JP6087476B1 - Biological reaction device using oxygen-enriched micro-nano bubbles and biological reaction method using this biological reaction device - Google Patents

Abstract

The problem of the biological reaction device of the present invention and the biological reaction method using this biological reaction device is to reduce the stress and damage to the microorganisms during the biological reaction, and the biological reaction using the microorganisms is efficient and economical. It is to be able to do it. In order to solve the above problems, the biological reaction device of the present invention and the biological reaction method using this biological reaction device are prepared by using a micro / nano bubble generator to add air to the biological culture solution extracted from the culture tank. It is characterized by containing the formed micro-nano bubbles (oxygen-enriched MNB), and refluxing the biological culture solution containing the oxygen-enriched MNB to the culture tank. As a method for adding oxygen-enriched MNB to the biological culture liquid extracted from the culture tank, 1) The biological culture liquid extracted from the culture tank is separated into a biological culture liquid from which the filtrate and the filtrate are removed by a filter. Any one of the method of containing oxygen-enriched MNB in the filtrate and 2) the method of directly adding oxygen-enriched MNB to the biological culture solution extracted from the culture tank can be employed.

Description

The present invention cultivates aerobic or facultative anaerobic microorganisms (hereinafter also referred to as “microorganisms”) to produce reaction products in the microorganisms or to propagate the microorganisms and the like. Regarding a biological reaction method using a reaction apparatus, a micro-nano bubble (hereinafter referred to as “micro-nano bubble” is referred to as “MNB”, “nano-bubble” is referred to as “nano bubble” is defined as “micro-nano bubble”) in a biological culture solution containing microorganisms or the like. NB ”,“ micro / nano bubbles formed from air with increased oxygen content ”may be referred to as“ oxygen-enriched MNB ”)), and biological reaction is efficiently performed It is.

Unlike chemical reactions, biological reactions are slow, but they do not use a lot of energy or chemicals, so they are mild and meaningful for the environment.
However, the biological reaction generally has a problem that the reaction is mild and slow. That is, for chemical reactions, a reaction within one hour is often sufficient, whereas in the case of biological reactions, a reaction time of several days or longer, several days or particularly several weeks or longer is required. There is also. For this reason, it is required to perform biological reactions efficiently and economically.

  As technologies for improving the efficiency of biological reactions, Patent Documents 1 to 3 promote the activation of microorganisms and the like by allowing MNB or NB formed from air to exist in the culture medium in the culture of microorganisms and the like. In addition, it is disclosed that the reaction efficiency of biological reactions, reduction of reaction time, and the like are attempted.

  Specifically, Patent Document 1 describes that air MNB and NB are mixed in the culture solution before the culture solution is supplied to the culture tank. It is described that air MNB is mixed before the liquid is supplied to the culture vessel. Further, in Patent Document 3, in a batch system, a culture solution is extracted from a culture tank, filtered through a bacterial cell filter to obtain a filtrate, and this filtrate is mixed with air MNB and refluxed to the culture tank. It is described.

  However, in an apparatus that includes air MNB and NB in the culture solution supplied to the culture vessel as disclosed in Patent Documents 1 and 2, an appropriate amount of the culture solution in the culture vessel is appropriate in the initial stage of the biological reaction. Although the content of MNB and / or NB in the culture medium in the culture tank cannot be properly maintained over the entire long-term biological reaction, the reaction of the biological reaction Efficiency, shortening of reaction time, etc. cannot be achieved sufficiently.

  Further, in Patent Document 3, as shown in FIG. 8, a culture solution is extracted from a culture tank 107 as a biological reaction tank and filtered through a cell filter 110 to obtain a filtrate, and micro-nano bubbles are generated in the filtrate. In the tank 115, an apparatus for generating and mixing air MNB by the micro / nano bubble generator 116 and returning it to the culture tank is described. In this apparatus, the MNB content of the culture solution in the culture tank is set to an appropriate value. Although it can be maintained, microorganisms and the like are subjected to stress and damage in the step of extracting the culture solution from the culture vessel, the step of filtering the culture solution with a cell strain filter, the step of returning the culture solution excluding the filtrate to the culture vessel, etc. Therefore, there exists a problem that activity of microorganisms etc. will fall.

Therefore, the present inventors made the oxygen content of the gas forming MNB higher than the oxygen content in the air (about 21%),
-Even if the amount of the biological culture solution extracted from the culture tank is reduced and the amount of MNB contained in the biological culture solution in the culture tank is reduced, high concentration oxygen that is easily absorbed can be supplied to microorganisms. Therefore, the activity of microorganisms etc. can be maintained,
〇 By reducing the amount of the biological culture solution withdrawn from the culture tank, stress and damage to microorganisms can be reduced and the energy required for circulation of the biological culture solution can be reduced.
〇 By reducing the amount of MNB contained in the biological culture, the energy required for driving the MNB generator can be reduced.
It has been found that there is a great merit, and the present invention has been achieved.

  In addition, tube pumps, diaphragm pumps, screw pumps with relatively little stress and damage to microorganisms, etc. as a pump for circulating the biological culture solution outside the culture vessel as the amount of the biological culture solution drawn out from the culture vessel decreases. It has also been found that a positive displacement pump such as a rotary pump can be suitably used, and this can further reduce stress and damage to microorganisms.

Japanese Patent No. 4805120 Japanese Patent No. 4956552 Japanese Patent No. 4146476

  The problem of the biological reaction device of the present invention and the biological reaction method using this biological reaction device is to reduce the stress and damage to the microorganisms during the biological reaction, and the biological reaction using the microorganisms is efficient and economical. It is to be able to do it.

  In order to solve the above-mentioned problems, the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus are characterized in that oxygen-enriched MNB is contained in a biological culture solution containing microorganisms and the like. is there.

  In addition, as a pump for circulating the biological culture solution outside the culture tank, by suitably using a positive displacement pump such as a tube pump, a diaphragm pump, a screw pump, a rotary pump, etc., which has relatively little stress and damage to microorganisms etc. The problem can be further solved.

