JP5563774B2 - Electric culture device - Google Patents

Description

The present invention relates to electrical culture device. In more detail, the present invention is a gaseous substance or volatile substances suitable electrical culture apparatus for culturing the microorganism producing, further, produce gaseous material and volatiles using this electric culture apparatus And a method for decomposing volatile harmful substances by microorganisms.

  As a new culturing technology for microorganisms, research on electroculturing technology is underway. Electric culture is a technique for culturing microorganisms while controlling the oxidation-reduction potential of the culture solution within the optimum range of the microorganisms to be cultured (see Patent Document 1). By controlling the oxidation-reduction potential of the culture solution to the optimum range of the microorganism to be cultured, only the microorganism can be selectively activated to improve various functions of the microorganism such as promotion of growth and substance resolution. it can. In addition, the electric culture includes a method of culturing while regenerating substances necessary for microorganisms by reducing substances oxidized by microorganisms or conversely reducing substances reduced (patents). Reference 2 and Patent Document 3).

  As a specific example of the electroculturing apparatus for carrying out the electroculturing technique, for example, an electroculturing apparatus shown in FIG. 10 is known (see Patent Document 1). An electroculture apparatus 101 shown in FIG. 10 includes two tanks (a culture tank 107 and a counter electrode tank 108) partitioned by an ion exchange membrane 106, a working electrode 109 and a counter electrode 110, a working electrode 109, and a counter electrode 110. The culture vessel 107 contains the culture solution 104 containing the redox substance 103 and the working electrode 109 is immersed in the culture solution 104, and the counter electrode vessel 108 is electrolyzed. The liquid 104a is accommodated, the counter electrode 110 is immersed in the electrolyte 104a, the microorganism 102 to be cultured is added to the culture liquid 104, and a potential difference is applied between the working electrode 109 and the counter electrode 110 by the power source 112. However, the microorganism 102 is cultured. 10, the reference electrode 111 is immersed in the culture solution 104, and the working electrode 109, the counter electrode 110, and the reference electrode 111 are connected to a potentiostat 112, which is a three-electrode type potential control device. Thus, the oxidation-reduction potential of the culture solution 104 in the culture tank 107 can be set strictly.

JP 2008-54646 A JP 2006-55134 A JP 2007-89580 A

  However, since the conventional electric culture apparatus has not a sealed structure, when an electric culture is performed on a microorganism that produces a gaseous substance, the gaseous substance produced by the microorganism is outside the electric culture apparatus. It was difficult to accurately analyze the amount of gaseous substances produced by the microorganisms and to recover without leakage.

  In addition, when electroculturing is performed on microorganisms that produce volatile substances, the same problems as when electroculturing is performed on microorganisms that produce gaseous substances have occurred. That is, the gas component produced by volatilization of the volatile substance produced by this microorganism is volatilized out of the electric culture apparatus, so that the amount of the volatile substance produced by this microorganism can be accurately analyzed or leaked. It was difficult to recover.

  In addition, when conducting culturing for microorganisms that decompose volatile harmful substances while decomposing volatile harmful substances added to the culture solution, volatile harmful substances that are decomposition targets are also present. There was a problem that it was volatilized out of the electroculture apparatus and the decomposition process could not be performed efficiently.

  In addition, since the conventional electroculture apparatus did not have a sealed structure, even if nitrogen gas or the like was flowed into the electroculture apparatus, the gas was volatilized out of the electroculture apparatus, and the culture environment of microorganisms was anaerobic It was difficult to control the sex. Therefore, conventionally, the culture environment is controlled to be anaerobic by, for example, housing an electric culture device in an anaerobic box having an airtight structure made of acrylic and flowing nitrogen gas or the like through the anaerobic box. However, if the electroculturing apparatus is accommodated in an anaerobic box or the like having a closed structure, there is a problem that management of the electroculturing apparatus itself, for example, a process of adding a substance to the culture solution becomes extremely complicated. Further, when culturing again after the completion of the electroculture, it is necessary to sterilize the anaerobic box together with the electrocultivation apparatus, and the labor is extremely complicated.

The present invention has an object to provide a can Ru electric culture apparatus culturing microorganisms without leaking gaseous substances and volatile substances produced by microorganisms outside the electric culture device.

  It is another object of the present invention to provide a substance production method capable of efficiently recovering gaseous substances and volatile substances produced by microorganisms without leaking out of the electric culture apparatus.

  Furthermore, an object of the present invention is to provide a method for decomposing substances that can be efficiently decomposed using microorganisms without causing leakage of volatile harmful substances to the outside of the electric culture apparatus.

  In order to solve such a problem, the inventors of the present application have studied about culturing microorganisms by sealing a conventional electric culture apparatus. As a result, it has been found that simply having a conventional electroculture apparatus with a sealed structure may adversely affect the growth state of microorganisms. As a result of studying this cause, the inventors of the present application have found that the gas generated from the counter electrode tank of the electric culture apparatus dissolves in the culture solution in the culture tank, which may adversely affect the growth state of the microorganism. I found it.

Therefore, the inventors of the present application have further studied, and the gaseous components generated from the counter electrode without causing leakage of gaseous substances and volatile substances produced by microorganisms to the outside of the electric culture apparatus, and the culture solution in the culture tank It conceived a configuration that no new electric culture device to blend into, and completed the present invention.

