JP2012200200A - Method for controlling metabolic pathway of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To control the pyruvic acid metabolism pathway of a microorganism, in association with the method for controlling the metabolism pathway of the microorganism.SOLUTION: The metabolism of the microorganism, having a starting point of pyruvic acid is controlled by making an electrode in contact with a culturing environment comprising the microorganism producing metabolites from the pyruvic acid, and culturing while controlling the potential of the electrode. More specifically, the method is provided by using the microorganism having the metabolism pathways having a reducing pathway of reducing the pyruvic acid to lactic acid and an oxidation pathway of oxidizing the pyruvic acid to an acetyl coenzyme (A), as a controlling objective microorganism, making the electrode in contact with the culturing environment containing the microorganism, and by culturing the microorganisms while controlling the potential of the electrode to the reducing potential to prompt the reducing reaction in the reducing pathway and to decrease the oxidation reaction in the oxidizing pathways. Oppositely, by culturing while controlling the potential of the electrode to the oxidizing potential, the reducing reaction in the reducing pathway is decreased and the oxidation reaction in the oxidizing pathway is prompted.

Description

  The present invention relates to a method for controlling metabolic pathways of microorganisms. More specifically, the present invention relates to a method for controlling a metabolic pathway of a microorganism that produces a metabolite from pyruvic acid.

  It is well known that microorganisms can produce various metabolites via various metabolic pathways. For example, E. coli is a microorganism that produces metabolites from pyruvate. More specifically, it is a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A. Acetyl coenzyme A is finally converted into acetic acid and ethanol (see FIG. 1 and Non-Patent Document 1).

J. Bacteriol (2000), vol182, 3, p620-626

  If the reduction reaction of pyruvic acid to lactic acid in the metabolic pathway of E. coli can be promoted, the amount of lactic acid useful as a raw material for polylactic acid, which is a biodegradable plastic, can be increased. Conversely, if the oxidation reaction of pyruvate to acetyl coenzyme A in the oxidation pathway can be promoted, the production amount of ethanol useful as a biofuel can be increased.

  In addition, the hydrolyzing bacterium is a microorganism having a function of producing hydrogen necessary for hydrogen-utilizing methane bacterium during growth in the methane fermentation tank. On the other hand, however, the hydrolyzing bacterium has an oxidation pathway of pyruvate to acetyl coenzyme A as a metabolic pathway, and acetic acid produced by the final conversion of acetyl coenzyme A is the acid of the methane fermenter. Can be a factor. Therefore, if the oxidation reaction of pyruvic acid to acetyl coenzyme A in the oxidation pathway can be reduced, it becomes possible to reduce the amount of acetic acid produced that can cause rancidity in the methane fermenter, and long-term stabilization of the methane fermenter Can also be realized.

  Thus, it is considered that various advantages can be produced by controlling the metabolic pathway starting from pyruvic acid of microorganisms.

  Accordingly, an object of the present invention is to provide a method for controlling a metabolic pathway starting from pyruvic acid of a microorganism.

  As a result of intensive studies by the inventors of the present invention in order to solve such problems, the amount of lactic acid produced is increased by bringing the electrode into contact with a culture environment containing E. coli and controlling the potential of the electrode to a reduction potential. It was found that the production amount of acetic acid and ethanol decreased. Conversely, it has been found that by controlling the electrode potential to the oxidation potential, the production amount of acetic acid and ethanol increases while the production amount of lactic acid decreases.

  Furthermore, the inventors of the present application usually do not produce almost anything by bringing the electrode into contact with a culture environment containing Thermotoga maritima, which is a hyperthermophilic hydrolyzing bacterium, and controlling the potential of the electrode to a reduction potential. It was also found that the amount of acetic acid decreased as lactic acid was produced.

  Based on these findings, the inventors of the present invention made metabolic pathways starting from pyruvate by bringing the electrodes into contact with the culture environment and controlling the potential of the microorganisms that produce metabolites from pyruvate. As a result, it has been found that it is possible to control the above, and various studies have been made to complete the present invention.

  That is, the method for controlling a metabolic pathway of a microorganism of the present invention starts with pyruvic acid of a microorganism by bringing the electrode into contact with a culture environment containing a microorganism that produces a metabolite from pyruvic acid and culturing while controlling the potential of the electrode. I try to control the metabolism.

  Here, in the metabolic pathway control method for microorganisms of the present invention, a microorganism having as a metabolic pathway a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A is set as a control target microorganism. It is preferable to promote the reduction reaction in the reduction pathway and reduce the oxidation reaction in the oxidation pathway by bringing the electrode into contact with a culture environment containing microorganisms and culturing while controlling the potential of the electrode to the reduction potential.

  In the method for controlling metabolic pathways of microorganisms of the present invention, a microorganism having as a metabolic pathway a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A is defined as a microorganism to be controlled. It is preferable to reduce the reduction reaction in the reduction pathway and promote the oxidation reaction in the oxidation pathway by bringing the electrode into contact with the culture environment containing

  According to the present invention, it is possible to artificially control a metabolic pathway starting from pyruvic acid for a microorganism that produces a metabolite from pyruvic acid. Therefore, it is possible to perform control for increasing or decreasing the production amount of the metabolite from this metabolic pathway according to desired conditions.

  For example, the electrode is brought into contact with a culture environment containing Escherichia coli, and the potential of the electrode is controlled to the reduction potential, thereby promoting the reduction reaction of pyruvic acid to lactic acid in the reduction pathway, and polylactic acid, a biodegradable plastic, The production amount of lactic acid useful as a raw material can be increased. Conversely, by controlling the electrode potential to the oxidation potential, it is possible to promote the oxidation reaction of pyruvate to acetyl coenzyme A in the oxidation pathway and increase the amount of ethanol useful as a biofuel. Become.

  Further, the electrode is brought into contact with a culture environment containing hydrolyzing bacteria, and the potential of the electrode is controlled to the reduction potential, thereby reducing the oxidation reaction of pyruvate to acetyl coenzyme A in the oxidation pathway, and pyruvate in the reduction pathway Since the reduction reaction of lactic acid to lactic acid can be promoted, for example, when the culture environment is a methane fermenter, it is possible to reduce the amount of acetic acid produced that can cause the methane fermenter to lose acid, It is also possible to achieve long-term stabilization by preventing the rancidity.

