JP5832732B2 - Method for culturing microorganisms producing nitrite - Google Patents

Description

The present invention relates to a method for culturing a nitrite-producing microorganism .

  The nitrogen cycle by microorganisms in the natural environment is shown in FIG. As shown in FIG. 20, nitrogen has various oxidation-reduction reactions in the natural environment and exists in various forms.

  At present, various methods for treating ammonium ions, nitrate ions, and nitrite ions contained in wastewater (drainage) using the nitrogen cycle by such microorganisms have been proposed (see, for example, Patent Document 1). .

JP 2000-237771

When wastewater treatment is performed using a nitrogen cycle by microorganisms,
It is important that microorganisms having a function necessary for the treatment are selectively present at a high concentration in the treatment tank to function. However, in the conventional wastewater treatment method using the nitrogen cycle by microorganisms, there is a limit to allowing the microorganisms having the functions necessary for treatment to function selectively in a treatment tank at a high concentration. It was desired.

The present invention is selective for a nitrous acid-reducing microorganism that reduces nitrite ions to nitrogen gas or a nitrite-oxidizing microorganism that oxidizes nitrite ions to nitrate ions, which are nitrogen metabolism microorganisms that are particularly important in the nitrogen cycle by microorganisms. An object of the present invention is to establish a method for activating a nitrogen-metabolizing microorganism that can be made to function in a treatment tank or the like at a high concentration, and to provide a method for culturing a nitrite-producing microorganism using this method And

  As a result of intensive studies by the inventors of the present invention to solve this problem, paddy soil is added to the culture solution, and the electrode is immersed in the culture solution to which the redox substance is added to control the potential of the electrode to a certain value. Thus, it was found that the rate at which nitrite ions are reduced to nitrogen gas is significantly accelerated as compared with the case where no potential is applied to the electrode. It is also clear that controlling the electrode potential to a value different from the above significantly accelerates the rate at which nitrite ions are oxidized to nitrate ions compared to when no potential is applied to the electrodes. became.

  In addition, the microbial flora of the culture solution and the rate at which nitrite ions are oxidized to nitrate ions are significantly increased when the culture is performed at a potential at which the rate at which nitrite ions are reduced to nitrogen gas is significantly promoted. Genetic analysis was performed on the microflora of the culture medium when the culture was carried out under the controlled potential. As a result, the microbial flora of the culture solution when cultivated by controlling the potential at which the rate at which nitrite ions are reduced to nitrogen gas is remarkably accelerated has nitrite reduction ability (denitrification ability). It became clear that the number of microorganisms per se increased as compared with the case where the number of microorganisms was not applied. In addition, in the microflora of the culture medium when the culture is performed at a potential at which the rate at which nitrite ions are oxidized to nitrate ions is remarkably accelerated, a group of microorganisms capable of oxidizing nitrite dominate. It was also found that the number of microorganisms itself increased as compared with the case where no potential was applied.

  From these results, the potential of the electrode immersed in the culture solution was used as an index of the reduction rate of nitrite ion to nitrogen gas or the oxidation rate of nitrate ion of nitrite ion when nitrite ion was dissolved in the culture solution. By controlling the above, it has been found that microorganisms having nitrite reducing ability and microorganisms having nitrite oxidation ability, which are nitrogen-metabolizing microorganisms, can be selectively activated (growth promotion and further function improvement). The present invention has been completed through repeated studies.

Based on such knowledge, the method for culturing a nitrite-producing microorganism of the present invention includes a nitrogen-metabolizing microorganism and nitrogen metabolism of at least one of a nitrite-reducing microorganism having a nitrite-reducing ability and a nitrite-oxidizing microorganism having a nitrite-oxidizing ability. The rate of reduction of nitrite ions when the electrode is immersed in a culture solution containing an organic substance and a redox substance as a carbon source for microorganisms and nitrite ions are dissolved in the culture solution is higher than when no potential is applied to the electrode. A nitrite-producing microorganism that produces nitrite ions is added to a culture solution containing nitrogen-metabolizing microorganisms activated by culturing the nitrogen-metabolizing microorganisms while applying a potential that improves to the electrode. The generated nitrite ions are reduced or reduced by activated nitrogen-metabolizing microorganisms, and the growth of nitrite is avoided while avoiding growth inhibition due to toxicity of nitrite ions. So that culturing the thing. Therefore, the growth of the nitrite-producing microorganism can be favorably cultured without being inhibited by the toxicity of the nitrite ions produced by the nitrite-producing microorganism itself.

Here, in the method for culturing a nitrous acid-producing microorganism of the present invention, it is preferable that the electrolyte solution is brought into contact with the culture solution via an ion exchange membrane, and the electrode paired with the electrode immersed in the culture solution is immersed in the electrolyte solution. . Moreover, it is preferable to contact the electrode which makes a pair with the electrode immersed in the culture solution through the ion exchange membrane. In this case, the oxidation-reduction substance in the culture solution can be prevented from coming into contact with the electrode paired with the electrode immersed in the culture solution, and the potential (oxidation-reduction potential) of the culture solution can be easily controlled.

Further, in the method for cultivating a nitrite-producing microorganism of the present invention, the potential applied to the electrode is set to a potential that improves the rate of generation of nitrogen gas as a result of improving the rate of reduction of nitrite ions, thereby reducing nitrite. Microorganisms can be activated. The potential applied to the electrode is preferably set to a larger potential on the negative side than -0.6 V including -0.6 V on the basis of the silver / silver chloride electrode potential.

Further, in the method for culturing a nitrite-producing microorganism of the present invention, the potential applied to the electrode is set to a potential that improves the nitrate ion production rate as a result of improving the reduction rate of nitrite ions. Can be activated. The potential applied to the electrode is preferably set to a potential larger on the plus side than +0.6 V including +0.6 V on the basis of the silver / silver chloride electrode potential.

According to the culture method of a nitrite-producing microorganism of the present invention, it is possible to first activate a nitrogen-metabolizing microorganism. In other words, it is possible to selectively promote the growth of nitrite-reducing microorganisms or nitrite-oxidizing microorganisms, and to selectively improve the nitrite-reducing ability of nitrite-reducing microorganisms or the nitrite-oxidizing ability of nitrite-oxidizing microorganisms. It becomes. Therefore, for example, using a sample containing various microorganisms such as a soil sample, nitrite-reducing microorganisms or nitrite-oxidizing microorganisms are selectively grown to be present at a high concentration in the culture solution. By selectively improving the nitrite reducing ability of the microorganism or the nitrite oxidizing ability of the nitrite oxidizing microorganism, it becomes possible to make the nitrite ion treating ability of the culture solution itself as a microbial treatment tank extremely high. .

Since the activated nitrite-reducing microorganisms or nitrite-oxidizing microorganisms are used to oxidize or reduce nitrite ions produced by nitrite-producing microorganisms, the concentration thereof is reduced. The growth is not hindered by the toxicity of nitrite ions produced by the microorganisms themselves, and the nitrite-producing microorganisms can be cultured well. For example, the growth of a microorganism having nitrate respiration ability (for example, Escherichia coli, etc.) that reduces nitrate ions to nitrite ions by respiration is inhibited by the nitrite ions generated by the nitrate respiration. By using this method, it is possible to suppress the growth inhibition by nitrite ions and to perform good culture.