  In the present invention, the oxygen content of the gas forming the MNB is made higher than the oxygen content of air (about 21%), thereby reducing the amount of the biological culture solution withdrawn from the culture tank, and the biological culture solution contains Even if the amount of MNB to be reduced is decreased, a high concentration of easily absorbed oxygen in the MNB state can be supplied to the microorganism and the like, and the activity of the microorganism and the like can be maintained.

  Furthermore, by reducing the amount of the biological culture solution extracted from the culture tank, it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution.

  Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.

In addition, tube pumps, diaphragm pumps, screw pumps with relatively little stress and damage to microorganisms, etc. as a pump for circulating the biological culture solution outside the culture vessel as the amount of the biological culture solution drawn out from the culture vessel decreases. Further, a positive displacement pump such as a rotary pump can be suitably used, and this can further reduce stress and damage to microorganisms.
As described above, the present invention is excellent in that a biological reaction using microorganisms or the like can be performed efficiently and economically.

It is a schematic diagram which shows 1st Embodiment of the biological reaction apparatus of this invention. It is sectional drawing which shows the outline | summary of the micro-nano bubble generator of a water flow system used in 1st Embodiment. It is sectional drawing which shows the outline | summary of the oxygen enrichment means used in 1st Embodiment. It is a schematic diagram which shows 2nd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows 3rd Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows 4th Embodiment of the biological reaction apparatus of this invention. It is a schematic diagram which shows the biological reaction apparatus used by the Example and comparative example of this specification. It is FIG. 1 of patent document 3 (patent 4146476 gazette) which is a prior art example.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

First, general items of the biological reaction apparatus and biological reaction method of the present invention will be described.
The bioreactor of the present invention and the bioreaction method using the bioreactor include reaction products produced by microorganisms such as brewing, fermentation, etc., for producing foods, chemicals, chemicals, etc., bioethanol production using biomass, etc. It is useful not only for the production of but also for the growth of microorganisms and the like.

  In the biological reaction of the present invention, in a culture solution containing microorganisms or the like housed in a culture tank, reaction products are produced by microorganisms or the like, and the microorganisms are grown using the culture solution as a nutrient source.

  As the culture solution in the present invention, a culture solution containing a saccharide and a nitrogen source is used. As saccharides, saccharides such as maltose, sucrose, glucose, fructose, and mixtures thereof are usually used, and the concentration of saccharides in the culture solution is not particularly limited, but is set to 0.1 to 10 w / v%. preferable. As the nitrogen source, ammonium chloride, ammonium sulfate, corn steep liquor, yeast extract, meat extract, peptone, or the like is used, and it is preferably set to 0.1 to 10 w / v%. Furthermore, it is preferable to add vitamins, inorganic salts, and the like to the culture solution as needed in addition to the saccharides and the nitrogen source.

  Examples of the microorganism in the present invention include aerobic and facultative anaerobic microorganisms such as koji molds such as Aspergillus, Bacillus natto, acetic acid bacteria, yeasts, and lactic acid bacteria conventionally used in technical fields such as brewing and fermentation. Various aerobic and facultative anaerobic microorganisms created by gene recombination technology can be used. Examples of the cells include animal cells for producing physiologically active peptides or proteins used as antibody drugs, particularly genetically modified animal cells.

Although the addition concentration to the culture solution of microorganisms etc. is not specifically limited, It is preferable to set it as 0.5-10 g / L, and it is more preferable to set it as 3.0-6.0 g / L.

Next, features of the biological reaction apparatus and biological reaction method of the present invention will be described.
As described above, the first feature of the present invention is that the biological culture liquid extracted from the culture tank contains oxygen-enriched MNB, and the biological culture liquid containing the oxygen-enriched MNB is added to the culture tank. To reflux.

  Thus, by making the oxygen content rate of the gas forming MNB higher than the oxygen content rate of air (about 21%), the amount of the biological culture solution extracted from the culture tank is reduced, and the biological culture solution contains Even if the amount of MNB is decreased, high-concentration oxygen that is easily absorbed in the MNB state can be supplied to the microorganisms in the culture tank, and the activity of the microorganisms can be maintained.

  Furthermore, by reducing the amount of the biological culture solution extracted from the culture tank, it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution.

  Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.

  In the present invention, the matter of setting the oxygen content of the gas forming MNB higher than the oxygen content of air (about 21%) and setting the amount of the biological culture solution extracted from the culture tank to be lower will be further described. To do.

  As an MNB generating apparatus for adding MNB to a biological culture liquid extracted from a culture tank, a system driven by a water stream, which can generate a large amount of MNB economically, as outlined in FIG. Since the (water flow system) type is usually used, first, a case of using such a water flow type MNB generator will be described.

  In the water flow type MNB generator, the biological culture liquid extracted from the culture tank is supplied under pressure, and the turbulent flow is generated by reducing the diameter of the pipe and increasing the flow velocity. MNB is generated by the water flow by supplying the water. Hereinafter, the amount of the biological culture solution extracted from the culture tank in one minute may be referred to as “liquid circulation amount” (mL / min), and the liquid circulation with respect to the amount of biological culture solution (mL) accommodated in the culture tank. The ratio of the amount may be referred to as “liquid circulation ratio” (%).

  By reducing the liquid circulation rate, it is possible to reduce the stress and damage received by microorganisms and the like due to the liquid circulation. On the other hand, with the decrease in the liquid circulation rate, the water flow type MNB generator is driven to generate MNB. Since the source water flow is weakened, the amount of MNB generated decreases, and the dissolved oxygen concentration (hereinafter, also referred to as “dissolved oxygen concentration”) of the biological culture solution decreases. In addition, since the amount of MNB that can be contained in the liquid is naturally limited, the amount of MNB that can be supplied per hour to the biological culture solution extracted from the culture tank is reduced due to a decrease in the amount of liquid circulation. Then, the dissolved oxygen concentration decreases. As described above, there is a limit to efficiently and economically performing a biological reaction using microorganisms or the like only by reducing the liquid circulation rate, but this problem can be solved by increasing the oxygen content of MNB. Can be solved.

