JP7494025B2 - Agitator-free bioreactor using fine/ultra-fine bubbles containing gas useful for culturing microorganisms or cells, and a bioreaction method using said bioreactor - Google Patents

Description

The present invention relates to a biological reaction apparatus that cultures microorganisms or cells (hereinafter also referred to as "microorganisms, etc.") to cause the microorganisms, etc. to produce reaction products or to grow the microorganisms, etc., and a biological reaction method using this biological reaction apparatus, which is characterized by stirring a biological culture solution (hereinafter also referred to as "biological culture solution") that contains a culture solution and microorganisms, etc., contained in a culture tank without using an agitator.

Biological reactions are different from chemical reactions in that the reactions themselves are slow, but they do not use a lot of energy or chemicals, making them gentle and beneficial to the environment.

However, biological reactions generally have the problem of being slow. That is, while a chemical reaction is often sufficient within an hour, biological reactions can take anywhere from a few hours to several days, or even several weeks in particularly long cases. For this reason, there is a demand for biological reactions to be carried out efficiently and economically.

In Patent Documents 1 to 4, the present inventors have proposed using micro-nano bubbles of oxygen-containing gas to efficiently and economically carry out biological reactions of microorganisms, etc.

In addition, in the claims and specification of this patent, conventional "microbubbles" and "nanobubbles" are referred to as "fine bubbles" and "ultrafine bubbles", respectively.

However, in the biological reaction apparatus and biological reaction method of Patent Documents 1 to 4, a stirrer is used to stir the biological culture solution contained in the culture tank,
a) Stress or damage reduces the activity of microorganisms or inhibits their growth.
b) There are some areas where it is difficult to sterilize the medium before biological reaction (the seal of the agitator's rotating shaft, the bearing of the rotating shaft, the agitator blades, baffles, baffles, etc.), making it difficult to prevent contamination.
c) Problems arise such as the costs involved in installing, operating, maintaining and managing the mixer.

In addition, Patent Documents 5 and 6 propose a method for culturing microorganisms and the like that does not use an agitator. However, Patent Document 5 uses a special structure installed in the main body of the culture tank in a special culture called fed-batch culture of bacteria (increasing the amount of medium as the bacteria grow), and natural circulation is performed by utilizing the specific gravity difference between the liquid inside and outside the inner cylinder. In Patent Document 6, cell culture is performed using a special reaction tank in which the lower parts of a pair of vertical culture cylinders are connected, and the culture liquid is stirred by alternately blowing gas from a gas blowing means connected to the lower parts of both culture cylinders. In this way, the stirring methods disclosed in Patent Documents 5 and 6 cannot be adopted in biological reaction devices and biological reaction methods that use general culture tanks because the structure inside the culture tank is complicated and it is difficult to prevent the inclusion of unwanted bacteria.

The present inventors have discovered the following and have made the present invention.
1) In conventional biological reaction apparatuses and biological reaction methods, agitators are used for the purposes of i) finely shearing the gas bubbles supplied to the culture tank, and ii) uniformly mixing the biological culture solution in the culture tank. However, in the biological reaction apparatuses and biological reaction methods using fine or ultrafine bubbles of oxygen-containing gas as described in Patent Documents 1 to 4, the above purpose i) is already sufficiently achieved, and therefore if the above purpose ii) can be achieved, there is little need to use agitators.
2) The objective of ii) above can be achieved economically and efficiently by discharging the biological culture solution containing the fine and ultra-fine bubbles, which is to be returned to the culture tank, toward the culture tank using a multi-nozzle equipped with multiple outlets arranged to discharge in multiple directions at angles between 0° and 90° with respect to the central axis toward the culture tank.
3) As the gas to be contained in the fine and ultra-fine bubbles, in the cultivation of <aerobic or facultative anaerobic microorganisms, etc.> as described in Patent Documents 1 to 4, an oxygen-containing gas is used. However, the invention is not limited to this. In the cultivation of <obligate anaerobic microorganisms, etc.>, a nitrogen-containing gas is used. In the cultivation of microorganisms <organic matter synthesizing microorganisms, etc.> that produce organic matter (amino acids, organic acids, proteins, etc.) from carbon gases derived from natural gas (carbon dioxide, methane, etc.), carbon dioxide, methane, and other carbonaceous gases derived from natural gas are used. In this manner, cultivation can be carried out without the use of a stirrer.