It is a figure which shows the concept of the activation method of the nitrogen metabolism microorganisms of this invention. It is sectional drawing which shows an example of the bioreactor concerning 1st Embodiment A. It is sectional drawing which shows an example of the bioreactor concerning 1st Embodiment B. It is sectional drawing which shows an example of the bioreactor concerning 1st Embodiment C. It is sectional drawing which shows an example of the bioreactor concerning 1st Embodiment D. It is sectional drawing which shows an example of the bioreactor concerning 2nd embodiment. It is sectional drawing which shows an example of another structure of a bioreactor. It is a conceptual diagram of the apparatus used for experiment. It is a figure which shows the structure of the small container of the apparatus used for experiment. It is a figure which shows the oxidation-reduction characteristic of an ammonium ion. It is a figure which shows the oxidation-reduction characteristic of nitrite ion. In Example 3, it is a figure which shows the time-dependent change of various substances in the case of no electricity supply. In Example 3, it is a figure which shows the time-dependent change of various substances in the case of + 0.2V. It is a figure which shows the time-dependent change of the various substances in Example 3 in the case of + 0.6V. In Example 3, it is a figure which shows the time-dependent change of various substances in the case of -0.6V. It is a figure which shows the fixed_quantity | quantitative_assay result of the total bacteria count in Example 4. FIG. It is a figure which shows the quantification result of mirK gene in Example 4. It is a figure which shows the quantification result of nirS gene in Example 4. It is a figure in Example 5 which shows the result of the microflora analysis in a culture solution. It is a figure which shows the oxidation-reduction reaction in connection with the nitrogen cycle by the microorganisms in a natural environment.

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

  In FIG. 1, an example of embodiment of the activation method of the nitrogen metabolism microorganisms of this invention is shown notionally. The method for activating a nitrogen-metabolizing microorganism according to the present invention comprises oxidizing at least one of the nitrogen-metabolizing microorganism 2, the organic substance 5, and the nitrite-reducing microorganism 2a having a nitrite-reducing ability and the nitrite-oxidizing microorganism 2b having a nitrite-oxidizing ability. When the electrode 9 (working electrode 9) is immersed in the culture solution 4 containing the reducing substance 3 and the nitrite ions are dissolved in the culture solution 4, the reduction rate of nitrite ions does not apply a potential to the electrode 9 The nitrogen-metabolizing microorganism 2 is cultured while applying a potential that is further improved to the electrode 9.

  In the present invention, as the nitrogen-metabolizing microorganism 2, at least one of a nitrite-reducing microorganism 2a having a nitrite-reducing ability and a nitrite-oxidizing microorganism 2b having a nitrite-oxidizing ability is included in the culture solution 4.

  The microorganism 2a having the ability to reduce nitrite is not particularly limited as long as it is a microorganism having the ability to reduce nitrite ions, but the microorganism having the ability to reduce (denitrify) nitrite ions to nitrogen gas is not limited. Particularly preferred, for example, it is preferable to use microorganisms belonging to the genera Pseudomonas, Alicycliphilus and Tissierella.

  The microorganism 2b having the ability to oxidize nitrite is not particularly limited as long as it has the ability to oxidize nitrite ions to nitrate ions, but it is preferable to use microorganisms belonging to the genus Burkholderia. is there.

  In the present invention, as the nitrogen-metabolizing microorganism 2, a commercial product or a cultured product of an isolated microorganism may be used, but a microorganism-containing soil sample such as paddy soil, a sludge sample, or the like can be used. That is, since the microorganism 2a having the ability to reduce nitrite and the microorganism 2b having the ability to oxidize nitrite are generally present in soil and sludge, the soil sample and the sludge sample are added to the culture solution 4. By doing so, the nitrogen-metabolizing microorganisms 2 can be selectively grown to be present in the culture solution 4 at a high concentration. In other words, nitrogen-metabolizing microorganisms can be accumulated and cultured from soil samples and sludge samples.

The composition of the culture solution 4 may be the composition of a medium generally used for culturing the nitrogen-metabolizing microorganism 2, and is not particularly limited. For example, KH ₂ PO ₄ , MgSO ₄ A medium containing 7H ₂ O, CaCl ₂ .2H ₂ O, and trace elements (Zn, Co, Mn, Cu, Mo, Ni, Se, B, Fe) can be used.

  The organic substance 5 is not particularly limited as long as it is a substance that can be a carbon source of the nitrogen-metabolizing microorganism 2. For example, a carbonate, propionic acid, or the like can be preferably used.

  The redox substance 3 may be any substance that reversibly undergoes a redox reaction with the electrode 9 immersed in the culture solution 4 and that does not exhibit toxicity to the nitrogen-metabolizing microorganism 2. Moreover, it is preferable to use a substance that is not directly involved in the metabolism of nitrogen-metabolizing microorganisms. For example, the chelating agents diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), tetraethylenetriamine (TET), ethylenediamine (EDA), diethylenetriamine (DETA), citric acid, oxalic acid, crown ether, nitrilotetraacetic acid, Edetate disodium, edetate sodium, edetate trisodium, penicillamine, pentetate calcium trisodium, pentetate, succimer and edetate trientine, preferably iron ions coordinated to EDTA, potassium ferrocyanide, sodium anthraquinone disulfonate, etc. Quinone compounds, viologen derivatives such as methyl viologen, and the like can be used, but are not limited thereto.

  In addition, since the quinone compound is a substance known as one of the soil components, the redox material 3 may be allowed to be present in the culture solution 4 by adding a soil sample or a sludge sample. That is, the redox material 3 may be sufficiently contained in the soil sample or the sludge sample. In such a case, it is not necessary to add the redox material 3 to the culture solution 4 separately.

  The amount of the redox substance 3 added to the culture solution 4 is preferably 0.1 to 100 mM, more preferably 1 to 10 mM, and even more preferably several mM. If the amount of the redox substance 3 added is too small, it may be difficult to control the redox potential of the culture solution 4. Moreover, when too much, it may not melt | dissolve in the culture solution 4, or it may have a bad influence on the growth of microorganisms.

In the present invention, the ratio of the oxidant and reductant of the redox substance 3 is controlled by controlling the potential of the electrode 9, and the potential of the culture solution 4 (redox potential) is controlled. The oxidation-reduction potential of the culture solution 4 is expressed by the Nernst equation of Equation 1.
[Formula 1] E = E ₀ + RT / nF × ln (C _ox / C _red )

In Equation 1, E is the redox potential of the culture solution 4, E ₀ is the standard redox potential of the redox substance 3, R is the gas constant, T is the absolute temperature, n is the number of reaction electrons, F is the Faraday constant, C _ox Is the concentration of the oxidized form of the redox substance 3, and C _red is the concentration of the reduced form of the redox substance 3. That is, the oxidation-reduction potential of the culture solution 4 itself is controlled by controlling the potential of the electrode 9 to be constant by controlling the ratio of the oxidant concentration of the redox substance 3 contained in the culture solution 4 and the concentration of the reduced product. can do.