  In the present invention, in order to perform a biological reaction efficiently and economically using a water flow type MNB generator, the liquid circulation rate is set low, thereby reducing the stress and damage to microorganisms and the like due to the liquid circulation. And the fall of the dissolved oxygen concentration accompanying this can be ensured by setting the oxygen content rate of MNB high.

  The upper limit of the liquid circulation ratio is preferably less than 48%, more preferably 40% or less, further preferably 30% or less, and most preferably 20% or less. If the liquid circulation ratio is too large, such as 48% or more, the stress and damage to microorganisms and the like due to the liquid circulation will increase. Moreover, as a lower limit of a liquid circulation ratio, 1% or more is preferable and 10% or more is more preferable. If the liquid circulation ratio is too small, less than 1%, the amount of MNB generated decreases, and it becomes difficult to ensure a decrease in dissolved oxygen concentration even if the oxygen content of MNB is set high.

  In the present invention, even when an MNB generator other than the water flow method is used, the amount of MNB generated by the MNB generator is reduced to reduce the stress and damage received by microorganisms, and the dissolved oxygen concentration associated therewith Can be ensured by setting the oxygen content of MNB high.

  The second feature of the present invention is that, as described above, by using oxygen-enriched MNB, the amount of the biological culture solution extracted from the culture vessel can be reduced, and the biological culture solution is circulated outside the culture vessel. As a pump to be used, a positive displacement pump such as a tube pump, a diaphragm pump, a screw pump, a rotary pump, or the like, which has relatively little stress and damage to microorganisms or the like is preferably used.

  The use of such a positive displacement pump can further reduce stress and damage to microorganisms.

  The “micro-nano bubbles” of the present invention means “micro bubbles” and / or “nano bubbles”. While “normal bubbles” rapidly rise in water and burst and disappear on the surface, microbubbles with a diameter of 50 μm or less called “microbubbles” shrink in water and disappear. Together with free radicals, “nanobubbles”, which are ultrafine bubbles having a diameter of 100 nm or less, are generated, and these “nanobubbles” remain in water for a certain amount of time.

  In the present invention, bubbles having a number average diameter of 100 μm or less are referred to as “micro bubbles”, and bubbles having a number average diameter of 1 μm or less are referred to as “nano bubbles”. As a method for measuring the bubble size of microbubbles, image analysis method, laser diffraction scattering method, electrical detection band method, resonance mass measurement method, optical fiber probe method, etc. are generally used, and the method of measuring the bubble size of nanobubbles In general, a dynamic light scattering method, a Brownian motion tracking method, an electrical detection band method, a resonance mass measurement method, and the like are generally used.

  “Nano bubbles”, which are extremely fine bubbles, are also called “ultra fine bubbles”. Currently, in the ISO (International Organization for Standardization), the creation of an international standard for fine bubble technology is being considered, and once the international standard is created, the name of “nanobubble”, which is currently commonly used, There is a possibility that it will be unified into “Ultra Fine Bubble”.

  As the micro / nano bubble generating device, a known or commercially available device can be used. For example, after a sufficient amount of gas is dissolved in water at a certain high pressure, the dissolved gas is released by releasing the pressure. "Pressurized dissolution type microbubble generator" that creates supersaturation conditions, and the phenomenon that bubbles are subdivided by turbulent flow generated by water flow by stirring and mixing a mixed fluid consisting of liquid and gas as a water flow. A “loop flow type bubble generating nozzle” using the above can be used.

  Examples of nanobubble generators include, for example, JP 2007-31690 A, JP 2006-289183 A, JP 2005-245817 A, JP 2007-136255 A, and JP 2009-39600 A. Those described can be used.

It is preferable to use a water flow type device as the micro / nano bubble generation device because a large amount of MNB can be generated economically.
In order to obtain air with an increased oxygen content, known oxygen enrichments such as PSA method using adsorbent, VSA method, water electrolysis method, cryogenic separation method, membrane separation method, chemical adsorption method, etc. Although means can be used, it is preferable to use an oxygen-enriched film from an economical viewpoint.

  The upper limit of the oxygen content of the oxygen-enriched MNB is preferably less than 60%, more preferably 55% or less, still more preferably 50% or less, and most preferably 45% or less. If the oxygen concentration of the oxygen-enriched MNB is excessively increased to 60% or more, the stress and damage to microorganisms and the like due to the oxidizing action of oxygen will increase. Further, the lower limit value of the oxygen concentration of the oxygen-enriched MNB is preferably 23% or more, more preferably 25% or more, further preferably 27% or more, and most preferably 30% or more. If the oxygen concentration of the oxygen-enriched MNB is too small, such as less than 23%, the dissolved oxygen concentration decreases, making it difficult to increase the activity of microorganisms such as microorganisms.

As described above, the first feature of the present invention is that the biological culture liquid extracted from the culture tank contains oxygen-enriched MNB, and the biological culture liquid containing the oxygen-enriched MNB is added to the culture tank. The following two methods can be adopted as a method of adding oxygen-enriched MNB to the biological culture solution extracted from the culture tank.
1) A method in which a biological culture liquid extracted from a culture tank is separated into a biological culture liquid from which the filtrate and the filtrate are removed by a filter, and this filtrate contains oxygen-enriched MNB.
2) A method in which oxygen-enriched MNB is directly contained in a biological culture solution extracted from a culture tank without using a filter.

  In the above method 1), oxygen-enriched MNB is blown into the filtrate that does not substantially contain microorganisms. Therefore, the microorganisms are not subjected to stress or damage in the step of blowing oxygen-enriched MNB. In some cases, stress and damage may occur during the filtration process. Further, since the amount of the filtrate separated in the filtration step is small (the amount of the filtrate is usually about 1/10 to 1/100 of the amount of the biological culture liquid extracted from the culture tank), In order to supply a sufficient amount of oxygen-enriched MNB to the biological culture solution, it may be necessary to increase the amount of the biological culture solution extracted from the culture tank, or to increase the amount of oxygen-enriched MNB blown in, There is a possibility that the operating cost of the apparatus becomes high and the stress and damage to which microorganisms are subjected are also increased.