The bioreactor of the present invention, which was developed based on the ideas 1) to 3) above, and the bioreaction method using this bioreactor, are characterized in that, without using an agitator, a biological culture solution containing fine and ultrafine gas bubbles (hereinafter also referred to as "fine bubbles") containing gases useful for culturing microorganisms, such as air, oxygen, nitrogen, carbon dioxide, carbon dioxide gas derived from natural gas, and carbon dioxide gas emitted from thermal power plants (hereinafter also referred to as "useful gases") is discharged in multiple directions into a culture tank using a multi-nozzle. This solves the problems a) to c) above without reducing the activity of microorganisms, and by using a multi-nozzle, agitation can be performed economically and efficiently.

Patent No. 5985114 Japanese Patent No. 6087476 Patent No. 6138390 Patent No. 6499203 Japanese Patent Application Laid-Open No. 6-327460 JP 2012-115232 A

The objective of the bioreactor of the present invention (hereinafter also referred to as the "bioreaction apparatus of the present invention") and the bioreaction method using this bioreactor (hereinafter also referred to as the "bioreaction method of the present invention" and collectively referred to as the "bioreaction apparatus and method of the present invention") is to provide a bioreactor and a bioreaction method that can sufficiently stir the biological culture liquid contained in the culture tank without using an agitator, maintain the activity of microorganisms, etc., and by not using an agitator, can simplify the internal structure of the culture tank and biological reactor, improve cleanability, and prevent bacterial contamination, and ultimately perform stirring economically and efficiently.

In order to solve the above problems, the biological culture solution is extracted from a culture tank and returned to the culture tank after containing fine bubbles. In this case, the biological culture solution containing fine bubbles of a gas containing a useful gas is discharged in multiple directions in the culture tank using a multi-nozzle, thereby stirring the biological culture solution without using a stirrer. Specifically, the biological culture solution is a biological culture tank that contains a biological culture solution, an extraction pipe that extracts the biological culture solution from the culture tank, and a multi-nozzle that is arranged between the extraction pipe and the culture tank and returns the biological culture solution to the culture tank. The multi-nozzle is characterized by having a fine bubble generator that causes the biological culture solution to contain fine bubbles of a gas containing a useful gas, and a plurality of discharge ports provided at the outlet on the culture tank side, and discharging the biological culture solution containing fine bubbles (hereinafter also referred to as "biological culture solution containing fine bubbles") from the multiple discharge ports in multiple directions that are 0° to 90° with respect to the direction of the culture tank side of the central axis of the multi-nozzle.

In the present invention, as described above, by using a multi-nozzle to discharge the biological culture solution containing fine bubbles of a gas containing a useful gas in multiple directions within the culture tank, the biological culture solution contained in the culture tank can be sufficiently stirred without using an agitator, and the activity of microorganisms, etc. can be maintained.

Furthermore, in the present invention, by not using an agitator as described above, the internal structure of the culture tank/bioreaction device can be simplified, and cleaning properties can be improved and contamination by bacteria can be prevented.
Furthermore, by not using an agitator, it is possible to solve the following conventional problems: a) microorganisms etc. become stressed and damaged, which reduces their activity; b) there are areas that are difficult to sterilize before a biological reaction (the seal part of the agitator's rotating shaft, the bearing part of the rotating shaft, the agitating blades, baffles/baffles, etc.), making it difficult to prevent the introduction of bacteria (contamination); and c) the costs involved in installing, operating, maintaining, and managing the agitator.

In this way, by using a multi-nozzle without using a stirrer, the biological culture solution contained in the culture tank can be stirred economically and efficiently.