  The electrode 9 is appropriately made of a material capable of reversibly oxidizing / reducing the redox material 3. Specifically, a carbon electrode such as a carbon plate or glassy carbon, a platinum electrode, or the like can be used, but is not limited thereto.

  The potential applied to the electrode 9 (the oxidation-reduction potential of the culture solution 4) is determined by the following procedure. That is, nitrite ions are dissolved in the culture solution 4 and a culture test is performed without applying an electric potential to the electrode 9 to determine the reduction rate of nitrite ions. The rate of nitrite ion reduction can be determined by measuring the time course of nitrite ions during the culture process. Then, various potentials are applied to the electrode 9 and a similar culture test is performed to determine the reduction rate of nitrite ions. If the reduction rate of nitrite ions is improved as compared with the case where the culture test is performed without applying a potential to the electrode 9, the potential corresponds to a potential that can activate the nitrogen-metabolizing microorganism 2. And when the generation rate of nitrogen gas is improving as a result of the improvement of the reduction | decrease rate of nitrite ion, the electric potential is the electric potential which can activate the nitrite reduction microorganisms 2a among the nitrogen metabolism microorganisms 2. It corresponds to. Moreover, when the production | generation rate of nitrate ion is improving as a result of improving the reduction | decrease rate of nitrite ion, the electric potential is the electric potential which can activate the nitrite oxidation microorganisms 2b among the nitrogen metabolism microorganisms 2. It corresponds to.

  According to the experiments by the present inventors, the potential at which nitrite-reducing microorganisms 2a among nitrogen-metabolizing microorganisms 2 can be activated is present at -0.6V to less than + 0.2V based on the silver / silver chloride electrode potential reference. And if it is set to -0.6V, it is confirmed that the nitrite reduction microorganisms 2a among the nitrogen metabolism microorganisms 2 can be activated reliably. In addition, since it was confirmed that the nitrite-reducing microorganism 2a can be activated at -0.6V, the nitrite-reducing microorganism 2a can be activated by setting the potential at which the electrode 9 can easily give electrons to the culture solution 4. it is conceivable that. Therefore, it is preferable that the potential of the electrode 9 is set to a larger potential on the negative side than -0.6 V including -0.6 V on the basis of the silver / silver chloride electrode potential. If it is too high, the electrolysis of water will occur and the pH of the culture solution 4 will fluctuate, which may inhibit the growth of the nitrite-reducing microorganism 2, so that it is more preferably -0.6V to -1.0V. is there.

  Further, according to the experiments by the inventors of the present application, the potential at which the nitrite oxidizing microorganism 2b among the nitrogen-metabolizing microorganisms 2 can be activated is from +0.2 V to +0.6 V on the basis of the silver / silver chloride electrode potential. It is confirmed that, if it is present and +0.6 V, the nitrite oxidizing microorganism 2b among the nitrogen metabolizing microorganisms 2 can be reliably activated. Further, since it was confirmed that the nitrite oxidizing microorganism 2b can be activated at +0.6 V, it is considered that the redox microorganism 2a can be activated by setting the potential at which the electrodes 9 can easily extract electrons from the culture solution 4. It is done. Therefore, it is preferable that the potential of the electrode 9 is set to a potential larger on the plus side than +0.6 V including +0.6 V on the basis of the silver / silver chloride electrode potential, but if the potential is increased too much on the plus side. Since electrolysis of water occurs and the pH of the culture solution 4 fluctuates and there is a risk of inhibiting the growth of the nitrite oxidizing microorganism 2, it is more preferably + 0.6V to + 1.0V. Here, if the potential is larger than +0.7 V on the plus side, nitrite ions are electrically oxidized and decreased, so that nitrite ions are likely to be insufficient. Therefore, when growing the nitrite oxidizing microorganism 2b, it is preferable to set it to + 0.6V to + 0.7V. However, when the nitrite-oxidizing microorganism 2b is present in the culture solution 4 at a sufficiently high concentration, the potential of the nitrite-oxidizing microorganism 2b is larger than that of + 0.7V, and the oxidation by the nitrite-oxidizing microorganism 2b is electrically performed. Nitrite ions may be reduced in combination with oxidation.

  Therefore, according to the method for activating a nitrogen-metabolizing microorganism of the present invention, a microbial sample capable of mixing nitrite-reducing microorganisms 2a and nitrite-oxidizing microorganisms 2b is used as the nitrogen-metabolizing microorganism 2, such as a soil sample or a sludge sample. Even in this case, either the nitrite-reducing microorganism 2a or the nitrite-oxidizing microorganism 2b can be selectively activated.

  In the method for activating a nitrogen-metabolizing microorganism of the present invention, the nitrogen-metabolizing microorganism 2 may be activated while adding nitrite ions to the culture solution 4. If the cell density of 2 is sufficiently high and it is not necessary to grow it, the nitrogen-metabolizing microorganism 2 may be activated by applying only a potential without adding nitrite ions. For example, a potential is applied to the culture solution 4 in which the cell density of the nitrogen-metabolizing microorganism 2 is maintained at a high concentration to activate the nitrogen-metabolizing microorganism 2, and a nitrite ion-containing solution such as a nitrite ion-containing waste solution is used. The nitrite ion concentration in the nitrite ion-containing solution may be efficiently reduced by introducing it into the culture solution 4.

  In the method for activating a nitrogen-metabolizing microorganism of the present invention, an electrode 10 (counter electrode 10) that forms a pair with the electrode 9 is required to control the potential of the electrode 9 (working electrode 9). Here, when the redox substance 3 comes into contact with the counter electrode 10, the controllability of the redox potential of the culture solution 4 is lowered. In order to easily control the potential, it is preferable to provide the ion exchange membrane 6 between the working electrode 9 and the counter electrode 10.

  In the following description, together with the method for activating the nitrogen-metabolizing microorganism of the present invention when the ion exchange membrane 6 is provided between the working electrode 9 and the counter electrode 10, biotechnology for treating nitrite ion-containing liquid using this method is used. Regarding the configuration of the reactor, the first embodiment will be described based on FIGS. 2 to 5, and the second embodiment will be described based on FIG. 6.

<First embodiment>
The method for activating a nitrogen-metabolizing microorganism according to the first embodiment includes at least one of the nitrogen-metabolizing microorganism 2 of at least one of a nitrite-reducing microorganism 2a having a nitrite-reducing ability and a nitrite-oxidizing microorganism 2b having a nitrite-oxidizing ability. When the working electrode 9 is immersed in the culture solution 4 containing the organic substance 5 and the redox substance 3, and the nitrite ions are dissolved in the culture solution 4, the potential for reducing the nitrite ions does not apply a potential to the working electrode 9. Nitrogen-metabolizing microorganisms 2 are cultured while applying a potential that improves more than the case to the working electrode 9. In the present embodiment, the electrolyte solution 4a is brought into contact with the culture solution 4 via the ion exchange membrane 6, and the electrode 10 (counter electrode 10) paired with the working electrode 9 is immersed in the electrolyte solution 4a.