  In the method 2), oxygen-enriched MNB is blown into the biological culture solution extracted from the culture tank containing microorganisms, so that the microorganisms are stress-damaged in the step of blowing oxygen-enriched MNB. In some cases, as in the above method 1), stress damage may be reduced in the filtration step. Further, in the above method 1), even when it is necessary to increase the amount of oxygen-enriched MNB, if the above method 2) is used, the oxygen-enriched MNB is directly added to the biological culture solution extracted from the culture tank. Therefore, it is not necessary to increase the amount of the biological culture solution, and the operating cost of the apparatus is not increased, and the stress and damage to the microorganisms are not increased.

  The biological reaction apparatus employing the method 1) will be described based on the first embodiment of the present invention shown in FIG. 1, and the biological reaction apparatus employing the method 2) will be illustrated in FIG. A description will be given based on the second embodiment of the invention.

○ First embodiment (FIG. 1)
First, a first embodiment of the present invention will be described with reference to FIG.
The first embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and includes oxygen-enriched MNB in a biological culture solution as follows.
a) The culture solution 1 is supplied to the culture tank 2.
b) The valve 12 is closed, the valve 13 and the valve 14 are opened, the culture tank pump 8 is driven, and the biological culture solution 3 containing the culture solution, microorganisms and the like is extracted from the culture vessel 2 and supplied to the filter 4.
c) The biological culture solution B (that is, the biological culture solution in which microorganisms and the like are concentrated) separated by the filter 4 and excluding the filtrate is returned to the culture tank 2.
d) The filtrate A separated by the filter 4 is stored in the micro / nano bubble generating tank 6, and oxygen enriched MNB is contained by the micro / nano bubble generating device 7a.
g) The return pump 9 is driven to return the filtrate D containing oxygen-enriched MNB to the culture tank 2.
h) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
i) When the biological reaction has sufficiently progressed, the valve 13 is closed, the valve 12 and the valve 14 are opened, the culture tank pump 8 is driven, and the reaction product generated in the culture tank 2 is collected together with the filtrate A. Store in the filtrate storage tank 5.

  The filter 4 includes a filtration membrane and a container that accommodates the filtration membrane. The filtration membrane may be an organic membrane or an inorganic membrane. The shape of the filtration membrane may be any shape such as a flat membrane, a hollow fiber membrane, and a spiral type. Among these, a hollow fiber membrane module is preferable. Any of the pressure type shapes can be employed.

  As the filtration method, cross flow filtration using a hollow fiber membrane module is preferable. In this filtration method, a culture solution containing reaction products, microorganisms, and the like is filtered while being supplied to the inside of the hollow fiber membrane, and the filtrate is taken out from the outside. Microorganisms deposited inside the hollow fiber membrane And so on, so that a stable filtration state can be maintained over a long period of time.

  When performing cross-flow filtration using a hollow fiber membrane module, it is necessary to flow the liquid to be filtered into the hollow fiber membrane at a flow rate of a certain level or more in order to scrape membrane dirt. However, in the present invention, since the biological culture solution containing microorganisms and the like to be filtered contains oxygen-enriched MNB, even if it is flowed at a lower flow rate than usual, it is possible to scrape membrane dirt, Can significantly reduce the stress and damage to them.

  Specifically, in general cross-flow filtration, steady operation is performed at a circulation flow rate of about 1 to 2 m / s when using an organic membrane and about 1 to 3 m / s when using a ceramic membrane. However, by adding oxygen-enriched MNB to the biological culture solution, it is possible to maintain low filtration resistance by reducing membrane contamination, so that the same flux (the amount of membrane filtered water per unit time / unit membrane area) can be obtained. The necessary circulation flow rate can be reduced to about 0.2 to 1.5 m / s. In addition, when operating at the same circulation flow rate, the flux can be increased by about 1.2 to 2.0 times.

  As the filtration membrane, an organic polymer compound can be preferably used from the viewpoints of separation performance and water permeation performance, and stain resistance. Examples include polyethylene resins, polypropylene resins, polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins, polyacrylonitrile resins, cellulose resins, and cellulose triacetate resins. A mixture of these resins as the main component may be used. Polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins and polyacrylonitrile resins, which are easy to form in solution and have excellent physical durability and chemical resistance, are preferred. A vinylidene chloride resin or a resin containing the vinylidene fluoride resin as a main component is more preferably used because it has a characteristic of having both chemical strength (particularly chemical resistance) and physical strength.

  Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferably used. Furthermore, the polyvinylidene fluoride resin may be a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride. Examples of vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.

  The average pore diameter of the filtration membrane can be appropriately determined according to the purpose and situation of use, but it is preferable that it is small to some extent, and it is usually preferably 0.01 μm or more and 1 μm or less. When the average pore diameter of the hollow fiber membrane is less than 0.01 μm, components such as microorganisms, such as sugars and proteins, and membrane dirt components such as aggregates thereof block the pores, and stable operation cannot be performed. In consideration of the balance with water permeability, it is preferably 0.02 μm or more, and more preferably 0.03 μm or more. In addition, when it exceeds 1 μm, the film surface smoothness and the shearing force due to the flow of the film surface, and the peeling of dirt components from the pores by physical cleaning such as backwashing and air scrubbing are insufficient, and stable operation is possible. Disappear.

  Moreover, when an average pore diameter approaches the magnitude | size of microorganisms etc., these may block | close a pore directly. In addition, there may be cases where broken cells of microorganisms or cultured cells in the fermented liquid are killed to produce cell crushed materials. In order to avoid pore clogging by these crushed materials, the average pore diameter is 0.4 μm. The following is preferable, and 0.2 μm or less is preferable.

  Here, the average pore diameter of the filtration membrane can be determined by measuring and averaging the diameters of a plurality of pores observed by scanning electron microscope observation at a magnification of 10,000 times or more. Preferably, 10 or more, preferably 20 or more pores are randomly selected, the diameters of these pores are measured, and the number average is obtained. When the pores are not circular, it is also preferable to use an image processing device or the like to obtain a circle having an area equal to the area of the pores, that is, an equivalent circle, and obtain the equivalent circle diameter as the pore diameter. it can.

