JP6357360B2 - Electric culture device - Google Patents

Description

  The present invention relates to a microorganism electroculturing apparatus, that is, an electroculturing apparatus suitable for culturing microorganisms such as chemically autotrophic bacteria while controlling the oxidation-reduction potential of a culture solution, and enables particularly efficient culture. The present invention relates to an electric culture apparatus.

  Chemical autotrophic bacteria, such as iron-oxidizing bacteria, proliferate by immobilizing carbon dioxide through the transfer of electrons between inorganic electron donors and electron acceptors, that is, the energy associated with redox reactions. Yes. Chemical autotrophic bacteria can be grown by electroculturing with an electron donor supplied electrochemically.

  Electroculturing can be carried out by placing an electrode in the culture medium and continuously supplying an electron donor, which is an energy source necessary for the growth of chemically autotrophic bacteria, at an appropriate concentration. Compared to normal culture with only air supply, it is said that efficient culture is possible, and further technological development is underway. For example, in Patent Document 1, as a method for predicting the growth process of chemically autotrophic bacteria by electroculture, a predetermined arithmetic expression is used from the bacterial concentration, bacterial concentration change, electron donor concentration, consumption rate, current value, and the like. A method for predicting the above is disclosed. By the method of patent document 1, it is supposed that the characteristic of the mass production system of a chemical autotrophic bacterium can be grasped, and the optimal environmental condition of continuous culture can be grasped.

  Patent Document 2 discloses an electroculture method in which a voltage is intermittently applied to a culture solution and a static potential of the culture solution is measured in a state where no voltage is applied. According to the method of Patent Document 2, it is possible to measure a static potential (potential between electrodes when no current flows between the electrodes) that cannot be measured by the conventional method of continuously applying a voltage to the electrodes. Based on the above, it is possible to estimate the concentration of redox species in the culture solution, accurately grasp the growth status of microorganisms, and feed back to the control of voltage application. It is said that voltage control is possible for the incubator.

Japanese Patent Laid-Open No. 11-113565 JP 2005-198509 A

  However, in the method of Patent Document 1, in order to derive optimal culture conditions, factors such as potential control by adjusting the voltage, current value, and distance between electrodes, and the amount of nutrient added, etc. are set in detail, and further fed back. It is necessary to set repeatedly many times until it is optimized by the control. In addition, the method of Patent Document 2 requires a large apparatus, and the optimization of culture conditions requires repeated feedback control as in Patent Document 1 and requires a complicated control mechanism.

  In other words, in the conventional electroculture apparatus, the position (distance between electrodes), voltage, and current of the electrodes are accurately set in order to accurately set the oxidation-reduction potential of nutrient salts (electron donors, etc.) that serve as the energy source of microorganisms. Therefore, there is a problem that the control system becomes complicated.

Further, for example, when iron-oxidizing bacteria that grow using divalent iron ions (Fe ²⁺ ) as nutrient salts are electrocultured, (i) the iron-oxidizing bacteria receive donation of electrons from Fe ²⁺ and undergo oxidation-reduction reactions in the microorganism. As it is used, it is oxidized to trivalent iron ions (Fe ³⁺ ), and (ii) Fe ³⁺ accepts electrons at the electrode, is reduced to Fe ²⁺ , and becomes a nutrient salt of the bacteria again. However, due to problems such as the degree of agitation of the culture solution and the potential between the electrodes, Fe ³⁺ combines with oxygen in the culture solution before accepting electrons from the electrode, so that it becomes iron oxide and precipitates and adheres to the electrode surface. In some cases, the growth of iron-oxidizing bacteria is suddenly decelerated due to precipitation in the culture tank. In this case, in order to restart the culture, the electrode is washed with hydrochloric acid, and depending on the degree of precipitation, electrode replacement and removal of the sediment in the culture tank are necessary. It was a big problem.

  Accordingly, an object of the present invention is an electroculturing apparatus used for electroculturing in which microorganisms are cultured by placing an electrode in a culture solution and applying a voltage, and there is no need for a complicated control system for redox potential. It is another object of the present invention to provide a culture apparatus capable of efficiently cultivating an electrode, in which metal oxides do not easily adhere to an electrode and complicated maintenance is not required.

  In the electric culture, the present inventors have studied to increase the electrode area by using a plate electrode as a working electrode in order to efficiently contact nutrient salts and microorganisms with the working electrode. It has been found that there is a problem that it is obstructed and as a result, adhesion of oxides such as iron oxide increases. Therefore, the present invention has been accomplished by variously examining the form of the working electrode.

