecular oxygen using electrons extracted from the substrate to generate water molecules. There are no particular restrictions on the presence or absence of coenzyme and cofactor requirements.

A substance necessary for the enzyme to exert its catalytic activity may be immobilized on the electrode base material together with the enzyme. For example, in the case of an enzyme that requires a coenzyme or the like to exert the catalytic activity of the enzyme, a substance required for activation can be immobilized together with the enzyme on the electrode substrate. On the other hand, when only the enzyme is immobilized on the electrode substrate, a member for supplying the substance necessary for activation to the enzyme immobilized on the electrode substrate may be provided. As for the enzyme that requires a cofactor, the enzyme to be immobilized may be in the apo form or the holo form, but it must be configured so that it can exert its activity in the electrode reaction. Therefore, when it is held on the electrode base material as the apo form, the enzyme in the apo form is changed to the active holo form by providing a member for supplying a cofactor to the enzyme immobilized on the electrode base material. It is necessary to provide a member for conversion. Further, an electron mediator that mediates the catalytic reaction of the enzyme and the transfer of electrons between the electrodes may be immobilized on the electrode substrate together with the enzyme.

The biobattery diaphragm of the present invention has ionic conductivity capable of allowing protons and the like to pass through, and as long as it has the property of not allowing the negative electrode side constituents and the positive electrode side constituents other than ions such as protons to permeate, the material and the material thereof. There are no restrictions on the shape, etc. For example, a cellulose membrane or the like can be used, and a solid electrolyte membrane can be used. Examples of the solid electrolyte film include a solid film having an ion exchange function such as a strong acid group such as a sulfone group, a phosphoric acid group, a phosphone group, and a phosphine group, a weak acid group such as a carboxyl group, and an organic polymer having a polar group. By way of example, but not limited to these. Specifically, a cellulose membrane, and Nafion (registered trademark), which is a copolymer of tetrafluoroethylene and perfluoro [2- (fluorosulfonylethoxy) propylvinyl ether]: tetrafluoroethylene and perfluoro [2- (fluorosulfonylethoxy) propylvinyl ether], etc. Perfluorocarbon sulfonic acid (PFS) -based resin film can be used.

The fuel for the biobattery of the present invention is not particularly limited as long as it is a substance capable of producing an organic acid while being oxidized by a redox reaction carried out by an enzyme immobilized on the negative electrode. The production of an organic acid includes not only the direct catalytic action of the enzyme but also the production of the organic acid by the subsequent non-enzymatic action on the product produced by the catalytic action of the enzyme. Therefore, the fuel can be appropriately selected according to the type of enzyme immobilized on the negative electrode. When using a multi-step reaction in which two or more types of enzymes are combined, an organic acid may be produced at any of the steps, preferably a fuel produced by the organic acid as the final product of the multi-step reaction. Is the subject of the present invention. The fuel is preferably a biomass fuel. Biomass means resources derived from living organisms, and these may be themselves, but processed ones are preferable.

For example, as the fuel, saccharides can be used regardless of whether they are monosaccharides, disaccharides, or polysaccharides. Examples of monosaccharides include erythrose with 4 carbon atoms, threose, elittlerose, arabinose with 5 carbon atoms, xylose, ribose, lyxose, ribulose, glucose with 6 carbon atoms, galactose, talose, mannose, sorbose, fructose, taga sauce, sorbose and the like. Can be mentioned. Examples of the disaccharide include maltose, lactose, sucrose and the like, and examples of the polysaccharide include starch, glycogen, cellulose and the like. Further, alcohols such as methanol, ethanol and isopropyl alcohol, aldehydes such as formaldehyde and acetaldehyde, neutral amino acids such as alanine, valine, leucine and glycine, peptides and polyamino acids such as proteins are exemplified. There is no particular limitation as long as it is converted into a more acidic product by catalytic action.

The fuel is supplied as a fuel solution dissolved in a suitable solvent. The solvent is an aqueous medium, and in addition to distilled water, a suitable buffer solution may be used. Examples of the buffer solution component contained in the buffer solution include imidazole, carbonate, phosphate, borate, tartrate, citrate, tris (hydroxymethyl) aminomethane (TRIS), 4- (2-hydroxy). Ethyl) -piperazin-1-ethane sulfonic acid (HEPES), 3-morpholinopropane sulfonic acid (MOPS) and the like can be exemplified. These may be used alone, but two or more kinds may be used in combination. However, when the pH adjusting unit operates, the concentration and pH of the buffer solution component will be prepared as a replacement fuel solution described later.

Even if the fuel solution contains substances necessary for enzymes such as coenzymes and cofactors to exert their catalytic activity, and substances necessary for coupling the catalytic reaction of enzymes such as electron mediators with the electrode reaction. Good. For example, when NAD or NADP-dependent enzyme is used as the electrode catalyst of the negative electrode, it can be composed of NAD or NADP, NADH or NADPH oxidase such as diaphorase, and an appropriate electron mediator. Further, when a PQQ-dependent enzyme is used as the electrode catalyst of the negative electrode, it can be configured to include PQQ, an appropriate electronic mediator, and the like.

The fuel solution is supplied to the enzyme on the negative electrode located inside the biobattery. The fuel solution is supplied by the fuel solution supply unit. The form of the fuel solution supply unit is not limited as long as the fuel solution can be supplied to the enzyme on the negative electrode. A fuel solution storage space may be provided inside the biobattery to store the fuel solution and to supply the fuel solution stored inside the biobattery so as to be in contact with the negative electrode. Then, the fuel may be supplied to the fuel solution storage space from the outside of the biobattery by a pump or the like. Further, a supply flow path and a supply hole for supplying the fuel solution to the fuel solution storage space, and a discharge flow path and a discharge hole for collecting the used fuel solution after contact with the negative electrode and discharging the used fuel solution from the fuel solution storage space are provided. It may be provided. Further, the fuel solution may be supplied to the fuel solution storage space when the biobattery is assembled.

The biobattery of the present invention includes a pH adjuster. In a biobattery that uses an enzyme electrode as a negative electrode to oxidize fuel and take out electrons, an organic acid is generated on the negative electrode side in response to the oxidation of the fuel by the enzyme. Since such an organic acid is generated in a large amount as compared with the concentration of the buffer solution component contained in the electrolytic solution, the fuel solution, or the like, the pH on the negative electrode side shifts to acidic. As a result, the pH deviates from the optimum range of the enzyme, and the output of the biobattery decreases due to the decrease in the enzyme activity. In the biobattery of the present invention, the pH adjusting unit adjusts the pH fluctuation in the negative electrode due to the organic acid generated by the oxidation of the fuel by the negative electrode, and reduces the enzyme activity caused by the pH fluctuation due to the organic acid on the negative electrode side. The enzyme, which is an electrode catalyst, can prevent and stably and sustainably maintain its catalytic activity in a suitable environment.

The pH adjusting unit supplies the negative electrode with a replacement fuel solution containing the fuel and a buffer solution having a concentration of 0.5 times or more that of the fuel and which is adjusted to be alkaline, and replaces the fuel solution with a used fuel solution. It can be configured by a fuel solution exchange unit. Further, the negative electrode can be composed of a negative electrode impregnated portion which contains a buffer solution component having a concentration of 0.5 times or more that of the fuel supplied to the negative electrode and impregnates the buffer solution which is adjusted to be alkaline. The pH adjusting unit may be composed of either a fuel solution exchange unit or a negative electrode impregnated unit, or may be composed of both.

The replacement fuel solution supplied by the fuel solution exchange unit configured as the pH adjuster contains the fuel and a buffer solution component of 0.5 times or more, preferably 1 time or more of the concentration of the fuel, and is adjusted to be alkaline. Has been done. The fuel concentration in the replacement fuel solution at this time is preferably 0.5 M or more. The fuel solution further preferably contains a buffer solution component having a concentration of 0.75 M or more, particularly preferably 2.0 M or more. Here, the buffer solution component refers to a component having a buffering action that prevents a change in hydrogen ion concentration due to an acid or base, and is preferably a weak acid and a salt thereof, or a mixture of a weak acid and a salt thereof. The concentration of the buffer component is, for example, the total concentration of the acid and its salt when the acid and its salt exhibit a buffering effect, and the combination of the base and its salt is the concentration of the base and its salt. Refers to the total concentration. As the concentration of fuel contained in the fuel solution increases, the amount of organic acid produced by the oxidation of the fuel increases and the pH fluctuation also increases. A strong buffering action is required to return the pH inclined to the acidic side to the optimum pH of the enzyme due to a large amount of organic acid. On the other hand, since the buffering action of the buffer solution becomes stronger as the concentration of the buffer solution increases, it is preferable to set the concentration of the buffer solution component contained in the replacement fuel solution according to the fuel concentration.

The liquidity of the replacement fuel solution is adjusted to be alkaline, i.e. pH above 7. However, depending on the type of enzyme, the three-dimensional structure of the enzyme may change and be inactivated in a strongly alkaline environment, so the pH is preferably adjusted to the range of 12 or less. In particular, the pH is preferably adjusted in the range of 8-11. As a result, the enzyme can be maintained in a state suitable for exerting its activity without inactivating the enzyme.

The buffer component contained in the replacement fuel solution supplied by the fuel solution exchange unit is not limited as long as it can buffer the alkaline region.