It is a figure which shows the metabolic pathway of colon_bacillus | E._coli. It is a figure which shows the metabolic pathway of Thermotoga maritima (Thermotoga maritima). It is sectional drawing which shows an example of the electroculture apparatus concerning 1st Embodiment A. It is sectional drawing which shows an example of the electroculture apparatus concerning 1st Embodiment B. It is sectional drawing which shows an example of the electroculture apparatus concerning 1st Embodiment C. It is sectional drawing which shows an example of the electroculture apparatus concerning 1st Embodiment D. It is sectional drawing which shows an example of the electroculture apparatus concerning 2nd embodiment. It is sectional drawing which shows an example of the other structure of the electroculture apparatus for implementing this invention. It is a figure which shows a time-dependent change of a microbial cell density (Example 1). It is a figure which shows the starch remaining amount in a culture solution (Example 1). It is a figure which shows the gas generation amount (Example 1). It is a figure which shows the time-dependent change of pH of a culture solution (Example 1). It is a figure which shows the time-dependent change of the acetic acid concentration of a culture solution (Example 1). It is a figure which shows the time-dependent change of the lactic acid density | concentration of the culture solution in a culture test (Example 1). It is a figure which shows the measurement result of the electric current value during a culture | cultivation period (Example 1). It is a figure which shows the lactic acid density | concentration of a culture solution (Example 2). It is a figure which shows the acetic acid and ethanol density | concentration of a culture solution (Example 2). It is sectional drawing which shows the structure of the electroculture apparatus used in the Example. It is a figure which shows the structure of the small container of the electroculture apparatus used in the Example.

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

  The microorganism metabolic pathway control method of the present invention is based on pyruvic acid of microorganisms by bringing the electrode into contact with a culture environment containing microorganisms that produce metabolites from pyruvic acid and culturing while controlling the potential of the electrode. It tries to control metabolism.

  More specifically, a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A is set as a control target microorganism, and an electrode is attached to the culture environment containing the microorganism. By contacting the cells and culturing while controlling the electrode potential to the reduction potential, the reduction reaction in the reduction pathway is promoted and the oxidation reaction in the oxidation pathway is reduced. Conversely, by culturing while controlling the potential of the electrode to the oxidation potential, the reduction reaction in the reduction pathway is reduced and the oxidation reaction in the oxidation pathway is promoted.

  The microorganism to which the present invention can be applied is not particularly limited as long as it is a microorganism that produces a metabolite from pyruvic acid. For example, preferably, Escherichia coli and hydrolyzing bacteria are mentioned, but other lactic acid bacteria that perform heterolactic fermentation (for example, Lactobacillus brevis, Lactobacillus fermentum, Lactococcus cremoris, L. buchnerii, L. cellobisous, Lactobacillus casei, Lactococcus lactis subsp. Lactobacillus casei, Lactobacillus curvantus, Lactobacillus plantarum, Sporolactobacillus inulinus, Lactococcus lactis, Enterococcus faecalis, Pdeiococcus damnosus, Streptococcus acidominimus, Streptococcus agalactiae, Streptococcusequinus, Stroccoccusequinus, Strocto ilus, Streptococcus uberis, etc.), Bacillus subtilis, Bacillus caldolyticus, Bacillus megaterium, Carnobacterium divergens, Lactosphaera pasteurii, Oenococcus oeni, Enterococcus faecium, Klebsiella pneumoniae, Clostridium perfringens, Clostriella thermoacter marcescens, Thermus thermophilus, Thermus caldophilus, Geobacillus stearothermophilus, Thermoanaerobacter ethanolicus, Bacillus caldolyticus, Nocardia asteroids, Selenomonas ruminantium, Actinomyces viscosus, Butyrivibrio fibrisolvens, Staphylococcus aureus, Rhizopusto

  Here, the present invention will be described based on the metabolic pathway of E. coli exemplified as a suitable microorganism (FIG. 1). Pyruvate is produced from glucose via phosphoenolpyruvate. This pyruvic acid is reduced to lactic acid via a reduction pathway, and is oxidized to acetyl coenzyme A (acetyl-CoA) via an oxidation pathway. Acetyl coenzyme A is converted to ethanol via acetyl aldehyde, and is converted to acetic acid via acetyl-P. That is, Escherichia coli is a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A.

  When the electrode is brought into contact with a culture environment containing E. coli and the potential of the electrode is controlled to the reduction potential, the reduction reaction from pyruvate to lactic acid in the reduction pathway is promoted, and the oxidation reaction from pyruvate to acetyl coenzyme A in the oxidation pathway Will decline. Thereby, the production amount of lactic acid increases, and the production amounts of acetic acid and ethanol decrease. In this case, a large amount of lactic acid useful as a raw material for polylactic acid, which is a biodegradable plastic, can be produced in E. coli.

  On the contrary, when the potential of the electrode is controlled to the oxidation potential, the reduction reaction from pyruvate to lactic acid in the reduction pathway decreases, and the oxidation reaction from pyruvate to acetyl coenzyme A in the oxidation pathway is promoted. Thereby, the production amount of lactic acid decreases, and the production amounts of acetic acid and ethanol increase. In this case, a large amount of ethanol useful as a biofuel can be produced in E. coli.

  Next, among the hydrolyzing bacteria exemplified as preferred microorganisms, the present is based on the metabolic pathway (Fig. 2) of Thermotoga maritima (hereinafter, also referred to as thermotoga), which is a hyperthermophilic hydrolyzing bacterium. The invention will be described. Pyruvate is produced from glucose via glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate. This pyruvate is oxidized to acetyl coenzyme A (acetyl-CoA) via an oxidation pathway. Acetyl coenzyme A is converted to acetic acid. Usually, lactic acid is hardly produced from pyruvic acid, but lactic acid can be produced from pyruvic acid depending on the culture conditions. Therefore, Thermotoga is a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A.