  The configuration of a bioreactor using the method for activating a nitrogen-metabolizing microorganism according to the first embodiment is illustrated in FIGS. The bioreactor 1 shown in FIGS. 2 to 5 has one of two tanks partitioned by an ion exchange membrane 6 as a culture tank 7 and the other tank as a counter electrode tank 8. While the liquid 4 is accommodated, the working electrode 9 and the reference electrode 11 are immersed, and the counter electrode tank 8 is accommodated with the electrolytic solution 4a and the counter electrode 10 is immersed, and the working electrode 9, the counter electrode 10, and the reference electrode 11 are immersed. Is connected to the constant potential setting device 12. The culture solution 4 contains at least one of the nitrogen-metabolizing microorganism 2, the redox substance 3, and the organic substance 5 of the nitrite-reducing microorganism 2 a having nitrite-reducing ability and the nitrite-oxidizing microorganism 2 b having nitrite-oxidizing ability. Thus, the nitrogen-metabolizing microorganisms are cultured while the potential at which the reduction rate of nitrite ions when the nitrite ions are dissolved in the culture solution 4 is higher than when no potential is applied to the working electrode 9 is applied to the working electrode 9. It is supposed to be.

  Thus, 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, and the potential of the culture solution 4 (oxidation-reduction potential) can be set to a desired value. Can be strictly controlled. 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.

  Further, the ion exchange membrane 6 allows not only the redox substance 3 contained in the culture solution 4 but also the nitrogen-metabolizing microorganisms 2 and the organic matter 5 to remain in the culture solution 4 without permeating the counter electrode tank 8, and for culturing. Ions contained in the liquid 4 or ions contained in the electrolytic solution 4 a are permeated to generate an ionic current, and a reaction that complements the oxidation-reduction reaction occurring in the working electrode 9 is generated in the counter electrode 10. Thereby, the ratio of the oxidant and reductant of the redox substance 3 is stably maintained in the culture solution 4, and the potential of the culture solution 4 can be easily controlled over a long period of time. As the ion exchange membrane 6, a material that does not allow the redox material 3 (oxidant and reductant) to pass through is appropriately selected and used.

  Here, in the bioreactor 1 shown in FIGS. 2 to 5, the culture tank 7 has a sealed structure. Therefore, it is easy to control the anaerobic atmosphere of the culture solution 4. That is, since the culture tank 7 has a sealed structure, free oxygen does not enter, so if the free oxygen in the culture tank 7 is substantially eliminated before the culture is started, the anaerobic state is maintained during the culture period. Subsequently, the function of the nitrogen-metabolizing microorganism 2 can be made to function well.

  Moreover, in the bioreactor 1 shown in FIGS. 2-5, the to-be-processed liquid introduction part 18 which introduce | transduces a nitrite ion containing liquid is provided in the culture tank 7, and the nitrite ion containing liquid is added to the culture liquid 4 of the culture tank 7. FIG. I am trying to introduce it. However, the method of introducing the nitrite ion-containing liquid into the culture tank 7 is not limited to this method.

  Furthermore, in the bioreactor 1 shown in FIGS. 2 to 5, the treatment liquid in the culture tank 7 (the nitrite ion concentration of the nitrite ion-containing liquid is reduced below the liquid level of the culture liquid 4 in the culture tank 7. A treatment liquid discharge pipe 16a that guides the treated liquid) together with the culture liquid 4, and the treatment liquid is cultured from the inside of the culture tank 7 by the treatment liquid discharge means 16 that can be opened and closed by a valve 16b. The liquid 4 is discharged together. However, the method for discharging the treatment liquid is not limited to this method.

  Here, in the bioreactor of the present invention, it is preferable to put a carrier capable of supporting the nitrogen-metabolizing microorganism 2 in the culture solution 4. In this case, the nitrogen-metabolizing microorganisms 2 are supported on the carrier, and the treatment liquid and the culture solution 4 are discharged so as to keep the carrier in the culture solution 4, whereby the activated nitrogen-metabolizing microorganisms 2 are stored in the culture tank 7. The function can be utilized for a long time without discharging to the outside. In addition, a filtration membrane or a filtration device for separating the carrier from the treatment solution and the culture solution 4 is provided at a position where the treatment solution and the culture solution 4 pass before being discharged from the treatment solution discharge means 16. It is preferable that the carrier is separated from the treatment liquid discharged from the discharge means 16 and the culture liquid 4 by filtration. As the carrier, a material that can carry the nitrogen-metabolizing microorganism 2 and easily absorbs water, such as a sponge, can be used, but is not limited thereto. For example, a carrier carrying microorganisms may be prepared in advance by a entrapping immobilization method in which the nitrogen-metabolizing microorganisms 2 are immobilized in advance using a gel such as a photocurable resin, and this may be introduced into the culture solution 4.

  Moreover, in the bioreactor 1 shown in FIGS. 2-5, the culture tank 7 is made into the airtight structure, and the nitrogen gas which retains in the space (head space) above the liquid level of the culture solution 4 of the culture tank 7 is culture tank 7 A nitrogen gas discharge pipe 15a led to the outside, and a nitrogen gas discharge means 15 capable of opening and closing the nitrogen gas discharge pipe 15a by a valve 15b. The gas recovery means 15 discharges nitrogen gas that can stay in the head space. However, when the nitrite oxidizing microorganism 2b is activated, no nitrogen gas is generated. In this case, the nitrogen gas discharging means 15 may not be provided. Further, the nitrogen gas staying in the head space may be guided out of the culture tank 7 only by the nitrogen gas discharge pipe 15a.

  A means for adding and supplying a substance to the culture solution 4 may be provided separately from the treatment liquid introduction unit 18. Specifically, an openable and closable substance introduction tube that can add and supply substances to the culture solution 4 from the outside of the culture tank 7 may be provided. In this case, nutrient sources, neutralizing agents, substances necessary for substance production, and the like can be added to the culture solution 4 as necessary. Of course, the microorganism 2 can be supplied from the introduction tube, and the redox material 3 and the organic matter 5 can be introduced. Moreover, gas (nitrogen gas etc.) for making it anaerobic conditions can also be supplied. However, the means for adding and supplying the substance to the culture solution 4 does not necessarily have to be provided, and the treatment liquid introducing unit 18 may be used together as a means for adding and supplying the substance to the culture solution 4. In addition, substances added and supplied to the liquid to be treated may be mixed and supplied to the culture tank 7. Further, a means for adding and supplying a substance to the electrolytic solution 4a may be provided.

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

  Hereinafter, the case of using the bioreactor shown in FIG. 2 will be described as a first embodiment A, the case of using the bioreactor shown in FIG. 3 will be described as a first embodiment B, and the bioreactor shown in FIG. The case of using the bioreactor will be described as a first embodiment C, and the case of using the bioreactor shown in FIG.

(First embodiment A)
In the bioreactor 1 shown in FIG. 2, a sealed container 20 is used as a culture tank 7, and a sealed small container 21 that can be accommodated in the container 20 is used as a counter electrode tank 8. The small container 21 is at least partly an ion exchange membrane. 6 and the culture tank 7 is provided with a treatment liquid introducing section 18, a treatment liquid discharge means 16 and a nitrogen gas discharge means 15.