An imidazole compound is preferably exemplified as a buffer component. The imidazole compound is not particularly limited as long as it is a compound having an imidazole ring having a buffering effect on hydrogen ion concentration, in addition to imidazole (CAS No. 288-32-4, C ₃ H ₄ N ₂ , MW = 68.08). Further, the imidazole buffer solution may contain sodium chloride, ethylenediaminetetraacetic acid dinatrim, magnesium acetate and the like.

A strong basic compound can be added to the replacement fuel solution supplied by the fuel solution exchange unit in addition to the buffer solution component. As the strongly basic compound, for example, hydroxides of alkaline earth metal and alkali metal salt are preferable, and in particular, they are strongly basic compounds of sodium hydroxide, potassium hydroxide, calcium hydroxide and barium hydroxide. Is preferable. As a result, the presence of a strongly basic compound in addition to the buffer component improves the buffering capacity on the alkaline side, so that pH fluctuations due to the organic acid generated on the negative electrode side can be effectively buffered, and particularly high. Even at high output using a concentrated fuel, the pH on the negative electrode side can be effectively adjusted, and the negative electrode side can be maintained in a state suitable for exerting the activity of the enzyme that is the electrode catalyst of the negative electrode. it can.

Further, in the replacement fuel solution supplied by the fuel solution exchange unit, substances necessary for enzymes such as coenzymes and cofactors to exert their catalytic activities, and catalytic reactions and electrodes of enzymes such as electron mediators. The substance required for conjugation with the reaction may be included.

The form of the fuel solution exchange unit is not particularly limited as long as the replacement fuel solution can be exchanged with the used fuel solution to the enzyme on the negative electrode arranged inside the biobattery. After a predetermined operation of the biobattery, all or part of the used fuel solution after fuel oxidation can be configured to be replaced with a replacement fuel solution, preferably all of the used fuel solution. Exchange. For example, the fuel solution exchange unit may be configured as the same member as the fuel solution supply unit described above, or may be configured as a separate member. When the fuel solution exchange unit is configured as the same member as the fuel solution supply unit, the used fuel solution is discharged from the fuel solution storage space through the discharge hole after a predetermined operation of the biobattery, and then the used fuel solution is discharged. The replacement fuel solution can be configured to be supplied into the fuel storage space through the supply holes. It can also be configured to supply a replacement fuel solution to the fuel storage space when assembling the biobattery.

The replacement fuel solution can be supplied by the fuel solution exchange unit after a predetermined operation of the biobattery, that is, after oxidation of a certain amount of fuel by the negative electrode. For example, the amount of organic acid products produced by the oxidation of fuel at the negative electrode can be estimated from the amount of power generated by the biobattery, and when the estimated amount of organic acid produced exceeds a certain standard, the fuel solution is supplied. It can be configured as follows. Further, a pH measuring unit may be provided to supply the fuel solution when the pH exceeds a certain standard, or the fuel solution may be supplied after the biobattery has been operated for a predetermined time. You may.

The fuel solution exchange unit may be configured to operate at each exchange of the fuel solution of the biobattery, or may be configured to operate at regular intervals instead of every exchange. When the fuel solution exchange unit is configured to operate at regular intervals, the buffer solution component concentration and pH are adjusted as the above replacement fuel solution when exchanging the fuel solution other than when the fuel solution exchange unit is operated. It may be configured to supply the normally used fuel solution which is not used by the fuel solution supply unit. The initial fuel solution supplied when the bio-cell is operated may be a normally used fuel solution whose buffer solution concentration and pH are not adjusted as the above replacement fuel solution, or the buffer component concentration and pH may be high. A solution having the same composition as the above replacement fuel solution may be used.

The fuel solution exchange unit is preferably configured to operate repeatedly. By repeatedly operating the pH adjuster when exchanging the fuel solution, pH fluctuations due to the organic acid generated on the negative electrode side can be continuously buffered, and the negative electrode side is in a state suitable for exerting the activity of the enzyme that is the electrode catalyst of the negative electrode. Can be maintained for a long period of time.

The buffer solution in which the negative electrode is impregnated by the negative electrode impregnating part configured as the pH adjusting part contains a buffer solution component having a concentration of 0.5 times or more, preferably 1 times or more the concentration of the fuel, and is adjusted to be alkaline. .. The fuel concentration in the fuel liquid at this time is preferably 0.5 M or more. The buffer solution more preferably contains 1 M or more of a buffer solution component. Further, the pH is adjusted to exceed 7, the pH is preferably adjusted to the range of 12 or less, and the pH is particularly preferably adjusted to the range of 8 to 11.

The buffer solution component contained in the buffer solution in which the negative electrode is impregnated by the negative electrode impregnated portion is not limited as long as it can buffer the alkaline region. An imidazole compound is preferably exemplified as a buffer component. Further, a strong basic compound can be added in addition to the buffer solution component, which can further effectively prevent the pH on the negative electrode side from becoming acidic, and the environment on the negative electrode side can be adjusted to the negative electrode enzyme. It can be maintained in an appropriate range.

The form of the negative electrode impregnated portion is not particularly limited as long as the negative electrode, which is a component of the biobattery, particularly the region in which the enzyme of the negative electrode is immobilized can be uniformly impregnated in the buffer solution. After a predetermined operation of the biobattery, the buffer can be configured to impregnate all or part of the negative electrode with the buffer, preferably the entire area of the enzyme immobilized on the negative electrode. For example, a buffer solution storage space that stores a buffer solution inside the biobattery and supplies the buffer solution stored inside the buffer solution so as to be in contact with the negative electrode, and a buffer solution storage space from the outside of the battery cell of the biobattery by a pump or the like. Can be configured to supply a buffer solution. A supply path and a supply hole for supplying the buffer solution to the buffer solution storage space, and a discharge path and a discharge hole for collecting the buffer solution after impregnation with the negative electrode and discharging the buffer solution from the buffer solution storage space may be provided. The negative electrode impregnated portion may be configured as a part or all of the fuel solution supply portion and the fuel solution exchange portion described above as the same member, or may be configured as separate members. For example, the fuel solution storage space and the buffer solution storage space are configured as the same member, and only the supply path and supply hole for supplying the buffer solution and the discharge path and discharge hole for discharging are combined with the fuel solution supply unit and the fuel solution exchange unit. May be configured as a separate member. Further, a buffer tank filled with a buffer solution is provided, the negative electrode is taken out from the biobattery after a predetermined operation, the taken out negative electrode is immersed in the buffer solution tank for a predetermined time, and then the negative electrode is reassembled into the battery cell. You may.

The impregnation of the negative electrode into the buffer solution by the negative electrode impregnating portion can be performed after a predetermined operation of the biobattery, that is, after oxidation of a certain amount of fuel by the negative electrode. For example, the amount of organic acid products produced by the oxidation of fuel at the negative electrode can be estimated from the amount of power generated by the biobattery, and when the estimated amount of organic acid produced exceeds a certain standard, the negative electrode is impregnated into the buffer solution. Can be configured to: Further, a pH measuring unit may be provided so that the negative electrode is impregnated with the buffer solution when the pH exceeds a certain standard, and the negative electrode is impregnated with the buffer solution after the biobattery has been operated for a predetermined time. It may be configured.

The negative electrode impregnated portion is preferably operated when the fuel solution of the biobattery is replaced. When operating at the time of replacement of the fuel solution, it may be configured to operate at each replacement, or may be configured to operate at regular intervals instead of every replacement.

The negative electrode impregnated portion is preferably configured to operate repeatedly. In particular, by repeatedly operating when the fuel solution is replaced, pH fluctuations due to the organic acid generated on the negative electrode side can be continuously buffered, and the negative electrode side remains in a state suitable for exerting the activity of the enzyme that is the electrode catalyst of the negative electrode for a long period of time. Can be maintained over.

By providing the pH adjusting unit in this way, it is possible to prevent the pH on the negative electrode side from tilting to acidic due to the organic acid generated when the enzyme, which is the electrode catalyst of the negative electrode, oxidizes the fuel. Even if the pH on the negative electrode side tends to be acidic, the pH on the negative electrode side can be returned to the optimum pH range of the enzyme. The optimum pH of an enzyme is the optimum pH at which the enzyme exerts its function. In the case of an enzyme as an electrode catalyst for the negative electrode of a biocell, the pH varies depending on the type of enzyme, but is generally pH 6.5 to 9.5. Most of the enzymes have a pH of 7 to 9, preferably pH 7.5 to 8.5. By setting the pH on the negative electrode side to the optimum pH range of the enzyme, the enzyme that is the electrode catalyst of the negative electrode can be maintained in the active state. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved. Therefore, it is possible to provide a biobattery that exhibits excellent battery characteristics with the above configuration. Further, the pH on the negative side can be adjusted simply by replacing the used fuel solution with a fuel solution having an adjusted buffer component concentration and pH, or by impregnating the buffer solution having an adjusted buffer component concentration and pH. There is also an advantage that it can be performed by a simple mechanism and does not lead to an increase in size and complexity of the biocell.

(Preparation of Biobattery of the Present Invention)
The biobattery of the present invention can be produced based on a known method. For example, it comprises manufacturing a negative electrode and a positive electrode, manufacturing a battery cell, and manufacturing a battery cell stack if necessary.

The negative electrode, which is an enzyme electrode, is produced by preparing an enzyme solution and immobilizing the enzyme on the electrode substrate. When the positive electrode is used as the enzyme electrode, the enzyme solution is prepared and the enzyme is immobilized on the electrode base material in the same manner as the negative electrode.