That is, the electroculture apparatus of the present invention includes two tanks partitioned by an ion exchange membrane, a working electrode and a counter electrode, and a power source that provides a potential difference between the working electrode and the counter electrode. One of the two tanks is used as a culture tank, and a culture solution containing a redox substance is stored therein, and the working electrode is immersed in the culture solution, and the other of the two tanks is used as a counter electrode tank and an electrolyte solution is stored therein. In addition, the counter electrode is immersed in the electrolytic solution, the microorganism to be cultured is added to the culture solution, and the microorganism is cultured while applying a potential difference between the working electrode and the counter electrode by a power source. The container is a culture tank, and a small container with a sealed structure that can be accommodated in the container is a counter electrode tank. The small container is provided with an ion exchange membrane at least in part and discharges gas generated from the counter electrode tank to the outside a discharge tube, the culture solution Contact with electrolyte through on-exchange membrane, culture medium in contact with counter electrode through ion-exchange membrane, and / or working electrode in contact with electrolyte through ion-exchange membrane By doing so, it is possible to generate an ionic current between the working electrode and the counter electrode. Here, the porous working electrode contacts the ion exchange membrane, the culture solution contacts the ion exchange membrane at the contact surface between the working electrode and the ion exchange membrane, and the culture solution and the working electrode pass through the ion exchange membrane. You may make it contact with electrolyte solution and / or a counter electrode. In addition, the porous counter electrode contacts the ion exchange membrane, the electrolyte solution contacts the ion exchange membrane at the contact surface between the porous counter electrode and the ion exchange membrane, and the electrolyte solution and the counter electrode contact the ion exchange membrane. It may be in contact with the culture medium and / or the working electrode.

Therefore, according to the electric culture apparatus of the present invention, since the culture tank has a sealed structure that prevents the gas generated from the counter electrode tank from entering the culture tank, gaseous substances (microorganisms produced in the culture tank are produced). Gas components produced by volatilization of volatile substances produced by microorganisms and microorganisms), and the growth of microorganisms is adversely affected by the gas generated from the counter electrode tank without leaking out of the culture tank Nothing will happen.

Next, the substance production method of the present invention is caused by culturing microorganisms that produce gaseous substances or microorganisms that produce volatile substances using the electric culture apparatus of the present invention, and the gaseous substances or volatile substances are volatilized and generated. The gas component is retained in a space above the liquid level of the culture solution in the culture tank, and this is recovered by the gas recovery means of the electric culture apparatus .

As described above, according to the electric culture apparatus of the present invention, the gaseous substance generated in the culture tank does not leak out of the culture tank, and the gas generated from the counter electrode tank does not adversely affect the growth state of the microorganism. It will not be done. Therefore, according to the substance production method of the present invention, while eliminating the adverse effect on the microorganism to be cultured due to the gas generated from the counter electrode, the gaseous substance produced by the microorganism to be cultured and the volatile substance produced by the microorganism to be cultured The gas component generated by volatilization can be efficiently collected by being retained in the space above the liquid level of the culture solution in the culture tank without leaking out of the culture tank.

Then, substance production method of the present invention, culturing a microorganism producing volatiles by electrical cultivation apparatus of the invention, the culture solution was collected volatiles produced by microorganisms from the culture fluid collecting means electrically culture apparatus To recover from.

As described above, according to the electric culture apparatus of the present invention, the gaseous substance generated in the culture tank does not leak out of the culture tank, and the gas generated from the counter electrode tank does not adversely affect the growth state of the microorganism. It will not be done. Therefore, according to the substance production method of the present invention, the volatile substance produced by the culture target microorganism is cultured without leaking out of the culture tank while eliminating the adverse effect on the culture target microorganism due to the gas generated from the counter electrode. It can be efficiently recovered from the culture medium in the tank.

Next, in the material decomposition treatment method of the present invention, the microorganisms that decompose volatile harmful substances are cultured by the electroculture apparatus of the present invention, and the volatile harmful substances added to the culture solution in the culture tank are decomposed. Like to do.

According to the electric culture apparatus of the present invention, gaseous substances in the culture tank do not leak out of the culture tank, and the gas generated from the counter electrode tank does not adversely affect the growth state of the microorganism. Therefore, according to the material decomposition treatment method of the present invention, the volatile harmful substance added to the culture solution is leaked out of the culture tank while eliminating the adverse effect on the growth state of the microorganisms caused by the gas generated from the counter electrode. It is possible to efficiently perform the decomposition process without any problem.

Incidentally, in the electric cultivation apparatus of the invention, the porous counter electrode without being electrolytic solution accommodated in the counter electrode reservoir may also be in contact with the ion-exchange membrane. In this case as well, it is possible to carry out electroculture.

  Here, the electric culture apparatus of the present invention preferably includes a gas recovery means for recovering a gaseous substance staying in a space above the liquid level of the culture solution in the culture tank.

  Moreover, it is preferable that the electroculture apparatus of the present invention includes a culture solution collecting means for collecting the culture solution in the culture tank.

According to electric culture device of the present invention, while eliminating adverse effects on the state of growth of microorganisms due to gas generated from the counter electrode, the gaseous substances and microorganisms generated gaseous materials (microorganisms produced in the culture tank is It is possible to carry out the electro-culture of microorganisms without leaking the gas component produced by volatilization of the produced volatile substance) to the outside of the culture tank. Therefore, it is possible to accurately analyze the relationship between the potential applied during culture and the production amount of gaseous substances and volatile substances by the microorganisms to be cultured.

  Moreover, according to the electric culture apparatus of this invention provided with the gas collection | recovery means which collect | recovers the gaseous substance which retains in the space above the liquid level of the culture solution in a culture container, the gaseous substance generated in the culture tank ( Gaseous substances produced by microorganisms and gas components produced by volatilization of volatile substances produced by microorganisms can be collected without waste, and the collection efficiency can be improved.

  Furthermore, according to the electric culture apparatus of the present invention equipped with the culture solution collecting means for collecting the culture solution in the culture vessel, the volatile substances produced by the microorganisms in the culture vessel can be recovered without waste and the recovery efficiency can be improved. It can be.