  When the electrode is brought into contact with a culture environment containing thermotoga and the potential of the electrode is controlled to the reduction potential, the reduction reaction from pyruvate to lactic acid in the reduction pathway is promoted, and the oxidation reaction from pyruvate to acetyl coenzyme A in the oxidation pathway Will decline. As a result, Thermotoga, which normally produces little lactic acid, reduces pyruvic acid to produce lactic acid and reduces the amount of acetic acid produced. As a result, for example, when the culture environment is a methane fermenter, it is possible to reduce the amount of acetic acid produced that can cause rancidity in the methane fermenter, and to prevent long-term stabilization by preventing rancidity in the methane fermenter Is possible.

  In addition, lactic acid bacteria that perform heterolactic fermentation are microorganisms that have a reduction pathway that reduces pyruvate to lactic acid and an oxidation pathway that oxidizes pyruvate to acetyl coenzyme A as metabolic pathways, and the potential of the electrode is the reduction potential. Thus, the reduction reaction from pyruvate to lactic acid in the reduction pathway is promoted, and the oxidation reaction from pyruvate to acetyl coenzyme A in the oxidation pathway decreases. Conversely, by setting the potential of the electrode to the oxidation potential, the reduction reaction from pyruvate to lactic acid in the reduction pathway decreases, and the oxidation reaction from pyruvate to acetyl coenzyme A in the oxidation pathway is promoted. In heterolactic fermentation, acetic acid and ethanol are produced via acetyl coenzyme A. Therefore, as in the case of Escherichia coli, it is possible to produce more lactic acid bacteria useful as a raw material for polylactic acid, which is a biodegradable plastic, by setting the electrode potential to a reduction potential. Conversely, by setting the electrode potential to the oxidation potential, it is possible to produce more ethanol useful as a biofuel in lactic acid bacteria.

  Further, even in a lactic acid bacterium that performs homolactic fermentation, a by-product such as acetic acid can be produced in a very small amount depending on the culture conditions and the like. The effect of can be produced. Even if the metabolic pathway from pyruvic acid of lactic acid bacteria performing homolactic fermentation is limited to the metabolic pathway from pyruvic acid to lactic acid, the potential of the electrode is reduced to pyruvic acid to lactic acid. The production of lactic acid can be increased by promoting the reduction reaction. Conversely, by setting the electrode potential to the oxidation potential, the reduction reaction from pyruvic acid to lactic acid can be reduced to reduce the amount of lactic acid produced.

  Here, in the present invention, the control of the electrode potential to the reduction potential is realized by making the electrode potential smaller than the oxidation-reduction potential of the culture environment. Conversely, control of the electrode potential to the oxidation potential is realized by making the electrode potential larger than the oxidation-reduction potential of the culture environment.

  That is, when the oxidation-reduction potential of the culture environment is A (V) and the electrode potential is X (V), the control of the electrode potential X to the reduction potential is realized by setting X <A. Conversely, the control of the electrode potential X to the oxidation potential is realized by setting X> A.

  Specifically, the control of the electrode potential X to the reduction potential is preferably X ≦ −0.6 (V) on the basis of the silver / silver chloride electrode potential, and X <−0.6 (V ) And more preferably X ≦ −0.8 (V). However, if the electrode potential X is set too low, for example, electrolysis of water occurs in the culture environment, so that the input electric energy can be consumed in addition to the control of the metabolic pathway. Therefore, X ≧ −1.4 (V ), X ≧ −1.2 (V) is more preferable, and X ≧ −1.0 (V) is more preferable.

  It should be noted that, by setting X ≦ −0.6 (V), when hydrolyzing bacteria are controlled microorganisms, it is possible to obtain an effect of shortening the growth start time of the microorganisms by experiments of the present inventors. It has been confirmed. Further, by setting X ≦ −0.8 (V), in addition to the above-mentioned effects, there are an effect of promoting the decomposition of organic matter, a significant increase in the amount of hydrogen generation, and an effect of maintaining the neutrality of the pH of the culture environment. Therefore, when the hydrolyzing bacterium is a control target microorganism, it can be said that X ≦ −0.6 (V), particularly X ≦ −0.8 (V) is extremely suitable.

  Next, the control of the electrode potential X to the oxidation potential is preferably X ≧ −0.3 (V) on the basis of the silver / silver chloride electrode potential, and X> −0.3 (V). It is more preferable that X ≧ 0 (V). However, if the electrode potential X is increased too much on the positive side, electrolysis of water from the culture environment and the like occur, and the input electric energy can be consumed in addition to the control of metabolites. Therefore, X ≦ + 1.4 (V ), X ≦ + 1.2 (V) is more preferable, and X ≦ + 1.0 (V) is more preferable.

  Here, an oxidation-reduction substance that does not inhibit the metabolism of microorganisms may be added to the culture environment (culture solution). By adding a redox substance, the potential of the culture environment can be easily controlled. As the redox substance, iron ions, potassium ferrocyanide, quinone compounds such as sodium anthraquinone disulfonate, viologen derivatives such as methyl viologen, and the like can be used. In addition, when using an iron ion as a redox substance, it is preferable to coordinate an iron ion to a chelating agent so that an iron ion can exist stably in a culture solution. Examples of the chelating agent include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), tetraethylenetriamine (TET), ethylenediamine (EDA), diethylenetriamine (DETA), citric acid, oxalic acid, crown ether, nitrilotetraacetic acid, Mention may be made of any chelating agent capable of coordinating iron ions such as disodium edetate, sodium edetate, trisodium edetate, penicillamine, trisodium pentetate calcium, pentetate, succimer and trientine edetate.

  When controlling the electrode potential, the electrode (working electrode) is brought into contact with the culture environment (for example, a culture solution), and the electrode (counter electrode) paired with this electrode is brought into contact with the culture environment via the ion exchange membrane. You may make it make it. In addition, the counter electrode and the ion exchange membrane may be in direct contact with each other or indirectly through an electrolytic solution. It is preferable that the ion exchange membrane can be retained in the culture solution without allowing the redox material to permeate. In this case, the potential of the entire culture solution can be easily controlled. However, since the potential around the working electrode is controlled without providing an ion exchange membrane, the effects of the present invention can be achieved. Therefore, the use of an ion exchange membrane is not essential. Further, a dialysis membrane may be used instead of the ion exchange membrane.