The enzyme solution can be prepared by dissolving or dispersing the enzyme in a suitable solvent. The solvent is an aqueous medium, and in addition to distilled water, a suitable buffer solution may be used. Examples of the buffer solution component contained in the buffer solution include imidazole, carbonate, phosphate, borate, tartrate, citrate, tris (hydroxymethyl) aminomethane (TRIS), 4- (2-hydroxy). Ethyl) -piperazin-1-ethane sulfonic acid (HEPES), 3-morpholinopropane sulfonic acid (MOPS) and the like can be exemplified. These may be used alone, but two or more kinds may be used in combination.

Immobilization of the enzyme on the electrode substrate can be performed by using any known method. For example, immobilization is performed by a carrier bonding method by covalent bonding, physical adsorption, ionic bonding, biochemical specific bonding such as an antibody, a cross-linking method using a reagent having two or more functional groups, a comprehensive method of encapsulation in a gel, or the like. be able to. In addition, these may be combined, and it is desirable to appropriately select an enzyme immobilization method that is optimized for each enzyme.

As an omnibus method, a lattice type encapsulating in a gel having a network-like three-dimensional structure such as various natural polymers and synthetic polymers can be used. Examples of the gel include natural polymers such as agarose, agaropectin, arabic rubber, colorginan, collagen and gelatin, and synthetic polymers such as polyacrylamide-based polymers, polyvinyl alcohol-based polymers, polyvinylpyrrolidone-based polymers and polyvinyl ether-based polymers. Etc. can be preferably used, and the use of a hydrophilic polymer is preferable.

Further, a microcapsule type enclosed by a semipermeable membrane such as a dialysis membrane, a liposome type enclosed by a liquid membrane such as phospholipid, and the like can be used. In addition, the enzyme may be immobilized in the crystalline state.

Particularly preferred is physical adsorption, which can be carried out by bringing an enzyme solution in which the enzyme is dissolved or dispersed in an appropriate solvent into contact with the surface of the electrode substrate. Contact can be performed by a known method such as spraying, dropping, impregnation or the like. Further, a coating method such as a spin coating method, a spray method, a screen method, a dip coating method, or a blade method can be used.

Coenzymes such as NAD, NADP, PQQ, NADH oxidases such as diaholase or NADPH oxidases required when NAD or NADP-dependent enzymes are used as electrode catalysts, and electron mediators as enzyme solutions mixed with enzymes. It may be immobilized, or it may be prepared as a separate solution and immobilized on the electrode substrate before or after the enzyme is immobilized.

The battery cell is composed of a set of a negative electrode, a positive electrode, and a diaphragm, and is manufactured by laminating the negative electrode, the diaphragm, and the positive electrode in this order. Specifically, the negative electrode and the positive electrode are arranged so as to face each other with the diaphragm interposed therebetween, and the negative electrode and the positive electrode are connected by an external circuit. Fuel is supplied to the negative electrode side, and oxygen in the atmosphere can be taken in from the positive electrode side. The pH adjusting unit can be provided inside the battery cell, and a part or all of the members constituting the pH adjusting unit may be provided inside the battery cell.

A battery cell is the smallest unit of a biobattery, and a battery cell stack can be produced by electrically connecting a plurality of battery cells in series and accumulating them. The shape of the battery cell stack may be a flat shape in which the battery cells are arranged in a plane, or may be a laminated shape in which the battery cells are stacked.

(Operating mode of the biobattery of the present invention)
In the operating mode of the biobattery of the present invention, an enzyme immobilized on an electrode base material oxidizes a fuel to generate an organic acid and also produces an electron and a proton. Then, these electrons are transferred to the electrode base material via the electron mediator. For example, when NAD or NADP-dependent glucose dehydrogenase is used as the enzyme, the fuel glucose is oxidized to produce gluconolactone. With the oxidation reaction of the fuel, the coenzyme NAD or NADP is reduced to NADH or NADPH, respectively. NADH or NADPH is oxidized by NADH or NADPH oxidase such as diaholase. The electron mediator receives the electrons generated by the oxidation of NADH or NADPH and transfers them to the conductive substrate.

Subsequently, the electrons are transferred from the negative electrode to the positive electrode through an external circuit, so that the chemical energy of the fuel extracted from the substrate by the enzyme is converted into electrical energy. Protons generated with electrons in the fuel oxidation reaction move to the positive electrode side through the diaphragm. On the positive electrode, protons produce water by the reaction of oxygen taken in from the atmosphere and electrons moving through an external circuit.

Then, after the predetermined operation of the biobattery, the pH adjustment unit is operated to replace the fuel solution after fuel oxidation with a fuel solution containing a buffer solution component having a predetermined concentration or more and adjusted to be alkaline, and / or fuel oxidation. The latter negative electrode is impregnated with a buffer solution containing a buffer solution component having a predetermined concentration or higher and adjusted to be alkaline. It prevents the pH on the negative electrode side from tilting to acidic due to the organic acid generated when the enzyme that is the electrode catalyst of the negative electrode oxidizes the fuel, and even if the pH on the negative electrode side tilts to acidic. The pH on the negative electrode side can be returned to the optimum pH range of the enzyme. By setting the pH on the negative electrode side to the optimum pH range of the enzyme, the enzyme that is the electrode catalyst of the negative electrode can be maintained in the active state. As a result, stable and sustainable operation of the bio-battery can be realized, power generation efficiency, voltage holding time and battery capacity can be improved, and thus durability can be improved.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Reference example 1. Verification of the negative electrode internal configuration of the biobattery 1 A. Outline In this reference example, the negative electrode internal configuration of the biobattery 1 used in the following examples, comparative examples, and reference examples will be described. One of the factors for improving the output of the bio-battery 1 is the improvement of the cell voltage. In the following examples, comparative examples, and reference examples, two types of negative electrode internal configurations were appropriately used, focusing on the redox potential.

Composition 1 / PQQGDH-m PMS system As an electrode catalyst for the negative electrode 22, PQQ-dependent glucose dehydrogenase (hereinafter abbreviated as "PQQGDH"), which is an enzyme with inferior potential but high catalyst rotation speed (hereinafter abbreviated as "PQQGDH") (catalyst rotation speed: 4000 s) Combination of -1) and 1-methoxy-5-phenazinemethsulfate (hereinafter abbreviated as "mPMS") as a mediator

Composition 2 / NADGDH-ACNQ system As an electrode catalyst for the negative electrode 22, there is little potential loss, but the catalyst rotation speed is slower than the above PQQGDH, and another type of enzyme, diaphorase, is also required. NAD-dependent glucose dehydrogenase (hereinafter, A combination of (abbreviated as "NADGDH") (catalyst rotation speed: 500 s-1) and 2-amino-3-chloro-1,4-naphthoquinone (hereinafter abbreviated as "ACNQ") as a mediator.

B. Specific Configuration Example of Bio-Battery 1 Below, a specific configuration example of the PQQGDH-mPMS system and the NADGDH-ACNQ system bio-battery 1 will be described in detail, and FIG. 1 shows the measurement results of the redox potentials of both. ..

B-1. The procedure for manufacturing the PQQGDH-mPMS positive electrode 21, the diaphragm 3, and the negative electrode 22 will be described. The water used in the production was all purified by the ultrapure water production device Direct-Q UV manufactured by Millipore.

a. Preparation of positive electrode 21 a-1. Preparation of positive electrode enzyme solution Bilirubin oxidase (Bilirubin Oxidase: Amano enzyme, BO-3, hereinafter abbreviated as "BOD") was used as the positive electrode enzyme. BOD is dissolved in an appropriate amount of 1 M imidazole pH 4.6, and the concentration is adjusted to 100 mg / ml by converting the absorbance 1 = 1.0 mg / ml from the 280 nm absorbance measurement, and the concentration of potassium ferricyanide to 50 mM. The prepared solution was used as a positive enzyme solution.

a-2. Fixation of Enzyme on Electrode Base Material 51 μl of the positive electrode enzyme solution prepared above was applied to each electrode base material, and this was used as the positive electrode 21. Two positive electrodes 21 were used. As the electrode base material, a carbon cloth cut into 10 mm × 10 mm with a cutter and coated with activated carbon powder was used. To apply the activated carbon powder, mix the activated carbon powder, polyvinylidene fluoride (PVDF), and 1-methyl-2-pyrrolidinone (NMP) in a mortar, and then smear an appropriate amount on both sides of the carbon cloth with a spatula for 8 hours at 60 ° C. This was done by drying the above.