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

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

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

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

  According to the bioreactor 1 shown in FIG. 2, since the nitrite ion-containing liquid can be treated in the culture liquid 4 containing the activated nitrogen-metabolizing microorganisms 2, the nitrite ion concentration of the nitrite ion-containing liquid is set. It can be reduced efficiently.

(First embodiment B)
In the bioreactor 1 shown in FIG. 3, two tanks opened by partitioning a container 23 whose upper side is opened by an ion exchange membrane 6 are formed, and the upper open part of one tank as the culture tank 7 is a gas. It is assumed that it is blocked by an impermeable film or a gas impermeable member 24. Similarly, the upper open portion of the other tank as the counter electrode tank 8 is also closed by the gas impermeable film or the gas impermeable member 24. The culture tank 7 is provided with a treatment liquid introducing portion 18, a treatment liquid discharge means 16 and a nitrogen gas discharge means 15. In the bioreactor 1 shown in FIG. 3, it is not necessary to completely partition the head space of the culture tank 7 and the head space of the counter electrode tank 8 as long as the container 23 is sealed.

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

  According to the bioreactor 1 shown in FIG. 3, the nitrite ion-containing solution can be treated in the culture solution 4 containing the activated nitrogen-metabolizing microorganisms 2, as in the bioreactor 1 shown in FIG. The nitrite ion concentration of the nitrate ion-containing liquid can be efficiently reduced.

(First embodiment C)
In the bioreactor 1 shown in FIG. 4, two containers 25a and 25b each having an opening below the liquid level of the liquid to be accommodated are connected to each other through the ion exchange membrane 6 through the opening to form a U-shaped container 25. The one container 25a is used as a culture tank 7 with a sealed structure, and the other container 25b is also used 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. According to the bioreactor 1 shown in FIG. 4, the nitrite ion-containing solution can be treated in the culture solution 4 containing the activated nitrogen-metabolizing microorganisms 2, as in the bioreactor 1 shown in FIG. The nitrite ion concentration of the nitrate ion-containing liquid can be efficiently reduced.

(First embodiment D)
In the bioreactor 1 shown in FIG. 5, 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 to connect the H-shaped container 26. The one container 26a is used as a culture tank 7 with a sealed structure, and the other container 26b is also used 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. According to the bioreactor 1 shown in FIG. 5, since the nitrite ion-containing solution can be treated in the culture solution 4 containing the activated nitrogen-metabolizing microorganisms 2, as in the bioreactor 1 shown in FIG. The nitrite ion concentration of the nitrate ion-containing liquid can be efficiently reduced.

<Second Embodiment>
The method for activating a nitrogen-metabolizing microorganism according to the second embodiment includes at least one of the nitrogen-metabolizing microorganism 2 of at least one of the nitrite-reducing microorganism 2a having a nitrite-reducing ability and the nitrite-oxidizing microorganism 2b having a nitrite-oxidizing ability. When the working electrode 9 is immersed in the culture solution 4 containing the organic substance 5 and the redox substance 3, and the nitrite ions are dissolved in the culture solution 4, the potential for reducing the nitrite ions does not apply a potential to the working electrode 9. Nitrogen-metabolizing microorganisms 2 are cultured while applying a potential that improves more than the case to the working electrode 9. In this embodiment, the counter electrode 9 is brought into contact with the culture solution 4 via the ion exchange membrane 6. That is, the method for activating the nitrogen-metabolizing microorganism in the first embodiment is different in that the counter electrode 10 is brought into direct contact with the ion exchange membrane 6 without using the electrolytic solution 4a. However, since the ionic current flows through the ion exchange membrane 6 between the working electrode 9 and the counter electrode 10 without using the electrolytic solution 4a as in the first embodiment, the nitrogen according to the second embodiment. According to the method for activating metabolic microorganisms, it is possible to obtain the same effect by controlling the potential of the working electrode 9 as in the first embodiment.

  A configuration of a bioreactor using the method for activating a nitrogen-metabolizing microorganism according to the second embodiment is illustrated in FIG. In the bioreactor 1 shown in FIG. 6, the working electrode 9 and the reference electrode 11 are arranged in a sealed container 50 having at least a part of the ion exchange membrane 6, and the counter electrode 10 is arranged outside the container 50. 50, the culture solution 4 is accommodated, and the working electrode 9 and the reference electrode 11 are immersed in the culture solution 4. At least a part of the ion exchange membrane 6 of the container 50 is contained when the culture solution 4 is accommodated in the container 50. It is assumed that 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 provided at a position where it can come into contact with the ion exchange membrane 6. In the bioreactor 1 shown in FIG. 6, an opening 50 a is provided below the liquid level of the culture solution 4 in the container 50, the opening 50 a is closed with the ion exchange membrane 6, and the ion exchange membrane outside the container 50. It is assumed that the counter electrode 10 is disposed in contact with at least a part of the surface of 6. That is, in the bioreactor 1 shown in FIG. 6, the entire container 50 functions as the culture tank 7.

  Therefore, according to the bioreactor 1 shown in FIG. 6, since the container 50 has a sealed structure, it is easy to control the anaerobic atmosphere of the culture tank 7 as in the first embodiment.

  In addition, in the bioreactor 1 shown in FIG. 6, the treatment liquid introduction part 18, the treatment liquid discharge means 16 and the nitrogen gas discharge means 15 are provided as in the first embodiment. The treatment liquid introduction method, the treatment liquid discharge method, and the nitrogen gas discharge method are not limited to those using these means. Similarly to the first embodiment, a carrier capable of supporting the nitrogen-metabolizing microorganisms 2 may be placed in the culture solution 4, or the treatment solution and the culture solution 4 may be discharged before being discharged from the treatment solution discharge means 16. Is provided with a filtration membrane or a filtration device that separates the carrier from the treatment liquid and the culture liquid 4, and the carrier is filtered and separated from the treatment liquid discharged from the treatment liquid discharge means 16 and the culture liquid 4. You may make it do. Furthermore, as in the first embodiment, means for adding and supplying substances to the culture solution 4 may be provided.

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

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

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

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

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

  For example, as shown in FIG. 7, the culture solution 4 and the electrolyte 4a are separated not by the ion exchange membrane 6 but by the impervious member 40 that does not allow any permeation of ions or microorganisms, or the culture vessel 7 and the counter electrode vessel 8 are separated. It may be formed in a separate container, and the culture solution 4 and the electrolyte 4a may be brought into contact (liquid junction) via the salt bridge 41 (agar or the like containing a saturated electrolyte solution such as KCl). Also in this case, the flow of ion current is allowed by the salt bridge 41 and the permeation of the redox material 3 to the counter electrode tank 8 can be prevented, so that the same effect as in the above embodiment can be obtained.