  As shown in FIG. 1, in 1st Embodiment, the filtrate A which is the liquid made to contain MNB in the MNB generator 7a is driven out of the MNB generation tank 6 by driving the liquid supply pump 10, and the MNB generator In addition to being supplied to 7a, air C having an increased air concentration obtained by the oxygen enrichment means is supplied to the MNB generator 7a.

  As the MNB generating apparatus 7a used in the first embodiment, as shown in FIG. 2, an apparatus that can generate a large amount of MNB economically and that is driven using a water stream (water stream system) is used. In the MNB generator 7a, the filtrate A is supplied from the inlet 21 of the nozzle in a state where pressure is applied, and turbulence is generated in the throat 22 while reducing the diameter of the pipe and increasing the flow velocity. In this state, air C having an increased oxygen content is supplied from the gas inlet 24, mixed with the filtrate A in the suction part 23, becomes MNB by the water flow, and MNB of the air having an increased oxygen content is supplied from the outlet 25. The filtrate D containing is discharged and supplied to the macro / nano bubble generating tank 6.

  By adjusting the flow rate of the filtrate A and the air C with an increased oxygen content supplied to the MNB generator 7a, the amount and size of the MNB can be adjusted.

  In the first embodiment, in order to obtain air C having an increased oxygen content supplied to the MNB generator 7a, oxygen enrichment means using an oxygen enriched film as shown in FIG. 3 is used. To do.

  In the oxygen-enriching means using this oxygen-enriched film, basically, the container 31 provided with the oxygen-enriched film 30 discharges the air introduction part 33 and the air F having a low oxygen content at both ends. Part 34, the air pressurized by the intake fan 32 is vented from the air introduction part 33 to the oxygen-enriched film 30, and the air C with an increased oxygen content is discharged from the outlet part 35, Air F having a low oxygen content is discharged from the outlet 34.

  In the first embodiment, by increasing the oxygen content of the gas forming the MNB higher than the oxygen content of air (about 21%), the amount of the biological culture liquid extracted from the culture tank is reduced, and the biological culture liquid Even if the amount of MNB contained in is reduced, the activity of the microorganisms and the like can be maintained by supplying the microorganisms with a high concentration of oxygen that is easily absorbed in the MNB state. Furthermore, by reducing the amount of the biological culture solution extracted from the culture tank, it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution.

Moreover, in 1st Embodiment, positive displacement pumps, such as a tube pump, a diaphragm pump, a screw pump, and a rotary pump with comparatively little stress and damage given to microorganisms etc., are suitably used as the culture tank pump 8 and the return pump 9. This also makes it possible to further reduce the stress and damage to microorganisms.
Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.

○ Second embodiment (Fig. 4)
Next, a second embodiment of the present invention will be described with reference to FIG.
The second embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and contains the oxygen-enriched MNB in the biological culture as follows.
a) The culture solution 1 is supplied to the culture tank 2.
b) The valve 15 is closed, the valve 16 is opened, and the culture tank pump 8 is driven to extract the biological culture solution 3 containing microorganisms and the like from the culture tank 2 and supply it to the micro / nano bubble generation tank 6.
c) The biological culture solution 3 is stored in the micro / nano bubble generating tank 6 and oxygen enriched MNB is contained by the micro / nano bubble generating device 7a.
d) The return pump 9 is driven to return the biological culture solution G containing the oxygen-enriched MNB to the culture tank 2.
e) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
f) When the biological reaction has sufficiently progressed, the valve 16 is closed, the valve 15 is opened and the culture tank pump 8 is driven, and the reaction product generated in the culture tank 2 is collected together with the filtrate A to obtain the filtrate. Store in storage tank 5.

  The method of 1) (first embodiment) and the method of 2) (second embodiment), which are methods for adding oxygen-enriched MNB to the biological culture solution extracted from the culture tank, are the types of microorganisms and the like. It is preferable to adopt a method in which the stress and damage to the microorganisms are generally reduced depending on the conditions of the biological reaction.

  In the present invention, as a means for containing an oxygen-enriched MNB in a biological culture solution, the biological culture solution extracted from the culture tank mentioned as the first feature point contains the oxygen-enriched MNB and is returned to the culture tank. Although means (hereinafter referred to as “first means”) is used, other means may be used in combination.

  When the first means is used alone, it may take time to set the MNB content in the biological culture solution in the culture tank to an appropriate value. Includes means for containing oxygen-enriched MNB in the culture medium supplied to the culture tank (hereinafter referred to as “second means”), means for containing oxygen-enriched MNB in the biological culture medium in the culture tank (hereinafter referred to as “second means”). , "Third means") and the like. In particular, the second means is preferable as a means used in combination with the first means because the microorganisms and the like are not subjected to stress or damage due to the blowing of MNB.

○ Third embodiment (FIG. 5)
Next, a third embodiment of the present invention will be described with reference to FIG.
The third embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and uses the second means in combination with the first embodiment (using the first means) of the present invention.

In the third embodiment, the oxygen-enriched MNB is contained in a culture solution such as a microorganism as follows.
a) The culture solution 1 supplied to the culture tank 2 is caused to contain oxygen-enriched MNB (culture solution E containing oxygen-enriched MNB) by the micro / nano bubble generator 7b.
b) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
c) When the content of micro-nano bubbles in the biological culture solution 3 at the beginning of the biological reaction is small, or when the content of micro-nano bubbles in the biological culture solution 3 decreases due to progress of the biological reaction, b in the first embodiment ) To g) By adding the oxygen-enriched MNB to the filtrate A obtained by filtering the biological culture solution 3 and returning to the culture tank 2, the content of micro-nano bubbles in the biological culture solution 3 Adjust to an appropriate value.
d) When the biological reaction is sufficiently advanced, the valve 13 is closed, the valve 12 and the valve 14 are opened, the culture tank pump 8 is driven, and the reaction product generated in the culture tank 2 is collected together with the filtrate A. Store in the filtrate storage tank 5.

