JP5730668B2 - Alcohol production method and apparatus using microorganisms - Google Patents

Description

  The present invention relates to an alcohol production method and apparatus using microorganisms. More specifically, the present invention relates to an alcohol production method and apparatus suitable for producing alcohol using a microorganism belonging to the genus Clostridium that performs acetone-butanol-ethanol fermentation (ABE fermentation).

  Various researches have been conducted for a long time on substance production methods using microorganisms, and representative examples thereof include alcohol production methods using alcohol fermentation by Saccharomyces yeasts or the like. In recent years, as a method for producing butanol, which is expected as a next-generation biofuel, alcohol (butanol, ethanol) using acetone, butanol, ethanol fermentation (ABE fermentation) of microorganisms of the genus Clostridium (Clostridium) is used. Production methods are attracting attention.

  As a specific metabolic pathway of ABE fermentation, FIG. 22 shows the metabolic pathway of ABE fermentation of Clostridium acetobutylicum. In ABE fermentation, saccharides such as glucose are consumed, and organic acids such as acetic acid and butyric acid are mainly produced in the logarithmic growth phase (organic acid production phase). Butanol, acetone and ethanol are mainly produced in the stationary growth phase. It is produced at a ratio of 6: 3: 1 (solvent production phase). That is, butanol and ethanol can be produced simultaneously from saccharides or the like as alcohol, and butanol can be produced predominantly (see, for example, Non-Patent Document 1).

D.T.JONES. 1981. Solvent Production and Morphological changes in Clostridium acetobutylicum. Applied and Environmental Microbiology

  However, the alcohol production method utilizing ABE fermentation of microorganisms belonging to the genus Clostridium has a low alcohol production amount (yield) relative to the organic substances such as saccharides that have been added, and an improvement thereof has been desired.

Then , this invention provides the method and apparatus which can improve the production amount (yield) of alcohol significantly compared with the past, when producing alcohol using the microorganism of the genus Clostridium which performs ABE fermentation. Objective.

  In order to solve this problem, the inventors of the present application have conducted extensive research. As a result, when alcohol is produced using Clostridium acetobutylicum, which is a microorganism belonging to the genus Clostridium that performs ABE fermentation, It was found that by adding viologen, although the growth of microorganisms is inhibited, the amount of alcohol produced with respect to the substrate such as saccharide added can be improved as compared with the case where methylviologen is not added. In addition, methyl viologen added to the culture solution was reduced during the culture process. From this finding, the inventors of the present application added methyl viologen to the culture solution, so that methyl viologen withdraws electrons from the metabolic pathway of ABE fermentation, and as a result, methyl viologen is reduced and alcohol productivity is improved. Estimated.

  The inventors of the present application conducted further studies based on the above findings in order to further increase the productivity of alcohol. As a result, alcohol productivity is improved by immersing the electrode in the culture medium, controlling the potential of the electrode within a certain range, and culturing Clostridium acetobutylicum while electrically oxidizing the reduced methylviologen, In particular, they have found that butanol productivity is greatly improved.

The present inventors have, based on the above findings, is not limited to Clostridium acetobutylicum, the microorganisms in general Clostridium performing ABE fermentation, fermentation pathway electrically oxidizing the electronic medium material capable of extracting electrons from metabolic pathway It has been found that the possibility of significantly improving alcohol productivity can be obtained by culturing while continuing to extract electrons from the present invention, and various studies have been made to complete the present invention.

  The alcohol production method of the present invention is a method of producing alcohol from an organic substance using an alcohol-producing microorganism that produces alcohol as a metabolite in fermentation, and an electronic medium substance is added to a culture solution containing the alcohol-producing microorganism and the organic substance, The electronic medium substance is a substance that can be reduced by alcohol-producing microorganisms, and the alcohol-producing microorganisms are cultured while oxidizing by applying an electric potential to an electrode immersed in a culture solution of a reduced substance of the electronic medium substance that is reduced by the alcohol-producing microorganisms. I try to produce alcohol.

  Here, in the alcohol production method of the present invention, it is preferable that the electrolyte solution is brought into contact with the culture solution via an ion exchange membrane, and the electrode paired with the electrode immersed in the culture solution is immersed in the electrolyte solution.

  The alcohol production method of the present invention in which an electrolyte solution is brought into contact with a culture solution via an ion exchange membrane, and an electrode paired with the electrode immersed in the culture solution is immersed in the electrolyte solution, specifically, by the following alcohol production apparatus Can be implemented. That is, the alcohol production apparatus of the present invention is an apparatus for producing alcohol from an organic substance using an alcohol-producing microorganism that produces alcohol as a metabolite in fermentation, having a counter electrode tank and a sealed structure partitioned by an ion exchange membrane. A culture vessel, a culture solution contained in the culture vessel, an electrolyte solution contained in a counter electrode vessel, a working electrode immersed in the culture solution, a counter electrode immersed in the electrolytic solution, a working electrode and a counter electrode And an electric medium substance capable of reducing alcohol-producing microorganisms, organic substances, and alcohol-producing microorganisms in a culture solution. In addition, the reduced form of the electron medium substance produced by reduction by the alcohol-producing microorganism is oxidized by applying a potential difference between the working electrode and the counter electrode with a constant potential setting device. By culturing a reluctant alcohol-producing microorganism it is assumed that the production of alcohol.

  The alcohol production apparatus of the present invention is an apparatus for producing alcohol from an organic substance using an alcohol-producing microorganism that produces alcohol as a metabolite in fermentation, and can be accommodated in a sealed container as a culture tank and in the container A small container with a closed structure as a counter electrode tank, a culture solution stored in the culture tank, an electrolyte solution stored in the counter electrode tank, a working electrode immersed in the culture solution, and an electrolyte solution A constant potential setting device in which the counter electrode, the working electrode and the counter electrode are connected, and an alcohol recovery means for recovering alcohol produced in the culture tank, wherein the small container is at least at a position in contact with the culture solution. It is assumed that a part of it is equipped with an ion exchange membrane, and the culture medium contains alcohol-producing microorganisms, organic substances, and an electronic medium substance that can be reduced by alcohol-producing microorganisms, and is returned by alcohol-producing microorganisms. The reduced form of electronic medium occurring substance is being assumed that the produced while oxidized to give a potential difference culturing the alcohol-producing microorganism in an alcohol between the working electrode and the counter electrode at a constant potential setting device.

  In the alcohol production method of the present invention, it is preferable that an electrode paired with an electrode immersed in the culture solution via an ion exchange membrane is brought into contact with the culture solution.

  The alcohol production method of the present invention in which an electrode paired with an electrode immersed in a culture solution is brought into contact with the culture solution via an ion exchange membrane can be specifically carried out by the following alcohol production apparatus. That is, an apparatus for producing alcohol from an organic substance using an alcohol-producing microorganism that produces alcohol as a metabolite in fermentation, a container provided with at least a part of an ion exchange membrane, a culture solution contained in the container, and a culture A working electrode immersed in the liquid, a counter electrode in contact with at least a part of the outer surface of the container of the ion exchange membrane, a constant potential setting device in which the working electrode and the counter electrode are connected, and An electronic medium having an alcohol recovery means for recovering the produced alcohol, wherein the ion exchange membrane is provided at a position in contact with the culture solution in the container, and the alcohol-producing microorganism, the organic substance, and the alcohol-producing microorganism can be reduced in the culture solution. The reductant of the electron medium material that is produced by reduction by the alcohol-producing microorganism is contained between the working electrode and the counter electrode with a constant potential setting device. While oxidizing giving by culturing alcohol-producing microorganism is intended to produce the alcohol.

  Here, in the alcohol production method and apparatus of the present invention, the alcohol-producing microorganism is preferably a Clostridium microorganism that performs acetone-butanol-ethanol fermentation (ABE fermentation).

  In the alcohol production method and apparatus of the present invention, the electronic medium substance is at least one of a viologen and a viologen derivative, the ion exchange membrane is an anion exchange membrane, and the alcohol producing microorganism is acetone / butanol / ethanol fermentation (ABE). Preferably, the microorganism of the genus Clostridium is cultured at a high density by setting the potential of the electrode (working electrode) to +0.3 V to +0.9 V on the basis of the silver / silver chloride electrode potential. In this case, the productivity of butanol produced by high-density culture of Clostridium microorganisms is significantly improved, and the productivity of ethanol is also improved.

  Furthermore, in the alcohol production method and apparatus of the present invention, at least one of viologen and a viologen derivative is used as the electronic medium material, an anion exchange membrane is used as the ion exchange membrane, and acetone / butanol / ethanol fermentation (ABE) is used as the alcohol producing microorganism. Fermentation) Clostridium microorganisms are used, and the Clostridium microorganisms are included in the culture solution at the cell density before entering the logarithmic growth phase. The potential of the electrode (working electrode) is based on the potential of the silver / silver chloride electrode. After shifting the growth stage of the microorganism of the genus Clostridium to +0.3 V to +1.2 V to the stationary phase, the potential of the electrode (working electrode) is set to +0.3 V to +0.9 V on the basis of the silver / silver chloride electrode potential reference. It is preferable to culture the microorganisms at high density. In this case, microorganisms belonging to the genus Clostridium can be transferred to the stationary phase at an early stage to increase the density, and the accumulation of organic acid is suppressed, so that a state suitable for alcohol production is formed. Then, by performing high-density culture with the potential of the electrode (working electrode) being +0.3 V to +0.9 V, butanol productivity is remarkably improved and ethanol productivity is also improved.

  Here, in the alcohol production method and apparatus of the present invention, at least one of viologen and a viologen derivative is used as the electronic medium substance, an anion exchange membrane is used as the ion exchange membrane, and acetone, butanol, ethanol fermentation ( A microorganism of the genus Clostridium performing ABE fermentation) is included in the culture solution at the cell density before entering the logarithmic growth phase, and the potential of the electrode (working electrode) is based on the silver / silver chloride electrode potential standard. After moving the growth stage of the microorganism belonging to the genus Clostridium to +0.9 V to +1.2 V in the stationary phase, the potential of the electrode (working electrode) is set to +0.3 V to +0.6 V on the basis of the silver / silver chloride electrode potential reference. It is particularly preferred to culture the microorganisms of the genus at high density. In this case, microorganisms belonging to the genus Clostridium can be transferred to the stationary phase earlier to increase the density, the final cell density can be increased, and the accumulation of organic acids is suppressed, making it suitable for alcohol production. A state is formed. Then, by performing high-density culture with the potential of the electrode (working electrode) being +0.3 V to +0.6 V, butanol productivity is significantly improved and ethanol productivity is also improved.

  Incidentally, since Clostridium microorganisms are obligately anaerobic bacteria, there are reports that growth is inhibited in an oxidizing atmosphere (+0.04 V or more), and that a reducing environment is suitable for growth and alcohol (butanol) production. (Mutharasan, GRaR, Altered electron flow in a reducing environment in Clostridium acetobutylicum Biotechnology Letters 1988. 10 (2): p. 129-132). On the other hand, in the present invention, an oxidizing potential is applied to form an oxidizing atmosphere in order to electrically oxidize the reductant of the electronic medium substance. Thus far, no example has been reported that shows that alcohol (butanol) production has been promoted under an oxidizing atmosphere. In addition, it has not been reported at all that the control to the oxidation potential suppresses the accumulation of organic acids during the growth process and forms a state suitable for alcohol (butanol) production, and the early transition to the stationary phase occurs. . Therefore, the present invention is based on a novel finding that has not existed before.