  Further, according to the material production method of the present invention, while eliminating the adverse effect on the growth state of microorganisms caused by the gas generated from the counter electrode, gaseous substances generated in the culture tank (gaseous substances produced by microorganisms and microorganisms) Can be collected without leaking out of the culture tank. Therefore, the microorganism-derived gaseous substance generated in the culture tank can be recovered without waste and the recovery efficiency can be improved.

  Furthermore, according to the substance production method of the present invention, volatile substances produced by microorganisms in the culture tank are leaked out of the culture tank while eliminating adverse effects on the growth state of the microorganisms caused by the gas generated from the counter electrode. Can be recovered without any problems. Therefore, the volatile substances produced by the microorganisms can be recovered without waste and the recovery efficiency can be improved.

  In addition, according to the material decomposition treatment method of the present invention, the volatile harmful substances added to the culture solution are removed from the culture tank while eliminating the adverse effects on the growth state of the microorganisms caused by the gas generated from the counter electrode tank. It can be decomposed without leakage. Therefore, the decomposition efficiency of volatile harmful substances by microorganisms can be improved.

It is sectional drawing which shows 1st embodiment of the electroculture apparatus of this invention. It is sectional drawing which shows the other example of 1st embodiment of the electroculture apparatus of this invention. It is sectional drawing which shows 2nd embodiment of the electroculture apparatus of this invention. It is sectional drawing which shows 3rd embodiment of the electroculture apparatus of this invention. It is sectional drawing which shows 4th embodiment of the electroculture apparatus of this invention. It is a figure which shows the electroculture apparatus used in the Example. It is a figure which shows the form of the small container as a counter electrode tank. It is a figure which shows the result of having measured the cell density with respect to culture | cultivation time. It is a figure which shows the result of having measured the ethanol concentration of the culture solution with respect to culture | cultivation time. It is sectional drawing which shows the conventional electroculture apparatus.

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

  The electrocultivation method of the present invention contains a culture solution containing a redox substance with one of the two chambers partitioned and opened by an ion exchange membrane as a culture vessel and immersing the working electrode in the culture solution. The other tank is used as a counter electrode tank to store the electrolyte solution, and the counter electrode is immersed in the electrolyte solution, the microorganism to be cultured is added to the culture tank, and the microorganism is removed while applying a potential difference between the working electrode and the counter electrode. In an electroculturing method for culturing, a culture vessel is sealed, and microorganisms are cultured while preventing gas generated from the counter electrode vessel from entering the culture vessel.

  Specifically, the electroculture method of the present invention can be carried out by the electroculture apparatus shown in FIGS. The electric culture apparatus 1 of the present invention includes two tanks (a culture tank 7 and a counter electrode tank 8) partitioned and opened by an ion exchange membrane 6, a working electrode 9, a counter electrode 10, and a working electrode 9 and a counter electrode. 10 and a power source 12 for providing a potential difference between them, a culture solution 4 containing the redox substance 3 is accommodated in the culture tank 7, and a working electrode 9 is immersed in the culture solution 4. The electrolytic solution 4a is accommodated, the counter electrode 10 is immersed in the electrolytic solution 4a, the microorganism 2 to be cultured is added to the culture solution 4, and a potential difference is generated between the working electrode 9 and the counter electrode 10 by the power source 12. In the electric culture apparatus for culturing microorganisms while being applied, the culture tank 7 has a sealed structure that prevents gas generated from the counter electrode tank 8 from entering the culture tank 7.

  The electroculture apparatus 1 of the present invention is an improvement over the conventional electroculture apparatus (FIG. 10) disclosed in Japanese Patent Application Laid-Open No. 2008-54646, and is divided into two pieces separated and opened by an ion exchange membrane 6. It has a tank (the culture tank 7 and the counter electrode tank 8), the working electrode 9 and the counter electrode 10, and a power source 12 for applying a potential difference between the working electrode 9 and the counter electrode 10. The culture solution 4 containing the substance 3 is accommodated and the working electrode 9 is immersed in the culture solution 4. The counter electrode tank 8 is accommodated with the electrolyte solution 4 a and the counter electrode 10 is immersed in the electrolyte solution 4 a. 4 is a conventional electroculture apparatus in which the microorganism 2 to be cultured is added and the microorganism 2 is cultured while a potential difference is applied between the working electrode 9 and the counter electrode 10 by the power source 12. It is the same as the device.

  The difference between the conventional electric culture apparatus shown in FIG. 10 and the electric culture apparatus of the present invention is that the culture tank 7 has a sealed structure. Thereby, the gas generated from the counter electrode 10 does not dissolve in the culture solution 4 in the culture tank 7. Therefore, while eliminating the adverse effect of the gas generated from the counter electrode 10 on the growth state of the microorganism 2, the gaseous substance or volatile substance produced by the microorganism 2 to be cultured, or the additive (gaseous state) in the culture tank 7 It becomes possible to perform the electric culture of the culture target microorganism 2 without leaking substances or volatile substances) out of the culture tank 7.

  Hereinafter, the embodiment of the electroculture apparatus of the present invention will be described in more detail.

  A first embodiment of the electroculture apparatus of the present invention is shown in FIG. In the electroculture apparatus 1 shown in FIG. 1, a sealed container 20 is used as a culture tank 7, a sealed small container 21 that can be accommodated in the container 20 is used as a counter electrode tank 8, and 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 tank) to the outside of the container 20 is provided.