  When the nitrite ion-containing solution is treated in the culture solution 4 containing the nitrite-reducing microorganism 2a activated by the method for activating the nitrogen-metabolizing microorganism of the present invention, nitrate ions are contained in the nitrite ion-containing solution. It may be included. In this case, the nitrite reducing microorganism 2a can efficiently reduce both nitrite ions and nitrate ions to nitrogen gas. In addition, the nitrite ion-containing liquid may contain an organic substance 5 that is a carbon source of the nitrogen-metabolizing microorganism 2. In this case, the organic substance 5 contained in the nitrite ion-containing liquid can be reduced or oxidized while being decomposed by the nitrogen-metabolizing microorganism 2 as a carbon source, and is contained in the nitrite ion-containing liquid. The TOC concentration can be reduced simultaneously with the reduction of the nitrite ion concentration.

  In addition, the nitrite-reducing microorganism 2a or the nitrite-oxidizing microorganism 2b activated by the method for activating a nitrogen-metabolizing microorganism of the present invention is used to oxidize or reduce nitrite ions produced by a nitrite-producing microorganism. By reducing the concentration, the growth of the nitrite-producing microorganism can be favorably cultured without being inhibited by the toxicity of the nitrite ions produced by the nitrite-producing microorganism itself. For example, microorganisms having nitrate respiration ability that generate nitrite ions by performing nitrate respiration using nitrate ions as the final electron acceptors are widely present in nature. Specifically, for example, the genus Alcaligenes, the genus Agrobacterium, the genus Aquaspirillum, the genus Azospirillum, the genus Bacillus, the genus Blastobacter, the Bradyrizo Genus Bradyrhizobium, genus Branhamella, genus Chromobacterium, genus Cytophaga, genus Enterobacter, genus Escherichia, genus Flavobacterium, Flexibacter genus, Halobacterium genus, Hyphomicrobium genus, Kingella genus, Klebsiella genus, Lysobacter genus, Neisseiria genus, Paracoccus genus Propionibacterium Propionibacterium, Pseudomonas, Rhizobium, Wolinella, Rhodopseudomonas, Thiobacillus, Nitrosomonas, Thiospira Genus, Thiosphera genus and the like. Where Escherichia coli, Bradyrhyzobium japonicum, Klebsiella pneumonia, Enterobacter aerogenes, Thiobacillus thioparum ys, and Thiobacillus thioparum ys ) Etc. are microorganisms having nitrate respiration ability, in particular, microorganisms in which nitrite ions are likely to be accumulated in the culture solution due to nitrate respiration. Growth is likely to be hindered due to the biotoxicity of nitrite ions and difficult to culture. However, the nitrite-reducing microorganisms 2a or the nitrite-oxidizing microorganisms 2b activated by the method for activating the nitrogen-metabolizing microorganisms of the present invention are used to oxidize or reduce nitrite ions produced by these microorganisms and to reduce their concentrations. These microorganisms can be well cultivated (accumulated culture) without the growth being hindered by the toxicity of nitrite ions produced by these microorganisms. It becomes.

  In particular, when the nitrite oxidizing microorganism 2b is activated by the method for activating a nitrogen-metabolizing microorganism of the present invention, nitrite ions can be oxidized into nitrate ions by the function of the microorganisms. Nitrate ions are supplied to the above microorganisms having nitrate respiration ability, nitrate respiration is performed, nitrite ions generated by nitrate respiration are supplied to the nitrite oxidizing microorganism 2b, and nitrite ions are oxidized to nitrate ions. And the circulation route of nitrite ion can be established. Therefore, a microorganism having nitrate respiration ability can be efficiently cultured together with the nitrite oxidizing microorganism 2b. When the nitrite-reducing microorganism 2a is activated by the method for activating the nitrogen-metabolizing microorganism of the present invention, although the above-mentioned circulation path is not established, nitrate ions are periodically or optionally supplied to the culture solution, Nitrogen respiration can be performed by a microorganism having nitrate respiration ability and cultured with the nitrite-reducing microorganism 2a.

  Further, the nitrite-producing microorganism may be a bacterium (denitrifying bacterium) that converts nitrate ions into nitrogen via nitrite ions alone. That is, by using the nitrite-reducing microorganism 2a or the nitrite-oxidizing microorganism 2b activated by the method for activating the nitrogen-metabolizing microorganism of the present invention, a bacterium that converts nitrate ions to nitrogen via nitrite ions alone (deoxidation). By reducing or oxidizing the nitrite ions produced by the reduction of nitrate ions by nitrifying bacteria, the concentration of the nitrite ions is reduced, so that the growth of the nitrite ions is not hindered by the toxicity of the nitrite ions produced by the denitrifying bacteria. It becomes possible to culture the denitrifying bacteria well. Therefore, it is possible to satisfactorily culture (accumulate culture) some of the denitrifying bacteria that were conventionally difficult to culture. In addition, the culture solution 4 contains bacteria (denitrifying bacteria) that convert nitrate ions to nitrogen via nitrite ions and nitrite-reducing microorganisms 2a, and was activated by the method for activating nitrogen-metabolizing microorganisms of the present invention. By promoting the nitrite reduction reaction using the nitrite-reducing microorganism 2a, the growth of the denitrifying bacteria can be improved, and further, some of the denitrifying bacteria that have conventionally been difficult to culture can be successfully cultured. Since the entire denitrification process can be promoted, it is possible to improve the efficiency of a water treatment process such as waste water containing nitrate ions and nitrite ions using the denitrification reaction.

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

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

<Experimental equipment>
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). 200 mL of culture solution 4 was stored. 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.

  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 (manufactured by DuPont, Nafion K) is processed by thermocompression bonding with a heat sealer to form a bag-like container 21 having an upper opening, and the bag 21 contains the electrolyte 4a. 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 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 9 were drawn out of the container 20 through a silicone rubber stopper provided on the lid 30. 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 treatment liquid discharge pipe 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 liquid 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 treatment liquid discharge pipe 16 was connected to a syringe so that the culture solution 4 could be collected.

<Culture solution>
The culture tank 7 contained a culture solution having the following composition. In the following composition, KHCO ₃ and propionic acid function as a carbon source of the microorganism 2.
[Culture solution composition]
・ KHCO ₃ : 500ppm
・ KH ₂ PO ₄ : 27 ppm
・ MgSO ₄ · 7H ₂ O: 300 ppm
・ CaCl ₂ · 2H ₂ O: 180 ppm
・ Propionic acid: 1 mM
Trace elements (Zn, Co, Mn, Cu, Mo, Ni, Se, B, Fe): 1 mL

<Electrolyte>
It was set as the same component as the culture solution 4 except not containing the redox substance 3 (anthraquinone 2, 6- disulfonic acid: AQDS) mentioned later.

Example 1
The redox properties of ammonium ions were investigated.

By adding 10 mM NH ₄ Cl as ammonium ion to the culture solution 4, using the working electrode 9 as a carbon electrode (manufactured by BAS) and the counter electrode 10 as a platinum rod electrode (manufactured), cyclic voltammetry using the above experimental apparatus Redox characteristics were measured (sweep speed 100 mV / s). The results are shown in FIG. From the results shown in FIG. 10, it was clarified that ammonium ions are not oxidized and reduced in the range of −1.5V to + 1.5V.