According to the alcohol production method and the alcohol production apparatus of the present invention, when alcohol is produced using a microorganism of the genus Clostridium that performs ABE fermentation, the production amount (yield) of alcohol with respect to the input organic matter is greatly improved compared to the conventional method. In particular, the production amount (yield) of butanol, which is expected to increase as a next-generation biofuel, can be significantly improved as compared with the prior art. Therefore, it is possible to increase the conversion rate of the input organic substance into alcohol, particularly the conversion rate into butanol, and reduce the cost for alcohol production.

It is a figure which shows the concept of the alcohol production method of this invention. It is sectional drawing which shows an example of the alcohol production apparatus concerning 1st Embodiment A. It is sectional drawing which shows an example of the alcohol production apparatus concerning 1st Embodiment B. It is sectional drawing which shows an example of the alcohol production apparatus concerning 1st Embodiment C. It is sectional drawing which shows an example of the alcohol production apparatus concerning 1st Embodiment D. It is sectional drawing which shows an example of the alcohol production apparatus concerning 2nd embodiment. It is sectional drawing which shows an example of the other structure of the alcohol production apparatus for enforcing the alcohol production method of this invention. It is a figure which shows the result of having examined the influence on the proliferation of the Clostridium bacteria by addition of various electronic media (Example 1). It is a figure which shows the time-dependent change of glucose concentration about the influence on the metabolism of the Clostridium bacterium by addition of an electronic medium (Example 1). It is a figure which shows the acetic acid concentration of a culture solution after completion | finish of culture | cultivation, butyric acid concentration, ethanol concentration, and butanol concentration about the influence on the metabolism of Clostridium bacteria by addition of an electronic medium (Example 1). (Example 1) which is a figure which shows the amount of hydrogen and the amount of carbon dioxide of the vapor phase part of a vial container after completion | finish of culture | cultivation about the influence on the metabolism of Clostridium bacteria by addition of an electronic medium. FIG. 10 is a conceptual diagram of an apparatus used in Example 4. It is a conceptual diagram of the apparatus used in Example 2. It is a figure which shows the oxidation-reduction characteristic of methyl viologen (MV). (Example 2) which is a figure which shows the time-dependent change of glucose concentration about the influence on metabolism of the Clostridium bacterium by addition of an electronic medium and electric potential application. (Example 2) which is a figure which shows the time-dependent change of pH about the influence on the metabolism of the Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 2) which is a figure which shows the time-dependent change of a butyric acid density | concentration about the influence on the metabolism of the Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 2) which is a figure which shows the time-dependent change of an acetic acid density | concentration about the influence on the metabolism of the Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 2) which is a figure which shows the time-dependent change of a butanol density | concentration about the influence on the metabolism of the Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 2) which is a figure which shows the time-dependent change of ethanol concentration about the influence on metabolism of the Clostridium bacterium by addition of an electronic medium and electric potential application. It is a figure which shows the result examined about the influence on the proliferation of the Clostridium bacterium by addition of an electronic medium and electric potential application (Example 3). It is a figure which shows the metabolic pathway of ABE fermentation of Clostridium acetobutylicum (Clostridium acetobutylicum). (Example 4) which is a figure which shows the result examined about the influence on the proliferation of the Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 4) which is a figure which shows the time-dependent change of glucose concentration about the influence on the proliferation and metabolism of Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 4) which is a figure which shows the time-dependent change of an acetic acid density | concentration about the influence on the proliferation and metabolism of Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 4) which is a figure which shows the time-dependent change of a butyric acid density | concentration about the influence on the proliferation and metabolism of Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 4) which is a figure which shows the time-dependent change of a butanol density | concentration about the influence on the proliferation and metabolism of Clostridium bacteria by addition of an electronic medium and electric potential application. (Example 4) which is a figure which shows the amount of hydrogen and the amount of carbon dioxide which were collect | recovered by the gas pack about the influence on the proliferation and metabolism of Clostridium bacteria by addition of an electronic medium and electric potential application. It is a figure which shows the MV density | concentration dependence of butanol production (Example 5). FIG. 4 is a diagram showing changes in natural potential of a culture solution in the culture test of Example 4. FIG. 6 is a graph showing changes in current value in the culture test of Example 4. FIG. 4 is a diagram showing changes in natural potential of a culture solution in the culture test of Example 2. FIG. 4 is a diagram showing changes in current value in the culture test of Example 2.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

  In FIG. 1, an example of embodiment of the alcohol production method of this invention is shown notionally. The alcohol production method of the present invention is a method for producing alcohol from an organic substance using an alcohol producing microorganism 2 that produces alcohol as a metabolite in fermentation, wherein an electronic medium substance is added to a culture solution containing the alcohol producing microorganism 2 and the organic substance. The electron medium substance is a substance that can be reduced by the alcohol-producing microorganism 2, and a potential is applied to the electrode 9 (working electrode 9) in which a reduced form of the electron medium substance generated by reduction by the alcohol-producing microorganism 2 is immersed in the culture solution. The alcohol-producing microorganism 2 is cultured while being oxidized to produce alcohol.

  Examples of the alcohol-producing microorganism 2 include various yeasts such as Saccharomyces that perform alcohol fermentation, Clostridium microorganisms that perform ABE fermentation, etc. In particular, it is preferable to use Clostridium microorganisms such as Clostridium acetobutylicum that perform ABE fermentation. However, it is not particularly limited as long as it is an alcohol-producing microorganism that produces alcohol as a metabolite in fermentation. The microorganism is not limited to a natural product, and may be an artificial product to which an artificial operation such as gene recombination is added.

  The cell density in the culture solution of the alcohol-producing microorganism 2 is preferably set to a cell density in the stationary growth phase or close to it. In the alcohol production method of the present invention, the amount of alcohol produced per cell of the alcohol-producing microorganism 2 can be improved. Therefore, by adding the maximum number of bacteria that can be cultured in the culture tank or a number close to that to the culture solution, the effect of improving the alcohol production amount according to the present invention can be maximized. In addition, when a Clostridium microorganism that performs ABE fermentation is used, solvent (acetone, butanol, ethanol) is produced in the stationary growth phase in ABE fermentation. Therefore, the cell density of the culture solution is set to the stationary growth phase. By setting the corresponding cell density, alcohol can be produced by the microorganism immediately after the start of culture (high-density culture).

  However, it is not an essential condition that the cell density in the culture solution of the alcohol-producing microorganism 2 is equal to or close to the cell density in the stationary stationary phase, and the initial cell density is less than the stationary stationary phase, for example, logarithm. You may make it implement the alcohol production method of this invention as less than a growth phase. According to the alcohol production method of the present invention, the growth of alcohol-producing microorganisms can be promoted, so that the growth stage can be shifted to the stationary phase at an early stage. In addition, by suppressing the production of organic acids and other by-products accumulated at the growth stage, the effect of improving the alcohol production is maximized, combined with the effect of forming a state suitable for alcohol production. Can be made. For example, when a Clostridium microorganism that performs ABE fermentation is used, the cell density of the culture solution corresponds to the growth stage before the transition to the stationary phase, for example, the growth stage before the transition to the logarithmic growth phase. Even if the cell density is equivalent, the growth stage reaches the stationary phase, which is the solvent generation phase, at an early stage, and the organic acid accumulation is suppressed and a state suitable for alcohol production is formed. Alcohol production can be carried out by maximizing the productivity of alcohol of microorganisms. Such an embodiment is also included in the alcohol production method of the present invention.

  The organic substance is a substrate (alcohol production source material) for obtaining alcohol which is a metabolite in fermentation of the alcohol-producing microorganism 2, and is appropriately determined according to the alcohol-producing microorganism 2 to be used. For example, when using a microorganism belonging to the genus Clostridium that performs ABE fermentation, it is possible to use monosaccharides such as glucose and polysaccharides such as starch, cellulose, and xylan as substrates, as well as paper, wheat straw, and rice straw. Various types of biomass waste such as plant fiber such as food waste, agricultural waste, shochu distillation waste liquid, mixture of surplus sludge (activated sludge) garbage, starch waste liquid, waste palm fiber, etc. can be widely used as substrates it can. By using such biomass waste, the cost for alcohol production can be further reduced.

  The amount of organic matter added to the culture solution is not particularly limited. For example, when using a Clostridium microorganism that performs ABE fermentation, glucose may be added in an amount of 10 to 100 mM, preferably about 50 mM, but the amount is not necessarily limited. The organic substance may be added before the start of alcohol production (before the start of alcohol-producing microorganisms) or during the production of alcohol. In either case, the organic substance is consumed by the alcohol-producing microorganism 2 to produce alcohol, and the conversion amount of the organic substance into alcohol can be increased.

  The electronic medium material is not particularly limited as long as the alcohol-producing microorganism 2 can be reduced without inhibiting the growth of the alcohol-producing microorganism 2, and is appropriately determined according to the alcohol-producing microorganism 2 to be used. Even if it inhibits the growth of the alcohol-producing microorganism 2, it may be used as long as the alcohol-producing microorganism 2 can be reduced.

  For example, when a Clostridium microorganism that performs ABE fermentation is used, it is preferable to use at least one of a viologen and a viologen derivative as the electron medium substance. As the viologen derivative, methyl viologen, ethyl viologen, benzyl viologen and the like can be used, and it is preferable to use methyl viologen. By using these substances, these substances are reduced during the culture process and the growth of Clostridium microorganisms is inhibited, but the alcohol productivity is significantly improved over the adverse effects of the growth inhibition. An effect can be produced. Further, growth inhibition (delay of transition to the logarithmic growth phase) can be canceled by controlling the oxidation potential in the growth process described later. Therefore, by controlling to the oxidation potential in the growth process, only the remarkable improvement effect of alcohol productivity by using these substances as the electron medium substance can be exhibited.

  In addition, it has been reported that the productivity of ethanol using glucose as a substrate is changed by adding an electronic medium such as neutral red and menadione for yeasts of the genus Saccharomyces that perform alcoholic fermentation (produce ethanol). (Analytica Chimica Acta 597 (2007) 67-74, Zhao et al, The defferent behaviors if three oxidative mediators in probing the redox activities of the yeast Saccharomyces cerevisiae). Therefore, when using yeast of the genus Saccharomyces that performs alcoholic fermentation (production of ethanol), neutral red, menadione, or the like can be used as the electronic medium material.

  The optimum amount of the electron medium substance added to the culture medium varies appropriately depending on the type of the electron medium substance, the fermentation reaction efficiency of the microorganism, the cell density, and the like. It may be about 0.5 to 10 mM or 1 to 10 mM, preferably 0.5 to 5 mM, more preferably 0.5 to 2 mM, and even more preferably several mM. If the concentration of the electronic medium substance is too low, it is difficult to obtain the effect of improving alcohol productivity. On the other hand, if the concentration of the electronic medium substance is too high, the growth is excessively inhibited depending on the type of the electronic medium substance, and it becomes difficult to obtain the effect of improving the alcohol productivity. For example, when the electron medium substance is a viologen derivative and the concentration thereof is 0.5 to 2 mM, particularly 1 to 2 mM, the microorganisms belonging to the genus Clostridium can exhibit an extremely remarkable alcohol (butanol) productivity improvement effect.