FIG. 1 is a schematic diagram showing an overview of a first embodiment of a biological reaction apparatus and method of the present invention. FIG. 1 is a schematic diagram showing an overview of a second embodiment of the biological reaction apparatus and method of the present invention. FIG. 1 is a schematic cross-sectional view showing an overview of a multi-nozzle used in the biological reaction device and method of the present invention. FIG. 4 is a schematic cross-sectional view showing the X-X' cross section of FIG. FIG. 4 is a schematic external view showing the left end surface of FIG. 3 . FIG. 1 is a schematic diagram for explaining the acute angle formed by the transport pipeline and the discharge pipeline in the biological reaction apparatus and method of the present invention. FIG. 1 is a schematic diagram for explaining the acute angle formed by the transport pipeline and the discharge pipeline in the biological reaction apparatus and method of the present invention. FIG. 11 is a schematic diagram showing an overview of a third embodiment of the biological reaction apparatus and method of the present invention. FIG. 2 is a schematic cross-sectional view showing a microbubble generating device used in the bioreactor and method of the present invention. FIG. 1 is a schematic diagram showing the appearance of a microbubble generating device preferably used in the biological reaction device and method of the present invention. FIG. 2 is a schematic cross-sectional view showing the operating state of the micro-bubble generating device. FIG. 1 is a schematic diagram showing a conventional biological reaction device.

The following describes in detail the embodiments of the present invention with reference to the attached drawings, but the present invention is not limited to these.

<General aspects of the bioreactor and method of the present invention>
First, general aspects of the bioreactor and method of the present invention will be described.

The bioreactor and method of the present invention can be suitably used for culturing <aerobic or facultative anaerobic microorganisms, etc.>. Specifically, it can be applied not only to the production of reaction products by microorganisms, such as the production of food, pharmaceuticals, chemicals, etc. by brewing and fermentation, and the production of bioethanol using biomass, but also to the proliferation of microorganisms, etc. When culturing <aerobic or facultative anaerobic microorganisms, etc.>, a gas containing oxygen is used as the useful gas.

Biological reactions using <aerobic or facultative anaerobic microorganisms, etc.> involve causing the microorganisms to produce reaction products or grow in a culture solution that contains the microorganisms, etc., contained in a culture tank, using the culture solution as a nutrient source.

The culture medium for <aerobic or facultative anaerobic microorganisms, etc.> contains sugars and a nitrogen source. As sugars, typically, maltose, sucrose, glucose, fructose, mixtures of these, ethanol, etc. are used, and the concentration of sugars in the culture medium is not particularly limited, but is preferably 0.1 to 10 w/v%. As nitrogen sources, ammonium chloride, ammonium sulfate, corn steep liquor, yeast extract, meat extract, peptone, etc. are used, and is preferably 0.1 to 10 w/v%. Furthermore, in addition to the sugars and nitrogen sources, it is preferable to add vitamins, inorganic salts, etc. to the culture medium as necessary.

Examples of aerobic or facultative anaerobic microorganisms that can be used include aerobic and facultative anaerobic microorganisms such as Aspergillus and other koji molds, natto bacteria, acetic acid bacteria, yeasts, and lactic acid bacteria that have been conventionally used in technical fields such as brewing and fermentation, as well as various aerobic and facultative anaerobic microorganisms created by genetic engineering. Examples of cells that can be used include animal cells, particularly genetically modified animal cells, for producing physiologically active peptides or proteins used as antibody drugs.

Furthermore, the bioreactor and method of the present invention can also be used to cultivate obligate anaerobic microorganisms, such as bifidobacteria, whose growth is inhibited by exposure to atmospheric levels of oxygen. When culturing obligate anaerobic microorganisms, nitrogen is used as the useful gas.

Furthermore, the biological reaction apparatus and method of the present invention can also be used to cultivate <organic matter synthesizing microorganisms, etc.> that produce organic matter such as amino acids, organic acids, and proteins from carbon dioxide gases (carbon dioxide, methane gas) derived from natural gas and carbon dioxide gas discharged from thermal power plants. When culturing <organic matter synthesizing microorganisms, etc.>, useful gases used are carbon dioxide gases derived from natural gas and carbon dioxide gas discharged from thermal power plants.

<Fine bubbles used in the biological reaction device and method of the present invention>
Next, the microbubbles used in the bioreactor and method of the present invention will be described.