  The microorganism metabolic pathway control method of the present invention is carried out by, for example, an electroculture apparatus shown in FIGS. Hereinafter, the first embodiment will be described based on FIGS. 3 to 6, and the second embodiment will be described based on FIG. 7.

<First embodiment>
The method for controlling the metabolic pathway of microorganisms according to the first embodiment is performed by introducing a microorganism that produces a metabolite from pyruvate into a culture solution, bringing the culture solution and an electrolyte solution into contact with each other via an ion exchange membrane, and The working electrode and the reference electrode are brought into contact, the counter electrode is brought into contact with the electrolyte, the working electrode, the counter electrode and the reference electrode are connected to a constant potential setting device, and the potential of the working electrode is controlled by a three-electrode system to It tries to control the metabolic pathway.

  The method for controlling the metabolic pathway of microorganisms according to the first embodiment is performed by, for example, the electric culture apparatus 1 shown in FIGS. That is, in the electroculture apparatus 1 shown in FIGS. 3 to 6, one of the two tanks partitioned by the ion exchange membrane 6 is a culture tank 7, and the other tank is a counter electrode tank 8. 7 contains the culture solution 4 containing microorganisms and redox substances, and the working electrode 9 and the reference electrode 11 are immersed therein. The counter electrode tank 8 contains the electrolyte solution 4a and the counter electrode 10 is immersed therein. The working electrode 9, the counter electrode 10 and the reference electrode 11 are connected to a constant potential setting device 12, and the constant potential setting device 12 controls the potential of the working electrode 9 in a three-electrode system.

  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.

  Moreover, in the electric culture apparatus 1 shown in FIGS. 3-6, the gas which stays in the space (head space) above the liquid level of the culture solution 4 in the culture tank 7 is outside the culture tank 7 (the electric culture apparatus 1). A gas exhaust pipe 15a that leads to the outside of the culture tank 7 is provided, and the gas in the culture tank 7 is recovered by a gas recovery means 15 that can be opened and closed by a valve 15b. However, the gas recovery method is not limited to this method. For example, without providing the gas recovery means 15, an opening is provided in the upper part of the culture tank 7, the opening is closed with an elastic material such as synthetic rubber (for example, silicone rubber), and the syringe is injected into the elastic material that closes the opening. You may make it collect | recover gas from a head space with a needle. The elastic material such as synthetic rubber closes the hole when the injection needle is pulled out. Therefore, when the gas is not collected, the gas does not leak from the culture tank 7 even if the injection needle is pulled out. By recovering the gas in this way, it is possible to recover the hydrogen generated when the metabolic pathway is controlled for microorganisms that produce gas and volatiles, for example, hydrolyzing bacteria that produce hydrogen, without leakage. . Then, the hydrogen gas that can be retained in a large amount in the head space can be recovered without leakage due to the remarkable increase effect of the amount of hydrogen generation that can be achieved when metabolic pathway control is performed on hydrolyzing bacteria. In addition, when the metabolite is a volatile substance such as ethanol or acetic acid, it is possible to recover the volatile matter staying in the head space.

  Furthermore, in the electric culture apparatus 1 shown in FIG. 3 to FIG. 6, 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. The culture solution 4 is collected from the inside of the culture tank 7 by a culture solution collecting means 16 provided with a tube 16a and opening and closing the culture solution discharge tube 16a by a valve 16b. However, the method for collecting the culture solution 4 is not limited to this method. For example, without providing the culture solution collecting means 16, an opening may be provided in the culture tank 7, closed with an elastic material such as synthetic rubber, and the culture solution 4 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 is immersed in the culture solution 4 to collect the culture solution 4 through the tube. By recovering the culture solution 4 in this manner, the metabolite of the microorganism 2 contained in the culture solution 4 can be recovered.

  In addition to the gas recovery means 15 and the culture medium collection means 16, means for adding and supplying substances to the culture medium 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 also be supplied from this introduction tube. 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 gas recovery means 15 and the culture solution collecting means 16 may be used together as 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.

  Hereinafter, the case where the electroculture apparatus shown in FIG. 3 is used will be described as a first embodiment A, and the case where the electroculture apparatus shown in FIG. 4 is used will be described as a first embodiment B, which is shown in FIG. The case where the electroculturing apparatus is used will be described as a first embodiment C, and the case where the electroculturing apparatus shown in FIG. 6 is used will be described as a first embodiment D.

(First embodiment A)
The electric culture apparatus 1 shown in FIG. 3 uses a sealed container 20 as a culture tank 7 and a sealed small container 21 that can be accommodated in the container 20 as a counter electrode tank 8. The small container 21 is at least partially ion-exchanged. In addition to the membrane 6, a gas discharge pipe 22 that discharges gas (gas generated from the counter electrode 10) to the outside of the container 20 is provided. The wiring connecting the counter electrode 10 and the constant potential setting device 12 passes through the gas exhaust pipe 22. In the electroculture device 1 shown in FIG. 3, the wiring connecting the counter electrode 10 and the constant potential setting device 12 passes through the gas discharge pipe 22, but is not necessarily limited to this configuration. May be connected to the constant potential setting device 12 without passing through the gas discharge pipe 22.

  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. It may be configured.

  Thus, 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 in contact with the culture broth 4. In other words, the culture solution 4 comes into contact with the electrolyte solution 4 a through the ion exchange membrane 6.

  Here, in the first embodiment A, it is preferable that the small container 21 has a sealed structure. In this case, when a useful gas is generated from the counter electrode 10 by providing a gas discharge pipe that discharges the gas generated in the small container 21 to the outside of the container 20, the gas is recovered without omission. Can do. However, the small container 21 does not necessarily have a sealed structure.