  The electron medium substance is preferably added to the culture solution in the form of an oxidant or a form containing an oxidant (that is, a form in which an oxidant and a reductant coexist). Alternatively, it may be added to the culture solution in the form of a reduced form. That is, even when added in the form of a reductant, the reductant is oxidized one after another by oxidizing the electrode 9 immersed in the culture solution, and electrons from the metabolic pathway in the fermentation of alcohol-producing microorganisms. Can be pulled out.

  The composition of the culture solution is appropriately determined according to the alcohol-producing microorganism to be used, but a culture solution that does not substantially contain a substance that can cause an oxidation or reduction reaction electrically, such as the above-mentioned electronic medium substance. It is preferred to use. For example, when a Clostridium microorganism that performs ABE fermentation is used, a thioglycolate medium, a TYA medium, or the like may be used. For example, when it is intended to grow a Clostridium microorganism that performs ABE fermentation and shift it to a stationary phase at an early stage, it is preferable to use a thioglycolate medium, and a Clostridium microorganism that performs ABE fermentation. Is preferably used in a stationary phase or a cell density close to the stationary phase, and a TYA medium containing acetic acid is preferably used, but is not limited thereto.

  The electronic medium substance in the culture solution has an action of extracting electrons from the metabolic pathway in the fermentation of the alcohol-producing microorganism 2. Therefore, the oxidant of the electron medium substance is completely converted (reduced) into a reduced form in the process of culturing the alcohol-producing microorganism 2 (alcohol production process) and depleted. In the present invention, the oxidant of the electron medium substance is converted (reduced) in the process of culturing the alcohol-producing microorganism 2 (alcohol production process) by applying a potential to the electrode 9 (working electrode 9) immersed in the culture solution. The reduced form of the generated electronic medium substance is oxidized and regenerated to an oxidized form, thereby continuously supplying the oxidized form of the electronic medium substance to the alcohol-producing microorganism 2. Thereby, in the process of culturing the alcohol-producing microorganism 2 (alcohol production process), the alcohol productivity is improved by continuously extracting electrons from the metabolic pathway in the fermentation of the alcohol-producing microorganism 2 and affecting the metabolic reaction in some way. be able to.

  As the electrode 9, an electrode capable of oxidizing a reduced form of the electron medium substance is appropriately used. Specifically, a carbon electrode such as a carbon plate or glassy carbon, a platinum electrode, or the like can be used, but is not limited thereto.

  The potential applied to the electrode 9 can vary depending on the type of alcohol-producing microorganism, the cell density (growth stage) of the alcohol-producing microorganism, the type of the electronic medium material, the material of the electrode 9, etc. The minimum potential and the maximum potential of the electrode 9 can be derived by the following procedure. That is, an electron medium substance is added to a culture solution containing alcohol-producing microorganisms at a cell density corresponding to the stationary phase of growth, and the electrode 9 is brought into contact with each other at various potentials within a range where the reductant of the electron medium substance is oxidized. Perform high-density culture test. In addition, as a comparative test, a high-density culture test is performed without adding an electron medium substance to the culture solution and without contacting the electrode 9 (without applying a potential). Then, the range of the potential at which the alcohol production increases is more apparent than in the comparative test. The minimum value in this potential range is the minimum potential for increasing the alcohol production amount, and the maximum value is the maximum potential for increasing the alcohol production amount. In addition, various potentials within a range in which the reductant of the electron medium substance is oxidized by adding the electron medium substance to the culture solution containing the alcohol-producing microorganisms at the cell density before entering the logarithmic growth phase and contacting the electrode 9 Perform the culture test at In addition, as a comparative test, the culture test is performed without adding an electron medium substance to the culture solution and without contacting the electrode 9 (without applying a potential). Then, the range of potentials at which growth promotion (early transition to the stationary phase due to early transition to the logarithmic growth phase) occurs becomes clearer than the comparative test. The minimum value in this potential range is the minimum potential at which the growth of the alcohol-producing microorganism is promoted, and the maximum value is the maximum potential at which the growth of the alcohol-producing microorganism is promoted.

  According to the experiments of the present inventors, for microorganisms belonging to the genus Clostridium that perform ABE fermentation, a viologen derivative is used as the electronic medium material, the microorganisms belonging to the genus Clostridium are included in the culture solution at a stationary cell density, and carbon is used as the electrode 9. When an electrode is used, an effect of increasing ethanol production is obtained by setting it to +0.3 V or more, and a significant effect of increasing butanol production and an increase in ethanol production by setting it to +0.6 V or more. It has been confirmed that an effect can be obtained. From this, it is considered that there is a minimum potential between +0.3 V and +0.6 V where the butanol production amount is higher than that in the comparative test. Therefore, in this case, the potential of the electrode 9 may be +0.3 V or higher, preferably higher than +0.3 V, more preferably +0.6 V or higher.

  For clostridium microorganisms that perform ABE fermentation, a viologen derivative is used as an electronic medium substance, and the clostridium microorganisms are included in the culture solution at a cell density before entering the logarithmic growth phase. When it is used, it has been confirmed that the effect of promoting the growth of microorganisms belonging to the genus Clostridium is observed at +0.3 V to +1.2 V (the early transition effect to the stationary phase due to the early transition to the logarithmic growth phase). Specifically, the growth inhibitory effect (delay of transition to the logarithmic growth phase) by using a viologen derivative as an electronic medium substance is cultured while controlling the potential of the electrode 9 to + 0.3V to + 1.2V. The period of transition to the logarithmic growth phase can be canceled, and is comparable to the case of culturing without energization without using a viologen derivative as an electronic medium substance. In particular, by setting it to +0.9 V to +1.2 V, it is possible to obtain a growth promoting effect that is superior to that of culturing without energization without using a viologen derivative, and further culturing without energization without using a viologen derivative. The final cell density can be set to the same level as that of the case. It should also be noted that by using a viologen derivative as the electron medium material and culturing while controlling the potential of the electrode between +0.3 V and +1.2 V, the generation of organic acids accumulated during the growth process can be suppressed. is there. This creates a very suitable state for the production of alcohol (butanol). In other words, a precursor state capable of improving alcohol productivity in the stationary phase (solvent generation phase) is formed. As described above, in the growth process of Clostridium microorganisms, the potential of the electrode 9 is preferably +0.3 V to +1.2 V, more preferably +0.6 V to +1.2 V, and +0.9 V to +1. More preferably, it is 2V.

  And after controlling the electric potential of the electrode 9 to promote the growth of microorganisms belonging to the genus Clostridium and shifting to the stationary phase, the butanol production can be increased by setting to +0.3 to +0.9 V, and +0.3 V It has been confirmed that the amount of butanol produced can be increased particularly by setting it to ~ + 0.6V. Therefore, after controlling the potential of the electrode 9 to promote the growth of Clostridium microorganisms and shifting to the stationary phase, the potential of the electrode 9 may be +0.3 to +0.9 V, and +0.3 V to It is preferably + 0.6V. In addition, when the above-described oxidation potential is applied from a state in which the cell density is lower than that in the logarithmic growth phase to a high density state through the growth process, in the stationary phase (solvent generation phase), even at +0.3 V A significant improvement in butanol productivity can be obtained. That is, it is possible to obtain the merit that the effect of improving butanol productivity can be obtained while the input electric energy is reduced by controlling to a lower potential.

  In the stationary phase (solvent generation phase), when the potential of the electrode 9 was +1.2 V, the butanol productivity tended to be slightly lower than when no potential was applied. On the other hand, since the productivity of butanol can be improved at +0.9 V as compared with the case where no potential is applied, the productivity of butanol is higher than when no potential is applied between +0.9 V and less than +1.2 V. It is considered that there may be a potential that becomes an improved boundary. However, in alcohol production, it is advantageous in terms of energy to lower the applied potential, and more significant butanol is obtained when the potential is lower than +0.9 V (for example, +0.6 V). Considering that the productivity improvement effect is achieved, it can be said that the potential of the electrode 9 in the stationary phase is desirably +0.9 V or less.

  In summary, for the microorganisms of the genus Clostridium that perform ABE fermentation, when the viologen derivative is used as an electronic medium material and alcohol production is performed by culturing the microorganisms of the genus Clostridium at high density, the potential of the electrode 9 is + 0.3V to + 0.9V, but from the viewpoint of significantly improving butanol productivity, it is preferably more than 0.3V to + 0.9V, more preferably + 0.6V to + 0.9V. Is more preferable, and it can be said that + 0.6V is more preferable.

  In addition, for clostridium microorganisms that perform ABE fermentation, a viologen derivative is used as an electronic medium material, and the microorganisms of the genus clostridium are included in the culture solution at a cell density before transitioning to the logarithmic growth phase and shifted to the stationary phase. In this case, the potential of the electrode 9 may be +0.3 to +1.2 V, preferably +0.6 V to +1.2 V, and more preferably +0.9 to +1.2 V. . When alcohol production is performed by high-density culture after shifting to the stationary phase, the potential of the electrode 9 is preferably +0.3 V to +0.9 V, and is preferably +0.3 V to +0.6 V. Is more preferable. Most preferably, for clostridium microorganisms that undergo ABE fermentation, a viologen derivative is used as the electron medium material, and the clostridium microorganisms are contained in the culture solution at the cell density before transitioning to the logarithmic growth phase and shifted to the stationary phase. When the alcohol is produced by high-density culture after shifting to the stationary phase, the potential of the electrode 9 should be + 0.3V to + 0.6V. It is. In this case, all of the effects of the early growth of the logarithmic phase, the improvement of the final cell density and the suppression of the accumulation of organic acids are achieved, and a state that is extremely suitable for alcohol production is formed. Alcohol productivity of microorganisms of the genus is improved, and these effects are combined to produce an extremely remarkable alcohol productivity improvement effect.

  Therefore, the production of alcohol (butanol) can be carried out very efficiently by continuous culture such as fed-batch culture using Clostridium microorganisms.

  In addition, since the change of the minimum electric potential and the maximum electric potential by the material of the electrode 9 is thought to be slight, the remarkable increase effect of the butanol production amount in the present invention is also exhibited when an electrode other than the carbon electrode is used. it is conceivable that.

  The alcohol production method of the present invention is carried out using, for example, an alcohol production apparatus shown in FIGS. Hereinafter, the first embodiment of the alcohol production apparatus of the present invention will be described based on FIGS. 2 to 5, and the second embodiment of the alcohol production apparatus of the present invention will be described based on FIG. 6.

<First embodiment>
The alcohol production method according to the first embodiment is a method of producing alcohol from an organic substance 3 using an alcohol producing microorganism 2 that produces alcohol as a metabolite in fermentation, and a culture solution containing the alcohol producing microorganism 2 and the organic substance 3 Electrode medium material 5 is added to 4, and the electron medium material 5 is a substance that can be reduced by the alcohol-producing microorganism 2, and an electrode obtained by immersing the reductant of the electron medium substance 5 generated by reduction by the alcohol-producing microorganism 2 in the culture solution 4 9 (working electrode 9) is applied with an electric potential to oxidize the alcohol-producing microorganism 2 to produce alcohol. In the present embodiment, the electrolyte solution 4a is brought into contact with the culture solution 4 via the ion exchange membrane 6, and the electrode 10 (counter electrode 10) paired with the working electrode 9 is immersed in the electrolyte solution 4a.