b. Preparation of negative electrode 22 b-1. Preparation of Negative Enzyme Solution The negative enzyme PQQGDH used here is a PQQ-dependent soluble glucose dehydrogenase (hereinafter abbreviated as "sPQQGDH") derived from the Acinetobacter calcoaceticus NBRC12552 strain. The production method and sequence information of such an enzyme are disclosed in Japanese Patent Application Laid-Open No. 2013-45647. Specifically, the sPQQGDH gene (GeneID: X15871) was inserted into the multicloning site (NdeI / BamHI) of the vector pET-22b (+). Escherichia coli BL21 (DE3) strain was transformed using the pET-22b (+) vector into which the sPQQGDH gene was inserted, and the resulting colonies were inoculated into 300 ml of LB / Amp (including ampicillin 50 μg / ml) medium, 37 Inoculated overnight at ° C. Next, add 20 L of LB / Amp medium to the jar fermenter, add 200 ml of the preculture solution, incubate at 37 ° C for about 1 hour (until OD = 0.1), and add 0.01 mM IPTG to induce protein expression. It was sprinkled and cultured with shaking at 28 ° C overnight. The culture solution was centrifuged, and the precipitate from which the supernatant had been removed was cryopreserved at -80 ° C. 5 g of the cryopreserved protein-expressing cells were suspended in 15 ml of PBS buffer. On ice, crushing was performed 10 times at 15 W for 15 seconds using an ultrasonic crusher XL2000 (MISONIX). The crushed solution was centrifuged at 4 ° C. and 5000 rpm for 20 minutes, and the separated supernatant was filtered with a Cellulose Acetate 0.45 μm filter (ADBANTEC) as a sample. An open column (Bio-Rad) was packed with 10 ml of histidine tag purification resin: TALON (Clontech) and equilibrated with 5 times the bed volume of equilibration buffer (PBS + 50 mM NaCl). The pretreated sample is applied to a column, washed with 5 times the bed volume of wash buffer (PBS + 50 mM NaCl + 10 mM imidazole), and then 3 times the bed volume of elution buffer (PBS + 50 mM NaCl). Elution with +150 mM imidazole). The recovered eluate is concentrated using Amicon Ultra-4 (Millipore), and a dialysis buffer (10 mM Tris-HCl (pH 7.5) (pH 7.5) +) is used using a microdialysis machine low-speed type and a dialysis cup MWCO1200 (both Bio-Tec). 0.1 mM CaCl ₂ ) was replaced every hour and dialyzed for a total of 2 hours. The dialysis sample was centrifuged at 4 ° C. and 15000 rpm for 5 minutes, and the separated supernatant was reconcentrated to 20 mg / ml or more. At the time of use, CaCl ₂ was added to 1 mM and PQQ to 1 μM, and the mixture was incubated at 4 ° C. for 30 minutes or longer. Further, a solution prepared by dissolving in 1 M imidazole pH 8.0 and adjusting the concentration of sPQQGDH to 1 mg / ml and mPMS to 30 mM was used as a negative enzyme solution.

b-2. Fixation of Enzyme on Electrode Base Material 51 μl of the negative electrode enzyme solution prepared above was applied to each electrode base material, and this was used as the negative electrode 22. Two negative electrodes 22 were used. As the electrode base material, a carbon cloth cut into 10 mm × 10 mm with a cutter was used.

c. Septum 3
The cellulose membrane was used as the diaphragm 3.

B-2. The procedure for manufacturing the NADGDH-ACNQ positive electrode 21, the diaphragm 3, and the negative electrode 22 will be described. The water used in the production was all purified by the ultrapure water production device Direct-Q UV manufactured by Millipore.

a. Preparation of Positive Electrode 21 The positive electrode 21 was produced in the same manner as the above-mentioned PQQGDH-mPMS system.

b. Preparation of negative electrode 22 b-1. Preparation of Enzyme Solution The negative electrode enzyme NADGDH used here is NAD-dependent glucose dehydrogenase (Amano Enzyme, GLUCDH “Amano” 2, hereinafter abbreviated as “NADGDH”). Dissolve in an appropriate amount of 1M imidazole pH 8.7, NADGDH 10 mg / ml, diaholase (Toyo Boseki DAD301) 20 mg / ml, and NADH (β-Nicotinamide-adenine dinucleotide reduced, Wako Pure Chemical Industries Wako 305-50451) Was used as a negative enzyme solution, the concentration of which was adjusted to 10 mM.

b-2. Fixation of negative electrode enzyme on electrode substrate
51 μl of the negative electrode enzyme solution prepared above was applied to the electrode base material to which 60 mM ACNQ was previously applied, and this was used as the negative electrode 22. Two negative electrodes 22 were used. The electrode base material used was a carbon cloth cut into 10 mm × 10 mm with a cutter.

c. Septum 3
The cellulose membrane was used as the diaphragm 3.

Reference example 2. Verification of optimum pH of enzymes used for positive and negative electrodes (PQQGDH-mPMS system)
A. Outline In this reference example, the pH dependence of the enzyme used for the positive electrode 21 and the negative electrode 22 was verified from the dynamics of the hydrogen ion concentration in the reaction system of the positive electrode 21 and the negative electrode 22.

B. Verification In the biobattery 1, an enzyme that reduces O ₂ , H ⁺ and e ^- to H ₂ O is used for the positive electrode 21, and for example, the BOD used in Reference Example 1 can be used. The anode 22, an organic acid by oxidizing biomass glucose etc., H ^+, e ^- enzymes that produce is used, for example, glucose dehydrogenase, more particularly, the PQQGDH or the like used in Reference Example 1 Can be used.

Here, FIG. 2 shows an example of examining the pH dependence of the enzyme activities of the above BOD (positive electrode enzyme) and PQQGDH (negative electrode enzyme) in a test tube. As can be understood from this result, regardless of the type of enzyme used, since the positive electrode 21 is a reaction that consumes H ⁺ , it is essentially preferable to have an environment rich in H ⁺ (low pH), and conversely. Since the negative electrode 22 is a reaction that produces H ⁺ , it is essentially preferable that the environment is H ⁺ deficient (high pH).

Reference example 3. Verification of the effect of pH environment on biobattery output (configuration 1 / PQQGDH-mPMS system)
A. Outline In this reference example, the pH of the positive electrode 21 and the negative electrode 22 was set based on the result of the reference example 2, and the influence of the pH environment on the output of the biobattery 1 was examined.

B. Method B-1. The configuration of the bio-battery 1 and the procedure for manufacturing the bio-battery 1 using the configuration B-1-1. Preparation of each configuration The preparation of the positive electrode 21, the negative electrode 22, and the fuel solution will be described. The water used in the production was all purified by the ultrapure water production device Direct-Q UV manufactured by Millipore.

a. Preparation of positive electrode 21 a-1. Preparation of enzyme solution The BOD used in Reference Example 1 was used as the positive electrode enzyme. BOD was dissolved in an appropriate amount of 1 M Sodium phosphate buffer, and the concentration was adjusted to 100 mg / ml by converting the absorbance at 1 = 1.0 mg / ml from the 280 nm absorbance measurement, and this was used as the positive enzyme solution.

a-2. Fixation of Enzyme on Electrode Base Material 51 μl of the positive electrode enzyme solution prepared above was applied to each electrode base material, and this was used as the positive electrode 21. Two positive electrodes 21 were used. As the electrode base material, a carbon cloth cut into 10 mm × 10 mm with a cutter and coated with activated carbon powder was used. To apply the activated carbon powder, mix the activated carbon powder, polyvinylidene fluoride (PVDF), and 1-methyl-2-pyrrolidinone (NMP) in a mortar, and then smear an appropriate amount on both sides of the carbon cloth with a spatula for 8 hours at 60 ° C. This was done by drying the above.

b. Preparation of negative electrode 22 b-1. Preparation of Enzyme Solution As the negative electrode enzyme, the glucose dehydrogenase derived from the Acinetobacter calcoaceticus NBRC12552 strain described in Reference Example 1 was used. Details of the method for producing such an enzyme are as described in Reference Example 1. At the time of use, CaCl ₂ was added to 1 mM and PQQ to 0.8 mM to the sample in which the supernatant was reconcentrated to 20 mg / ml or more, and the mixture was incubated at 4 ° C. for 30 minutes or more. Further, a solution adjusted to PQQGDH 1 mg / ml, 30 mM mPMS, and 1 M Sodium Phosphate Buffer pH 7.0 was used as a negative enzyme solution.

b-2. Fixation of Enzyme on Electrode Base Material 51 μl of the negative electrode enzyme solution prepared above was applied to each electrode base material, and this was used as the negative electrode 22. Two negative electrodes 22 were used. As the electrode base material, a carbon cloth cut into 10 mm × 10 mm with a cutter was used.

c. Preparation of Septum 3 A cellulose film was used as the diaphragm 3 that separates the positive electrode 21 and the negative electrode 22.

d. Preparation of fuel solution
It was prepared at 1 M D-Glucose and 1 M Sodium Phosphate Buffer pH 7.0 and used as a fuel solution.

B-1-2. Assembly of Battery Cell The battery cell 1a of the biobattery 1 was produced by combining the positive electrode 21, the negative electrode 22, and the diaphragm 3 produced above. This will be described with reference to FIG. 3, which schematically shows the configuration of the battery cell 1a produced here. The battery cell 1a shown in FIG. 3 has two 3 mmφ round holes (fuel supply holes) 110 formed in a 2 mm thick acrylic plate 100a for supplying a fuel solution, and 10 mm × 10 in a 5 mm thick acrylic plate 100b. The one with a square hole 111 of mm was used. Holes were made in the four sides of the square hole 111 for screwing. Titanium mesh 103 (Alfa Aesar 40921) as a current collector is 10 mm x 40 mm, 0.4 mm thick SUS net 10 mesh 102 is 14 mm x 14 mm, and 0.5 mm thick silicon sheet 101a (AS ONE, etc.) is used as a spacer. A cellulose film (Wako Pure Chemical Industries, Ltd. biochemical dialysis film) as a diaphragm 3 with a square hole of mm × 16 mm or a silicon sheet 101b with a thickness of 1 mm with a square hole of 10 mm × 10 mm. 047-30941) was used. These were laminated in the order of FIG. 3 so as to have a positive electrode 21 | diaphragm 3 | negative electrode 22, and screwed on all sides to assemble the battery cell 1a. When the biobattery 1 is driven, a fuel solution is introduced into the negative electrode 22 from a round hole, and the atmosphere is sent to the positive electrode 21 by a diaphragm pump ((5.5 L-air / min, KNF LAB, LABOPORT N86 KT.18)). It was configured as follows.