  Moreover, it is preferable that the culture tank 7 (container 20) has a sealed structure. In this case, it is easy to control the culture tank 7 to anaerobic conditions. Furthermore, when microorganisms produce gas, it is preferable that the culture tank 7 has a sealed structure and includes the gas recovery means 15 described above. In this case, the gas produced by the microorganisms can be easily recovered without leaking out of the container 20 and while preventing an increase in pressure in the container 20 due to the gas. However, it is not an essential condition that the culture tank 7 has a sealed structure. That is, when microorganisms produce gas and gas is generated from the liquid surface of the culture solution 4, contact of free oxygen to the liquid surface of the culture solution 4 is suppressed, and the anaerobic condition of the culture tank 7 can be maintained. In such a case, the culture tank 7 may not have a sealed structure.

  The working electrode 9 that can be used in the present embodiment is not particularly limited, and an electrode capable of causing an oxidation reaction or a reduction reaction on its surface, such as a carbon electrode or a platinum electrode, can be used as appropriate. As the counter electrode 10, an electrode capable of generating a reaction that complements the oxidation reaction or the reduction reaction in the working electrode 9, such as a carbon electrode or a platinum electrode, can be used as appropriate.

(First embodiment B)
In the electroculture apparatus 1 shown in FIG. 4, 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. That is, the electroculturing apparatus 1 shown in FIG. 4 has substantially the same configuration as that of FIG. 3 except for the partition configuration of the culture tank 7 and the counter electrode tank 8 by the ion exchange membrane 6. The same effects as when using the apparatus can be achieved.

  In the electroculture device 1 shown in FIG. 4, the counter electrode tank 8 may remain open, but it has a sealed structure like the culture tank 7, and the gas generated in the counter electrode tank 8 is discharged from the counter electrode tank 8. A gas discharge pipe for discharging may be provided. By comprising in this way, when useful gas generate | occur | produces from the counter electrode 10, this can be collect | recovered without a leak.

  4, the head space of the culture tank 7 and the head space of the counter electrode tank 8 may be communicated with each other, or may be separated from each other by a partition wall or the like.

  Further, the culture tank 7 preferably has a sealed structure and includes the gas recovery means 15 and the like, as in the first embodiment A, but these configurations are not necessarily employed.

  In the electroculture apparatus 1 shown in FIG. 4, as the gas impermeable membrane 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, the ion exchange membrane 6 can be used, but is not limited thereto.

  The material of the container 23 that can be used in this embodiment is the same as that of the first embodiment A. The working electrode 9 and the counter electrode 10 that can be used in this embodiment are the same as those in the first embodiment A.

(First embodiment C)
The electroculture apparatus 1 shown in FIG. 5 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 via the ion exchange membrane 6 at the opening. 25, one container 25a is used as a culture tank 7 with a sealed structure, and the other container 25b is opened as a counter electrode tank 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. That is, the electroculture apparatus 1 shown in FIG. 5 has substantially the same configuration as that of FIG. 4 except for the configuration of the container 25. Therefore, the same effect as that obtained when the electroculture apparatus shown in FIG. 4 (and FIG. 3) is used can be obtained.

  Here, the opening of the other container 25b of the electroculture apparatus 1 in the first embodiment C is, for example, when the end of the other container 25b is completely opened, as shown in FIG. This means that the case includes a gas discharge pipe 22 that discharges the gas generated in the counter electrode tank 8 outside the counter electrode tank 8 while having a sealed structure like the one container 25a. By providing the gas discharge pipe 22, when useful gas is generated from the counter electrode tank 8, it can be easily recovered without leakage.

  Further, the culture tank 7 preferably has a sealed structure and includes the gas recovery means 15 and the like, as in the first embodiment A, but these configurations are not necessarily employed.

  The material of the container 25 that can be used in this embodiment is the same as that of the first embodiment A. The working electrode 9 and the counter electrode 10 that can be used in this embodiment are the same as those in the first embodiment A.

(First embodiment D)
The electroculture apparatus 1 shown in FIG. 6 is an H-shaped container in which two containers 26a and 26b each having an opening below the liquid level of the liquid to be stored are connected via the ion exchange membrane 6 through the opening. 26 is formed, one container 26a is used as a culture tank 7 with a sealed structure, and the other container 26b is opened as a counter electrode tank 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. That is, the electroculture apparatus 1 shown in FIG. 6 has substantially the same configuration as that of FIG. 4 except for the configuration of the container 25. Therefore, the same effect as that obtained when the electroculture apparatus shown in FIG. 4 (and FIG. 3) is used can be obtained.

  Here, the opening of the other container 26b of the electroculture apparatus 1 in the first embodiment D is, for example, when the upper part of the other container 26b is completely opened, as shown in FIG. This means that the case includes a gas discharge pipe 22 that discharges the gas generated in the counter electrode tank 8 outside the counter electrode tank 8 while having a sealed structure like the one container 25a. By providing the gas discharge pipe 22, when useful gas is generated from the counter electrode tank 8, it can be easily recovered without leakage.

  Further, the culture tank 7 preferably has a sealed structure and includes the gas recovery means 15 and the like, as in the first embodiment A, but these configurations are not necessarily employed.

  The material of the container 26 that can be used in the present embodiment is the same as that in the first embodiment A. The working electrode 9 and the counter electrode 10 that can be used in this embodiment are the same as those in the first embodiment A.

<Second Embodiment>
In the method for controlling the metabolic pathway of microorganisms according to the second embodiment, a microorganism producing a metabolite from pyruvic acid is introduced into a culture solution, the working electrode and the reference electrode are brought into contact with the culture solution, and the culture solution and the counter electrode are used. Contact with the ion exchange membrane, connect the working electrode, the counter electrode, and the reference electrode to a constant potential setting device, and control the potential of the working electrode in a three-electrode system to control the metabolic pathway of microorganisms. Yes. That is, the method for controlling the metabolic pathway of microorganisms in the first embodiment is different only in that the counter electrode is brought into direct contact with the ion exchange membrane without using an electrolytic solution.

  However, since the ion current flows between the working electrode 9 and the counter electrode 10 via the ion exchange membrane 6 without using the electrolytic solution 4a as in the first embodiment, the microorganism according to the second embodiment. According to this metabolic pathway control method, it is possible to obtain the same effect by controlling the potential of the working electrode 9 as in the first embodiment.