  The alcohol production method according to the first embodiment is performed by, for example, the alcohol production apparatus 1 shown in FIGS. That is, the alcohol production apparatus 1 shown in FIGS. 2 to 5 has one of the two tanks partitioned by the ion exchange membrane 6 as a culture tank 7 and the other tank as a counter electrode tank 8. 7 contains the culture solution 4 and is immersed in the working electrode 9 and the reference electrode 11, and the counter electrode tank 8 is filled with the electrolyte solution 4 a and the counter electrode 10 is immersed in the working electrode 9 and the counter electrode 10. The reference electrode 11 is connected to the constant potential setting device 12. Then, the culture medium 4 contains the alcohol-producing microorganism 2, the organic substance 3, and the electronic medium substance 5 that can be reduced by the alcohol-producing microorganism, and the potential of the working electrode is controlled by the constant potential setting device 12 in a three-electrode system. The alcohol-producing microorganism 2 is cultured to produce alcohol while oxidizing the reduced form of the electron medium material 5 generated by reduction by the alcohol-producing microorganism 2.

  In this way, by controlling the potential of the working electrode 9 by the three-electrode method, the potential of the working electrode 9 can be strictly controlled to the set potential. Specifically, a potential difference between the working electrode 9 and the reference electrode 11 is measured by a constant potential setting device (potentiostat) 12, and the working electrode 9 and the counter electrode 10 are adjusted so that the potential difference reaches the set potential. A current is passed between them so that no current flows through the reference electrode 11 serving as a reference. The details of the potential control by the three-electrode method are described in, for example, pages 6 to 9 of Electrochemical Measurement Method (above), Technical Bulletin Publishing Co., Ltd., 1st edition 15 printing, published in June 2004. ing. However, when the potential of the working electrode 9 can be controlled only by the voltage between the working electrode 9 and the counter electrode 10, the three-electrode system may not be used.

  In addition, the ion exchange membrane 6 allows the microorganism 2, the electronic medium material 5, and the organic substance 3 contained in the culture solution 4 to remain in the culture solution 4 without passing through the counter electrode tank 8, and is included in the culture solution 4. Ions or ions contained in the electrolyte solution 4 a are transmitted to generate an ionic current, and a reduction reaction that complements the oxidation reaction that occurs at the working electrode 9 is generated at the counter electrode 10. Thereby, the reductant of the electronic medium material 5 is easily oxidized stably over a long period of time. As the ion exchange membrane 6, a material that does not transmit the electron medium material 5 (oxidant and reductant) is appropriately selected and used.

  Here, as described above, when alcohol production is performed using a Clostridium microorganism that performs ABE fermentation, it is preferable to use at least one of viologen and a viologen derivative as an electronic medium substance, In this case, it is preferable to use an anion exchange membrane as the ion exchange membrane 6. When a proton exchange membrane or a cation exchange membrane is used, viologen and viologen derivatives are adsorbed on the ion exchange membrane 6 and cannot oxidize on the working electrode 9, but when an anion exchange membrane is used. The viologen and the viologen derivative are not adsorbed on the ion exchange membrane 6, and the above functions required for the ion exchange membrane 6 are maintained.

  Depending on the type of the electron medium material 5, depending on the applied potential, the reduction reaction at the counter electrode 10 may not occur. Therefore, when the potential is applied within a range where the reduction reaction at the counter electrode 10 does not occur. May omit the ion exchange membrane 6. Even if a reduction reaction occurs at the counter electrode 10, the effect of the present invention can be obtained as long as an oxidation reaction on the working electrode 9 side continues to occur. Therefore, the installation of the ion exchange membrane 6 is not essential.

  Moreover, in the alcohol production apparatus 1 shown in FIGS. 2 to 5, the culture tank 7 has a sealed structure. Thereby, it is possible to prevent the alcohol produced in the culture tank 7 from volatilizing and diffusing outside the container. Moreover, there exists an advantage that it is easy to control the anaerobic atmosphere of the culture solution 4 by making the culture tank 7 into a sealed structure. That is, by making the culture tank 7 a sealed structure, if the free oxygen in the culture tank 7 is substantially eliminated before the culture is started, no entry of free oxygen into the culture tank 7 occurs. Fermentation proceeds satisfactorily while maintaining the anaerobic atmosphere in the culture tank 7. In particular, since microorganisms belonging to the genus Clostridium are microorganisms that perform ABE fermentation in an anaerobic environment, it is extremely advantageous to cause the microorganisms belonging to the genus Clostridium to perform ABE fermentation by making the culture tank 7 closed in this way. It becomes composition.

  Furthermore, in the alcohol production apparatus 1 shown in FIG. 2 to FIG. 5, the culture solution discharge that guides the culture solution 4 in the culture vessel 7 to the outside of the culture vessel 7 below the liquid level of the culture solution 4 in the culture vessel 7. A culture solution 4 containing alcohol is collected from the inside of the culture tank 7 by a culture solution recovery means 16 provided with a tube 16a and opening and closing the culture solution discharge tube 16a by a valve 16b. The alcohol contained in the culture solution 4 can be recovered by appropriate purification by distillation or the like. However, the method for collecting the culture solution is not limited to this method. For example, the culture tank 7 may be provided with an opening and closed with an elastic material such as synthetic rubber, and the culture solution 4 containing alcohol may be collected by inserting a syringe needle. Alternatively, it may be connected to a syringe at one end of a tube having both ends opened, and the other end may be immersed in the culture solution 4 to collect the culture solution 4 containing alcohol through the tube.

  In addition, in the alcohol production apparatus 1 shown in FIGS. 2 to 5, the culture tank 7 has a sealed structure, and the gas staying in the space (head space) above the liquid level of the culture liquid 4 in the culture tank 7 is stored in the culture tank 7. A gas recovery means 15 is provided which includes a gas exhaust pipe 15a leading to the outside of the gas exhaust pipe 15a, and the gas exhaust pipe 15a can be opened and closed by a valve 15b. This gas recovery means 15 recovers the volatile matter of alcohol that can stay in the head space. However, the volatile component of the alcohol staying in the head space may be guided to the outside of the culture tank 7 only by the gas discharge pipe 15a, or an opening is provided in the upper part of the culture tank 7, and synthetic rubber or the like (for example, silicone rubber) This elastic material may be used to close the opening, and the elastic material that closes the opening may be stabbed with a syringe needle to recover the volatile content of alcohol from the headspace.

  In the alcohol production apparatus of the present invention, the culture medium recovery means 16 and the gas recovery means 15 are combined to form an alcohol recovery means. However, when no alcohol stays in the head space, the gas recovery means 15 is omitted, Only the recovery means 16 may be alcohol recovery means. Conversely, the culture medium recovery means 16 may be omitted, and only the volatile content of the alcohol remaining in the head space may be recovered.

  In addition to the alcohol recovery means, means for adding and supplying substances to the culture solution 4 may be provided. Specifically, an openable and closable substance introduction tube that can add and supply substances to the culture solution 4 from the outside of the culture tank 7 may be provided. In this case, nutrient sources, neutralizing agents, substances necessary for substance production, and the like can be added to the culture solution 4 as necessary. Of course, the microorganism 2 can be supplied from the introduction tube, or the organic substance 3 can be introduced. Moreover, gas (nitrogen gas etc.) for making it anaerobic conditions can also be supplied. However, the means for adding and supplying the substance to the culture solution 4 is not necessarily provided, and the alcohol recovery means may be used in combination as the means for adding and supplying the substance to the culture solution 4. Further, as described above, the injection needle of the syringe may be inserted into the elastic material, and the substance may be added to and supplied to the culture solution 4. Further, a means for adding and supplying a substance to the electrolytic solution 4a may be provided.

  Moreover, in the alcohol production apparatus 1 shown in FIGS. 2-5, the counter electrode tank 8 is made into a sealed structure, and the gas which stays in the space (head space) above the liquid level of the electrolyte solution 4a of the counter electrode tank 8 is countered. A gas discharge pipe 17a that leads to the outside of the electrode tank 8 is provided, and the gas discharge means 17 that can be opened and closed by a valve 17b releases the gas generated from the counter electrode 10 without leaking to the culture tank 7. Like to do. However, when the amount of gas generated in the counter electrode tank 8 is small, the gas discharge means 17 may not be provided. Of course, the valve 17b may be omitted and gas may be constantly discharged from the gas discharge pipe 17a. Moreover, it is not essential that the counter electrode tank 8 has a sealed structure, and an open structure may be used as long as the sealing property of the culture tank 7 is not impaired.

  Hereinafter, the case where the alcohol production apparatus shown in FIG. 2 is used will be described as a first embodiment A, and the case where the alcohol production apparatus shown in FIG. 3 is used will be described as a first embodiment B, which is shown in FIG. A case where an alcohol production apparatus is used will be described as a first embodiment C, and a case where an alcohol production apparatus shown in FIG.

(First embodiment A)
In the alcohol production apparatus 1 shown in FIG. 2, the sealed container 20 is used as the culture tank 7, the sealed container 21 that can be accommodated in the container 20 is used as the counter electrode tank 8, and the small container 21 is at least partially ion-exchanged. The membrane 6 is provided, and the culture tank 7 is provided with alcohol recovery means (gas recovery means 15 and culture solution collection means 16).

  By accommodating the small container 21 in the container 20, the small container 21 is immersed in the culture solution 4 accommodated in the container 20, and the ion exchange membrane 6 provided in at least a part of the small container 21 is the culture solution 4. Contact with. In other words, the culture solution 4 comes into contact with the electrolyte solution 4 a through the ion exchange membrane 6.

  The sealed container 20 as the culture tank 7 is a container having a size capable of accommodating the small container 21 with the sealed structure as the counter electrode tank 8, and the shape is not particularly limited. Examples of the material of the container include, but are not limited to, glass, plastic, an insulating metal, concrete, and the like. Moreover, you may make it use the container which formed the gas impermeable membrane material in the bag shape by the heat seal etc. as the culture tank 7. FIG.

  The small container 21 having a sealed structure as the counter electrode tank 8 is a container of a size that can be accommodated in the container 20 as the culture tank 7, and includes the ion exchange membrane 6 at least partially. Here, the small container 21 may be a bag-like container formed entirely by the ion-exchange membrane 6, but only one side of the bag-like container may be constituted by the ion-exchange membrane 6, or a part of one surface may be further formed. You may make it comprise only the ion exchange membrane 6. FIG. When the ion exchange membrane 6 is partially used, other portions may be made of the same material as that of the container 20, or may be made of a membrane material other than the ion exchange membrane 6, such as a gas-impermeable membrane material. The gas generated in the small container 21 may be configured not to leak into the container 20.

  As the counter electrode 10, an electrode made of a material capable of causing a reduction reaction that complements the oxidation reaction in the working electrode 9, such as a carbon electrode, can be used, but is not limited thereto.

  According to the alcohol production apparatus 1 shown in FIG. 2, it is possible to recover the entire amount of alcohol produced in the culture tank 7 without leaking out of the container while increasing the amount of alcohol produced by the alcohol producing microorganism 2. it can.