(Example 2)
The redox characteristics of nitrite ion were investigated.

By adding 10 mM of KNO ₂ as nitrite ion to the culture solution 4, using the working electrode 9 as a carbon electrode (manufactured by BAS) and the counter electrode 10 as a platinum rod electrode (self-made), cyclic voltammetry using the above experimental apparatus Redox characteristics were measured (sweep speed 100 mV / s). The results are shown in FIG. From the results shown in FIG. 11, it became clear that nitrite ions are oxidized at +0.7 V or higher, but are not oxidized and reduced in the range of −1.5 V to less than +0.7 V.

(Example 3)
2 g of paddy field soil collected from the microorganism 2 (Kukyukuri, April 18, 2009) and 2 mM of anthraquinone 2,6-disulfonic acid (AQDS) as the redox substance 3 were added to the culture solution 4. The working electrode 9 and the counter electrode 10 were carbon electrodes. In addition, 16 sponges made by cutting sponge (made by Towa Sangyo Co., Ltd., trade name: Pikaru-chan cube) into 200 mL culture solution 4 in 10 mm x 10 mm x 15 mm were added to culture solution 4 for the test period. The outflow and decrease of the microorganisms 2 during the withdrawal of the culture medium 4 inside were suppressed.

To the culture solution 4, 50 ppm of KNO ₂ was added as a nitrite ion source, and 50 ppm of (NH ₄ ) ₂ SO ₄ was added as an ammonium ion source. During the test period, KNO ₂ , (NH ₄ ) ₂ SO ₄ , KHCO _{3 as} a carbon source, propionic acid, and trace elements were added periodically. KHCO ₃ , propionic acid, and trace elements were periodically added in the same amounts as the initial values (18 days, 40 days, 60 days). Ammonium ions were not added after setting to about 100 ppm after 5 days. Nitrite ions were sampled and measured approximately every 5 days, and when a decrease was observed, operation was performed with the policy of adding them, and the KNO ₂ load was increased stepwise. Specifically, Day 5: 50 ppm, Day 5 to Day 20: 17.5 ppm, Day 21 to Day 32 35 ppm, Day 33 to Day 48: 70 ppm, Day 49 and after 210 ppm, 56 days Only the eyes increased the load significantly at 525 ppm.

Two injection needles were inserted into the silicone rubber stopper provided in the lid 30 and N ₂ gas was aerated for one hour from one of the injection needles to replace the head space above the liquid level of the culture solution 4 with N ₂ gas. . The culture solution 4 was stirred with a stirrer 34 and a culture test was performed at 37 ° C.

The conditions for the culture test were as follows.
・ No power supply (73 mV)
・ + 0.2V
・ + 0.6V
-0.6V

  The ammonium ion concentration, nitrite ion concentration, and nitrate ion concentration of the culture solution during the test period are ion chromatography (product name: ICS-1500, manufacturer: DIONEX), TOC concentration is TOC analyzer (model number TNC-6000, device name Automatic) TOC analyzer, manufacturer: Toray).

  FIG. 12 shows the test results without energization, FIG. 13 shows the test results at +0.2 V, FIG. 14 shows the test results at +0.6 V, and FIG. 15 shows the test results at -0.6 V. In FIGS. 12 to 15, ♦ represents the ammonium ion concentration, x represents the TOC concentration, ■ represents the nitrate ion concentration, and ▲ represents the nitrite ion concentration.

  In the case of no energization and +0.2 V, the concentration of nitrite ions only increased according to the amount of nitrite ions added. On the other hand, at +0.6 V, there was a tendency for the nitrate ion concentration to increase corresponding to the decrease in the nitrite ion concentration. In addition, at -0.6 V, significant bubble generation from the culture solution 4 was observed corresponding to the decrease in the nitrite ion concentration. From these results, at +0.6 V, nitrite oxidizing bacteria in the culture solution 4 are activated and nitrite ions are oxidized to nitrate ions, and at −0.6 V, nitrite reducing bacteria are activated and nitrite ions are converted to nitrogen. It was thought that it was reduced to gas.

Example 4
Genetic analysis was performed on the number of bacteria in the culture solution and irK and irS, which are genes responsible for the nitrite reduction reaction.

<DNA extraction from culture medium>
After completion of the test, 50 mL of the culture solution 4 was collected and centrifuged to collect the cells. Then, genomic DNA was extracted from the collected cells using Power Microbial Maxi DNA isolation kit (MO BIO), and finally 2 ml of purified DNA was obtained. As reference data, 50 mL of the culture solution 4 before the start of the test was collected, and genomic DNA was extracted as described above.

<Quantification of microorganisms>
Real-time PCR was performed using Light Cycler ST300 (Roche) in order to quantify microorganisms involved in nitrite reduction (denitrification process) contained in the culture solution.

  First, in order to measure the total amount of bacteria contained in the culture solution, 16Sr DNA was quantified by the Taq-Man probe method using a LightCycler TaqMan Master (Roche).

  NirK and irS genes that are genes responsible for the nitrite reduction reaction were quantified by the SYBR Green method using Light Cycler Fast start DNA Master SYBR Green I (Roche).

  For quantification, the Cp value (Crossing point, the point at which the second derivative of the fluorescence intensity increase curve reaches the maximum) is calculated by the second derivative maximum method, and the total number of bacteria is absolute quantified by comparing with the calibration curve. The relative quantification was performed with respect to nirS, assuming that the culture was performed without energization.

  A calibration curve was prepared by performing real-time PCR using a 10-diluted dilution series as a sample with a plasmid (pT7Blue) obtained by cloning the 16S rRNA gene of E. coli JM109 strain amplified with the primers used for real-time PCR as a standard substance.

  Primers, probes, and PCR conditions used in each experiment were as follows.

(1) Whole bacteria Primers and probes were as follows.
・ Uni340F: CCTACGGGRBGCASCAG (SEQ ID NO: 1)
・ Uni806R: GGACTACNNGGGTATCTAAT (SEQ ID NO: 2)
・ Uni516F (TaqMan probe): TGYCAGCMGCCGCGGTAAHACVNRS (SEQ ID NO: 3)
(5'6-FAM, 3'TAMRA)
PCR conditions were as follows, and (ii) to (iv) were performed 40 cycles.
・ (I) 95 ° C. for 10 minutes → (ii) 95 ° C. for 10 seconds → (iii) 50 ° C. for 45 seconds → (iv) 72 ° C. for 1 second

(2) NirK gene Primers were as follows.
NirK876: ATYGGCGGVAYGGCGA (SEQ ID NO: 4)
NirK1040: GCCTCGATCAGRTTRTGGTT (SEQ ID NO: 5)
PCR conditions were as follows, and (ii) to (iv) were performed 32 cycles.
(I) 95 ° C. for 10 minutes → (ii) 95 ° C. for 10 seconds → (iii) 60 ° C. for 30 seconds → (iv) 72 ° C. for 30 seconds

(3) NirS gene Primers were as follows.
NirSCd3aF: AACGYSAAGGARACSGG (SEQ ID NO: 6)
NirSR3cd: GASTTCGGRTGSGTCTTSAYGAA (SEQ ID NO: 7)
PCR conditions were as follows, and (ii) to (iii) were performed 40 cycles.
・ (I) 95 ° C. for 10 minutes → (ii) 95 ° C. for 10 seconds → (iii) 65 ° C. for 20 seconds

  The quantification result of all the bacteria is shown in FIG. 16, the quantification result of the nirK gene is shown in FIG. 17, and the quantification result of the mirS gene is shown in FIG.