○ Fourth embodiment (Fig. 6)
Next, a fourth embodiment of the present invention will be described with reference to FIG.
The fourth embodiment is a biological reaction apparatus for causing a microorganism or the like to generate a reaction product, and the second means and the third means are used in combination with the first embodiment (using the first means) of the present invention. Is.

In the fourth embodiment, the oxygen-enriched MNB is contained in a culture solution such as a microorganism as follows.
a) The culture solution 1 supplied to the culture tank 2 is caused to contain oxygen-enriched MNB (culture solution E containing oxygen-enriched MNB) by the micro / nano bubble generator 7b.
b) In this way, the biological reaction is advanced while stirring the biological culture solution 3 in the culture tank 2 with the culture tank agitator 11.
c) When the content of micro-nano bubbles in the biological culture solution 3 at the beginning of the biological reaction is small, or when the content of micro-nano bubbles in the biological culture solution 3 decreases due to progress of the biological reaction, b in the first embodiment ) To g), the filtrate A obtained by filtering the biological culture solution 3 is made to contain oxygen-enriched MNB and refluxed to the culture vessel 2, or the micronanobubble generator 7c is used to culture the culture vessel. By adding oxygen-enriched MNB to the biological culture solution 3 in 2, the content of the micro / nano bubbles in the biological culture solution 3 is adjusted to an appropriate value.
d) When the biological reaction is sufficiently advanced, the valve 13 is closed, the valve 12 and the valve 14 are opened, the culture tank pump 8 is driven, and the reaction product generated in the culture tank 2 is collected together with the filtrate A. Store in the filtrate storage tank 5.

  As the third embodiment and the fourth embodiment of the present invention, the first embodiment (using the first means) of the present invention, which is the combination of the second means, the second means, and the third means, respectively, has been described. However, similarly, the second embodiment (using the first means) of the present invention can be used in combination with the second means, the second means, and the third means, respectively, and the same effects can be obtained. It can be easily understood by a contractor.

  As described above, in the biological reaction apparatus of the present invention and the biological reaction method using this biological reaction apparatus, the oxygen content of the gas forming MNB is higher than the oxygen content of air (about 21%). By reducing the amount of the biological culture solution extracted from the culture tank and reducing the amount of MNB contained in the biological culture solution, a high concentration of easily absorbed oxygen in the MNB state is supplied to the microorganism. Etc. can be maintained.

  Furthermore, by reducing the amount of the biological culture solution extracted from the culture tank, it is possible to reduce stress and damage to microorganisms and the like, and to reduce the energy required for circulation of the biological culture solution.

In addition, as a pump for circulating a biological culture solution such as a pump for extracting the biological culture solution from the culture vessel, a pump for refluxing the biological culture solution containing oxygen-enriched MNB to the culture vessel, Tube pumps, diaphragm pumps, screw pumps, rotary pumps, and other positive displacement pumps that can be used with relatively little stress and damage on them. This can be further reduced.
Furthermore, by reducing the amount of MNB contained in the culture solution, the energy required for driving the MNB generator can be reduced.

As described above, the present invention is excellent in that a biological reaction using microorganisms or the like can be performed efficiently and economically.
Next, increasing the dissolved oxygen concentration while reducing the stress and damage to microorganisms, which is exhibited by setting the liquid circulation ratio low and setting the oxygen content of MNB high, which is a feature of the present invention. Examples and comparative examples will be described with reference to examples and comparative examples of effects such as the ability to efficiently and economically perform biological reactions using microorganisms. It is not limited by.

<Examples 1-2 and Comparative Examples 1-5>
In the following Examples 1-2 and Comparative Examples 1-5, microorganisms were cultured using an apparatus as schematically shown in FIG.
As the culture tank 2, a microorganism culture apparatus (microbe culture apparatus BMZ-P manufactured by Able Co., Ltd., internal volume 1000 ml) is used, and an aerobic microorganism [a standard strain of coryneform bacteria (corynebacterium glutamicum)], culture A biological culture solution 3 consisting of a solution [synthetic medium mainly composed of ammonium sulfate, concentration of glycolose: 4%] was accommodated, and the amount of the solution was adjusted to 500 mL. The initial bacterial concentration of the biological culture solution 3 was turbidity (OD610 value): 1.

  While culturing aerobic microorganisms with a culture temperature of 33 ° C., a culture pressure of 1 atm, and a rotation speed of the culture tank agitator 11 of 600 rpm, the culture tank pump 8 is driven to set the biological culture solution 3 from the culture tank 2 2 is extracted and supplied to a water flow type MNB generator 7a [a water flow type micro nano bubble generator manufactured by OK Engineering Co., Ltd., model number: OK-MB 200 ml] as shown in the schematic diagram of FIG. Was added to the culture tank 2. The gas C set to a constant oxygen content rate was supplied to the MNB generator 7a at a ventilation rate of 250 mL / min.

  Under these conditions, the aerobic microorganisms are cultured for 8 hours by changing the amount of circulating liquid and the oxygen content of gas C, and the turbidity of the bacterial concentration of the biological culture liquid 3 in the culture tank 2 after 8 hours has elapsed. And dissolved oxygen concentration was measured and it was set as Examples 1-2 and Comparative Examples 1-5. In Examples 1 and 2 and Comparative Examples 1 to 5, the amount of liquid circulation (mL / min), the ratio of liquid circulation (%), the oxygen content C of MNB (%), the turbidity of microbial concentration (value of OD660) and Table 1 shows the dissolved oxygen concentration (ppm). The “liquid circulation rate” refers to the rate of the liquid circulation rate (mL / min) per minute with respect to the amount (500 mL) of the biological culture solution 3 accommodated in the culture tank 2.

Hereinafter, Examples 1-2 and Comparative Examples 1-5 will be described.
First, in Comparative Examples 1 and 2, air is supplied to the water flow type MNB generator 7a, the oxygen content C of the MNB is 21%, the liquid circulation rate B is 48% in Comparative Example 1, and 16 in Comparative Example 2. %. When the liquid circulation rate B is reduced in this way, stress damage caused to the aerobic microorganisms due to the liquid circulation can be reduced, so that the bacterial concentration can be increased from 21 (OD610) to 25 (OD610). On the other hand, in a generally used water flow type MNB generator such as the MNB generator 7a, if the liquid circulation rate B is reduced, the amount of MNB generated itself decreases, so the dissolved oxygen concentration E is 6.9 (ppm). ) To 1.2 (ppm).