(First embodiment B)
In the alcohol production apparatus 1 shown in FIG. 3, two tanks opened by partitioning a container 23 whose upper side is opened by an ion exchange membrane 6 are formed, and an upper open part of one tank as the culture tank 7 is formed. The gas impervious film or the gas impervious member 24 is used. Similarly, the upper open portion of the other tank as the counter electrode tank 8 is also closed by the gas impermeable film or the gas impermeable member 24. The culture tank 7 is provided with alcohol recovery means (gas recovery means 15 and culture fluid collection means 16). In the alcohol production apparatus 1 shown in FIG. 3, it is not necessary to completely partition the head space of the culture tank 7 and the head space of the counter electrode tank 8 as long as the container 23 is sealed.

  As the gas impermeable film or the gas impermeable member 24, those generally used in various fields can be appropriately used. For example, examples of the gas impermeable member include, but are not limited to, glass, plastic, an insulating metal, concrete, and the like. Further, as the gas impermeable membrane, for example, an ion exchange membrane 6 can be used, but is not limited thereto.

  According to the alcohol production apparatus 1 shown in FIG. 3, as with the alcohol production apparatus 1 shown in FIG. 2, the alcohol produced in the culture tank 7 is removed from the container while increasing the amount of alcohol produced by the alcohol production microorganism 2. The entire amount can be recovered without leakage.

(First embodiment C)
The alcohol production apparatus 1 shown in FIG. 4 is a U-shaped container in which two containers 25a and 25b each having an opening below the liquid level of the liquid to be accommodated are connected to each other through the opening via the ion exchange membrane 6. 25, one vessel 25a is used as a culture tank 7 with a sealed structure, and the other vessel 25b is also used as a counter electrode vessel 8. In this case, the culture solution 4 and the electrolyte solution 4a are in contact with each other through the ion exchange membrane 6, and the space above the liquid level of the culture solution 4 in the culture tank 7 and the liquid level of the electrolyte solution 4a in the counter electrode tank 8 are used. Also, the upper space is spaced apart by the U-shaped structure of the container 25 itself. According to the alcohol production apparatus 1 shown in FIG. 4, the alcohol produced in the culture tank 7 is removed from the container while increasing the amount of alcohol produced by the alcohol production microorganism 2 as in the alcohol production apparatus 1 shown in FIG. 2. The entire amount can be recovered without leakage.

(First embodiment D)
The alcohol production apparatus 1 shown in FIG. 5 has an H-shaped container in which two containers 26a and 26b each having an opening below the liquid level of the liquid to be accommodated are connected via the opening through the ion exchange membrane 6. 26 is formed, and one vessel 26 a is used as a culture tank 7 with a closed structure, and the other vessel 26 b is also used as a counter electrode vessel 8. Also in this case, the culture solution 4 and the electrolyte solution 4a are in contact with each other through the ion exchange membrane 6, and the space above the liquid level of the culture solution 4 in the culture vessel 7 and the solution of the electrolyte solution 4a in the counter electrode vessel 8 are also shown. The space above the surface is separated by the H-shaped structure of the container 26 itself. According to the alcohol production apparatus 1 shown in FIG. 5, the alcohol produced in the culture tank 7 is placed outside the container while increasing the amount of alcohol produced by the alcohol production microorganism 2 as in the alcohol production apparatus 1 shown in FIG. 2. The entire amount can be recovered without leakage.

<Second Embodiment>
The alcohol production method according to the second embodiment is a method of producing alcohol from an organic substance 3 using an alcohol producing microorganism 2 that produces alcohol as a metabolite in fermentation, and a culture solution containing the alcohol producing microorganism 2 and the organic substance 3 Electrode medium material 5 is added to 4, and the electron medium material 5 is a substance that can be reduced by the alcohol-producing microorganism 2, and an electrode obtained by immersing the reductant of the electron medium substance 5 generated by reduction by the alcohol-producing microorganism 2 in the culture solution 4 9 (working electrode 9) is applied with an electric potential to oxidize the alcohol-producing microorganism 2 to produce alcohol. In this embodiment, the counter electrode 9 is brought into contact with the culture solution 4 via the ion exchange membrane 6. That is, it differs from the alcohol production method in the second embodiment in that the counter electrode 10 is brought into direct contact with the ion exchange membrane 6 without using the electrolytic solution 4a. However, since the ionic current flows through the ion exchange membrane 6 between the working electrode 9 and the counter electrode 10 without using the electrolytic solution 4a as in the first embodiment, the alcohol according to the second embodiment. According to the production method, the same effect can be obtained by controlling the potential of the working electrode 9 as in the first embodiment.

  The alcohol production method according to the second embodiment is performed by, for example, an alcohol production apparatus shown in FIG. In the alcohol production apparatus 1 shown in FIG. 6, the working electrode 9 and the reference electrode 11 are arranged in a sealed container 50 having at least a part of the ion exchange membrane 6, and the counter electrode 10 is arranged outside the container 50, The culture solution 4 is accommodated in the container 50 and the working electrode 9 and the reference electrode 11 are immersed in the culture solution 4, and the ion exchange membrane 6 of the container 50 is at least a part when the culture solution 4 is accommodated in the container 50. Is provided at a position where it can come into contact with the ion exchange membrane 6, and the counter electrode 10 is arranged in contact with at least a part of the surface of the ion exchange membrane 6 opposite to the contact surface of the culture solution 4. . In the alcohol production apparatus 1 shown in FIG. 6, an opening 50 a is provided below the liquid level of the culture solution 4 in the container 50, the opening 50 a is closed with the ion exchange membrane 6, and ion exchange outside the container 50 is performed. It is assumed that the counter electrode 10 is disposed in contact with at least a part of the surface of the film 6. That is, in the alcohol production apparatus 1 shown in FIG. 6, the entire container 50 functions as the culture tank 7.

  Therefore, according to the alcohol production apparatus 1 shown in FIG. 6, since the container 50 has a sealed structure, the alcohol produced in the culture tank 7 volatilizes and diffuses outside the container as in the first embodiment. Can be prevented. In addition, there is an advantage that the anaerobic atmosphere of the culture solution 4 can be easily controlled.

  In the alcohol production apparatus 1 shown in FIG. 6, as in the first embodiment, the gas recovery means 15 and the culture solution recovery means 16 are provided as the alcohol recovery means. The method and the culture solution collecting method are not limited to those using these means. Further, either the gas recovery means 15 or the culture medium recovery means 16 may be omitted. Furthermore, as in the first embodiment, means for adding and supplying substances to the culture solution 4 may be provided.

  Hereinafter, the details of the alcohol production apparatus 1 shown in FIG. 6 will be described. However, configurations other than those described below are substantially the same as those in the first embodiment, and a description thereof will be omitted.

  The container 50 has a sealed structure including at least a part of the ion exchange membrane 6. Examples of the material of the container 50 include, but are not limited to, glass, plastic, metal subjected to insulation treatment, concrete, and the like. In FIG. 6, the opening 50 a provided below the liquid level of the culture solution 4 in the sealed container 50 is closed by the ion exchange membrane 6, but the form and structure of the container 50 are particularly limited. Not. For example, the entire container 50 may be a bag-shaped container formed of the ion exchange membrane 6, or only one surface of the bag-shaped container may be formed of the ion-exchange membrane 6, or a part of one surface may be ion-exchanged. You may make it comprise only with the film | membrane 6. FIG. When the ion exchange membrane 6 is partially used, the other portions may be made of the above-mentioned material such as glass, or other membrane materials other than the ion exchange membrane 6, such as the culture solution 4 and the components in the culture solution 4. You may comprise by the film | membrane material which does not permeate | transmit both. In short, the culture solution 4 contained in the container 50 may be a container having a structure that can come into contact with the ion exchange membrane 6 constituting at least a part of the container 50.

  The counter electrode 10 is brought into contact with at least a part of the surface opposite to the contact surface of the ion exchange membrane 6 with the culture solution 4. In the present embodiment, the counter electrode 10 is a plate-like carbon electrode, but the shape and material of the counter electrode 10 are not limited to this, and the shape is that the contact with the ion exchange membrane 6 is essential. The electrode may be made of a material capable of causing a reduction reaction that complements the exchange of electrons with respect to the oxidation reaction at the working electrode 9. In the present embodiment, the counter electrode 10 has a larger area than the ion exchange membrane 6 so that the entire ion exchange membrane 6 is completely covered with the counter electrode 10, and the ion exchange membrane 6 and the counter electrode 10 are covered. The counter electrode 10 may be brought into contact with at least a part of the surface opposite to the contact surface of the ion exchange membrane 6 with the culture solution 4, and the entire ion exchange membrane 6 is not necessarily in contact with the counter electrode. The ion exchange membrane 6 and the counter electrode 10 do not have to be in contact with each other so as to be completely covered with 10. However, by completely covering the entire ion exchange membrane 6 with the counter electrode 10, the counter electrode 10 can function as a protective material for the ion exchange membrane 6, and the ion transmission surface from the culture solution 4 increases. As a result, there is an advantage that the potential controllability of the culture solution 4 can be improved, which is preferable. As a method of completely covering the entire ion exchange membrane 6 with the counter electrode 10, for example, an ion exchange is applied to block the opening 50 a by applying an adhesive around the opening 50 a of the container 50 and bonding the counter electrode 10. The entire membrane 6 and the counter electrode 10 may be brought into contact with each other, or an ion exchange membrane formed by applying an adhesive around the opening 50a of the container 50 and applying it to at least a part of the surface of the counter electrode 10 6 may be adhered, while the opening 50a is closed with the ion exchange membrane 6, and the entire ion exchange membrane 6 closing the opening 50a and the counter electrode 10 may be brought into contact with each other. Examples of the agent for coating and forming the ion exchange membrane 6 include a Nafion dispersion, but are not limited thereto. Alternatively, a Nafion dispersion may be applied to the surface of the counter electrode 10 and the ion exchange membrane 6 may be attached before the Nafion dispersion is dried. In this case, the adhesion and contact properties of the ion exchange membrane 6 to the surface of the counter electrode 10 can be made sufficient.

  Here, the counter electrode 10 is preferably a porous body. In this case, the gas generated at the contact surface between the ion exchange membrane 6 and the counter electrode 10 can easily pass through the surface opposite to the contact surface. In addition, the counter electrode 10 is made a porous body, and the ion exchange membrane 6 is attached using a Nafion dispersion liquid, whereby the ion exchange membrane 6 and the counter electrode 10 are separated by the penetration of the Nafion dispersion liquid into the pores of the porous body. The contact area can be increased to facilitate the progress of the electrochemical reaction, which is preferable.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the gist of the present invention.

  For example, as shown in FIG. 7, the culture solution 4 and the electrolyte 4a are separated not by the ion exchange membrane 6 but by the impervious member 40 that does not allow any permeation of ions or microorganisms, or the culture vessel 7 and the counter electrode vessel 8 are separated. It may be formed in a separate container, and the culture solution 4 and the electrolyte 4a may be brought into contact (liquid junction) via the salt bridge 41 (agar or the like containing a saturated electrolyte solution such as KCl). Also in this case, the flow of ion current is allowed by the salt bridge 41 and the permeation of the electron medium material 5 to the counter electrode tank 8 can be prevented, so that the same effect as in the above embodiment can be obtained.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

  The electrode potentials in this example are all based on the silver / silver chloride electrode potential.

Example 1
The effects of electronic media materials on the growth of Clostridium microorganisms were investigated.