The term "fine bubbles/ultrafine bubbles" (fine air bubbles) used in the bioreactor and method of the present invention means "fine bubbles" and/or "ultrafine bubbles." While "normal air bubbles" rise rapidly in the water and burst at the surface, disappearing, "fine bubbles," tiny air bubbles with a diameter of 50 μm or less, shrink in the water and disappear, and at that time, together with free radicals, generate "ultrafine bubbles," which are extremely tiny air bubbles with a diameter of 100 nm or less, and these "ultrafine bubbles" remain in the water for a relatively long time.

In the present invention, bubbles with a number-average diameter of 100 μm or less are called "fine bubbles", and bubbles with a number-average diameter of 1 μm or less are called "ultra-fine bubbles". Image analysis, laser diffraction scattering, electrical detection zone method, resonance mass measurement method, optical fiber probe method, etc. are commonly used methods for measuring the bubble diameter of "fine bubbles/ultra-fine bubbles" (microbubbles), and dynamic light scattering, Brownian motion tracking method, electrical detection zone method, resonance mass measurement method, etc. are commonly used methods for measuring the bubble diameter of nanobubbles.

<Features of the biological reactor of the present invention>
First, a conventional bioreactor for cultivating microorganisms and the like by incorporating fine bubbles of an oxygen-containing gas will be described.

As shown in FIG. 12, in a conventional biological reaction device, a culture tank pump 101 extracts biological culture liquid 103 from a culture tank 102, and a fine bubble generator 104, to which an oxygen-containing gas a is supplied, causes fine bubbles of oxygen-containing gas a to be contained in the biological culture liquid 103, which is then returned to the culture tank 102, while an agitator 105 stirs the biological culture liquid 103 in the culture tank 102. In addition, a filter (not shown) is disposed between the culture tank pump 101 and the fine bubble generator 104, and the filtrate separated from the biological culture liquid 103 extracted from the culture tank 102 is supplied to the fine bubble generator 104.

As shown in FIG. 1, the biological reaction apparatus of the present invention includes a culture tank 2 that contains a biological culture solution 1, an extraction pipeline 4 that extracts the biological culture solution 1 from the culture tank 2 by a culture tank pump 3 or the like, and a multi-nozzle 5 that is disposed between the extraction pipeline 4 and the culture tank 2 and returns the biological culture solution 1 to the culture tank 2.
The multi-nozzle 5 is equipped with a fine bubble generator 5-1 for adding fine bubbles of gas A containing a useful gas to the biological culture solution 1, and a plurality of outlets 5-2 provided at the outlet on the culture tank 2 side.
The nozzle is characterized in that a plurality of discharge ports 5-2 are provided so as to discharge the microbubble-containing biological culture solution in multiple directions at angles of 0° to 90° with respect to the forward direction of the central axis B of the multi-nozzle 5.

In the first embodiment of the biological reaction apparatus and method of the present invention shown in [Figure 1], a multi-nozzle 5 is provided at the bottom of the culture tank 2, but as in the second embodiment of the biological reaction apparatus and method of the present invention shown in [Figure 2], the multi-nozzle 5 can also be provided on the side of the culture tank 2.

In this way, when the microbubble-containing biological culture liquid of gas A containing useful gas is returned to the culture tank 2, it is discharged in multiple directions at 0° to 90° with respect to the forward direction of the central axis B of the multi-nozzle 5, thereby providing a biological reaction device and a biological reaction method that can maintain the activity of microorganisms and the like without using an agitator. Furthermore, by not using an agitator, it is possible to solve the conventional problems such as a) the activity of microorganisms and the like is reduced due to stress and damage to the microorganisms, b) the presence of places (seal parts of the rotating shaft of the agitator, bearing parts of the rotating shaft, agitator blades, baffles, baffles, etc.) that are difficult to sterilize before the biological reaction, making it difficult to prevent contamination, and c) the installation, operation, maintenance, and management of the agitator, which require costs. Furthermore, since there is no need to provide components such as baffles and baffles on the inner surface of the culture tank, the inner surface of the culture tank can be treated with Teflon (Teflon: registered trademark) to prevent adhesion of dirt and reduce the risk of contamination.