B-2. Measurement conditions At the time of measurement, 150 μl of a fuel solution prepared using a buffer solution having the same pH as the buffer solution on the negative electrode 22 side below is injected from the round hole for supplying the fuel solution, and the battery cell 1a prepared above is electronically charged. The battery output was measured by connecting to a load device (Kikusui Denshi PLZ164WA), increasing the load current by 0.5 mA, and measuring the stable voltage at each current value. As the buffer solution on the positive electrode 21 side and the negative electrode 22 side, 1 M Sodium phosphate Buffer combinations of various pHs shown in Table 1 below were used.

C. Results Table 1 shows the results of comparing the maximum power densities at various pH values. As a result, the combination of the negative electrode 22 having a pH of 8.0 and the positive electrode 21 having a pH of 4.3 had the highest maximum power density. When the negative electrode 22 had a pH of 8.8 and the positive electrode 21 had a pH of 4.3, enzyme aggregation was observed and the maximum power density also decreased.

Based on the results of this reference example, in the following examples and comparative examples, the pH of the enzyme-mediator solution impregnated in the electrodes is pH 4.6 for the positive electrode 21 and pH 4.6 for the negative electrode 22 in the PQQGDH-mPMS system, unless otherwise noted. The pH was 8.0, the first fuel to be added was pH 8.0, and in the NADGDH-ACNQ system, the positive electrode 21 was pH 4.6, the negative electrode 22 was pH 8.7, and the first fuel to be added was pH 8.7.

Comparative example 1. Verification of voltage holding time by exchanging fuel solution-1 (PQQGDH-mPMS system)
A. Outline In this comparative example, the voltage holding time of the PQQGDH-mPMS-based biobattery 1 was verified. At this time, verification was performed while exchanging the fuel solution.

B. Method B-1. Configuration of Biobattery 1 The configuration of biobattery 1 is the PQQGDH-mPMS system used in Reference Example 1, and the biobattery 1 was produced in the same manner as the PQQGDH-mPMS system of Reference Example 1.

B-2. Measurement condition
The first fuel solution was added to the battery cell 1a with an electrode area of 1 cm ² using the PQQGDH-mPMS system, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity. When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows. The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.0
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.0

C. Results The results are shown in FIG. In FIG. 4, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. The battery capacity in the first fuel solution was 2.0 mWh (4.5 mAh), but the battery capacity in the second fuel solution was 0.3 mWh (1.1 mAh), and the battery capacity (Wh) in the second fuel solution was about 1/6. Decreased to. It can be calculated that the first fuel solution contains 2.4 M × 78 μl (188 μmole) of glucose. Then, the first battery capacity was 4.5 mAh = 4.5 mC / s x 3600 s = 16.2 C = 16.2 C / 96500 C / mole = 168 μmole ^- electrons. Considering that dehydrogenation of glucose is a two-electron reaction, 168 μmole ^- electrons / (188 μmole ^- glucose x 2) = 0.45, and at least 45% of glucose (0.45 x 2.4 M = 1.1 M) is dehydrogenated and glucon. It is presumed to be acid. This is a concentration exceeding 1 M of the buffer solution component (imidazole) concentration of the fuel solution, and it can be estimated that the effect on pH is large. When exchanging the fuel solution, after the first power generation, the fuel solution was absorbed as much as possible with a pipettor, and the same fuel solution as the first time was put in to generate power, but since the electrode base material is carbon cloth, the inside of the electrode It is assumed that unreacted glucose and gluconic acid remain. Based on the glucose contained in the second fuel solution, it is estimated that 11% (0.26 M) of glucose was dehydrogenated by the same calculation, and it can be understood that the power generation efficiency is significantly reduced.

Comparative example 2. Verification of voltage holding time by exchanging fuel solution-2 (NADGDH-ACNQ system)
A. Outline In this comparative example, the voltage holding time of the NADGDH-ACNQ-based biobattery 1 was verified in the same manner as in Comparative Example 1. The NADGDH-ACNQ system is expected to have high output by optimizing the potential.

B. Method B-1. Configuration of Biobattery 1 The configuration of biobattery 1 is the NADGDH-ACNQ system used in Reference Example 1, and the biobattery 1 was produced in the same manner as the NADGDH-ACNQ system of Reference Example 1.

B-2. Measurement condition
The first fuel solution was added to the battery cell 1a with an electrode area of 1 cm ² using the NADGDH-ACNQ system, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity. When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows. The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 5, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, it was found that the battery capacity (Wh) in the second power generation was reduced to about 1/3.

Comparative example 3. Verification of voltage holding time by exchanging fuel solution-3 (NADGDH-ACNQ system)
A. Outline In this comparative example, following Comparative Example 2, the voltage holding time of the NADGDH-ACNQ-based biobattery 1 was verified. In this comparative example, the enzyme solution impregnated in the positive electrode 21 and the negative electrode 22 and the fuel solution were verified with a pH of 7.0.

B. Method B-1. Composition of bio-battery The composition of the bio-battery is the NADGDH-ACNQ system described in Reference Example 1, and the NADGDH-ACNQ of Reference Example 1 was prepared except that the positive electrode enzyme solution and the negative electrode enzyme solution were prepared with imidazole of pH 7.0. The biobattery 1 was produced in the same manner as the system.

Specifically, as the positive enzyme solution, a solution prepared by dissolving BOD at 100 mg / ml and potassium ferricyanide at pH 7.0 at 1 M imidazole to 50 mM was used. As the negative enzyme solution, a solution prepared by dissolving NADGDH at 10 mg / ml, diaholase at 20 mg / ml, and NADH at 1 M imidazole pH 7.0 to 10 mM was used.

B-2. Measurement conditions In the NADGDH-ACNQ system as in Comparative Example 2, the enzyme-mediator solution impregnated in the electrode and the fuel solution to be added first were both set to pH 7.0, and the first fuel solution was added and the electrode area was 1 cm. ^The first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a of ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 7.0
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 7.0

C. Results The results are shown in FIG. In FIG. 6, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, it was found that the battery capacity (Wh) in the second power generation was reduced to about 1/5.

Reference example 4. Verification of battery capacity reduction factors from electrodes (NADGDH-ACNQ system)
A. Outline In this reference example, it was verified whether the decrease in battery capacity in the second power generation due to the replacement of the fuel solution confirmed in Comparative Examples 1 to 3 was caused by the positive electrode 21 or the negative electrode 22.

B. Method B-1. Configuration of Bio-Battery 1 The configuration of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the NADGDH-ACNQ-based bio-battery 1 was produced in the same manner as in Comparative Example 2. Then, as shown in FIG. 7, the battery capacity is reduced by disassembling the battery cell 1a whose voltage has dropped due to the first power generation under a constant current load and replacing the positive electrode 21 or the negative electrode 22 with a new one. , Or the negative electrode 22 was verified. The exchange method of the positive electrode 21 and the negative electrode 22 is as follows.
1. In the second power generation, the negative electrode 22 is the same as the first one, the positive electrode 21 is replaced with a new one. 2. In the second power generation, the positive electrode 21 is the same as the first one, and the negative electrode 22 is new. Battery cell replaced with one

B-2. Measurement conditions Similar to Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity in the battery cell 1a having an electrode area of 1 cm ² . When the voltage dropped to 0.1 V, the positive electrode 21 or the negative electrode 22 was replaced and the fuel solution was replaced with the fuel solution for the second time as described above. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 8, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, in the battery cell 1a of "1" in which the positive electrode 21 was replaced and the negative electrode 22 was not replaced, the voltage dropped in a shorter time in the second power generation than in the first power generation, and the battery capacity (Wh) was also about. It turned out to drop to 1/5. In the battery cell 1a of "2" in which the negative electrode 22 was replaced and the positive electrode 21 was not replaced, the voltage was maintained in the second power generation as in the first time. From this, it was found that the negative electrode 22 is a factor of lowering the battery capacity.

Reference example 5. Verification from the internal components of the negative electrode, which is a factor that reduces battery capacity (NADGDH-ACNQ system)
A. Outline In this reference example, the factors of the battery capacity decrease due to the negative electrode 22 confirmed in the negative electrode 22 were examined in detail by focusing on the internal components of the negative electrode 22.

B. Method B-1. Configuration of Bio-Battery 1 The configuration of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the NADGDH-ACNQ-based bio-battery 1 was produced in the same manner as in Comparative Example 2. Then, the battery cell 1a whose voltage has dropped due to the first power generation with a constant current load is disassembled, the positive electrode 21 is used as it is for the second time, and the negative electrode 22 is also used for the second time, but the internal components of the negative electrode 22 are used. After impregnating a part of the negative electrode 22 with the negative electrode 22, the battery cell 1a was assembled, and it was verified which of the internal components of the negative electrode 22 caused the decrease in battery capacity. Table 2 summarizes the internal components of the negative electrode 22 impregnated into the negative electrode 22 during the second power generation.