That is, the object is to provide a culture vessel for culturing microorganisms in a culture solution, a working electrode for applying a voltage to the culture solution provided in the culture vessel, and a communication part partitioned by the culture vessel and an ion exchange membrane. A counter electrode tank connected via a counter electrode, a counter electrode provided in the counter electrode tank, and a voltage applying means for applying a voltage between the working electrode and the counter electrode, the working electrode, the counter electrode and The ion exchange membrane is arranged vertically, and the working electrode, the ion exchange membrane, and the counter electrode are arranged in a substantially horizontal direction, and the working electrode is connected to the voltage applying means, and a shaft portion and The electrode includes a flat plate portion electrically connected to the shaft portion and formed with a plurality of through holes, and the working electrode includes rotating means that rotates about the shaft portion. This is achieved by an electroculture device.

  In the electroculture apparatus having the above-described configuration, an electrode having a shaft portion and a flat plate portion as a working electrode, and having a plurality of through holes formed in the flat plate portion, and rotating the electrode, the culture in the culture tank Inhibition of the flow of the liquid can be prevented and stirring of the culture liquid can be promoted. Thereby, the electrochemical reaction by a nutrient salt and a working electrode is accelerated | stimulated, and adhesion of the above metal oxides can be prevented. In addition, since the distance between the working electrode and the counter electrode (distance between the electrodes) periodically changes due to the rotation of the plate portion of the working electrode, regardless of the redox potential of the nutrient in the culture solution, The distance between the electrodes is periodically adjusted to a suitable range. Therefore, it is possible to efficiently carry out the electroculture without performing complicated control of the redox potential.

  Preferred embodiments of the electroculture device of the present invention are as follows.

  (1) The flat plate portion of the working electrode is a mesh-shaped flat plate. Inhibition of the flow of water in the culture tank can be further prevented. Moreover, the surface area of an electrode can be increased and an electrochemical reaction can be promoted more efficiently.

  (2) The counter electrode is an electrode having a shaft portion connected to the voltage application means, and a flat plate portion electrically connected to the shaft portion and formed with a plurality of through holes, and the counter electrode is Rotating means that rotates about the shaft portion is provided. The counter electrode is also an electrode having a flat plate portion in which a plurality of through holes are formed. By rotating the electrode, the liquid flow in the counter electrode tank can be prevented from being obstructed, and the electrochemical reaction at the counter electrode can be promoted. Electroculture can be carried out automatically. In addition, the rate of change in the distance between the working electrode and the counter electrode can be increased, and it is possible to cope with the oxidation-reduction potentials of nutrient salts in various culture solutions.

  (3) The flat plate portion of the counter electrode is a mesh-shaped flat plate. Inhibition of the flow of water in the counter electrode tank can be further prevented. Moreover, the surface area of an electrode can be increased and an electrochemical reaction can be promoted more efficiently.

(4) When the working electrode is rotated by the rotating means, the maximum value (d _max ) of the distance (d) between the portion of the working electrode closest to the counter electrode and the portion of the counter electrode closest to the working electrode is The working electrode and the counter electrode are provided so as to be 1.5 times or more the minimum value (d _min ) of the distance (d). Periodic changes in the distance between the working electrode and the counter electrode (distance between the electrodes) become larger, and it is possible to cope with the oxidation-reduction potentials of nutrient salts in various culture media.

  (5) The apparatus further includes discharge means for discharging a culture solution containing the cultured microorganism during culture of the microorganism, and supply means for newly supplying the culture solution according to the amount of the discharged culture solution. Continuous culture can be performed at a constant microorganism concentration, and more efficient microorganism production can be performed.

  According to the electroculture apparatus of the present invention, the flat plate portion in which a plurality of through holes of the working electrode are formed rotates, thereby preventing the flow of the culture solution in the culture tank and promoting the stirring of the culture solution. Therefore, the electrochemical reaction between the nutrient salt and the working electrode is promoted, and adhesion of the metal oxide can be prevented. In addition, since the distance between the working electrode and the counter electrode (distance between the electrodes) changes periodically, the distance between the electrodes is periodically in a suitable range regardless of the oxidation-reduction potential of nutrients in the culture medium. Therefore, electroculturing can be performed efficiently without complicated control of the redox potential.