  Therefore, in Examples 1-2 and Comparative Examples 3-5, the liquid circulation ratio B was maintained at 16% as in Comparative Example 2, and the stress damage caused to the aerobic microorganisms by the liquid circulation was reduced. The oxygen content C was increased to 30%, 40%, 60%, 80% and 100%, respectively, and the dissolved oxygen concentration E was increased.

  Table 2 shows the relationship between the oxygen content C (%) of MNB and the bacterial concentration D (OD610) in Examples 1 and 2 and Comparative Examples 3 to 5. In Table 2, the vertical axis represents MNB oxygen content C (%) and bacteria concentration D (OD610), and the MNB oxygen content of Comparative Example 2, Examples 1-2, and Comparative Examples 3-5 The rate C (%) and the bacterial concentration D (OD610) are shown as broken lines.

  As can be seen from Table 2, as the oxygen content C of MNB increases from 21% (Comparative Example 2) to 30% (Example 1) to 40% (Example 2), the bacterial concentration D increases. However, the concentration D of oxygen in the MNB turns to a decreasing trend with the oxygen content C of the MNB at around 40% as a peak, and is lower than 21% (Comparative Example 2) at 60% (Comparative Example 2). . This is considered to be because when the oxygen content C of MNB becomes too high, the aerobic microorganisms are subjected to stress damage due to the oxidizing action of oxygen.

From these Examples 1-2 and Comparative Examples 1-5, in order to perform a biological reaction using microorganisms efficiently and economically,
1) The liquid circulation ratio B is set low to reduce the stress and damage to microorganisms due to the liquid circulation,
2) The decrease in dissolved oxygen concentration E associated with setting the liquid circulation rate B low, and the oxygen content rate C of MNB being set high, the dissolved oxygen concentration E while avoiding stress damage caused by oxygen to microorganisms. By increasing
It can be seen that the bacterial concentration in the biological reaction using microorganisms can be increased, and that the biological reaction using microorganisms can be performed efficiently and economically.

  Next, a method for culturing microorganisms or the like using oxygen-enriched MNB that satisfies the above items 1) and 2) will be specifically described. In addition, since culture | cultivation conditions, such as microorganisms, depend on the kind of microorganisms, the scale of a culture apparatus, the structure of a culture apparatus, etc., as shown in FIG. 7 used in the said Examples 1-2 and Comparative Examples 1-5. An explanation will be given while exemplifying a case of culturing microorganisms or the like using a culture apparatus for various microorganisms or the like.

1. Regarding the above 1) In the culture apparatus for microorganisms and the like shown in FIG. 7, the biological culture solution 3 is extracted from the culture tank 2 by the culture tank pump 8 and supplied to the water flow type MNB generator 7a. Oxygen-enriched MNB is contained and refluxed to the culture tank 2. Since microorganisms and the like are greatly stressed and damaged when passing through the MNB generating device 7a, the "stress and damage to which the microorganisms are subjected" is "the pressure of the biological culture solution 3 at the inlet of the MNB generating device 7a". (Hereinafter referred to as “inlet pressure”) can be evaluated as an index.

  Table 3 shows the liquid circulation rate (%) and inlet pressure (MPa) measured by changing the driving force of the culture tank pump 8 in the culture apparatus for microorganisms and the like shown in FIG. The pressure is plotted with the vertical axis.

  Since the resistance to stress and damage varies depending on the microorganisms to be cultured, if an appropriate upper limit value of the inlet pressure according to the type of microorganisms is examined in advance and a database is created, an appropriate inlet pressure is set from the beginning of the culture. be able to. If such a database has not been created, an appropriate inlet pressure can be initially set, and the inlet pressure can be adjusted by judging the amount of stress and damage based on the culture conditions of microorganisms, etc. it can.

  For example, although the inlet pressure was initially set to 0.075 MPa, when the stress and damage received by the microorganisms are large and the culture does not proceed smoothly, the driving force of the culture tank pump 8 is decreased, Adjustment is made so that the inlet pressure is reset to 0.04 MPa. This adjustment can reduce stress and damage to microorganisms and the like, but since the liquid circulation ratio is reduced from 30% to 20%, the dissolved oxygen concentration in the biological culture medium is reduced.

2. Regarding 2) As described in 1) above, when the inlet pressure is reduced in order to reduce the stress and damage to which microorganisms and the like are subjected, the liquid circulation rate is reduced and the dissolved oxygen concentration of the biological culture solution is reduced. However, in order to compensate for this, it is necessary to reset the oxygen content of the MNB to be high.
The appropriate oxygen content of MNB can be set as follows.

First, regarding the dissolved oxygen concentration, the following general formula (1) is known.
OTR = KLA × (Cs−C) (1)
In this equation (1),
OTR: Oxygen transfer rate (mg / L · h)
KLA: Mass transfer capacity coefficient (/ h)
Cs: Saturated solubility of oxygen in water (mg / L)
C: Oxygen solubility in water (mg / L).

Even if the inlet pressure and the liquid circulation ratio are lowered, it is necessary to keep the OTR at a constant value in order to keep the dissolved oxygen concentration of the biological culture solution at a constant value.
The value of OTR is determined by the variables “KLA” and “Cs” and the constant “C”. Of these, “Cs” can be expressed as a function of the oxygen content as shown in the following general formula (2).
Cs = [(X ÷ 100) × P ÷ H] × MO ₂ (2)
In this equation (2),
X: oxygen content (%)
P: Operating pressure of the culture tank (atm)
H: Henry's constant (atm · m ³ / mol)
MO ₂ : Molecular weight of oxygen (g / mol).