  The method for controlling the metabolic pathway of microorganisms according to the second embodiment is performed by, for example, an electroculture apparatus shown in FIG. In the electroculture apparatus 1 shown in FIG. 7, the working electrode 9 and the reference electrode 11 are arranged in a sealed container 5 having at least a part of the ion exchange membrane 6, and the counter electrode 10 is arranged outside the container 5. The culture solution 4 is accommodated in the container 5, 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 4 is at least a part when the culture solution 4 is accommodated in the container 5. 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 electric culture device 1 shown in FIG. 7, an opening 5 a is provided below the liquid level of the culture solution 4 in the container 5, the opening 5 a is closed with the ion exchange membrane 6, and ion exchange outside the container 5 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 electric culture apparatus 1 shown in FIG. 7, the entire container 5 functions as the culture tank 7.

  Therefore, according to the electroculture apparatus 1 shown in FIG. 7, since the container 5 has a sealed structure, no gas leaks from the container 5. Further, as in the first embodiment, there is an advantage that the inside of the container 5 can be easily controlled to an anaerobic condition.

  7 is provided with the gas recovery means 15 and the culture medium collection means 16 as in the first embodiment, but as described above, the gas recovery method and the culture medium collection are provided. The method is not limited to those using these means. Further, as in the first embodiment, a means for adding and supplying a substance to the culture solution 4 may be provided.

  Hereinafter, the details of the electroculture apparatus 1 shown in FIG. 7 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 5 has a sealed structure including at least a part of the ion exchange membrane 6. Examples of the material of the container 5 include, but are not limited to, glass, plastic, metal subjected to insulation treatment, concrete, and the like. In FIG. 7, the opening 5a provided below the liquid level of the culture solution 4 in the sealed container 5 is closed by the ion exchange membrane 6. However, the form and structure of the container 5 are particularly limited. Not. For example, the entire container 5 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 accommodated in the container 5 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 5.

  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. And a material capable of proceeding with a reduction reaction that complements the exchange of electrons with respect to the oxidation reaction at the working electrode 9, that is, the oxidation reaction can proceed when the reduction reaction occurs at the working electrode 9. A material electrode may be used. 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. If 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, the culture solution 4 is passed through the ion exchange membrane 6. Therefore, it is not always necessary to bring the ion exchange membrane 6 and the counter electrode 10 into contact with each other so that the entire ion exchange membrane 6 is completely covered with the counter electrode 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, ion exchange is performed by closing the opening 5 a by applying an adhesive around the opening 5 a of the container 5 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 5a of the container 5 and applying it to at least a part of the surface of the counter electrode 10 By bonding 6, the counter electrode 10 may be brought into contact with the entire ion exchange membrane 6 that closes the opening 5 a while closing the opening 5 a with the ion exchange membrane 6. 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.

  Moreover, although the container 5 functioning as the culture tank 7 is preferably provided with a sealed structure and the gas recovery means 15 and the like, as in the first embodiment, these configurations are not necessarily employed. Further, the counter electrode 10 outside the container 5 may be covered with a gas impervious member to form a sealed structure, and a gas recovery means for recovering the gas generated from the counter electrode 10 may be provided. By comprising in this way, when useful gas generate | occur | produces from the counter electrode 10, this can be collect | recovered without a leak.

  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. 8, 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 same effect as the above-described embodiment can be obtained.

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

<Experimental equipment>
(1) Electroculture apparatus used in Example 1 In Example 1, an experiment was performed using the electroculture apparatus shown in FIG. One of two 250 mL glass vials (manufactured by Duran) is used as a culture tank 7 and the other is used as a counter electrode tank 26b, and 2 through a cation exchange membrane (Nafion K, manufactured by DuPont) 106 at the lower opening. Two vials were connected to form an H-shaped container 26. The culture tank 7 was covered, and a silicone rubber stopper was provided on the upper surface of the lid to ensure a sealed product when wiring and electrodes were passed.

  The gas generated from the culture tank 7 during the culture period was recovered in an aluminum sampling bag (manufactured by GL Sciences, trade name: aluminum bag, 1 L) connected to the gas recovery means 15.

  The counter electrode tank 8 accommodated the electrolyte solution 4a and the counter electrode 10 and was immersed in the electrolyte solution 4a. The counter electrode tank 8 was also covered, and a silicone rubber plug was provided on the upper surface of the cover, and the gas discharge pipe 22 was passed through the silicone rubber plug. Then, a wiring for connecting the counter electrode 10 and the potential control device 12 was passed through the gas exhaust pipe 22. Both ends of the gas discharge pipe 22 are opened, and one end is disposed inside the counter electrode tank 8 and the other end is disposed outside the counter electrode tank 8.

  The working electrode 9 was accommodated in the culture tank 7 and immersed in the culture solution 4, and the wiring from the working electrode 9 to the potential control device 12 was drawn out of the culture tank 7 through a silicone rubber plug. The reference electrode 11 (silver / silver chloride electrode (RE-1B, BAS Co., Ltd.)) was inserted into a silicone rubber stopper from the outside of the culture tank 7 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, the culture solution 4 was stirred with a stirring bar.

  The working electrode 9 was a carbon electrode (size: 7.5 cm × 2.5 cm). The counter electrode 10 was a carbon electrode (size: 6.5 cm × 1.2 cm).

(2) Electroculture apparatus used in Example 2 In Example 2, an experiment was 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 culture solution 4 was put up to about the eighth minute of the container 20. 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 formed into a bag shape (hereinafter referred to as a bag 21) by molding the ion exchange membrane 6. The form of the small container 21 used in the experiment is shown in FIG. More specifically, an ion exchange membrane (Nafion K, manufactured by DuPont) is processed by thermocompression bonding with a heat sealer to form a bag-like container 21 having an open top, and the electrolyte solution 4a is accommodated in the bag 21 and a pair thereof is accommodated. The electrode 10 was accommodated and immersed in the electrolyte solution 4a. Then, a wiring 31 for connecting the counter electrode 10 and the constant potential setting device 12 was passed through the gas exhaust pipe 22. Both ends of the gas discharge pipe 22 are open, and one end is disposed inside the small container 21 and the other end is disposed outside the container 20, so that gas generated in the small container 21 is discharged outside the container 20. It was made to be. The opening at the top of the bag-like small container 21 was closed with a silicon adhesive 32.