It is the schematic which shows an example of the electroculture apparatus of this invention. It is the schematic which shows an example of the working electrode of the electroculture apparatus of this invention. It is the schematic which shows an example of the suitable aspect of the electroculture apparatus of this invention. It is the schematic for demonstrating the distance of the working electrode and counter electrode of the electroculture apparatus of this invention. It is a figure which shows the growth curve of the example of culture | cultivation of the microorganisms by the electric culture apparatus of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic view showing an example of the electroculture apparatus of the present invention. 1 includes a culture vessel 10 for culturing microorganisms in a culture solution containing nutrient salts and the like, a working electrode 11 for applying a voltage to the culture solution provided in the culture vessel 10, and a culture vessel 10. And a counter electrode 30 connected via a communication part 20 partitioned by an ion exchange membrane 21, a counter electrode 31 provided in the counter electrode 30, and a voltage applying means for applying a voltage between the working electrode 11 and the counter electrode 31 As a potential control device 40. The working electrode 11 is an electrode having a shaft portion 12 connected to the potential control device 40 and a flat plate portion 13 that is electrically connected to the shaft portion 12 and has a plurality of through holes (in a mesh shape in FIG. 1). is there. The working electrode 11 is connected to the shaft portion 12 as a rotating means, and is installed so as to rotate around the shaft portion 12. In FIG. 1, the direction of rotation is indicated by an arrow, but the direction of rotation is not particularly limited. For example, the direction of rotation may be opposite to the arrow in FIG. Also good. In FIG. 1, the working electrode 11 is installed in the vertical direction, but may be installed in the horizontal direction.

  The aeration apparatus 15 is installed in the culture tank 10, and in the case of an aerobic microorganism, for example, air is fed in the direction of an arrow from a compressor or the like, and air is supplied into the culture solution. Thereby, oxygen and carbon dioxide required for the growth of microorganisms can be supplied to the culture solution. An oxygen cylinder, a carbon dioxide cylinder or the like may be connected to the ventilation device 15. The culture tank 10 may be provided with a device installed in a normal culture device such as a pH electrode, a dissolved oxygen meter, an oxidation-reduction potentiometer, and a stirrer (not shown).

In the electric culture, for example, in the case of an iron-oxidizing bacterium that grows using divalent iron ions (Fe ²⁺ ) as a nutrient salt, (i) the iron-oxidizing bacterium receives an electron donation from Fe ²⁺ and undergoes a redox reaction in the microorganism. In addition to being oxidized to trivalent iron ions (Fe ³⁺ ), (ii) the Fe ³⁺ accepts electrons at the working electrode, is reduced to Fe ²⁺ , and becomes a nutrient salt of bacteria again. Grows. At this time, the ion exchange membrane prevents iron ions from flowing into the counter electrode layer and coming into contact with the counter electrode. Further, any potential control device 40 that applies a voltage to the working electrode may be used. For example, a galvano potentiometer can be used. The applied voltage is not particularly limited and can be adjusted according to the oxidation-reduction potential of the culture solution.

  In electrocultivation, in order to efficiently bring nutrient salts and microorganisms into contact with the working electrode, when the plate electrode is used so that the electrode area of the working electrode is increased, the flow of the culture solution in the culture tank may be hindered, and as a result In some cases, adhesion of metal oxides may increase. In the present invention, an electrode in which a plurality of through-holes are formed in the flat plate portion 13 as described above is used, and the electrode rotates to form a flow of the culture solution in the culture tank 10 to act on nutrient salts and microorganisms. Contact with the electrode 11 can be promoted, and adhesion of a metal oxide or the like can be prevented.

  FIG. 2 shows an enlarged schematic view of the working electrode 11 used in the electroculture apparatus of FIG. The flat plate portion 13 of the working electrode 11 has a rectangular mesh shape. The shaft portion 12 and the flat plate portion 13 are electrically connected by a method such as welding, soldering, or bolting. As the material for the shaft portion 12 and the flat plate portion 13, a material having high stability similar to that of the electrode used for electroculture is usually used. For example, a platinum steel material, a platinum plating titanium material, etc. are mentioned. If the diameter (φ) of the shaft portion 12 and the length (L), width (W) and thickness (T) of the flat plate portion 13 are strong enough not to be deformed when rotated in the culture solution, There is no restriction | limiting in particular, It can design according to the magnitude | size of a culture tank. Further, the shape of the flat plate portion does not have to be a rectangular shape, and may be, for example, a circle, an ellipse, or a polygon in a plan view.

  In FIG. 2, the flat plate portion 13 has a mesh shape, but may have a plurality of through holes, and may have a punching metal shape, for example. Since the flow of the culture solution in the culture tank is hardly hindered, the surface area of the electrode can be increased, and the electrochemical reaction can be promoted more efficiently, the mesh-like flat plate portion 13 as shown in FIG. preferable. There is no particular limitation on the size of the mesh.