  Moreover, the electric culture apparatus 1 shown in FIG. 1 has the gas collection | recovery means 15 which collect | recovers the gaseous substance which retains in the space above the liquid level of the culture solution 4 in the culture tank 7, and the culture solution 4 of the culture tank 7 And a culture solution collecting means 16 for collecting the sample.

  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, in the present embodiment, the entire small container 21 is a bag-shaped container formed by the ion exchange membrane 6, but the small container 21 is not limited to this form. For example, only one surface of the bag-like container may be constituted by the ion exchange membrane 6, or a part of one surface may be constituted by only the ion exchange membrane 6. 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 so that gas from the small container 21 (gas generated from the counter electrode tank 8) does not leak into the container 20.

  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 and the culture solution 4 provided in at least a part of the small container 21. And contact. Therefore, when a potential difference is applied between the working electrode 9 immersed in the culture solution 4 and the counter electrode 10 immersed in the electrolytic solution 4a, an ionic current is generated between the working electrode 9 and the counter electrode 10. And the oxidation-reduction potential of the culture solution 4 can be controlled.

  In addition, the working electrode 9 used in the present invention is immersed in the culture solution 4 and reversibly advances the oxidation-reduction reaction of the oxidation-reduction substance 3 as in the conventional electroculture apparatus. In the present embodiment, the working electrode 9 is a plate-like carbon electrode, but the shape and material of the working electrode 9 are not limited to this, and the point is that the redox substance 3 in the culture solution 4 is redox. What is necessary is just to set it as the electrode which can advance reaction reversibly.

  The counter electrode 10 used in the present invention proceeds with a reaction that complements the exchange of electrons with respect to the oxidation-reduction reaction at the working electrode 9 as in the case of a conventional electroculture device. In the present embodiment, the counter electrode 10 is a plate-like carbon electrode. However, the shape and material of the counter electrode 10 are not limited to this, and the point is that the electron is reduced with respect to the redox reaction in the working electrode 9. What is necessary is just to set it as the electrode which can advance the reaction which complements giving / receiving.

  In the present embodiment, a reference electrode 11 (for example, a silver / silver chloride reference electrode) is disposed inside the culture tank 7, and the reference electrode 11 is immersed in the culture solution 4, so that the operation is similar to that of a conventional electroculture device. The electrode 9, the counter electrode 10, and the reference electrode 11 are connected to a three-electrode potential control device (potentiostat) so that the potential of the culture solution 4 can be strictly controlled. As shown in FIG. The potential of the culture solution 4 can be controlled as long as a potential difference is applied between the working electrode 9 and the counter electrode 10 by the power source 12 without providing the reference electrode 11.

The ion exchange membrane 6 used in the present invention allows the oxidation-reduction substance 3 contained in the culture solution 4 to remain in the culture solution 4 without permeating the counter electrode 10 side, as in the case of the conventional electric culture apparatus, and the culture solution. 4 to transmit the ions contained in 4 to the counter electrode 10 side. Specific examples include a Nafion membrane that is a membrane that transmits only monovalent cations, but is not limited thereto. In addition, by using a Nafion membrane that is a membrane that transmits only monovalent cations, for example, when iron ions are used as the redox material 3, divalent iron ions and trivalent iron ions do not permeate. The redox material 3 can remain in the culture solution 4 without permeating the counter electrode 10 side. In addition, since the culture solution 4 normally contains monovalent cations such as hydrogen ions, sodium ions (Na ⁺ ), and potassium ions (K ⁺ ), these cations are transferred to the counter electrode 10 side. The ionic current is generated between the working electrode 9 and the counter electrode 10. As a result, a reaction that complements the transfer of electrons in the reaction that occurs at the working electrode 9 occurs at the counter electrode 10, and the redox potential of the culture solution 4 is controlled to be constant. For example, when an oxidation reaction occurs at the working electrode 9, a reduction reaction occurs at the counter electrode 10, and the oxidation-reduction potential of the culture solution 4 is controlled to be constant.

  The electrolyte solution 4a accommodated in the counter electrode tank 8 preferably contains a composition that can generate an ionic current between the working electrode 9 and the counter electrode 10, for example, sodium ions or potassium ions. . In addition, since the culture solution 4 normally contains sodium ions and potassium ions, the culture solution 4 containing these ions may be used as the electrolyte solution 4a.

  The oxidation-reduction substance 3 used in the present invention is a substance that can reversibly cause an oxidation-reduction reaction with the working electrode 9 immersed in the culture solution 4 as in the case of a conventional electric culture apparatus, and is a microorganism to be cultured. A substance that is not toxic to 2 can be used. For example, a general iron ion is mentioned as a soil component as mentioned above. Here, in order to allow iron ions to stably exist in the culture solution, it is preferable that iron ions be coordinated with the chelating agent and added to the culture solution. As the chelating agent, any chelating agent capable of coordinating iron ions can be used. For example, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), tetraethylenetriamine (TET), ethylenediamine (EDA), diethylenetriamine (DETA), citric acid, oxalic acid, crown ether, nitrilotetraacetic acid, disodium edetate, sodium edetate, trisodium edetate, penicillamine, trisodium pentetate calcium, pentetate, succil and edet Mention may be made of acid trientine. In addition to iron ions, quinone compounds such as potassium ferrocyanide and sodium anthraquinone disulfonate, and methyl viologen can be used. These substances also reversibly change into an oxidized form and a reduced form by an oxidation-reduction reaction. In particular, a quinone compound is a substance known as one of the soil components and is preferable. That is, by adding the soil itself to the culture solution, the oxidation-reduction potential of the culture solution may be controlled by the redox substance 3 contained in the soil. However, the oxidation-reduction substance 3 is not limited to the above-described substances.