  The small container 21 and the working electrode 9 were immersed in the culture solution 4, and the gas discharge pipe 22 of the small container 21 and the wiring of the working electrode 4 were drawn out of the container 20 through a silicone rubber stopper provided on the lid 30. The silver / silver chloride reference electrode 11 (RE-1B, BAS Co., Ltd.) was brought into contact with the culture solution 4 by being inserted from the outside of the container 20 through a silicone rubber stopper. The culture solution collection tube 16 was inserted from the outside of the container 20 through a silicone rubber stopper, so that one end thereof was 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. The other end of the culture solution collection tube 16 was connected to a syringe so that the culture solution 4 could be collected. During the culture, the culture solution 4 was stirred by the stirring bar 34.

  The working electrode 9 was a carbon electrode (size: 7.5 cm × 2.5 cm). The counter electrode 10 was a carbon electrode (size: 6.5 cm × 1.2 cm).

<Analysis method>

The cell density was calculated from the measured value (OD ₆₆₀ ).

  The starch concentration of the culture solution 4 was measured by the phenol / sulfuric acid method.

The amount of gas collected in the sampling bag of the electroculture apparatus 1 shown in FIG. 18 is analyzed using a water displacement method, and the composition of the gas is analyzed by gas chromatography (Varian, CR4900). Based on this, the amount of CO ₂ gas and the amount of H ₂ gas were calculated.

  The lactic acid concentration and the acetic acid concentration of the culture solution 4 were measured by ion chromatography (ICS-2000, manufactured by DIONEX).

  The ethanol concentration was measured by liquid chromatography (LachromeElite, Hitachi).

Example 1
As a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvic acid to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A, Thermotoga maritima DSM3109 (Thermotoga maritima DSM3109, hereinafter) (Referred to as Thermotoga).

A medium (JSCC237, Thermotoga medium) having the following composition is used as the culture solution 4, and Thermotoga is added to the culture solution 4 so that the initial cell density is 3.2 × 10 ⁶ cells / mL. 7 contained 250 mL.
[Medium composition]
Starch: 1.0g
KH ₂ PO ₄ : 0.5g
NaCl: 20 g
NiCl ₂ · H ₂ O: 0.002 g
East Extract: 0.5g
Artificial seawater: 250mL
Trace element solution: 10 mL
pH 6.5
[Composition of trace element solution (L ⁻¹ )]
Nitrilotriacetic acid 1.5g
MgSO ₄・ 7H ₂ O 3g
MnSO ₄・ 2H ₂ O 0.5g
NaCl 1.0g
FeSO ₄・ 7H ₂ O 0.1g
CoSO ₄・ 7H ₂ O 0.18g
CaCl ₂・ 2H ₂ O 0.1g
ZnSO ₄・ 7H ₂ O 0.18g
CuSO ₄・ 5H ₂ O 0.01g
H ₃ BO ₃ 0.01g
Na ₂ MoO ₄・ 2H ₂ O 0.01g
NiCl ₂・ 6H ₂ O 0.025g
Na ₂ SeO ₃・ 5H ₂ O 0.8mg
Na ₂ WO ₄ 0.8mg
KAl (SO ₄ ) ₂・ 12H2O 4mg
1L of water
[Artificial seawater]
MgSO ₄・ 7H ₂ O 7g
MgCl ₂・ 6H ₂ O 5.5g
NaCl 27.7g
CaCl ₂・ 2H ₂ O 2.25g
KCl 0.65g
NaBr 0.1g
H ₃ BO ₃ 30mg
SrCl ₂・ 6H ₂ O 15mg
Citric acid 10mg
KI 0.05mg
1L of water

  The electrolyte solution 4a was a NaCl solution (30 g / L).

  The culture temperature (culture solution temperature) was 80 ° C.

  As an oxidation-reduction substance, anthraquinone 2,6-disulfonic acid (AQDS) was added to the culture solution 4 so as to be 0.2 mM.

  The culture electrode was tested with the set potential of the working electrode 9 set to -0.8 V, -0.6 V, -0.3 V, 0 V, and +0.3 V on the basis of the silver / silver chloride electrode potential.

As a control, 0.5 g / L of Na ₂ S and L-cysteine was added to the culture solution 4 and a culture test was performed without energization. Since the oxidation-reduction potential of the culture solution 4 at this time was about −0.45 V, in this example, when the set potential was −0.8 V or −0.6 V, the potential of the working electrode 9 was The reduction potential is controlled, and when the set potential is set to −0.3 V, 0 V, and +0.3 V, the potential of the working electrode 9 is controlled to the oxidation potential.

  The culture tank 7 was purged with nitrogen, and the culture test was started after the culture tank 7 was placed under anaerobic conditions.

  FIG. 9 shows changes with time in cell density. By setting the set potential to -0.3V, -0.6V, and -0.8V, the final cell density can be made the same level as the control, and by setting it to -0.6V, -0.8V, There was a tendency for the time to start to be shorter than the control.

  The remaining amount of starch after the end of the culture test is shown in FIG. By setting the set potential at −0.8 V, the starch concentration tended to decrease more than the control. From this, it became clear that the decomposition amount of starch was increased by setting the set potential to −0.8V.

  The measurement result of the gas generation amount is shown in FIG. Although there was almost no difference in the amount of carbon dioxide generated depending on the conditions, it was revealed that the hydrogen generation amount was significantly increased over the control by setting the set potential to -0.8V.

  The change over time of the pH of the culture solution 4 is shown in FIG. It was revealed that the pH of the culture solution 4 can be maintained in the vicinity of 7 by setting the set potential to −0.8V.

  The time-dependent change of the acetic acid concentration of the culture solution 4 is shown in FIG. By setting the set potential to −0.6 V and −0.8 V, the concentration of acetic acid tends to be lower than that of the control.

  The time-dependent change of the lactic acid concentration of the culture solution 4 is shown in FIG. In the control, lactic acid was not detected over the whole culture period, but it was confirmed that lactic acid can be detected by setting the set potential to -0.6V and -0.8V.