  In FIG. 1, the counter electrode 31 is a general rod electrode, but as shown in FIG. 3, it is preferable that the counter electrode 31 is an electrode similar to the working electrode 11. 3, the counter electrode 31 ′ is electrically connected to the shaft portion 32 connected to the potential control device 40 and the shaft portion 32, and a flat plate in which a plurality of through holes (in FIG. 1, mesh shape) are formed. This is an electrode having a portion 33. The counter electrode 31 ′ is connected to the shaft portion 32 with a motor 34 as a rotating means, and is installed so as to rotate around the shaft portion 12. The details of the counter electrode 31 ′ are the same as those described for the working electrode 11. Similarly to the working electrode 11, the counter electrode 31 ′ having the flat plate portion 33 in which a plurality of through holes are formed is used, and by rotating the counter electrode 31 ′, obstruction of the liquid flow in the counter electrode tank 30 is also prevented. Electrochemical reaction can also be promoted, and electroculture can be carried out more efficiently.

  As for the flat plate portion 33 of the counter electrode 31 ′, the liquid flow in the counter electrode tank 30 can be further prevented from being obstructed, and the electrochemical reaction can be promoted more efficiently by increasing the surface area of the electrode. As in the case of the working electrode 11, it is preferably a mesh-like flat plate.

Furthermore, another advantage of rotating the flat plate portion 13 of the working electrode 11 is that the distance between the working electrode and the counter electrode (distance between the electrodes) periodically changes. FIG. 4 is a schematic diagram for explaining the distance between the electrodes. 4A1 shows that when the working electrode 11 in FIG. 1 rotates, the distance (d) between the portion of the working electrode 11 closest to the counter electrode 31 and the portion of the counter electrode 31 closest to the working electrode 11 is minimum. An arrangement with a value (d _min ) is shown, and FIG. 4 (a2) shows an arrangement with a distance (d) having a maximum value (d _max ). Thus, since the distance between the electrodes changes periodically, the distance between the electrodes periodically reaches a suitable range regardless of the oxidation-reduction potential of the nutrient in the culture solution. Therefore, it is possible to efficiently carry out the electroculture without performing complicated control of the redox potential.

FIG. 4B1 shows the distance between the portion of the working electrode 11 closest to the counter electrode 31 ′ and the portion of the counter electrode 31 ′ closest to the working electrode 11 when the working electrode 11 in FIG. d) shows an arrangement where the minimum value (d _min ) is shown, and FIG. 4B2 shows an arrangement where the distance (d) becomes the maximum value (d _max ). By rotating the counter electrode 31 ′, the maximum value (d _max ) and the minimum value (d _max ) of the distance (d) are compared with the case of the counter electrode 31 of the rod-shaped electrode (FIG. 1, FIG. 4 (a1), (a2)). d _min ) is increased, the rate of change can be increased, and more diverse redox potentials of nutrients in the culture solution can be handled.

The working electrode 11 and the counter electrode 31 (or 31 ′) are installed such that the maximum value (d _max ) of the distance (d) is 1.5 times or more the minimum value (d _min ) of the distance (d). It is preferable. Thereby, the periodic change of the distance (distance between electrodes) of a working electrode and a counter electrode becomes larger, and it can respond to the oxidation-reduction potential of the nutrient salt in a more various culture solution. The maximum value (d _max ) of the distance (d) is more preferably 1.5 to 10.0 times and further preferably 2.0 to 5.0 times the minimum value (d _min ) of the distance (d).

  The electroculturing apparatus of the present invention prevents metal oxide from adhering to the electrode and does not require complicated adjustment of the redox potential, so that it can be cultured for a long time without maintenance of the culture tank and the electrode, particularly continuous culture. Suitable for the system. That is, the electric culture apparatus of the present invention supplies a culture solution newly according to the discharge means for discharging the culture solution containing the cultured microorganism and the amount of the discharged culture solution during the cultivation of the microorganism. It is preferable to further provide a supply means. As a result, microorganisms such as chemical autotrophic bacteria such as iron-oxidizing bacteria that require a long culture time can be continuously cultured at a certain microorganism concentration, so that more efficient microorganism production can be carried out. it can.

  In FIG. 1 or FIG. 2, the culture tank 10 is provided with a discharge pipe 17 for discharging the culture solution containing microorganisms in the direction of the arrow and a supply pipe 16 for newly supplying the culture solution in the direction of the arrow. Yes. The discharge pipe 17 and the supply pipe 16 are connected to a culture liquid discharge pump and a culture liquid supply pump, respectively (not shown). The culture solution containing the discharged microorganisms is separated by filtering or centrifuging to collect the microorganisms. As a newly supplied culture solution, a culture solution separated from microorganisms after culture and / or a fresh culture solution can be used. In order to minimize the amount of nutrient used in the culture solution, for example, the nutrient concentration of the culture solution separated from the microorganism after culture (for example, in the case of iron-oxidizing bacteria, the concentration of divalent iron ions, etc.) By measuring and mixing the deficiency with a fresh culture solution or adding a nutrient salt, a newly supplied culture solution can be obtained. Thus, a useful microorganism can be efficiently produced by performing continuous culture using the electrocultivation apparatus of the present invention. In particular, since iron-oxidizing bacteria that can be used in a biomercury purification system can be cultured more efficiently than conventional methods, the purification method can be carried out efficiently.