  From the results shown in FIG. 16, it was revealed that the number of bacteria increased in all energized cases compared to the case without energization, and the increase in the number of bacteria was particularly remarkable at −0.6V.

  Further, from the results shown in FIG. 17 and FIG. 18, in all cases where electricity was applied, an increase in mirK and mirS was observed compared to the case where no electricity was supplied, and the tendency was particularly remarkable at −0.6V. .

(Example 5)
Microbial flora analysis of the culture broth after the test was completed.

The V3 region (about 200 bp) of the 16S rRNA gene was amplified by PCR. PCR was performed using Ex-taq polymerase (Takara bio, Japan) and the following primers prepared based on the following Escherichia coli-derived 16S rRNA gene (EMBL / GenBank / DDBJ accession number X80721).
・ Forward primer 5'-CCTACGGGAGGCAGCAG-3 '
(Equivalent to position 341 in the 16S rRNA gene from Escherichia coli, SEQ ID NO: 8)
・ Reverse primer 5'-ATTACCGCGGCTGCTGG-3 '
(Corresponds to position 517 in the 16S rRNA gene from Escherichia coli, SEQ ID NO: 9)

  A partial DNA fragment of about 200 bp 16S rDNA amplified by PCR was separated and purified by agarose electrophoresis. The purified DNA fragment was inserted into pT7Blue-T vector (Novagen), and JM109 was transformed. About 50 obtained transformants were randomly obtained, and the base sequence analysis of 16S rRNA gene clone was performed. The nucleotide sequence analysis was determined by the dideoxy nucleotide chain termination method using ABI PRISM BigDye terminator v3.0 cycle sequencing kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Homology search of the obtained 16S rRNA gene base sequence from the international base sequence database (GeneBank / EMBL / DDBJ) with respect to the DNA base sequence database was performed to estimate related microorganisms.

  The analysis result is shown in FIG. Despite the fact that the microbial species detected from the inoculated samples were very diverse as 37 types, there was a tendency for certain types of microorganisms to dominate by culturing while energizing. Specifically, at −0.6 V, microorganisms belonging to the genus Pseudomonas, Alicycliphilus and Tissierella tend to be dominant, and the dominant of these microorganisms is observed. It was considered that the rate of reducing nitrite ions to nitrogen gas was promoted by the conversion. At +0.6 V, microorganisms belonging to the genus Pseudomonas, Burkholderia and Pseudoxanthomonas tended to dominate. Here, microorganisms of the genus Pseudomonas and Pseudoxanthomonas are known to have nitrate reducing ability and nitrite reducing ability, and microorganisms of the genus Burkholderia are capable of oxidizing nitrite. Since it is known that the nitrite oxidation reaction is accelerated by + 0.6V, the growth of microorganisms of the genus Pseudomonas and Pseudoxanthomonas is promoted at + 0.6V. Nitrate reduction and nitrite reduction activities are weak, and it is considered that growth of microorganisms belonging to the genus Burkholderia was promoted to improve nitrite oxidation activity, and the nitrite oxidation reaction was promoted as a whole.

  From the above results, according to the method for activating a nitrogen-metabolizing microorganism of the present invention, a microorganism having nitrite reduction ability or a microorganism having nitrite oxidation ability is selectively grown and predominated in the culture solution. Thus, it has been clarified that the nitrite ion treatment ability (reduction or oxidation ability) of the culture solution can be greatly improved.

  By utilizing the method for activating nitrogen-metabolizing microorganisms of the present invention, nitrite-reducing microorganisms or nitrite-oxidizing microorganisms can be selectively grown from samples containing various microorganisms such as soil samples, and accumulated. Culture can be performed. Therefore, it contributes to the search for a new functional microorganism as a microorganism that is difficult to grow by a normal culture method. That is, the present invention can contribute to the search for microorganisms having a new or superior function from the conventional microbial resources that exist in nature.

DESCRIPTION OF SYMBOLS 1 Bioreactor 2 Nitrogen metabolism microorganism 2a Nitrite reduction microorganism 2b Nitrite oxidation microorganism 3 Redox substance 4 Culture solution 4a Electrolyte solution 5 Organic substance 6 Ion exchange membrane 7 Culture tank 8 Counter electrode tank 9 Working electrode 10 Counter electrode 12 Constant potential setting Device 15 Nitrogen gas discharge means 16 Process liquid discharge means 18 Liquid to be processed introduction section

Claims (7)

A culture solution containing a nitrogen-metabolizing microorganism of at least one of a nitrite-reducing microorganism having a nitrite-reducing ability and a nitrite-oxidizing microorganism having a nitrite-oxidizing ability, an organic substance that is a carbon source of the nitrogen-metabolizing microorganism, and a redox substance The nitrogen metabolism is applied while applying a potential to the electrode so that the reduction rate of nitrite ion when the electrode is immersed in the culture solution and the nitrite ion is dissolved in the culture medium is higher than when no potential is applied to the electrode. A nitrite-producing microorganism that produces nitrite ions is added to the culture solution containing the nitrogen-metabolizing microorganisms activated by culturing microorganisms, and the nitrite ions produced by the nitrite-producing microorganisms are activated. Reducing or oxidizing the generated nitrogen-metabolizing microorganism, and culturing the nitrite-producing microorganism while avoiding growth inhibition due to toxicity of nitrite ions The method of culturing nitrite-producing microorganisms, characterized. The method for cultivating a nitrite-producing microorganism according to claim 1, wherein an electrolyte solution is brought into contact with the culture solution via an ion exchange membrane, and an electrode paired with the electrode is immersed in the electrolyte solution. The method for culturing a nitrous acid-producing microorganism according to claim 1, wherein an electrode paired with the electrode is brought into contact with the culture solution via an ion exchange membrane. 4. The nitrite-reducing microorganism is activated by setting the potential applied to the electrode to a potential at which the generation rate of nitrogen gas is improved as a result of an increase in the reduction rate of nitrite ions. The culture | cultivation method of the nitrite production microorganisms as described in one. The method for cultivating a nitrite-producing microorganism according to claim 4, wherein the potential is set to a potential that is larger than -0.6V and including -0.6V on a silver / silver chloride electrode potential basis. The nitrite-oxidizing microorganism is activated by setting the potential applied to the electrode to a potential at which a nitrate ion generation rate is improved as a result of an improvement in a reduction rate of nitrite ions. A method for culturing a nitrite-producing microorganism as described in 1. above. The method for culturing a nitrite-producing microorganism according to claim 6, wherein the potential is set to a potential that is larger on the positive side than + 0.6V including + 0.6V on the basis of the silver / silver chloride electrode potential.