  The amount of the redox substance 3 added to the culture solution 4 is not particularly limited as long as the redox potential of the culture solution 4 can be controlled, but the concentration of the redox substance in the culture solution 4 is 0.1 to 100 mmol / L. Is preferable. If the redox substance concentration is too low, the redox potential may not be sufficiently controlled. If the redox substance concentration is too high, the redox substance 3 cannot be completely dissolved in the culture solution 4 or microorganisms 2 may cause growth inhibition.

The oxidation-reduction potential of the culture solution 4 is expressed by the Nernst equation shown in Equation 1.
[Formula 1] E = E ₀ + RT / nF × ln (C _ox / C _red )

In Equation 1, E is the redox potential of the solution, E ₀ is the standard redox potential of the redox material, R is the gas constant, T is the absolute temperature, n is the number of reaction electrons, F is the Faraday constant, and C _ox is the oxidant. And C _red is the concentration of the reductant. By controlling the ratio of the oxidant concentration of the redox substance 3 and the reductant concentration contained in the culture solution 4 to be constant, the oxidation of the culture solution 4 itself The reduction potential can be controlled. That is, the redox material 3 is an element that determines the redox potential of the culture solution 4, and the composition of the culture solution 4 hardly affects the redox potential.

  The tripolar potential control device 12 measures a potential difference between the working electrode 9 and the reference electrode 11, and passes a current between the working electrode 9 and the counter electrode 10 so that the measured potential difference reaches a set potential. Thus, no current flows through the reference electrode 11. In this way, the potential 9 of the working electrode 4 is controlled to a constant potential, and the ratio of the oxidant concentration to the reductant concentration of the redox substance 3 is controlled to be constant. Control is constant. Here, the oxidation-reduction potential of the culture solution 4 corresponds to the potential of the working electrode 9. Therefore, the set potential, that is, the potential of the working electrode 9 with respect to the reference electrode 11 corresponds to the oxidation-reduction potential of the culture solution 4. However, when the amount of current between the working electrode 9 and the counter electrode 10 necessary for setting the oxidation-reduction potential of the culture solution 4 is known in advance, a reference electrode 11 is provided as shown in FIG. The oxidation-reduction potential of the culture solution 4 can be controlled without any problem. In addition, when culturing while continuing to regenerate a substance necessary for the microorganism by reducing the substance oxidized by the microorganism or conversely reducing the reduced substance, the redox potential of the culture solution 4 There is no need to control, and a potential difference required to regenerate by reducing or oxidizing a substance oxidized or reduced by a microorganism may be provided between the working electrode 9 and the counter electrode 10.

  The microorganism 2 is activated by culturing the microorganism 2 while controlling the oxidation-reduction potential of the culture solution 4 in the culture tank 7. That is, the function of the microorganism group is enhanced by promoting the growth of the microorganism, and the function of the microorganism group is enhanced by increasing the proportion of the functioning microorganisms. In addition, metabolites may increase as a result of changes in the metabolic mechanism of microorganisms.

  Therefore, activation of the microorganism 2 can be promoted by culturing the microorganism 2 by the electric culture method and the electric culture apparatus of the present invention. And by making the culture tank 7 into the sealed structure, the gas component generated from the counter electrode 10 is not dissolved in the culture solution 4, and the growth state of the microorganism 2 can be improved.

  For example, when a microorganism that produces a gaseous substance is used as the culture target microorganism 2, the produced gaseous substance is added to the culture tank 7 while activating the microorganism 2 while increasing the production amount (production rate) of the gaseous substance. No leakage to the outside. The gaseous substance stays in a space in the culture tank 7 where the culture solution 4 does not exist, for example, a space (head space) above the liquid level of the culture solution 4 in the culture tank 7 in FIG. Gaseous material can be recovered from the space. Therefore, the substance produced by the microorganism 2 can be efficiently recovered.

  Examples of microorganisms that produce gaseous substances include methane-oxidizing bacteria that produce methane gas from hydrogen and carbon dioxide, but the microorganisms to be cultured in the present invention are not limited thereto.

  Further, for example, when a microorganism that produces a volatile substance is the microorganism 2 to be cultured, the produced volatile substance is increased while increasing the production amount (production rate) of the volatile substance by activating the microorganism 2. Leakage to the outside of the culture tank 7 is eliminated. And a volatile substance melt | dissolves in the culture solution 4, and a part is volatilized, for example, the space above the liquid level of the culture solution 4 of the culture tank 7 in FIG. Stay in space (headspace). Therefore, the culture solution 4 is collected, and only the volatile substance is separated and collected from the culture solution 4 by, for example, distillation, or the volatile substance that is volatilized and stays in the head space as a gaseous substance is collected. Thus, the substance produced by the microorganism 2 can be efficiently recovered.

  Examples of microorganisms that produce volatile substances include Clostridium acetobutylicum that produces ethanol, butanol, and acetone from glucose, but the microorganisms that are the subject of culture in the present invention are not limited thereto.

  Furthermore, the electro-cultivation apparatus of the present invention can be used for cases other than the production of substances by microorganisms. For example, when a harmful substance such as an environmental pollutant is decomposed by a microorganism, if the harmful substance has volatility, the harmful substance is removed from the culture tank 7 when the harmful substance is decomposed while culturing the microorganism. Volatilizes. Therefore, by culturing the microorganism 2 by adding a volatile harmful substance to the culture solution 4 by the electrocultivation method and the electroculture apparatus of the present invention in which the culture tank 7 has a sealed structure, the volatile harmful substance is cultured. The microorganism 2 can be decomposed without leaking to the outside of the tank 7.