  Next, the present invention will be specifically described with reference to examples.

1. Comparison of the Electroculture Apparatus of the Present Invention and the Conventional Electroculture Apparatus The culture solution (divalent iron inorganic salt medium) having the composition shown in Tables 1 and 2 is used as the electroculture apparatus of the present invention shown in FIG. and the charged into the electric culture device comprising a working electrode of rod-like, after sterilization, iron oxidizing bacteria (reeds di thio Bacillus ferrooxidans (Acidithiobacillus ferrooxidans) MON-1 strain) so that the initial microbial number 10 ⁶ cells / ml of And electroculture. The results are shown in FIG.

  As shown in FIG. 5, when the electroculture apparatus of the present invention was used, microorganisms grew logarithmically until the 11th day, whereas in the conventional electroculture apparatus, the working electrode rusted on the 11th day. As a result, the growth of microorganisms stopped. Therefore, it was shown that the electroculture apparatus of the present invention can perform electroculture more efficiently than the conventional electroculture apparatus.

In the culture using the electric culture apparatus of the present invention, after the 15th day of culture, a part of the culture solution containing the microorganisms is discharged, the microorganisms are collected aseptically, and then the culture solution is returned to the culture tank ( The decrease is supplemented with a fresh 9K basic medium), and the divalent iron concentration of the culture solution is measured. If the divalent iron concentration of the culture solution is less than 2%, the divalent iron concentration is 3%. The microorganism can be continuously produced over a long period of time by performing continuous culture in which FeSO ₄ · 7H ₂ O is added.

  In addition, this invention is not limited to the structure of said embodiment and Example, A various deformation | transformation is possible within the range of the summary of invention.

  The electroculture apparatus of the present invention enables efficient mass production of useful microorganisms without the need for complicated electrode maintenance and complicated control of redox potential.

DESCRIPTION OF SYMBOLS 10 Culture tank 11 Working electrode 12, 32 Shaft part 13, 33 Flat plate part 14, 34 Motor 15 Aeration apparatus 16 Supply pipe 17 Discharge pipe 20 Communication part 21 Ion exchange membrane 30 Counter electrode tank 31, 31 'Counter electrode 40 Potential control apparatus

Claims (6)

A culture vessel for culturing microorganisms in a culture solution;
A working electrode for applying a voltage to the culture medium provided in the culture tank;
A counter electrode tank connected via a communication section partitioned by the culture tank and an ion exchange membrane;
An electroculturing apparatus comprising a counter electrode provided in the counter electrode tank, and a voltage applying means for applying a voltage between the working electrode and the counter electrode,
The working electrode, the counter electrode and the ion exchange membrane are each arranged vertically, and the working electrode, the ion exchange membrane and the counter electrode are arranged in a substantially horizontal direction,
The working electrode is an electrode having a shaft portion connected to the voltage application means and a flat plate portion electrically connected to the shaft portion and having a plurality of through holes, and the working electrode is the shaft. An electroculturing apparatus comprising a rotating means that rotates about a section.   The electroculture apparatus according to claim 1, wherein the plate portion of the working electrode is a mesh-like plate. The counter electrode is an electrode having a shaft portion connected to the voltage application means, and a flat plate portion electrically connected to the shaft portion and formed with a plurality of through holes, and the counter electrode is the shaft portion. The electroculture apparatus according to claim 1, further comprising a rotation unit that rotates about the axis.   The electroculture apparatus according to claim 3, wherein the flat plate portion of the counter electrode is a mesh-like flat plate. When the working electrode is rotated by the rotating means,
The maximum value (d _max ) of the distance (d) between the portion of the working electrode closest to the counter electrode and the portion of the counter electrode closest to the working electrode is 1.5 times the minimum value (d _min ) of the distance (d). The electroculture apparatus according to any one of claims 1 to 4, wherein the working electrode and the counter electrode are installed so as to be described above. 6. A discharge means for discharging a culture solution containing the cultured microorganism during culture of microorganisms, and a supply means for newly supplying a culture solution according to the amount of the discharged culture solution. The electroculture apparatus according to any one of the above.

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