30 mL of a liquid thioglycolate medium was accommodated in a 100 mL glass vial, and Clostridium acetobutylicum NBCR13948 (Clostridium acetobutylicum NBCR13948) was added thereto so as to have an initial cell density of 1.0 × 10 ⁷ cells / mL. In the following description, Clostridium acetobutylicum NBCR13948 will be referred to as a Clostridium bacterium. The thioglycolate medium contains 27.8 mM (5 g / L) glucose. The composition of the thioglycolate medium used is shown below.
[Thioglycolate medium composition (g / L)]
L-cysteine: 0.5
・ Sodium chloride: 2.5
・ Glucose: 5.0
・ Yeast extract: 5.0
Casein peptone: 15.0
・ Thioglycolic acid: 0.5
・ PH 7.0

And the influence with respect to the proliferation of Clostridium bacteria when the following electronic media substance 2mM was added to the culture medium was examined.
・ Methylviologen (MV)
・ Anthraquinone-2,6-disulfonic acid disodium salt (AQDS)
・ Thionine ・ Fe (III) -EDTA
・ Neutral red ・ 2-hydroxy-1,4-naphthoquinone ・ 2,6-dichlorophenolindophenol

  The inside of the glass vial was replaced with nitrogen, and the inside of the glass vial was sealed to maintain a nitrogen atmosphere during the test period. Moreover, the test was implemented at 30 degreeC. As a comparative test, a similar test was performed without adding an electronic medium substance.

  The test results are shown in FIG. The cell density was calculated from the measured value obtained by separating the culture solution during the culturing process and using a spectrophotometer U-3010 (Hitachi) or microscopic observation. When AQDS or Fe (III) -EDTA was used as the electronic medium material, the same growth tendency was obtained as when no electronic medium material was added. In contrast, no growth was observed when neutral red, 2-hydroxy-1,4-naphthoquinone, or 2,6-dichlorophenolindophenol was used as the electronic medium material (data not shown). When MV was added as an electronic medium substance, the growth start time was greatly delayed, and the final cell density reached 37% when no electronic medium was added.

  In addition, when the test was carried out without adding the electronic medium substance, the medium that was light brown before the test turned yellow after the test as the cell density increased. On the other hand, when MV was added as an electronic medium substance and the test was performed, the medium that was light brown before the test changed to green after the test. Here, although MV is colorless in the oxidized form, but becomes blue in the reduced form, the change of the culture medium to green after the test is due to the blue and fungal cells caused by the reduced form of MV. It was thought that it was produced as a result of mixing yellow with the increase in density. From this, it was found that MV was reduced by Clostridium bacteria by culturing Clostridium bacteria by adding MV as an electron medium substance.

  Next, for the case where the electron medium substance was added and the case where the electron medium substance was not added, the glucose concentration of the medium during the test, the acetic acid concentration of the medium after the test, the butyric acid concentration, the ethanol concentration, and the butanol concentration The change with time was measured by liquid chromatography (Elite Lachrome, manufactured by Hitachi). The glucose concentration change is shown in FIG. 9, and the acetic acid concentration, butyric acid concentration, ethanol concentration, and butanol concentration are shown in FIG. The graph of FIG. 10 represents the acetic acid concentration, butyric acid concentration, ethanol concentration, and butanol concentration sequentially from the left. Moreover, Table 1 shows the sugar yield (%, mol-C / mol / C) of the metabolite under each condition.

  From the results shown in FIG. 9, it is understood that when AQDS, Fe (III) -EDTA is used as the electronic medium material, the same glucose consumption tendency as in the case where the electronic medium material is not added is observed. It was. On the other hand, when MV was used as the electronic medium substance, it was found that there was a large delay in the start of glucose consumption compared to the case where the electronic medium substance was not added.

  From the results shown in FIG. 10, when MV is used as the electronic medium material, the amount of acetic acid produced can be reduced to 0.5 times compared to the case where the electronic medium material is not added. It was found that the amount of butanol produced can be increased 1.5 times. Also, the amount of butyric acid produced was suppressed. In addition, since the final reached cell density was reduced by the addition of MV, the amount of butanol produced per cell was 4.6 times higher than when MV was not added. When AQDS or Fe (III) -EDTA was used as the electronic medium substance, there was no tendency for the butanol production to increase as compared with the case where no electronic medium substance was added.

  From the above results, in the alcohol production using Clostridium bacteria, by adding MV as an electron medium substance to the culture medium, compared with the case where no electron medium substance is added, it is a metabolite in ABE fermentation of Clostridium bacteria. It has been clarified that a certain butyric acid and acetic acid accumulation amount can be suppressed and a butanol production amount can be increased.

  In addition, after completion of the culture test, the gas in the gas phase portion in the vial was collected, and quantitative analysis of hydrogen and carbon dioxide was performed using a gas chromatography CR4900 (VARIAN) and a TCD detector. The results are shown in FIG. As a result of component analysis of the gas in the gas phase portion, the generated gas was mainly hydrogen and carbon dioxide. The amount of carbon dioxide generated did not change greatly depending on whether or not an electronic medium was added. On the other hand, the hydrogen generation amount tended to greatly decrease under the conditions where AQDS and MV were added as compared with the case where no hydrogen was added.

  Here, in the Clostridium bacteria used in this example, the reducing power (electrons) generated in the butanol production pathway is mainly used for hydrogen production in the growth phase, and the flow of electrons to this hydrogen production is suppressed. Can improve the productivity of butanol (Carolis J. Paredes, SWJ, Ryan S. Senger, Jacob R. Borden, Ryan Sillers, and Eleftherios T. Papoutsakis, Molecular Aspects of Butanol Fermentation. Bioenergy, 2008. ). From the three results of the decrease in cell density, the decrease in acetic acid and butyric acid yields, and the decrease in hydrogen production due to the addition of MV, the increase in butanol production under MV addition conditions was used for cell component synthesis and acetic acid / butyric acid synthesis. It was assumed that the carbon and electrons derived from glucose had shifted to butanol production. From the above, it has been clarified that MV has a growth inhibitory effect but can control metabolism based on adjustment of electron flow.

(Example 2)
In Example 1, it was considered that extraction of electrons from MV in the process of metabolism of Clostridium bacteria contributed to the improvement of alcohol (butanol) productivity. Then, it examined what kind of influence was exerted on the alcohol productivity of Clostridium bacteria by electrically oxidizing the reduced MV and regenerating and continuously supplying the depleted oxidized MV.

  FIG. 13 shows a sectional view of the experimental apparatus used in this example. Two 250 mL glass vials (manufactured by Duran) were connected via an anion exchange membrane (manufactured by Astom Co., Ltd., model No. Neocepta AMX) at the lower opening to form an H-shaped container 26. One of the glass vials was a culture tank 26a, and the other was a counter electrode tank 26b. The culture tank 26a and the counter electrode tank 26b are covered, and a silicone rubber stopper is provided on the upper surface of the cover so that the wiring connecting the electrodes (working electrode 9, counter electrode 10) and the constant potential setting device 12 is penetrated. The sealing inside the container was ensured. In addition, the culture tank 26a is provided with a culture solution collecting unit 52 for collecting the culture solution 4 so that the culture solution 4 can be appropriately collected during the test period.

In the culture tank 26a, 200 mL of TYA medium (natural potential +0.1 V) supplemented with 50 mM glucose and 2 mM MV was accommodated, and the working electrode 9 was immersed therein. The composition of the TYA medium was as follows.
[Composition of TYA medium (g / L)]
・ Glucose: 10.0
・ Tryptone: 6.0
-Yeast extract: 2.0
-Ammonium acetate: 3.0
-Potassium dihydrogen phosphate: 0.5
Magnesium sulfate heptahydrate: 0.3
・ Iron sulfate heptahydrate: 0.01
・ PH 7.0

  200 mL of electrolyte solution (NaCl solution: 0.58 g / L) was accommodated in the counter electrode tank 26b, and the counter electrode 10 was immersed therein.

  The wiring from the working electrode 9 in the culture tank 26a and the wiring from the counter electrode 9 in the counter electrode tank 26b were drawn to the outside of the container 26 through a silicone rubber plug and connected to the constant potential setting device 12, respectively. The reference electrode 11 (RE-1B, BAS Co., Ltd.) was inserted into the electrode insertion part 51 of the culture tank 26a and brought into contact with the culture solution 4, and the wiring was connected to the constant potential setting device 12. Thus, the potential of the working electrode 9 can be controlled strictly by connecting the working electrode 9, the counter electrode 10 and the reference electrode 11 to the three-electrode type constant potential setting device (potentiostat) 12.

  First, the working electrode 9 was a carbon electrode (manufactured by BAS), the counter electrode 10 was a platinum rod electrode (self-made), and the redox characteristics of MV were measured by cyclic voltammetry using the above experimental apparatus. The results are shown in FIG. From the results shown in FIG. 14, it was found that the reduced MV can be oxidized by controlling the potential of the working electrode to be larger than −0.6 V to the plus side.

Next, the working electrode 9 and the counter electrode 10 are used as carbon electrodes, and Clostridium acetobutylicum NBCR13948 in the culture solution 4 becomes 6.0 × 10 ⁸ cells / mL corresponding to the cell density in the stationary phase of growth. And a high density culture test was conducted. The culture conditions were as follows.
・ PH: 6.5
・ Culture temperature: 30 ℃
・ Culture period: 114 hours ・ Nitrogen atmosphere ・ Static bacteria test

The applied potential conditions were as follows.
・ No energization (no MV)
・ No energization (with MV)
・ + 0.6V (with MV)
・ + 0.3V (with MV)
-0.3V (with MV)
-0.5V (with MV)

  The time course of glucose concentration, pH, butyric acid concentration, acetic acid concentration, butanol concentration, and ethanol concentration of the medium during the high-density culture test was measured by liquid chromatography (Elite Lachrom, manufactured by Hitachi). FIG. 15 shows the glucose concentration change, FIG. 16 shows the pH change, FIG. 17 shows the butyric acid concentration change, FIG. 18 shows the acetic acid concentration change, FIG. 19 shows the butanol concentration change, and FIG. Shown in 15 to 20, the ◆ indicates the experimental result without energization (without MV), the ■ indicates the experimental result without energization (with MV), and the ▲ indicates the experimental result with +0.6 V (with MV). , X indicates the experimental result of +0.3 V (with MV), * indicates the experimental result of -0.3 V (with MV), and ● indicates the experimental result of -0.5 V (with MV) .

  From the results shown in FIG. 15 and FIG. 16, there was almost no difference in glucose consumption rate and pH change of Clostridium bacteria due to the difference in applied potential conditions.

  From the results shown in FIG. 17, it became clear that by setting the applied potential condition to +0.6 V (with MV), the amount of butyric acid accumulated can be suppressed compared to other applied potential conditions.

  From the results shown in FIG. 18, it is clear that the acetic acid accumulation amount can be suppressed when MV is added, as compared with the case where MV is not added, regardless of whether or not potential is applied.

  From the results shown in FIG. 19, it was found that by setting the applied potential condition to +0.6 V (with MV), the butanol production amount was significantly improved as compared with other applied potential conditions. Specifically, it became clear that the butanol production amount was 7 times as compared with no energization (no MV), and the butanol production amount was doubled when compared with other applied potential conditions.

  From the results shown in FIG. 20, it was clarified that by setting the applied potential condition to +0.6 V (with MV), the ethanol production amount is improved as compared with other applied potential conditions. Specifically, it has been clarified that the ethanol production amount is doubled compared to the case where no current is supplied (no MV), and the ethanol production amount is significantly increased as compared with other applied potential conditions. As for the ethanol production amount, it has been clarified that even when the applied potential condition is +0.3 V (with MV), the ethanol production amount can be improved slightly.