  Microorganisms that decompose volatile harmful substances include microorganisms that decompose volatile organochlorine compounds, such as tetrachlorethylene (PCE) dechlorination microorganisms (eg, Desulfitobacterium, Dehalococcoides, Dehalobacter, Geobacter, etc.), dichloroethylene (DCE) dechlorination Examples include microorganisms (such as Dehalococcoides) and vinyl chloride (VC) dechlorinated microorganisms (such as Dehalococcoides), but the microorganisms to be cultured and harmful substances to be decomposed in the present invention are not limited thereto. .

  In the case where it is desired to recover the entire amount from the culture solution 4 without volatilizing the volatile substance in the culture tank 7 or when the volatile harmful substance is decomposed, the gaseous substance is contained in the culture tank 7. The inside of the container 20 may be completely filled with the culture solution 4 without forming a staying space.

  Moreover, in this embodiment, the gas exhaust pipe 15a which guide | induces the gaseous substance which retains in the space (head space) above the liquid level of the culture solution in the culture tank 7 out of a culture tank (outside of an electric culture apparatus). The gas discharge pipe 15a can be opened and closed by a valve 15b, and the gaseous substance in the culture tank 7 is recovered by the gas recovery means 15. However, the gas recovery method is not limited to this method. For example, an opening is provided in the upper part of the culture tank 7 and the opening is closed with an elastic material such as a synthetic rubber (eg, silicone rubber). The substance may be recovered. An elastic material such as synthetic rubber closes the hole when the injection needle is pulled out, so that when the gaseous substance is not collected, the sealed state of the culture tank 7 is maintained even if the injection needle is pulled out.

  Furthermore, in the present embodiment, a culture solution discharge pipe 16 a that guides the culture solution 4 in the culture vessel 7 to the outside of the culture vessel 7 is provided 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 the culture solution collection means 16 that allows the discharge pipe 16a to be opened and closed by a valve 16b. However, the method for collecting the culture solution 4 is not limited to this method, and similarly to the above, the culture tank 7 is provided with an opening, closed with an elastic material such as synthetic rubber, and stabbed with a syringe needle and cultured. The liquid 4 may be collected. Alternatively, a tube having both ends opened may be connected to a syringe at one end, and the other end may be immersed in the culture solution 4 to collect the culture solution 4 through the tube.

  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, the nutrient source necessary for the microorganism 2, the substance necessary for the microorganism 2 to produce the target substance, and the state of the culture solution 4 that fluctuates with the growth and metabolism of the microorganism 2 are maintained in a constant state. In order to do so, a neutralizing agent, the culture solution 4 itself, and the like can be added as necessary. In addition, gas can be supplied to maintain the culture environment anaerobic or aerobic. However, the means for adding and supplying the substance to the culture solution 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. 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.

  According to the electric culture method and the electric culture apparatus of the present invention, since the culture tank 7 has a sealed structure, the gas supplied into the culture tank 7 does not leak out of the culture tank 7. Therefore, the culture environment can be easily controlled to be anaerobic or aerobic, and the culture environment of the culture solution 4 in the culture tank 7 can be managed as it is without housing the electric culture apparatus itself in a box as in the prior art. Therefore, the culture environment can be easily managed as compared with the conventional method in which the electric culture apparatus itself is accommodated in the box, and the time and labor for sterilizing the box when culturing again can be omitted.

  Next, a second embodiment of the electroculture apparatus of the present invention will be described. In the following embodiments, the reference electrode 11 is omitted, but the reference electrode 11 may be used to strictly control the oxidation-reduction potential of the culture solution 4. The electric culture apparatus 1a shown in FIG. 3 has two tanks opened by partitioning a container 23 whose upper side is opened by an ion exchange membrane 6, and the upper open part of one tank as the culture tank 7 is The gas impervious film or the gas impervious member 24 is used. In this case, the gas derived from the microorganisms generated from the culture tank 7 while preventing the gas generated from the counter electrode tank 8 from entering the culture tank 7 by only slightly modifying the conventional electric culture apparatus. The substance can be prevented from leaking from the culture tank 7.

  As the gas-impermeable film or gas-impermeable member, 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.

  The counter electrode tank 8 may be left open, but has a sealed structure like the culture tank 7 and includes a gas discharge pipe for discharging the gas generated in the counter electrode tank 8 to the outside of the counter electrode tank 8. It may be. In this case, since the gas generated from the counter electrode tank 8 can be discharged from a desired position, it can be recovered and reused.

  Next, a third embodiment of the electroculture apparatus of the present invention will be described. The electroculture apparatus 1b 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 through 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. And since one container 25a is made into the airtight structure, the gaseous substance derived from the microorganisms which generate | occur | produces from the culture tank 7 is culture | cultivating, preventing the gas generated from the counter electrode tank 8 invading into the culture tank 7. Leakage from the tank 7 can be prevented.

  Note that the opening of the other container 25b in the present embodiment refers to, for example, a case where the end of the other container 25b is completely opened, as well as a sealed structure similar to the one container 25a, while the counter electrode tank 8 is open. This also includes the case where a gas discharge pipe for discharging the gas generated in the above is discharged outside the counter electrode tank 8. When the gas discharge pipe is provided, the gas generated from the counter electrode tank 8 can be discharged from a desired position, so that it can be easily recovered and reused.

  Next, a fourth embodiment of the electroculture apparatus of the present invention will be described. The electroculture apparatus 1c shown in FIG. 5 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 accommodated are connected through the ion exchange membrane 6 at 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. And since one container 26a of the H-shaped container 26 is made into the sealed structure, the culture tank 7 becomes a sealed structure. Therefore, it is possible to prevent the gaseous substance derived from the microorganisms generated from the culture tank 7 from leaking from the culture tank 7 while preventing the gas generated from the counter electrode tank 8 from entering the culture tank 7.