B-2. Measurement conditions Similar to Comparative Example 2, the first fuel solution was added, and power was generated in the battery cell 1a having an electrode area of 1 cm ² by the first constant current load (2.5 mA / cm ² ). When the voltage dropped to 0.1 V, the negative electrode 22 was impregnated with the internal constituents as described above, and then the battery cell 1a was assembled again. Subsequently, the fuel solution was replaced with the second fuel solution, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² , and the voltage holding time was measured. The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 9, the horizontal axis shows a legend showing the internal components of the negative electrode 22 impregnated in the negative electrode 22 at the time of the second power generation, and the vertical axis shows the relative value with respect to the first voltage holding time of 100%. As a result, no matter which of the internal components of the negative electrode 22 such as the enzyme and the mediator was impregnated, none of them recovered until the voltage holding time of the first power generation.

Reference example 6. Verification from organic acids generated by fuel consumption, which is a factor that reduces battery capacity-1 (NADGDH-ACNQ system)
A. Outline In this reference example, the factors of the battery capacity decrease due to the negative electrode 22 confirmed in the reference example 4 were examined in detail by focusing on the organic acid generated by the fuel consumption. In Reference Example 5, we focused on the internal components of the negative electrode 22 and verified the factors that reduced the battery capacity. However, when the second power generation was performed, the voltage of the first time was impregnated with any of the internal components of the negative electrode 22 such as enzymes and mediators. Since it did not recover until the retention time, in this reference example, we focused on gluconic acid generated by the consumption of glucose as a fuel as another factor. In particular, as the first effect of gluconic acid, the effect of pH change due to gluconic acid production was examined.

B. Method B-1. Configuration of Bio-Battery 1 The configuration of Bio-Battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and is the same as in Comparative Example 2 except that the negative electrode enzyme solution was prepared with imidazole at pH 6.4 or pH 10.8. Biobattery 1 was produced.

Specifically, a negative enzyme solution prepared by dissolving NADGDH at 10 mg / ml, diaholase at 20 mg / ml, and NADH at 1 M imidazole pH 6.4 or pH 10.8 to 10 mM was used. did.

B-2. Measurement conditions Similar to the first power generation in Comparative Example 2, the prepared fuel solution was added to the same pH as the negative electrode enzyme solution, and a constant current load (2.5 mA / cm) was applied to the battery cell 1a having an electrode area of 1 cm ^{2. 2} ) was applied to verify the voltage holding time for one power generation. The composition of the fuel solution is as follows.
Fuel solution: 2.4 M glucose / 1 M imidazole pH 6.4, or
2.4 M glucose / 1 M imidazole pH 10.8

C. Results The results are shown in FIG. In FIG. 10, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, it was found that at pH 6.4, the voltage holding time was significantly reduced to about 1/10 of pH 10.8.

Reference example 7. Verification from organic acids generated by fuel consumption, which is a factor that reduces battery capacity-2 (NADGDH-ACNQ system)
A. Outline In this reference example, the factors of the battery capacity decrease due to the negative electrode 22 confirmed in the reference example 4 were examined in detail by focusing on the organic acid generated by the fuel consumption following the reference example 6. In Reference Example 5, we focused on the internal components of the negative electrode 22 and verified the factors that reduced the battery capacity. However, the voltage was held for the first time regardless of which of the internal components of the negative electrode 22 such as enzymes and mediators was impregnated during the second power generation. Since it did not recover until time, in this reference example, we focused on gluconic acid generated by the consumption of glucose as a fuel as another factor. In particular, as the second effect of gluconic acid, the effect of product inhibition by gluconic acid production was examined.

B. Method B-1. Configuration of Biobattery 1 The configuration of biobattery 1 is the NADGDH-ACNQ system used in Comparative Example 2. However, as the negative electrode 22, in order to verify the effect of gluconic acid on the battery capacity, it was prepared by impregnating gluconic acid (pH unadjusted or adjusted) in addition to the negative electrode enzyme solution prepared in the same manner as in Comparative Example 2. The biobattery 1 was produced in the same manner as in Comparative Example 2 except for the above. The details of the prepared negative electrode enzyme solution are as follows. It is presumed that the pH of the negative electrode 22 impregnated with gluconic acid whose pH has not been adjusted is inclined to the acidic side of around 5.

1. 1. Impregnated with unadjusted pH of gluconic acid
Dissolve NADGDH at 10 mg / ml, diaholase at 20 mg / ml, and NADH at 1 M imidazole pH 8.7 to 10 mM, followed by adding unadjusted gluconic acid at 0.36 M. What I did 2. Impregnated with pH-adjusted gluconic acid
NADGDH was dissolved at 10 mg / ml, diaholase at 20 mg / ml, and NADH at 1 M imidazole pH 8.7 to 10 mM, followed by gluconic acid adjusted to around pH 8 with NaOH. Addition to 0.36 M 3. No addition of gluconic acid (control)
As in Reference Example 1, NADGDH was dissolved at 10 mg / ml, diaholase at 20 mg / ml, and NADH at 10 mM at 1 M imidazole pH 8.7, and gluconic acid was not added.

B-2. Measurement conditions Similar to the first power generation in Comparative Example 2, a fuel solution is added, a constant current load (2.5 mA / cm ² ) is applied to the battery cell 1a having an electrode area of 1 cm ² , and the voltage generated by the first power generation. The retention time was verified. The composition of the fuel solution is as follows.
Fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 11, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, the voltage of "1" to which pH-adjusted gluconic acid was added decreased in a shorter time than that of "3" to which gluconic acid was not added, but in "2" where pH was adjusted, gluconic acid was added. The voltage holding time was equivalent to that of "3" without addition.

In view of the results of Reference Example 6, it was found that the decrease in battery capacity is not the effect of product inhibition by gluconic acid, but the effect of the change in pH in the electrode due to gluconic acid production on the voltage holding time.

Example 1. Verification of the effect of buffer impregnation on the negative electrode for pH adjustment (NADGDH-ACNQ system)
A. Outline In this example, since it was found in Reference Example 7 that the change in pH in the negative electrode 22 had a large effect on the voltage holding time, detailed verification was performed. In this example, the influence of the buffer solution used on the negative electrode 22 side on the voltage holding time and the battery capacity was verified. Since the buffer solution has a buffering action against the hydrogen ion concentration, the fluctuation of the pH in the negative electrode 22 due to the gluconic acid generated in the negative electrode 22 is reduced.

B. Method B-1. Configuration of Bio-Battery 1 The configuration of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the NADGDH-ACNQ-based bio-battery 1 was produced in the same manner as in Comparative Example 2. Then, as shown in FIG. 12, the battery cell 1a whose voltage has dropped due to the first power generation with a constant current load is disassembled, the positive electrode 21 is used as it is for the second time, and the negative electrode 22 is also used for the second time. After impregnating the negative electrode 22 with a part of the internal component and the buffer solution component of the negative electrode 22, the battery cell 1a is assembled, and which of the internal component component and the buffer solution component of the negative electrode 22 can recover the battery capacity. Was verified. The types of internal constituents and buffer components of the negative electrode 22 to be impregnated with the negative electrode 22 at the time of the second power generation, and their concentrations and liquid amounts are as follows.

1. 1. Buffer solution + internal components (impregnated with buffer solution, enzyme, mediator, and coenzyme)
Add 11 μl of 2.5 M imidazole pH 10.8, 5.5 μl of NADGDH 100 mg / ml, 11 μl of diahorase 200 mg / ml, 2.8 μl of NADH 200 mM, and 2 μl of ACNQ 60 mM, and mix them. 32.3 μl per negative electrode 22 Impregnated. Both of the two negative electrodes 22 were impregnated.
2. Buffer solution (impregnated with buffer solution only)
The negative electrode 22 was impregnated with 32 μl of 1 M imidazole pH 10.8 per sheet. Both of the two negative electrodes 22 were impregnated.
3. 3. Internal constituents (impregnated with enzymes, mediators, and coenzymes only)
NADGDH 100 mg / ml was added to 5.5 μl, diaholase 200 mg / ml was added to 11 μl, NADH 200 mM was added to 2.8 μl, ACNQ60 mM was added to 2 μl, and water was added to 11 μl to mix, and the negative electrode 22 was impregnated with 32 μl per sheet. Both of the two negative electrodes 22 were impregnated.
4. No impregnation (control)
Use the negative electrode 22 after the first power generation as it is

B-2.
In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity in the battery cell 1a having an electrode area of 1 cm ² . When the voltage dropped to 0.1 V, the negative electrode 22 was impregnated with the internal constituents as described above, and the battery cell 1a was assembled again. Subsequently, the fuel solution was replaced with a second fuel solution, and a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 10.8
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 10.8

C. Results The results are shown in FIG. In FIG. 13, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. “1” impregnated with buffer solution, enzyme, mediator, and coenzyme, and “2” impregnated with buffer solution only show the same voltage holding time as the first power generation, and the battery capacity is also the same. Met. From this result, it was found that the pH in the negative electrode 22 was lowered and the condition that the enzyme and the mediator could not function sufficiently became a factor for lowering the voltage holding time. It was also found that the battery capacity can be made the same for the second power generation by simply impregnating the negative electrode 22 with the buffer solution after the first power generation.