  From the above results, in alcohol production using Clostridium bacteria, by controlling the potential of the working electrode to +0.3 V and electrically oxidizing MVs reduced to Clostridium bacteria during the culture process, Clostridium The ethanol productivity of the genus bacterium can be improved, and the potential of the working electrode is controlled to +0.6 V, and the MV reduced to the clostridium bacterium is electrically oxidized during the culturing process. In addition to this improvement, it has become clear that a significant improvement effect of butanol productivity can be obtained.

  From the above results, there is a minimum potential at which butanol productivity starts to improve between + 0.3V and + 0.6V, and by setting it to + 0.6V, a remarkable butanol productivity improvement effect can be obtained with certainty. Furthermore, it was found that an effect of improving ethanol productivity can be obtained.

(Example 3)
The initial cell density of Clostridium acetobutylicum NBCR13948 was 1.0 × 10 ⁷ cells / mL, a culture test was performed under the same conditions as in Example 2, and the growth of Clostridium bacteria under applied potential conditions was examined. .

The applied potential conditions were as follows.
・ No energization (with MV)
・ + 0.6V (with MV)
・ + 0.3V (with MV)
・ 0.0V (with MV)
-0.3V (with MV)
-0.6V (with MV)

  The results are shown in FIG. At -0.6 V (with MV), Clostridium bacteria did not grow. In contrast, at +0.6 V (with MV) and +0.3 V (with MV), the growth rate tends to increase as compared with other applied potential conditions, and the effect is particularly +0.6 V (with MV). ).

  From this result, the applied potential is set to +0.3 V or higher, more preferably +0.6 V or higher, and culture is performed while oxidizing MV reduced by Clostridium bacteria, thereby increasing the growth rate of Clostridium bacteria, It became clear that the speed | rate which transfers a growth stage to the stationary phase which is a solvent (acetone, butanol, ethanol) production phase can be improved. That is, it has been clarified that it is possible to quickly and smoothly start up an alcohol production tank using Clostridium bacteria by increasing the speed of shifting the growth stage of Clostridium bacteria to the stationary phase.

  Therefore, by setting the initial cell density of Clostridium bacteria to less than the logarithmic growth phase and carrying out the alcohol production method of the present invention, the cell density of Clostridium bacteria can be rapidly increased to the cell density in the stationary phase of growth. It was revealed that alcohol production can be performed efficiently by shifting to the solvent production phase.

Example 4
A culture test similar to the culture test of Example 3 was conducted for further detailed examination.

  Experiments were performed using the electroculture apparatus shown in FIG. The container 20 as the culture tank 7 was a 250 mL glass vial (manufactured by Duran). The container 20 has a lid 30. A silicone rubber stopper was provided on the upper surface 30a of the lid 30 to ensure the hermeticity of the container 20 when wires, electrodes, and tubes were passed. In addition, a silicone rubber stopper is provided so that the injection needle can be pierced, and a hole generated by inserting the injection needle is closed when the injection needle is removed.

The small container 21 as the counter electrode tank 8 was cut into a semi-cylindrical shape by cutting a 15 mL plastic tube vertically and bonding an anion exchange membrane 6 (manufactured by Astom Co., Ltd., model number Neocepta AMX) to the cut surface. . The area of the anion exchange membrane 6 was 12 cm ² . The small container 21 contained the electrolyte solution 4a and the counter electrode 10 and was immersed in the electrolyte solution 4a. Then, the upper portion of the small container 21 was filled with a silicon adhesive and sealed.

  The small container 21 and the working electrode 9 were immersed in the culture solution 4, and the wiring between the working electrode 9 and the counter electrode 10 was drawn out of the container 20 through a silicone rubber wire provided on the lid 30. The silver / silver chloride reference electrode 11 (RE-1B, BAS Co., Ltd.) was inserted from below the two openings provided on the side surface of the container 20 and brought into contact with the culture solution 4. The working electrode 9, the counter electrode 10, and the reference electrode 11 were connected to a three-electrode constant potential setting device (potentiostat) 12 so that the potential of the working electrode 9 could be strictly controlled. During the culture period, the gas generated from the culture tank 7 was collected in an aluminum sampling bag 30 (manufactured by GL Sciences, trade name: aluminum bag, 1 L) installed on the upper part of the container 20.

  Both the working electrode 9 and the counter electrode 10 were carbon plates (7.5 cm × 2.5 cm).

  The culture solution 4 was prepared by adding 2 mM of MV to the same thioglycolate medium used in Example 1.

  The electrolyte solution 4a was the same as the thioglycolate medium used in Example 1 (MV not added).

After the potential of the culture solution 4 is stabilized at the set potential, 1% (2.5 mL) of a culture solution (cell density: 6.0 × 10 ⁸ cells / mL) containing Clostridium acetobutylicum NBCR13948 in a stationary phase ) Inoculated and verified the effect of energization in the culture process. Moreover, the test without the electricity supply by MV addition and non-addition was implemented as a control test.

  In the experiment, the culture solution temperature was set to 30 ° C., and the inside of the container 20 was set to a nitrogen atmosphere.

The applied potential conditions were as follows. Note that MV was added under potential application conditions.
・ No energization (MV added, non-added)
・ + 1.2V
・ + 0.9V
・ + 0.6V
・ + 0.3V
-0.6V
-0.8V

  The growth results of the bacterial cells are shown in FIG. When potentials of -0.6 V and -0.8 V were applied, no growth of Clostridium bacteria was observed. On the other hand, when a potential of +0.3 V to +1.2 V, which is an oxidation potential, was applied, the growth of Clostridium bacteria started after about 20 hours, and the growth started after 51.5 hours. Compared with the addition, the time required for the start of growth could be greatly shortened. From this, it became clear that the growth inhibited by the addition of MV recovered to the same extent as when MV was not added (no energization).

  In addition, when a high potential is applied (+ 0.9V, + 1.2V), the growth start time is slightly earlier than that when no energization is performed (no MV added), and the final cell density is also not energized. It was twice as much as (MV addition), and the same level as no energization (no MV addition).

  In addition, when the potentials of +0.6 V and +0.3 V were applied, the growth start time recovered to the same level as when there was no energization (no MV added), but the final cell density was not energized (MV added) It was suppressed to the same extent.

  Next, FIG. 24 shows the change in glucose concentration of the culture solution during the culture test, FIG. 25 shows the change in acetic acid concentration, FIG. 26 shows the change in butyric acid concentration, and FIG. 27 shows the change in butanol concentration.

  The glucose concentration decreased in synchronization with the growth, and the total consumption did not differ depending on the culture conditions (FIG. 24).

  As main metabolites of glucose, acetic acid, butyric acid, and butanol were detected under each condition (FIGS. 25, 26, and 27). Without energization (no MV added), metabolites concentrated on acetic acid and butyric acid, and the amount of butanol and ethanol accumulated was less than 1 mM. On the other hand, without energization (MV addition), although the accumulation start time was delayed, acetic acid and butyric acid accumulation was suppressed, and an improvement in butanol production was confirmed.

  In addition, when an oxidation potential is applied in the presence of MV, the period until the start of accumulation of metabolites may be significantly shortened as the growth (growth) is accelerated, compared to no energization (addition of MV). It became clear. Specifically, in the absence of electricity (addition of MV), it took about 60 hours to start production of butanol, whereas in the oxidation potential (+0.3 V to +1.2 V), production started after about 20 hours. It was revealed that the time required for butanol production can be shortened to 1/3 (FIG. 27). The final butanol concentration reached the maximum when +0.6 V and +0.3 V were applied, and increased to about 7 mM, which is about 7 times that of no energization (no MV added).

  Next, Table 2 shows the result of calculating the sugar yield from the culture solution after the test was completed.

  From the results shown in Table 2, it was confirmed that the yield of butanol with respect to sugar was improved in the case of no energization (addition of MV) compared to the case of no energization (no addition of MV). Further, by applying potentials of +0.3 V, +0.6 V, and +0.9 V, a further improvement effect of the sugar yield of butanol can be obtained, and the effect is particularly remarkable at +0.3 V and +0.6 V. It became clear that there was.

  After completion of the culture test, gas was collected from the gas bag, and quantitative analysis of hydrogen and carbon dioxide was performed using gas chromatography CR4900 (Varian) and a TCD detector. The results are shown in FIG. Although there was no significant difference in the amount of carbon dioxide generated under each condition, the amount of hydrogen generated decreased when no current was applied (adding MV) and when +0.6 V and +0.3 V were applied, as in Example 1. It was estimated that the addition of MV suppressed the flow of electrons to hydrogen production. On the other hand, when + 0.9V and + 1.2V were applied, the same amount of hydrogen production as when MV was not added was shown. From this, under these conditions, it was considered that the flow of electrons to the hydrogen production path was the same as when MV was not added, despite the presence of oxidized MV. Therefore, it was estimated that the electron flow pattern differs depending not only on the presence or absence of MV but also on the range of potential applied.

  As described above, the inhibitory effect on cell growth caused by the addition of MV (MV reductant) was suppressed, and the butanol productivity was further improved from both sides of butanol production start time and production amount.

  In addition, when + 0.6V and + 0.3V were applied, acetic acid / butyric acid accumulation was suppressed and the final cell density was suppressed lower than when + 1.2V and + 0.9V were applied.・ It was estimated that carbon and energy that were not used for butyric acid production and bacterial cell components shifted to butanol production.

(Example 5)
The same test as in Example 1 was performed at the following concentrations of MV, and the butanol concentration of the culture broth after completion of the culture was measured to examine the optimum amount of MV added.
・ No addition ・ 0.1 mM
・ 0.5 mM
1 mM
2 mM
5 mM
・ 10 mM

  The results are shown in FIG. It became clear that butanol concentration increased more than MV concentration 0.5 mM-10 mM rather than MV addition, and the effect was remarkable in the range of 0.5 mM-2 mM, especially the range of 1-2 mM. On the other hand, at the MV concentration of 0.1 mM, the effect of improving butanol concentration by adding MV was not obtained. Moreover, even if the MV concentration was higher than 10 mM, the effect of improving butanol concentration was not obtained.

  From the above results, the MV concentration may be 0.5 mM to 10 mM, preferably 0.5 mM to 5 mM, more preferably 0.5 to 2 mM, and 1 to 2 mM. It became clear that it was more preferable to do.

(Example 6)
Various considerations were made on Example 2 and Example 4 based on potential changes and current values during the culture test.

  FIG. 30 shows the change in the natural potential of the culture solution when no current is applied in the culture test of Example 4. The natural potential of the culture solution at the time of non-energization gradually decreases from −0.2 V at the start of culture regardless of the addition or non-addition of MV, and finally becomes almost stable at around −0.5 V, and then It showed a gradual increase at the end of culture.

  In Example 4, a change in medium color to blue, which is the reduced color of MV, was observed after about 30 hours with proliferation when no current was applied (adding MV). When energized, there is a difference in the color of the medium for each potential. At -0.6V and -0.8V, the medium appears blue due to electrical reduction from the beginning of the culture, and at + 0.6V and + 0.3V. A change to blue was observed with the growth of Clostridium bacteria. On the other hand, when a high potential (+1.2 V, +0.9 V) was applied to the redox potential of MV, no change in the medium color to blue was observed throughout the culture process.