  In the present embodiment, the opening of the other container 26b is not limited to the case where the container 26 is completely opened. This also includes the case where a gas discharge pipe for discharging outside the electrode tank 8 is provided. When the gas discharge pipe is provided, the gas generated from the counter electrode tank 8 can be discharged from a desired position, so that it can be easily recovered and reused.

  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, in the electroculture apparatus shown in FIGS. 1 to 5, the working electrode 9 and the counter electrode 10 are arranged apart from the ion exchange membrane 6, but the working electrode 9 and the counter electrode 10 with respect to the ion exchange membrane 6 are arranged. The position is not particularly limited. For example, electroculturing may be performed by bringing either one or both of the working electrode 9 and the counter electrode 10 into contact with the ion exchange membrane 6. For example, even if the ion exchange membrane 6 is sandwiched between the working electrode 9 and the counter electrode 10, the potential of the culture solution 4 can be controlled. When the working electrode 9 and the counter electrode 10 are brought into contact with the ion exchange membrane 6, the contact area between the working electrode 9, the ion exchange membrane 6 and the culture solution 4, the counter electrode 10, the ion exchange membrane 6 and the electrolytic solution 4a. In order to increase the contact area with the electrode and improve the progress of the electrochemical reaction, it is preferable that the electrode be a porous body.

  Further, when the ion exchange membrane 6 and the counter electrode 10 are brought into contact with each other, the electrochemical reaction can proceed without using the electrolytic solution 4a. Therefore, in this case, electroculturing may be performed without using the electrolyte solution 4a. In addition, when electroculturing is performed without using the electrolyte solution 4a, the counter electrode 10 is used as a porous body, and the gas generated at the contact surface between the ion exchange membrane 6 and the counter electrode 10 is opposite to the counter electrode 10. It is preferable to make it easy to pass through the surface.

  Further, a membrane filter that does not allow the microorganism 2 to pass is provided in the middle of the culture solution discharge pipe 16 of the culture solution collecting means 16, and only the culture solution 4 is cultured without discharging the microorganism 2 in the culture vessel 7 to the outside of the culture vessel 7. You may make it discharge | emit out of the tank 7. FIG. In this case, it is possible to recover the volatile substance contained in the culture solution 4, that is, the volatile substance produced by the microorganism 2, while holding the microorganism 2 in the culture tank 7 for a long period of time. In addition, since the microorganism 2 in the culture tank 7 can be retained for a long period of time, the ability to decompose the substance of the microorganism 2 can be demonstrated for a long period of time, and the volatile substance can be efficiently decomposed over a long period of time. Become.

  Moreover, the microorganisms that can be cultured in the electric culture apparatus of the present invention are not limited to the above-mentioned microorganisms. For example, hydrogen bacteria and photosynthetic bacteria that are gas-assimilating bacteria can be cultured. The electric culture apparatus of the present invention has a closed structure in which a culture tank that accommodates microorganisms to be cultured is sealed. Therefore, when cultivating microorganisms that require gaseous substances or volatile substances during growth, these substances can be used. It has an excellent advantage that it can be supplied to microorganisms without waste without leaking out of the culture tank. The hydrogen bacterium is a microorganism that grows while oxidizing carbon under aerobic conditions and fixing carbon dioxide using energy generated by the reaction, and can be used for producing useful substances from carbon dioxide. The photosynthetic bacterium is a microorganism that grows using hydrogen sulfide or mercaptan (cause of malodor) as an electron donor and carbon dioxide as a carbon source. The photosynthetic bacteria can also produce hydrogen using organic matter (waste) as a carbon source. Furthermore, microorganisms that decompose gaseous alkanes such as methane, ethane, and propane, and microorganisms that decompose C1 compounds such as methane, methanol, and formaldehyde that is a causative substance of sick house syndrome are also included in the present invention. It is possible to cultivate in an electrocultivation apparatus.

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

(Experimental device)
Experiments were performed using the electroculture apparatus 1 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 hermeticity when it was passed through wiring, electrodes, and tubes. 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. Specifically, a cation exchange membrane (Nafion K) is processed by thermocompression bonding with a heat sealer to form a bag-like container 21 having an upper opening, and the electrolytic solution 4a is accommodated in the bag 21 and a carbon electrode ( The counter electrode 10) was accommodated and immersed in the electrolyte solution 4a. Then, a wiring 31 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 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 (plate-like carbon electrode) are 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 are passed through a silicone rubber stopper provided on the lid 30. Pulled out 20 outside. The reference electrode 11 (RE-1B, BSS 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 potential controller (potentiostat) 12 so that the potential of the culture solution 4 could be strictly controlled. The other end of the culture solution collection tube 16 was connected to a syringe so that the culture solution could be collected.

Example 1
As the microorganism 2, Clostridium acetobutylicum NBRC13948 (hereinafter simply referred to as Clostridium) was used. This microorganism is an obligate anaerobic microorganism capable of producing ethanol, butanol, acetone, acetic acid and butyric acid from glucose under anoxic conditions. The culture temperature was 30 ° C. In addition, Fe (III) -EDTA was used as the redox material 3 and the concentration of the redox material in the culture solution 4 was 2 mM. Table 1 shows the components of the culture solution 4 used in the experiment. The composition of the electrolytic solution 4a was obtained by removing Fe (III) -EDTA from the composition shown in Table 1. The culture solution 4 is agitated with a stirrer 34, two injection needles are inserted into a silicone rubber stopper provided on the lid 30, and N ₂ gas is aerated for one hour from one of the injection needles, thereby reducing the head space to N. Replaced with ₂ gases.