  The measurement result of the current value during the culture period is shown in FIG. A current flowed to adjust to the potential environment set at the start of energization, and thereafter, the current showed a substantially constant value until the thermotoga grew into a stationary phase. From this, it was considered that no electron transfer occurred between AQDS added as a redox substance and Thermotoga during the growth process. Therefore, it has been clarified that the effect of the present invention does not depend on electron transfer between a redox substance such as AQDS and a microorganism. In addition, when the set potential was −0.8 V, it was confirmed that more reduction current was flowing in the later stage of the culture. Therefore, this increase in reduction current has some correlation with the increase in hydrogen generation amount. The possibility of having

  From the above results, by setting the set potential of the working electrode 9 to the reduction potential (−0.6 V, −0.8 V), the oxidation reaction of pyruvate to acetyl coenzyme A in the thermotoga oxidation pathway is reduced. It was shown that the amount of acetic acid produced can be reduced, and the reduction reaction of pyruvic acid to lactic acid in the reduction pathway is promoted to enable production of lactic acid. It was also revealed that an effect of shortening the time until the start of proliferation was obtained.

  Furthermore, it was found that by setting the set potential of the working electrode 9 to −0.8 V, the effect of promoting the decomposition of organic matter, the effect of significantly increasing the amount of hydrogen generation, and the effect of maintaining the neutrality of pH can be obtained. That is, it is clear that the excellent effect of maintaining the neutral region without causing an acidic shift of the pH of the culture solution 4 can be achieved while increasing the decomposition efficiency of organic matter and significantly increasing the hydrogen generation amount. became.

  In addition, since the hydrogen generation amount increased remarkably by setting the set potential to −0.8 V and the pH was maintained neutral, the reducing potential was reduced by setting the set potential to −0.8 V. It was suggested that may be released out of the system as hydrogen.

(Example 2)
A culture test was carried out using Escherichia coli as a microorganism having a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A as metabolic pathways.

Escherichia coli (E. coli JM109) was added to the culture solution 4 having the following composition so that the initial cell density was 8 × 10 ⁶ cells / mL, and 200 mL of this was stored in the culture tank 7.
[Composition of culture solution (L ⁻¹ )]
・ Glucose: 5.0g
・ KH ₂ PO ₄ : 4.2 g
・ NH ₄ Cl: 0.6 g
・ MgCl ₂ · 7H ₂ O: 0.08 g
・ Casamino acid: 5.0 g
・ Trace element solution: 1mL
[Composition of trace element solution (L ⁻¹ )]
_{· FeSO 4 · 7H 2 O:} 0.2g
_{· CuSO 4 · 5H 2 O:} 1.0g
・ NaMoO ₄ · 2H ₂ O: 0.034 g
・ CaCl ₂ · 2H ₂ O: 0.015 g
・ Na ₂ SeO ₄ : 0.5g

  The culture temperature (culture solution temperature) was 30 ° C.

  As an oxidation-reduction substance, anthraquinone 2,6-disulfonic acid (AQDS) was added to the culture solution 4 so as to be 0.2 mM.

  The pH of the culture solution 4 was 7.0.

  The culture electrode was tested with the working electrode 9 set at -1.0 V, -0.8 V, -0.6 V, -0.3 V, and 0 V based on the silver / silver chloride electrode potential.

  In this embodiment, when the set potential is −1.0 V, −0.8 V, and −0.6 V, the potential of the working electrode 9 is controlled to the reduction potential, and the set potential is − In the case of 0.3V and 0V, the potential of the working electrode 9 is controlled to the oxidation potential.

  The culture tank 7 was purged with nitrogen, and the culture test was started after the culture tank 7 was placed under anaerobic conditions.

  FIG. 16 shows the measurement result of the lactic acid concentration of the culture solution 4 after completion of the culture test. When the set potential is -1.0 V, -0.8 V, -0.6 V, the lactic acid concentration tends to increase. Conversely, when the set potential is -0.3 V, 0 V, the lactic acid concentration Tended to be lower.

  FIG. 17 shows the measurement results of the acetic acid and ethanol concentrations of the culture solution 4 after completion of the culture test. The ethanol concentration tends to decrease when the set potential is set to -1.0 V, -0.8 V, and -0.6 V, and tends to increase when the set potential is set to -0.3 V, 0 V. It was observed. In particular, when the set potential was set to 0 V, the tendency for the ethanol concentration to increase was remarkable. Regarding the acetic acid concentration, a tendency to decrease when the set potential was set to −0.8V was observed, and a tendency to increase when the potential was set to 0V was observed. For other set potentials, no significant difference in acetic acid concentration was observed.

  From the above results, it is possible to decrease the ethanol production amount and increase the lactic acid production amount by setting the electrode potential to the reduction potential, and conversely, the ethanol production amount by setting the electrode potential to the oxidation potential. It was revealed that the amount of lactic acid produced can be decreased while increasing the amount of lactic acid.

  As for acetic acid, the concentration decreased at the reduction potential of −0.8 V, and the concentration increased at the oxidation potential of 0 V. Therefore, the production amount decreased at the reduction potential, and the production amount increased at the oxidation potential. It became clear that there was a tendency to.

Claims (3)

It is characterized in that the metabolism of the microorganism starting from pyruvic acid is controlled by bringing the electrode into contact with a culture environment containing a microorganism that produces a metabolite from pyruvic acid and culturing while controlling the potential of the electrode. A method for controlling metabolic pathways of microorganisms.   The microorganism is a microorganism having, as metabolic pathways, a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A, and controlling the potential of the electrode to the reduction potential, The method for controlling a metabolic pathway of a microorganism according to claim 1, wherein the reduction reaction is promoted and the oxidation reaction in the oxidation pathway is reduced.   The microorganism is a microorganism having a reduction pathway for reducing pyruvate to lactic acid and an oxidation pathway for oxidizing pyruvate to acetyl coenzyme A as metabolic pathways, and controlling the potential of the electrode to an oxidation potential, The method for controlling a metabolic pathway of a microorganism according to claim 1, wherein the reduction reaction is reduced and the oxidation reaction in the oxidation pathway is promoted.