In addition, “KLA” can be expressed as a function of the liquid circulation ratio by obtaining the relationship with the liquid circulation ratio in the culture apparatus to be used. For example, Table 4 shows the liquid circulation ratio (%) and KLA (/ h) measured by changing the driving force of the culture tank pump 8 in the culture apparatus for microorganisms and the like shown in FIG. Although (x) and KLA are plotted with the vertical axis (y), an approximate expression such as Expression (3) can be obtained from this.
y = aln (x) -b (3)
In this formula:
y: KLA (/ h)
x: Liquid circulation ratio (%)
a and b are constants.

3. Control by the above 1) and 2) According to the above formulas (1) to (3), for example, the inlet pressure was initially set to 0.075 MPa, but the stress and damage received by microorganisms and the like are large, and the culture is smooth. Therefore, when the driving force of the culture tank pump 8 is decreased and the inlet pressure is adjusted to 0.04 MPa, the liquid circulation rate is decreased from 30% to 20%, and the dissolved oxygen concentration in the biological culture solution is reduced. To compensate for this, it is possible to determine how much the oxygen content of MNB needs to be increased to keep the dissolved oxygen concentration of the biological culture medium constant. As a setting ratio in the case of controlling the oxygen content, 70% to 130% is preferable, 80% to 120% is more preferable, 90% to 110% is further preferable, and 95% to 105% is most preferable. The “installation ratio” refers to the ratio of the control set value to the target value of the oxygen content obtained by the equations (1) to (3).

  Further, based on the above formulas (1) to (3), oxygen-enriched air is supplied to the device for driving the culture tank pump 8 and the MNB generator 7a in the culture device for microorganisms and the like as shown in FIG. Even if the inlet pressure and the liquid circulation rate are reduced by controlling the device, the dissolved oxygen concentration in the biological culture solution can be automatically maintained at a constant value, so that biological reactions using microorganisms can be performed efficiently and economically. Can be done automatically.

DESCRIPTION OF SYMBOLS 1 Culture liquid 2 Culture tank 3 Biological culture liquid containing culture liquid, microorganisms, etc. 4 Filter 5 Filtrate storage tank 6 Micro nano bubble generation tank 7a-7c Micro nano bubble generation apparatus 8 Culture tank pump 9 Return pump 10 Liquid supply pump 11 Culture Tank stirrer 12-16 Valve 21 Inlet part 22 Throat part 23 Suction part 24 Gas inlet 25 Outlet part 30 Oxygen-rich film 31 Container 32 Intake fan 33 Air introduction part 34 (Exhaust air with low oxygen content) Derived part 35 Derived part (discharges air with increased oxygen content) A Filtrate B Biological culture fluid excluding filtrate C Air with increased oxygen content D Filtrate with oxygen enriched MNB (filtrate + Air MNB with increased oxygen content)
E Culture medium containing oxygen-enriched MNB (culture medium + air MNB with increased oxygen content)
F Air with low oxygen content G Biological culture fluid containing oxygen-enriched MNB (biological culture fluid + MNB with air with increased oxygen content)
H biological culture solution 107 culture tank as biological reaction tank 110 microbial cell filter 115 micro-nano bubble generation tank 116 micro-nano bubble generation apparatus

Claims (12)

A culture vessel containing a culture solution and a biological culture solution containing aerobic or facultative anaerobic microorganisms ;
A micro-nano bubble generating device that contains micro-nano bubbles formed from air with an increased oxygen content in the biological culture solution extracted from the culture tank;
A conduit for refluxing the biological culture solution containing the micro / nano bubbles to the culture tank;
A biological reaction device comprising:
The amount of the biological culture liquid extracted from the culture tank and containing micro-nano bubbles and then refluxed to the culture tank is 1% to less than 48% of the amount of the biological culture liquid stored in the culture tank per minute. As well as setting
The biological reaction apparatus characterized by setting the oxygen content rate of the air which raised the said oxygen content rate to 23% or more and less than 60%.   The micro-nano bubble generating apparatus causes the biological culture liquid extracted from the culture tank using the extraction pump or the reflux pump to contain the micro-nano bubbles formed from the air having an increased oxygen content and refluxed to the culture tank. The biological reaction apparatus according to claim 1, wherein Between the culture tank and the micro-nano bubble generating device, a biological culture liquid extracted from the culture tank is disposed, and a filter for separating the filtrate and the biological culture liquid excluding the filtrate is disposed,
While making the filtrate contain micro-nano bubbles formed from air with an increased oxygen content by the micro-nano bubble generator,
The biological reaction apparatus according to claim 1 or 2, further comprising a conduit for refluxing the biological culture solution excluding the filtrate and the filtrate containing the micro / nano bubbles to the culture tank. .   The micro-nano bubbles are directly contained in a biological culture solution extracted from the culture tank without disposing a filter between the culture tank and the micro-nano bubble generator. The biological reaction apparatus as described.   The bioreactor according to any one of claims 1 to 4, wherein the micro / nano bubble generator is of a system driven using a water flow.   Volumes of tube pumps, diaphragm pumps, screw pumps, rotary pumps, etc., as pumps for extracting the biological culture liquid from the culture tank and / or pumps for returning the biological culture liquid containing the micro-nano bubbles to the culture tank The bioreactor according to any one of claims 1 to 5, wherein a type pump is used.   The biological reaction apparatus according to claim 6, wherein a tube pump is used as the positive displacement pump.   The biological reaction apparatus according to any one of claims 1 to 7, wherein the air having an increased oxygen content is obtained by passing air through an oxygen-enriched membrane.   The air having an increased oxygen content is obtained by mixing oxygen generated by any one of the PSA method, the VSA method, the cryogenic separation method, and the chemical adsorption method with air using a line mixer or the like. The biological reaction device according to any one of claims 1 to 8, wherein   The micro-nano bubble generator which makes the culture solution supplied to the said culture tank contain the micro-nano bubble formed from the air which raised oxygen content rate is further provided. Bioreactor.   The device according to any one of claims 1 to 10, further comprising a micro / nano bubble generating device that contains micro / nano bubbles formed from air with an increased oxygen content in the biological culture solution in the culture tank. Bioreactor. A reaction product of an aerobic or facultative anaerobic microorganism is obtained by the biological reaction device according to any one of claims 1 to 11, or an aerobic or facultative anaerobic microorganism is grown. , Biological reaction method.

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