  The energization potential of the culture solution 4, that is, the oxidation-reduction potential, was set to + 0.4V, + 0.1V, −0.2V, −0.5V, and −0.8V. In addition, as a control experiment, an experiment was also performed when no energization was performed.

  FIG. 8 shows changes with time in cell density when culturing was performed for 60 hours. In FIG. 8, ◆ indicates the experimental result without current supply, ■ indicates the experimental result of + 0.4V, ▲ indicates the experimental result of + 0.1V, × indicates the experimental result of −0.2V, * indicates The experimental result of -0.5V is shown, and ● indicates the experimental result of -0.8V. From the results shown in FIG. 8, it was revealed that the growth of C. acetobutylicum can be controlled by controlling the potential of the culture solution.

  Next, the ethanol concentration of the culture solution was measured by apparatus name: liquid chromatography (HITACHI, ELITE Lachrome). The results are shown in FIG. From the results shown in FIG. 9, it was confirmed that the ethanol production amount can be improved particularly in the case of +0.4 V compared to the case where no energization is performed. When the growth results shown in FIG. 8 and the measurement results of ethanol production shown in FIG. 9 are comprehensively determined, + 0.4V controls the intracellular metabolism of clostridium to improve the ethanol production per cell. It became clear that we could do it. As described above, it was confirmed that the relationship between the production amount of ethanol, which is a volatile substance, and the oxidation-reduction potential of the culture solution can be sufficiently analyzed by using the electroculture apparatus of the present invention.

From the above results, it was confirmed that the electroculture apparatus of the present invention can culture microorganisms while controlling the oxidation-reduction potential while having sealing properties.

1, 1a, 1b, 1c Electrical culture apparatus 2 Culture target microorganism 3 Redox material 4 Culture liquid 4a Electrolytic solution 6 Ion exchange membrane 7 Culture tank 8 Counter electrode tank 9 Working electrode 10 Counter electrode 12 Power supply (potential control device)
15 Gas recovery means 16 Culture fluid collection means 20 Container 21 Small container 22 Gas exhaust pipe 23 Container 24 Gas impermeable membrane, gas impermeable member 25 U-shaped structure container 25a One container constituting a U-shaped structure container 25b The other container 26 constituting the U-shaped structure container 26a The H-shaped structure container 26a The one container 26b constituting the H-shaped structure container The other container constituting the H-shaped structure container

Claims (10)

Two tanks partitioned by an ion exchange membrane, a working electrode and a counter electrode, and a power source for providing a potential difference between the working electrode and the counter electrode, and one tank of the two tanks A culture medium containing a redox substance is accommodated as a culture tank, the working electrode is immersed in the culture liquid, an electrolyte is accommodated while the other tank of the two tanks is used as a counter electrode tank, and the pair In an electric culture apparatus in which an electrode is immersed in the electrolytic solution, a microorganism to be cultured is added to the culture solution, and the microorganism is cultured while applying a potential difference between the working electrode and the counter electrode by the power source. The culture tank is a sealed container, and a small container having a sealed structure that can be accommodated in the container is used as the counter electrode tank. The small container includes at least a part of the ion exchange membrane and is generated from the counter electrode tank. gas And a gas discharge pipe for discharging to the outside of said container, said culture liquid is in contact with the electrolyte through the ion-exchange membrane, in contact with the counter electrode wherein the culture solution through the ion exchange membrane And / or that the working electrode is in contact with the electrolyte solution through the ion exchange membrane, thereby enabling generation of an ionic current between the working electrode and the counter electrode. An electroculture apparatus characterized by the above. The porous working electrode is in contact with the ion exchange membrane, the culture fluid is in contact with the ion exchange membrane at the contact surface between the working electrode and the ion exchange membrane, and the culture fluid and the working electrode are in contact with the ion exchange membrane. The electroculture apparatus according to claim 1, which is in contact with the electrolytic solution and / or the counter electrode via an ion exchange membrane. The porous counter electrode is in contact with the ion exchange membrane, the electrolyte solution is in contact with the ion exchange membrane at the contact surface between the porous counter electrode and the ion exchange membrane, and the electrolyte solution and the counter electrode are in contact with each other. The electroculture apparatus according to claim 1 or 2, wherein an electrode is in contact with the culture solution and / or the working electrode via the ion exchange membrane. The electroculture apparatus according to claim 1 or 2 , wherein the electrolyte solution is not accommodated in the counter electrode tank and the porous counter electrode is in contact with the ion exchange membrane. The electroculture apparatus according to any one of claims 1 to 4, wherein the small container is a bag-shaped container entirely formed of the ion exchange membrane. The electric culture apparatus according to any one of claims 1 to 5 , further comprising gas recovery means for recovering a gaseous substance staying in a space above the liquid level of the culture solution accommodated in the culture tank. The electroculture apparatus according to any one of claims 1 to 6 , further comprising a culture solution collecting unit that collects the culture solution contained in the culture tank. A microorganism that produces a gaseous substance or a microorganism that produces a volatile substance is cultured by the electric culture apparatus according to claim 6 , and a gas component generated by volatilization of the gaseous substance or the volatile substance is removed from the culture tank. A substance production method characterized in that the material is retained in a space above the liquid level of the culture solution, and is collected by the gas recovery means of the electric culture device. A microorganism that produces a volatile substance is cultured by the electric culture apparatus according to claim 7, and the volatile substance produced by the microorganism is recovered from the culture solution collected from the culture medium collecting means of the electric culture apparatus. A material production method characterized by A microorganism that decomposes volatile harmful substances is cultured by the electric culture apparatus according to any one of claims 1 to 7 , and the harmful substances added to the culture solution in the culture tank are decomposed. A material decomposition treatment method characterized by the above.