Example 2. Verification of the effect of buffer concentration in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, it was found that the voltage holding time was lowered because the pH in the negative electrode 22 was lowered due to the production of gluconic acid in Example 1 and the conditions were such that the enzyme and the mediator could not function sufficiently. Therefore, the effects of the buffer solution concentration in the fuel solution added when the fuel solution was replaced on the voltage holding time and the battery capacity were verified. As a measure for adjusting the pH inside the negative electrode 22 to be alkaline after the completion of the first power generation, an attempt was made to adjust the pH inside the negative electrode 22 with a fuel solution.

B. Method B-1. Configuration of Bio-Battery 1 The configuration of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the NADGDH-ACNQ-based bio-battery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement condition
As a measure to adjust the pH inside the negative electrode 22 to be alkaline after the end of the first power generation, we tried to adjust the pH inside the negative electrode 22 with a fuel solution, and added the fuel solution for the second time to the previous 2.4 M glucose / 1 M imidazole pH. The pH was changed from 8.7 to 1.5 M glucose / 2.5 M imidazole pH 10.8. In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity in the battery cell 1a having an electrode area of 1 cm ² . When the voltage dropped to 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7 or
1.5 M glucose / 2.5 M imidazole pH 10.8

The results are shown in FIG. In FIG. 14, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. By using 2.5 M imidazole pH 10.8 as a buffer solution for the second fuel solution, the same voltage holding time as the first one was shown in the second power generation, and the battery capacity was also improved. On the other hand, when 2.4 M glucose / 1 M imidazole pH 8.7 was used as the second fuel solution, a decrease in the voltage holding time was observed, but for gluconic acid produced in large quantities by increasing the glucose concentration. It is presumed that the pH adjustment function did not work sufficiently with the 1 M imidazole pH 8.7 buffer solution.

Example 3. Verification of the type of buffer solution in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, since it was found in Example 2 that the battery capacity can be maintained high by adjusting the pH in the negative electrode 22 with the fuel solution, the buffer solution concentration in the fuel solution added when the fuel solution is replaced is the voltage holding time. And the effect on the battery capacity was verified. As a measure for adjusting the pH inside the negative electrode 22 to be alkaline after the completion of the first power generation, the influence of the type of buffer solution of the fuel solution added at the time of replacement was examined.

B-1. Composition of Bio-Battery The composition of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the NADGDH-ACNQ-based bio-battery 1 was produced in the same manner as in Comparative Example 2 and in the same manner as in Reference Example 1.

B-2. Measurement condition
As a measure to adjust the pH inside the negative electrode 2 to alkaline after the end of the first power generation, we tried to adjust the pH inside the negative electrode 22 with the fuel solution, changed the type of buffer solution of the fuel solution added the second time, and verified the effect. did. Then, in the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was generated for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 0.9 M CHES pH 9.5,
2.4 M glucose / 0.7 M CAPS pH 10.0,
2.4 M glucose / 1 M imidazole pH 8.7,
2.4 M glucose / 1 M imidazole pH 10.8,
1.5 M glucose / 2.5 M phosphate pH 10.6, or
1.5 M glucose / 1 M imidazole pH 8.7 / 1.5 M NaOH

C. Results Results are shown in FIGS. 15 and 16. 15 and 16 show the voltage holding time (seconds) on the horizontal axis and the voltage (V) on the vertical axis, and FIG. 15 shows that the second fuel solution was 2.4 M glucose / 0.9 M CHES pH 9.5, 2.4 M. Results are shown for glucose / 0.7 M CAPS pH 10.0, 2.4 M glucose / 1 M imidazole pH 8.7, or 2.4 M glucose / 1 M imidazole pH 10.8, and 1.5 M glucose / 2.5 M phosphate pH 10.6, FIG. , 1.5 M glucose / 1 M imidazole pH 8.7 / 1.5 M NaOH results are shown. From FIG. 15, the 1 M imidazole buffer had a recovery effect on the battery capacity at pH 8.7 (1.0 mWh (1.8 mAh)) and pH 10.8 (1.0 mWh (1.8 mAh)) during the second power generation. The effect was small. On the other hand, CHES (0.3 mWh (0.5 mAh)), CAPS (0.3 mWh (0.5 mAh)), and phosphoric acid (0.3 mWh (0.5 mAh)) did not show a recovery effect on battery capacity. From FIG. 16, it was found that with 1 M imidazole buffer pH 8.7 / 1.5 M NaOH (2.5 mWh (4.0 mAh)), the same battery capacity as that of the first power generation can be obtained in the second power generation.

Example 4. Verification of the effect of high-concentration buffer solution and its pH in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, since it was found in Examples 2 to 3 that the battery capacity can be maintained high by adjusting the pH in the negative electrode 22 with the fuel solution, the buffer solution concentration in the fuel solution added when the fuel solution is replaced is a voltage. The effects on the holding time and battery capacity were examined. As a measure for adjusting the pH in the negative electrode 22 to be alkaline after the completion of the first power generation, the concentration of the buffer solution in the fuel solution added at the time of replacement was adjusted to a high concentration, and the effect of the pH was also verified.

B. Method B-1. Configuration of Biobattery 1 The configuration of the biobattery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the biobattery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement condition
As a measure to adjust the pH inside the negative electrode 22 to alkaline after the end of the first power generation, the buffer solution in the fuel solution is made high concentration (1.5 M glucose / 2.5 M imidazole), and the high concentration buffer solution is used for the voltage holding time and battery capacity. We examined the effect on pH and how pH affects it. In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 1.5 M glucose / 2.5 M imidazole pH 7.0,
1.5 M glucose / 2.5 M imidazole pH 8.7 or
1.5 M glucose / 2.5 M imidazole pH 10.8

C. Results The results are shown in FIG. In FIG. 17, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, a further significant improvement in battery capacity was obtained by setting the pH of the buffer solution of the fuel solution for the second time to 10.8. The battery capacity of the second power generation at pH 10.8 was 3.5 mWh (5.6 mAh). When this is calculated in the same manner as in Comparative Example 1, it can be calculated that 89% (1.3 M) of glucose is dehydrogenated by the same calculation based on the glucose contained in the second fuel. However, it should also be considered that the residual amount of glucose contained in the first fuel is added.

Example 5. Verification of the effect of buffer concentration in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, since it was found in Examples 2 to 4 that the battery capacity can be maintained high by adjusting the pH in the negative electrode 22 with the fuel solution, the buffer solution concentration in the fuel solution added at the time of fuel solution replacement holds the voltage. The effect on time and battery capacity was examined. As a measure for adjusting the pH in the negative electrode 22 to be alkaline after the completion of the first power generation, the influence of the buffer solution concentration was verified.

B. Method B-1. Configuration of Biobattery 1 The configuration of the biobattery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the biobattery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement condition
As a measure to adjust the pH in the negative electrode 22 to alkaline after the end of the first power generation, when the buffer solution in the fuel solution is set to a high pH (0.5 M glucose / imidazole pH 10.8), what is the concentration of the buffer solution? I verified whether it would affect it. In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 0.5 M glucose / 0.5 M imidazole pH 10.8
0.5 M glucose / 1 M imidazole pH 10.8,
0.5 M glucose / 2.5 M imidazole pH 10.8
0.5 M glucose / 3 M imidazole pH 10.8, or
0.5 M glucose / 4.2 M imidazole pH 10.8

C. Results The results are shown in FIG. In FIG. 18, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, there was an effect of improving the battery capacity in the case of imidazole 2.5 M as compared with the case of the fuel solution having an imidazole concentration of 1.0 M, but a slight decrease occurred at 3 M or more. From this, it was found that the buffer solution concentration had an optimum range.

Example 6. Verification of the effect of buffer concentration and pH in the fuel solution added when replacing the fuel solution (PQQGDH-mPMS system)
A. Outline In this example, in the NADGDH-ACNQ system, it was found in Examples 2 to 5 that the battery capacity can be maintained high by adjusting the pH in the negative electrode 22 and the buffer solution concentration with the fuel solution. Therefore, the fuel in the PQQGDH-mPMS system The effects of the buffer concentration and pH in the fuel solution added at the time of solution replacement on the voltage holding time and battery capacity were examined. Specifically, in the PQQGDH-mPMS system, as a measure to adjust the pH in the negative electrode 22 after the end of the first power generation to alkaline, the effect of increasing the pH and concentration of the buffer solution in the fuel solution is verified. did

B. Method B-1. Configuration of Biobattery 1 The configuration of biobattery 1 is the PQQGDH-mPMS system described in Comparative Example 1, and the biobattery 1 was produced in the same manner as in Comparative Example 1.

B-2. Measurement condition
In the PQQGDH-mPMS system, as a measure for adjusting the pH in the negative electrode 22 after the completion of the first power generation to be alkaline, the effect of increasing the pH and concentration of the buffer solution in the fuel solution to high was verified. In the same manner as in Comparative Example 1, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
First fuel solution: 1 M glucose / 1 M imidazole pH 8.0
Second fuel solution: 1 M glucose / 1 M imidazole pH 8.0,
1 M glucose / 2.5 M imidazole pH 8.0 or
1 M glucose / 2.5 M imidazole pH 10.8

C. Results The results are shown in FIG. In FIG. 19, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, the battery capacity of the second power generation was 0.9 mWh (2.2 mAh) for 1 M glucose / 1 M imidazole pH 8.0, 1 M glucose / 2.5 M imidazole pH 8.0, and 1 M glucose / 2.5 M imidazole pH 10.8, respectively. ), 1.1 mWh (2.7 mAh), 1.2 mWh (2.9 mAh). From this, it was confirmed that the higher the pH and concentration of the buffer solution in the fuel solution, the better the battery capacity.