  Next, the change in current value in the culture test of Example 4 is shown in FIG. When a potential was applied, an increase in current value was observed in a form synchronized with proliferation, and although there were large and small differences depending on the potential, a similar trend of current value variation was shown. When + 1.2V and + 0.9V were applied, a large amount of current flowed, the MV oxidation rate on the electrode was large, and the oxidation rate by the electrode exceeded the rate of MV reduction by Clostridium bacteria. It was estimated that it was in an almost oxidized state throughout the culture period. It is considered that a certain MV oxidant and reductant coexist at the time of electroculture at +0.6 V and +0.3 V due to the difference in oxidation rate. Here, reduced MV is highly reactive and exhibits cytotoxicity such as damage to DNA (Monk, PMS, The Viologens: Physicochemical Properties, Synthesis and Applications of the Salts of 4,4'-Bipyridine, 1999 .) It was considered that the growth was inhibited at −0.6 V and −0.8 V in which a significant amount of reduced MV was present from the beginning of the culture.

Next, the efficiency of energy (butanol) production increase with respect to electric energy input was calculated. The electric energy (ΔW _in ) input in each energization condition from the integration of the current values that flowed through the electroculture period of Example 4 was 1659.6 J (+1.2 V), 526.9 J (+0.9 V), 63. They were 3J (+ 0.6V) and 33.0J (+ 0.3V). As a result of comparing the amount of butanol produced at the end of the culture between non-energized (added MV) and each energized condition, the energy (ΔW _butanol ) calculated from the variation of the butanol produced was −735.8J (+1.2 V), 791.8J (+ 0.9V), 1234.6J (+ 0.6V), 1364.6J (+ 0.3V). Therefore, the energy efficiency in each energization condition of Example 4 is ΔW _butanol / ΔW _in = −0.44 (+1.2 V), 1.5 (+0.9 V), 19.5 (+0.6 V), 41.3 It was calculated as (+ 0.3V), and the efficiency was negative at + 1.2V, but it can be said that by energizing at a potential lower than that, it was possible to obtain an energy increase effect larger than the input electric energy. In Example 4, application of +0.3 V is optimal from the viewpoint of butanol production, and it becomes clear that this is an efficient culture technique showing an effect of increasing the production of energy (butanol) 41 times greater than the input electric energy by energization. It was.

In the _{above, W in} and _{W butanol} was obtained by the following formula. Further, Δ in the above means a difference between when a potential is applied and when no potential is applied.
W _in = Eap (voltage between electrodes: V) × C (amount of electricity: C)
W _butanol =
Butanol combustion heat (2675.9 kJ / mol) x butanol production (mol)

  Next, FIG. 32 shows changes in the natural potential of the culture solution when no current is applied in the culture test of Example 2. FIG. When the MV was added, the natural potential of the culture medium was around 0 V when MV was added, and around 0.1 V when the MV was not added. Both decreased gradually, and became -0.5 V when MV was added. . When MV was not added, it became around -0.4 and continued to increase thereafter.

Next, the change in current value in the culture test of Example 2 is shown in FIG. The value of the current that flowed during the culture test was smaller than that in Example 4. Then, from the integration of the current values that flowed until the end of the test, the input electric energy at the time of +0.6 V application where the butanol production increase effect was seen was calculated to be 0.27 C × 1.3 V = 0.35 J. At the end of the culture, the amount of energy derived from butanol increased by applying +0.6 V to the non-energized (MV addition) condition was calculated to be 2341.8 J. Therefore, energy efficiency ΔW _butanol / in Example 2 ΔW _in = 2158.1J / 0.35J = 6166 was calculated. From this result, it was clarified that a large amount of energy can be obtained with a small amount of electricity by the present invention.

  Next, the number of MV regenerations was calculated on the assumption that all of the current flowing during the test period was used for oxidation of reduced MV. Table 3 shows the calculation results.

  From the results shown in Table 3, it was revealed that no positive correlation was found between the number of MV regenerations and the butanol production promoting effect. From this, it was found that the increase or decrease in butanol production increase effect in the present invention cannot be explained only by continuous supply of oxidized MV.

DESCRIPTION OF SYMBOLS 1 Alcohol production apparatus 2 Alcohol production microorganism 3 Electronic medium substance 4 Culture solution 4a Electrolyte solution 5 Organic substance 6 Ion exchange membrane (anion exchange membrane)
7 Culture tank 8 Counter electrode tank 9 Working electrode 10 Counter electrode 12 Constant potential setting device 15, 16 Alcohol recovery means

Claims (12)

In a method of producing alcohol from organic matter using an alcohol producing microorganism that produces alcohol as a metabolite in fermentation,
An electronic medium substance is added to a culture solution containing the alcohol-producing microorganism and the organic substance, the electronic medium substance is a substance that can be reduced by the alcohol-producing microorganism, and the electronic medium substance produced by being reduced by the alcohol-producing microorganism A clostridium genus that produces alcohol by culturing the alcohol-producing microorganism while applying an electric potential to an electrode immersed in the culture solution to oxidize the reductant, and performing acetone, butanol, ethanol fermentation (ABE fermentation) as the alcohol-producing microorganism A method for producing alcohol, characterized in that at least one of viologen and a viologen derivative is used as the electronic medium substance . The alcohol production method according to claim 1, wherein an electrolyte solution is brought into contact with the culture solution via an anion exchange membrane, and an electrode paired with the electrode is immersed in the electrolyte solution. The method for producing alcohol according to claim 1, wherein an electrode paired with the electrode is brought into contact with the culture solution via an anion exchange membrane. Alcohol production method according to claim 2 or 3 potential before Symbol electrodes at a high density culturing a microorganism of the genus Clostridium as + -0.3V to + 0.9V with a silver-silver electrode potential reference chloride. The Clostridium microorganisms before Symbol Clostridium be included in the culture solution bacterial cells density before moving to the logarithmic growth phase, the potential of the electrode as + -0.3V to + 1.2V with a silver-silver electrode potential reference chloride The method for producing alcohol according to claim 2 or 3, wherein the microorganism of the genus Clostridium is cultured at a high density by setting the potential of the electrode to +0.3 V to +0.9 V after shifting the growth stage of the microorganism to a stationary phase. The Clostridium microorganisms before Symbol Clostridium be included in the culture solution bacterial cells density before moving to the logarithmic growth phase, the potential of the electrode as + 0.9V to + 1.2V with a silver-silver electrode potential reference chloride The method for producing alcohol according to claim 2 or 3, wherein the microorganism of the genus Clostridium is cultured at a high density by setting the potential of the electrode to +0.3 V to +0.6 V after the growth stage of the microorganism is shifted to a stationary phase. An apparatus for producing alcohol from organic matter using an alcohol producing microorganism that produces alcohol as a metabolite in fermentation,
A counter electrode vessel partitioned by an anion exchange membrane and a culture vessel having a sealed structure, a culture solution contained in the culture vessel, an electrolyte solution contained in the counter electrode vessel, and an action immersed in the culture solution An electrode, a counter electrode immersed in the electrolyte, a constant potential setting device in which the working electrode and the counter electrode are connected, and an alcohol recovery means for recovering alcohol produced in the culture vessel. Then, the alcohol-producing microorganism, the organic substance, and the electronic medium substance that can be reduced by the alcohol-producing microorganism are included in the culture solution, and the reduced form of the electronic medium substance that is reduced by the alcohol-producing microorganism is determined. by culturing said alcohol-producing microorganism while giving a potential difference is oxidized between the working electrode and the counter electrode at a potential setting device to produce an alcohol, said alcohol-producing microorganism is a Ton butanol-ethanol fermentation (ABE fermentation) and microorganism Clostridium performing, alcohol production device, wherein the electronic medium material is at least one of viologen and viologen derivatives. An apparatus for producing alcohol from organic matter using an alcohol producing microorganism that produces alcohol as a metabolite in fermentation,
A sealed container as a culture tank, a small container with a sealed structure as a counter electrode tank that can be stored in the container, a culture solution stored in the culture tank, and an electrolyte solution stored in the counter electrode tank; A working electrode immersed in the culture solution, a counter electrode immersed in the electrolytic solution, a constant potential setting device in which the working electrode and the counter electrode are connected, and alcohol produced in the culture vessel The small container is provided with an anion exchange membrane in at least a part of the position in contact with the culture solution, and the alcohol-producing microorganism, the organic matter, and the alcohol are included in the culture solution. An electron medium substance that can be reduced by the production microorganism, and a reduced product of the electron medium substance that is reduced by the alcohol-producing microorganism is generated between the working electrode and the counter electrode by the constant potential setting device. By culturing said alcohol-producing microorganism while oxidized giving much difference to produce alcohol, said alcohol-producing microorganism is a microorganism of the genus Clostridium performing acetone-butanol-ethanol fermentation (ABE fermentation), the electronic medium material An alcohol production apparatus characterized in that it is at least one of a viologen and a viologen derivative . An apparatus for producing alcohol from organic matter using an alcohol producing microorganism that produces alcohol as a metabolite in fermentation,
A container having at least a part of an anion exchange membrane, a culture solution contained in the container, a working electrode immersed in the culture solution, and at least a part of an outer surface of the container of the anion exchange membrane A counter potential electrode, a constant potential setting device in which the working electrode and the counter electrode are connected, and an alcohol recovery means for recovering alcohol produced in the container, and the anion exchange The membrane is provided at a position in contact with the culture solution of the container, and the alcohol-producing microorganism includes the alcohol-producing microorganism, the organic matter, and an electronic medium substance that can be reduced by the alcohol-producing microorganism. The alcohol production while oxidizing the reduced form of the electron medium substance generated by reduction by the constant potential setting device while applying a potential difference between the working electrode and the counter electrode To produce alcohol by culturing organisms, said alcohol-producing microorganism is a microorganism of the genus Clostridium performing acetone-butanol-ethanol fermentation (ABE fermentation), the electronic medium material is at least one of viologen and viologen derivatives Alcohol production equipment characterized by. Before SL potential of the working electrode is controlled to the silver-silver electrode at a potential reference chloride + -0.3V to + 0.9V according to any one of claims 7-9, wherein the clostridium microorganisms are densely cultured Alcohol production equipment. Microorganisms before Symbol Clostridia contained in the culture solution bacterial cells density before moving to the logarithmic growth phase, the potential of the working electrode is controlled to + -0.3V to + 1.2V with silver-silver chloride electrode potential reference After the growth stage of the Clostridium microorganism has shifted to the stationary phase, the potential of the working electrode is controlled to +0.3 V to +0.9 V on the basis of the silver / silver chloride electrode potential, and the microorganism of the Clostridium genus has a high density. The alcohol production apparatus according to any one of claims 7 to 9, which is cultured. Microorganisms before Symbol Clostridia contained in the culture solution bacterial cells density before moving to the logarithmic growth phase, the potential of the working electrode is controlled in + 0.9V to + 1.2V with silver-silver chloride electrode potential reference After the growth stage of the Clostridium microorganism has shifted to the stationary phase, the potential of the working electrode is controlled to +0.3 V to +0.6 V on the basis of the silver / silver chloride electrode potential, and the microorganism of the Clostridium genus has a high density. The alcohol production apparatus according to any one of claims 7 to 9, which is cultured.