In the biological reaction apparatus of the present invention, a filter is placed between the extraction pipeline 4 and the multi-nozzle 5 to separate the biological culture liquid 1 extracted from the culture tank 2 into a filtrate and the biological culture liquid excluding the filtrate, and this filtrate can be supplied to the multi-nozzle 5. In this way, the filtrate excluding the microorganisms, etc. is made to contain fine bubbles of gas A containing a useful gas, and discharged from the discharge port 5-2 of the multi-nozzle 5, thereby reducing the stress and damage suffered by the microorganisms, etc. The biological culture liquid excluding the filtrate is either recovered or returned to the culture tank 2 through a separate pipeline.

<Multi-nozzle used in the biological reaction device of the present invention>
Next, a preferred embodiment of the multi-nozzle used in the biological reaction apparatus of the present invention will be described with reference to [FIG. 3] to [FIG. 5].

As a suitable multi-nozzle 5, as shown in FIG.
1) the lower end 5-3 connected to the withdrawal pipeline 4;
2) Upper end portion 5-4 connected to culture tank 2;
3) A plurality of transport pipes 5-5 provided along the central axis B of the nozzle for dividing the biological culture solution 1 supplied to the lower end 5-3 and transporting it to the upper end 5-4 side;
4) a plurality of discharge pipelines 5-6 connected to each of the transport pipelines 5-5; and 5) a plurality of discharge ports 5-2 provided at the outlets of the discharge pipelines 5-6 on the culture tank 2 side.
The present invention is characterized in that the multiple discharge ports 5-2 are provided so as to discharge the microbubble-containing biological culture solution in multiple directions at an angle of 0° to 90° with respect to the direction of the central axis B toward the culture tank 2.

As described above, the multiple outlets 5-2 of the multi-nozzle 5 are usually provided to discharge the microbubble-containing biological culture fluid in multiple directions at 0° to 90° with respect to the direction of the culture tank 2 side of the central axis B. However, when the multiple outlets 5-2 are installed to protrude inward from the wall surface of the culture tank 2, the microbubble-containing biological culture fluid can also be discharged in a direction that is greater than 90° and less than 180° with respect to the direction of the culture tank 2 side of the central axis B. In this way, when the microbubble-containing biological culture fluid is discharged in a direction that is greater than 90° and less than 180° with respect to the direction of the culture tank 2 side of the central axis B, the microbubble-containing biological culture fluid collides with the inner wall surface of the culture tank 2, and the floating of microbubbles is suppressed, etc., so that the options for designing to appropriately stir the biological culture fluid 1 contained in the culture tank 2 can be increased.

[Fig. 4] is a schematic cross-sectional view showing the X-X' cross section of Fig. 3, and thus the multiple transport pipes 5-5 are arranged along the central axis B of the multi-nozzle. Also, [Fig. 5] is a schematic external view showing the left end face of [Fig. 3], and thus the multiple discharge ports 5-2 are preferably arranged symmetrically with respect to the central axis B of the multi-nozzle along the periphery of the upper end 5-4.

Although the fine bubble generating device 5-1 is not shown in FIG. 3, the fine bubble generating device 5-1 may be installed in the following manner.
a) An embodiment in which one fine bubble generating device 5-1 is provided between the lower end portion 5-3 and the transport pipe 5-5;
b) A mode in which a fine bubble generator 5-1 is provided in each discharge pipeline 5-6, or c) A mode in which a fine bubble generator 5-1 is provided in each transport pipeline 5-5, can be adopted.

The above embodiment a) is preferred from the viewpoint of installation, maintenance, and other costs, while the above embodiments b) and c) are preferred from the viewpoint of being able to individually adjust the amount of microbubbles in the biological culture solution 1 discharged from each discharge port 5-2.

<Discharge of biological culture solution containing fine bubbles>
In the multi-nozzle of the biological reaction device of the present invention, the multiple outlets are provided so as to discharge the microbubble-containing biological culture solution in multiple directions at angles of 0° to 90° relative to the forward direction of the central axis of the multi-nozzle. As a result, the biological culture solution contained in the culture tank can be sufficiently stirred by the microbubble-containing biological culture solution discharged from the multiple outlets without using a stirrer, and the activity of microorganisms, etc. can be maintained.