Comparative example 4. Verification of behavior when low-concentration fuel solution (NADGDH-ACNQ system)
A. Outline In this comparative example, the behavior of the decrease in battery capacity confirmed in Comparative Example 2 when the fuel concentration is low was verified.

B. Method B-1. Configuration of Biobattery 1 The configuration of the biobattery 1 is the NADGDH-ACNQ system described in Comparative Example 2, and the biobattery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement conditions The same experiment as in Comparative Example 2 was carried out with the fuel concentration reduced from 2.4 M to 1.0 M or 0.5 M. Similar to Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows.
Both 1st and 2nd fuel solutions: 0.5 M glucose / 1 M imidazole pH 8.7 or
Both 1st and 2nd fuel solutions: 1 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 20, the voltage holding time (seconds) is shown on the horizontal axis, and the voltage (V) is shown on the vertical axis. As a result, in the case of the low-concentration fuel solution, the decrease in battery capacity (Wh) in the second power generation was not noticeable. It is understood that when the fuel concentration in the fuel solution is the same as or lower than the buffer concentration, the battery capacity hardly decreases, and even if an organic acid is generated, the enzyme activity on the negative side does not decrease due to pH fluctuation. it can.

Example 7. Verification of the effects of fuel concentration and buffer concentration in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, based on the result that the buffer solution concentration was found to have an optimum range in Example 5, the influence of the relationship between the fuel concentration in the fuel solution and the buffer solution concentration on the battery capacity was verified.

B. Method B-1. Configuration of Biobattery 1 The configuration of the biobattery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the biobattery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement conditions In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is summarized in Tables 3 and 4 below.

The results are shown in Tables 3 and 4 below. Table 3 shows the average value and variation of the battery capacity due to the first power generation. Table 4 shows the battery capacity (mWh) in the second power generation by the combination of each fuel concentration and each buffer concentration in the second fuel solution. As a result, it was found that in order to maintain the battery capacity high, it is necessary to increase the buffer concentration as the fuel concentration in the fuel solution increases.

Example 8. Verification of the effect of buffer pH on the negative electrode when buffer is impregnated (NADGDH-ACNQ system)
A. Outline In this embodiment, it was found that the battery capacity can be made the same in the second power generation by simply impregnating the negative electrode 22 with the buffer solution after the first power generation in the first embodiment. Therefore, the influence of the pH of the buffer solution impregnated on the negative electrode 22 after the first power generation on the voltage holding time and the battery capacity was verified.

B. Method B-1. Composition of Bio-Battery The composition of the bio-battery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the bio-battery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement condition
Regarding the pH of the buffer solution impregnated in the negative electrode 22 after the first power generation, the effect on the battery capacity was verified under the three conditions of pH 7.0, pH 8.7, and pH 10.8. In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the negative electrode 22 was impregnated with 1 M imidazole pH 7.0, pH 8.7, or pH 10.8, and the battery cell 1a was reassembled. Subsequently, the fuel solution was replaced with a second fuel solution, and a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The case where the negative electrode 22 was not impregnated with the buffer solution after the first power generation was also examined in the same manner. The composition of the fuel solution is as follows.
1st fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7
Second fuel solution: 2.4 M glucose / 1 M imidazole pH 8.7

C. Results The results are shown in FIG. In FIG. 21, the voltage holding time is shown on the horizontal axis (seconds) and the voltage (V) is shown on the vertical axis. As a result, the average battery capacity from the first power generation is 2.5 mWh (3.7 mAh), while the battery capacity from the second power generation is 0.7 mWh (2.0 mAh), which is less than 1/3 without buffer impregnation. Decreased to. On the other hand, in the impregnation at pH 7.0, the battery capacity due to the second power generation is 1.1 mWh (2.0 mWh), 1.3 mWh (2.3 mAh) at pH 8.7, 1.8 mWh (3.1 mAh) at pH 10.8, and pH 7.0 or higher. It was found that the pH can be adjusted by impregnating the negative electrode 22 with the buffer solution, and the battery capacity is improved.

Example 9. Verification of the effects of fuel concentration and buffer concentration in the fuel solution added when replacing the fuel solution (NADGDH-ACNQ system)
A. Outline In this example, when the fuel concentration in the fuel solution is the same as or lower than the buffer concentration in Comparative Example 4, the battery capacity hardly decreases, and even if an organic acid is generated, it is on the negative side due to pH fluctuation. Since it was confirmed that the activity of the enzyme did not decrease, the effect of the relationship between the fuel concentration and the buffer concentration on the voltage holding time and the battery capacity was examined in more detail.

B. Method B-1. Configuration of Biobattery 1 The configuration of the biobattery 1 is the NADGDH-ACNQ system used in Comparative Example 2, and the biobattery 1 was produced in the same manner as in Comparative Example 2.

B-2. Measurement condition
As a measure to adjust the pH inside the negative electrode 22 to alkaline after the first power generation, try adjusting the pH inside the negative electrode 22 with a fuel solution, and add it to the first and second times to verify the relationship between the fuel concentration and the buffer solution concentration. The buffer solution concentration was 0.125 M, 0.25 M, 0.5 M, and 1 M with respect to 0.5 M of glucose. In the same manner as in Comparative Example 2, the first fuel solution was added, and the first constant current load (2.5 mA / cm ² ) was used to generate electricity for the battery cell 1a having an electrode area of 1 cm ² . When the voltage reached 0.1 V, the fuel solution was replaced with a second fuel solution. Subsequently, a second constant current load (2.5 mA / cm ² ) was applied to the battery cell 1a having an electrode area of 1 cm ² . The composition of the fuel solution is as follows, and the first and second fuel solutions used had the same imidazole concentration.
First fuel solution: 0.5 M glucose / 0.125 M imidazole pH 8.7,
0.5 M glucose / 0.25 M imidazole pH 8.7,
0.5 M glucose / 0.5 M imidazole pH 8.7,
0.5 M glucose / 1 M imidazole pH 8.7,
Second fuel solution: 0.5 M glucose / 0.125 M imidazole pH 8.7,
0.5 M glucose / 0.25 M imidazole pH 8.7,
0.5 M glucose / 0.5 M imidazole pH 8.7,
0.5 M glucose / 1 M imidazole pH 8.7,

C. Results The results are shown in FIG. In FIG. 22, the voltage holding time is shown on the horizontal axis (seconds) and the voltage (V) is shown on the vertical axis. As a result, the average battery capacity of the first power generation is 0.9 mWh (1.3 mAh), while the battery capacity of the second power generation is 0.5 Wh (0.7 mAh) at a buffer concentration of 0.125 M and 0.6 at 0.25 M. Wh (1.0 mAh), 0.5 M is 0.8 Wh (1.3 mAh), 1 M is 0.8 Wh (1.2 mAh), and if the buffer concentration is lower than the fuel concentration, the pH cannot be adjusted, and the second time. It was found that the battery capacity due to the power generation of It can be understood that it is preferable to adjust the buffer solution to a concentration of 0.5 times or more that of the fuel solution for effective pH adjustment.

The present invention relates to a biobattery and can be used in all fields in which a biobattery is required, particularly in industrial fields such as electronics, medical care, food, and the environment. Specifically, it can be applied to a power source of a portable device such as a desktop calculator or a small electronic device such as an implantable device such as a cardiac pacemaker.

Biobattery 1
Battery cell 1a
Positive electrode 21
Negative electrode 22
Septum 3

Claims (7)

A biobattery equipped with a negative electrode using an enzyme that oxidizes fuel to produce an organic acid as an electrode catalyst.
A pH adjusting unit for adjusting the pH fluctuation in the negative electrode due to the organic acid generated by the oxidation of the fuel by the negative electrode is provided.
The pH adjuster
(I) A replacement fuel solution containing the fuel and a buffer solution which is an imidazole compound having a molar concentration of 0.5 times or more and 6.0 times or less of the fuel and adjusted to be alkaline is supplied to the negative electrode and used. Fuel solution exchange part to replace the finished fuel solution, and
(Ii) Negative electrode impregnation in which the negative electrode is impregnated with a buffer solution containing a buffer solution which is an imidazole compound having a molar concentration of 0.5 times or more and 6.0 times or less of the fuel supplied to the negative electrode and which is adjusted to be alkaline. A biobattery composed of at least one selected from the parts. The biobattery according to claim 1, wherein the replacement fuel solution supplied to the negative electrode by the fuel solution exchange unit contains a buffer solution component of 0.75 M or more. The biobattery according to claim 1 or 2, wherein the buffer solution in which the negative electrode is impregnated by the negative electrode impregnated portion contains a buffer solution component of 1 M or more. The biobattery according to any one of claims 1 to 3, wherein the alkalinity is in the range of pH 8 to 11. The biobattery according to any one of claims 1 to 4 , wherein the buffer solution component has a buffering capacity adjusted by a strong basic compound. The biobattery according to any one of claims 1 to 5 , wherein the pH adjusting unit is configured to operate repeatedly when the fuel solution is replaced. The biobattery according to any one of claims 1 to 6 , wherein the enzyme is nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, or pyrroloquinoline quinone-dependent dehydrogenase.

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