In order to discharge the microbubble-containing biological culture liquid in multiple directions at angles between 0° and 90° relative to the forward direction of the central axis B of the multi-nozzle, the positional relationship between the pair of connected transport pipes 5-5 and discharge pipes 5-6 can be set as follows:

[Positional relationship A]
As shown in Figures 6 and 7, in each plane C including a pair of connected conveying pipelines 5-5 and discharge pipelines 5-6, the acute angle α formed by the conveying pipelines 5-5 and the discharge pipelines 5-6 is set to be greater than 90° and less than 180°.

This positional relationship A allows the direction of the microbubble-containing biological culture solution discharged from each outlet 5-2 to move in multiple directions away from the central axis B of the multi-nozzle, thereby stirring the biological culture solution 1 contained in the culture tank 2.

If the number of discharge ports 5-2 is n, the angles α1 to αn of each pair 1 to n of the connected conveying pipeline 5-5 and discharge pipeline 5-6 can be individually set as appropriate within the range of more than 90° and less than 180°.

[Positional relationship B]
As shown in FIG. 7, the acute angle β formed by each plane C including a pair of connected conveying pipelines 5-5 and discharge pipelines 5-6 and a plane D including the conveying pipelines and the central axis B of the multi-nozzle included in each plane C is set to be greater than 0° and less than 90°.

This positional relationship B allows the direction of the microbubble-containing biological culture solution discharged from each discharge port 5-2 to be in multiple directions around the central axis B, thereby stirring the biological culture solution 1 contained in the culture tank 2.

If the number of outlets 5-2 is n, there are n pairs of planes C and their corresponding planes D, and the angles β1 to βn in each pair can be individually set as appropriate within the range of more than 0° and less than 90°.

The angles α1 to αn and angles β1 to βn can be set appropriately through experiments, simulations, etc., depending on the shape of the culture tank 2, the number and installation positions of the discharge ports 5-2, etc., so that the biological culture solution 1 contained in the culture tank 2 can be appropriately stirred.

<Shape and structure of the culture tank>
The shape of the culture tank used in the bioreactor and method of the present invention can be a commonly used cylindrical, cubic, or rectangular shape, but from the viewpoint of uniform and homogeneous stirring, a cylindrical shape is preferred.

As the culture tank used in the bioreactor and method of the present invention, a vertical culture tank such as the first embodiment of the bioreactor and method of the present invention shown in [Fig. 1] and the second embodiment of the bioreactor and method of the present invention shown in [Fig. 2], or a horizontal culture tank such as the third embodiment of the bioreactor and method of the present invention shown in [Fig. 8] can be used, but it is preferable to use a horizontal culture tank. By using a horizontal culture tank as shown in [Fig. 8], the number of multi-nozzles, the installation position of the multi-nozzles, etc. can be designed by experiments, simulations, etc. so that the biological culture liquid 1 contained in the culture tank 2 can be appropriately stirred, and it becomes easier to achieve a desired stirring state. In addition, if the amount of fine bubbles supplied per unit volume of the biological culture liquid 1 is constant, when a horizontal culture tank is used, the amount of fine bubbles per surface of the biological culture liquid 1 can be reduced, so that the thickness of the bubbles formed on the surface of the biological culture liquid 1 can be reduced.

<Number of multi-nozzles installed>
In the biological reaction apparatus and method of the present invention, it is preferable that a plurality of multi-nozzles are provided. By providing a plurality of multi-nozzles, the number of multi-nozzles, the installation position of the multi-nozzles, etc. can be designed by experiments, simulations, etc. so that the biological culture solution 1 contained in the culture tank 2 can be appropriately stirred. It is possible to increase the options when performing the design, and it is easy to realize a desired stirring state.

Furthermore, by independently adjusting the amount of microbubble-containing biological culture fluid discharged from each of the multiple multi-nozzles, it is possible to further increase the design options for achieving the desired mixing state.

<Micro-bubble generating device installed in multi-nozzle>
As the fine bubble generating device provided in the multi-nozzle, a publicly known or commercially available water flow type fine bubble generating device can be used.

An example of a water flow type micro-bubble generator is shown in [Figure 9]. In this micro-bubble generator 6, biological culture liquid is supplied from the inlet 6-1 of the micro-bubble generator 6 under pressure, and turbulence is generated at the throat 6-2 while the flow rate is increased by narrowing the diameter of the pipe. In this state, gas A containing useful gas is supplied from the gas inlet 6-3, mixed with the biological culture liquid at the suction section 6-4, and turned into micro-bubbles by the water flow, and the biological culture liquid containing micro-bubbles of gas A containing useful gas is discharged from the outlet 6-5.

Moreover, it is preferable to use a fine-bubble generator in which the gas inlet 6-3 is a slit provided continuously on the side along a plane perpendicular to the central axis of the fine-bubble generator 6, as shown in [Fig. 10] to [Fig. 11]. When such a fine-bubble generator is used, as shown in [Fig. 11], the gas A containing useful gas blown in through the slit forms a continuous wide thin layer E of gas along the inner surface of the fine-bubble generator 6 and flows along the inner surface of the fine-bubble generator 6, and fine bubbles F are gradually formed, and a large amount of fine bubbles F is formed near the outlet portion 6-5, so that the efficiency of fine bubble generation can be improved and the fine bubble content of the biological culture liquid can be efficiently and sufficiently increased. Furthermore, the thin layer F acts as a cushion to prevent microorganisms, etc. from colliding with the inner surface of the fine-bubble generator 6, so that stress and damage to the microorganisms, etc. can be reduced.

＜Other＞
The upper limit of the oxygen content of the fine bubbles is less than 60%, preferably 55% or less, more preferably 50% or less, and most preferably 45% or less. If the oxygen content of the fine bubbles 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. In addition, the lower limit of the oxygen content of the fine bubbles is 23% or more, preferably 25% or more, more preferably 27% or more, and most preferably 30% or more. If the oxygen content of the fine bubbles is excessively reduced to less than 23%, the dissolved oxygen concentration will decrease, making it difficult to increase the activity of microorganisms and the like. In order to obtain a gas that forms fine bubbles with an increased oxygen content, it is usually preferable to increase the oxygen content of the gas using known oxygen enrichment means such as the PSA method, VSA method, etc. using an adsorbent, water electrolysis method, cryogenic separation method, membrane separation method, chemical adsorption method, etc., and from an economical point of view, it is preferable to use an oxygen enrichment membrane.

In addition, a positive displacement pump such as a tube pump, diaphragm pump, screw pump, or rotary pump can be preferably used as the culture tank pump 3 for extracting the biological culture solution 1 from the culture tank 2, as these pumps cause relatively little stress or damage to the microorganisms, etc.

<Summary>
As described above, the biological reaction apparatus and method of the present invention uses a multi-nozzle to discharge the biological culture solution containing fine bubbles of a gas containing a useful gas in multiple directions in the culture tank, without using a stirrer. The biological culture solution contained in the culture tank can be sufficiently stirred, and the activity of microorganisms, etc. can be maintained.

Furthermore, the bioreactor and method of the present invention do not use an agitator, which simplifies the internal structure of the culture tank and bioreactor, improving cleanability and preventing bacterial contamination.

By not using an agitator, problems such as a) stress and damage to microorganisms, which reduces their activity, b) difficulty in preventing contamination due to the presence of areas that are difficult to sterilize before biological reactions (seal parts of the agitator's rotating shaft, bearing parts of the rotating shaft, agitator blades, baffles, baffles, etc.), and c) the costs involved in installing, operating, maintaining, and managing the agitator can be solved.

In this way, by using a multi-nozzle without using a stirrer, the biological culture solution contained in the culture tank can be stirred economically and efficiently.

1 Biological culture liquid (containing culture liquid, microorganisms, etc.) 2 Culture tank 3 Culture tank pump 4 Extraction pipeline 5 Multi-nozzle 5-1 Fine bubble (fine bubble / ultra fine bubble) generator 5-2 (Multiple) discharge port 5-3 Lower end 5-4 Upper end 5-5 (Multiple) transport pipelines 5-6 (Multiple) discharge pipelines 6 Fine bubble generator 6-1 Inlet section 6-2 Throat section 6-3 Gas inlet 6-4 Suction section 6-5 Outlet section A Gas containing useful gas B (of multi-nozzle) Central axis C Each plane D including a pair of connected transport pipelines and discharge pipelines Plane E including the central axis B of the transport pipeline and multi-nozzle included in each plane C Wide thin layer F Fine bubble α In each plane C, the acute angle side angle β formed by the transport pipeline and discharge pipeline In each plane C, the acute angle side angle formed by the plane D including the central axis B of the transport pipeline and multi-nozzle included in each plane C 101 Culture tank pump 102 Culture tank 103 Biological culture solution 104 Microbubble generator 105 Agitator a Oxygen-containing gas

Claims (10)

A culture tank containing a biological culture solution containing a culture solution and microorganisms or cells;
A biological reaction apparatus comprising: an extraction pipe for extracting the biological culture solution from the culture tank; and a multi-nozzle disposed between the extraction pipe and the culture tank for returning the biological culture solution to the culture tank;
The above multi-nozzle is
a fine/ultra-fine bubble generator for adding fine/ultra-fine bubbles of a gas containing a gas useful for culturing the microorganism or cell to the biological culture solution; and a plurality of discharge ports provided at the outlet on the culture tank side,
The plurality of outlets are provided so as to discharge the biological culture solution containing the fine bubbles and ultra-fine bubbles in multiple directions at 0° to 90° with respect to the culture tank side direction of the central axis of the multi-nozzle ,
The above multi-nozzle is
A lower end connected to the withdrawal pipe line,
An upper end portion connected to the culture tank;
A plurality of transport pipes provided along a central axis of the multi-nozzle, which are connected to the lower end portion and divide the biological culture solution supplied to the lower end portion and transport it to the upper end portion; and
A plurality of discharge pipes are connected to the respective conveying pipes,
A biological reaction apparatus characterized in that the fine bubble/ultra-fine bubble generator is provided in each of the discharge pipelines or each of the transport pipelines . Between the extraction pipeline and the multi-nozzle, a filter is disposed to separate the biological culture solution extracted from the culture tank into a filtrate and the biological culture solution excluding the filtrate,
The biological reaction apparatus according to claim 1, wherein the filtrate is supplied to the multi-nozzle. The multi-nozzle includes the plurality of discharge ports provided at the outlets of the culture tank side of each of the discharge pipelines,
3. The biological reaction apparatus according to claim 1 or 2, characterized in that in each plane including the pair of connected conveying pipelines and the discharge pipeline, the acute angle formed by the conveying pipeline and the discharge pipeline is set to be greater than 90° and less than 180°. The biological reaction device according to claim 3, characterized in that each of the planes in the multi-nozzle is a plane that includes the central axis of the multi-nozzle. The biological reaction device according to claim 3, characterized in that each of the planes in the multi-nozzle does not include the central axis of the multi-nozzle, and the acute angle formed by each of the planes and a plane including the central axis of each of the transport pipes and the multi-nozzle is set to be greater than 0° and less than 90°. The biological reaction apparatus according to any one of claims 1 to 5 , wherein the culture tank is a horizontal culture tank. The biological reaction apparatus according to any one of claims 1 to 5 , wherein the culture tank is a vertical culture tank. The biological reaction apparatus according to any one of claims 1 to 7 , characterized in that a plurality of the multi-nozzles are provided. 9. The biological reaction apparatus according to claim 8, wherein the amount of the biological culture solution containing the fine bubbles and ultra-fine bubbles discharged from the plurality of multi-nozzles is independently adjusted for each of the plurality of multi -nozzles. A biological reaction method, comprising obtaining a reaction product of the microorganism or cell, or growing the microorganism or cell, by the biological reaction apparatus according to any one of claims 1 to 9 .

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