JP7546426B2 - A multi-nozzle for use in a biological reactor that uses fine and ultra-fine bubbles containing gases useful for culturing microorganisms or cells. - Google Patents

Description

The present invention relates to a multi-nozzle used in a biological reaction device to which fine and ultra-fine bubbles containing a gas useful for culturing microorganisms or cells (hereinafter also referred to as "microorganisms, etc.") are applied, more specifically, to a multi-nozzle used in a biological reaction device that causes fine and ultra-fine bubbles containing a gas useful for culturing microorganisms, etc. to be added to 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, and is used to stir the biological culture solution (hereinafter also referred to as "biological culture solution in the culture tank") contained in the culture tank without using a stirrer.

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 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 the claims and specifications of this patent, the conventional "micro-bubbles" and "nano-bubbles" are referred to as "fine bubbles" and "ultra-fine bubbles", respectively.

However, in the biological reaction apparatuses of Patent Documents 1 to 4, a stirrer is used to stir the biological culture solution 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 agitator.

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 addition, Patent Document 6 uses 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 a biological reaction device that uses a general culture tank because the structure inside the culture tank is complicated and it is difficult to prevent the inclusion of unwanted bacteria.

In addition, Patent Document 7 describes an adherent cell culture device in which, as shown in FIG. 21, a discharge nozzle 206 equipped with a baffle plate 205 is arranged inside an enlarged discharge port 204 in the culture solution of the culture tank 203 in which cells 202 attached to a carrier 201 are cultured, and a culture solution containing microbubbles is discharged from the discharge nozzle 206 so that an agitation flow is formed in the culture solution of the culture tank 203. However, in the adherent cell culture device of Patent Document 7, as described in paragraph [0005], in consideration of special circumstances when culturing adherent cells (when the culture solution is agitated by rotating the agitator blades, there is a risk that the cells 202 will be detached from the carrier 201 due to the mechanical shear force of the agitator blades), the above-mentioned special agitation method is adopted, and the agitation force of the culture solution in the culture tank 203 is small, which cannot be adopted in a general biological reaction device. Furthermore, since a discharge nozzle 206 etc. is provided in the culture solution in the culture tank 203, it is difficult to prevent the inclusion of unwanted bacteria, and this cannot be adopted in general biological reaction devices.

The present inventors have discovered the following and have made the present invention.
1) In conventional biological reactors, 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 biological reactors that use fine or ultrafine bubbles of oxygen-containing gas as described in Patent Documents 1 to 4, the purpose of i) above is already sufficiently achieved, and therefore if the purpose of ii) above 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 a plurality of outlets arranged to discharge in multiple directions at angles of 0° to 90° with respect to the central axis of the multi-nozzle 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 multi-nozzle of the present invention, which was developed based on the ideas 1) to 3) above, is characterized in that when it is used in a biological reaction device to which fine and ultra-fine bubbles containing gases useful for culturing microorganisms and the like are applied, it can discharge, without the use of an agitator, biological culture liquid containing fine and ultra-fine bubbles (hereinafter also referred to as "fine bubbles") of gases useful for culturing microorganisms and the like (hereinafter also referred to as "useful gases") such as air, oxygen, nitrogen, carbon dioxide, carbon dioxide gas derived from natural gas, and carbon dioxide gas emitted from thermal power plants, in multiple directions at angles of 0° to 90° with respect to the direction of the culture tank side of the central axis of the multi-nozzle. This solves the problems a) to c) above without reducing the activity of microorganisms and the like, and allows for economical and efficient stirring.

The multi-nozzle of the present invention has a structure as defined in claims 1 to 13 of the claims, and a) uses the water flow of the biological culture solution extracted from the culture tank and circulated to generate fine bubbles of useful gas, and b) discharges the biological culture solution containing the fine bubbles of useful gas (hereinafter also referred to as "biological culture solution containing fine bubbles") from multiple outlets in multiple directions at angles of 0° to 90° with respect to the direction of the central axis of the multi-nozzle toward the culture tank, thereby stirring the biological culture solution in the culture tank and uniformly mixing the biological culture solution including the fine bubbles of useful gas in the culture tank. The nozzle of the present invention is called a multi-nozzle because it has a structure in which multiple nozzles each equipped with a transport pipe/discharge pipe/discharge port are bundled together.

Patent No. 5985114 Patent No. 6087476 Patent No. 6138390 Patent No. 6499203 Japanese Patent Application Publication No. 6-327460 JP 2012-115232 A JP 2011-120535 A

The objective of the multi-nozzle of the present invention is to provide a multi-nozzle that, when applied to a biological reactor using fine bubbles of gas containing a useful gas, can adequately stir the biological culture liquid in the culture tank without using an agitator, maintaining the activity of microorganisms, etc., and by not using an agitator, can simplify the internal structure of the culture tank of the biological reactor, improve cleanability, and prevent contamination by bacteria, thereby enabling economical and efficient stirring.

To solve the above problems, the multi-nozzle of the present invention is equipped with a micro-bubble generator that causes the biological culture solution to contain micro-bubbles of a gas that contains a useful gas, and multiple outlets provided at the outlet on the culture tank side, and can stir the biological culture solution in the culture tank by discharging the micro-bubble-containing biological culture solution from the multiple outlets in multiple directions at angles of 0° to 90° with respect to the direction of the central axis of the multi-nozzle toward the culture tank.

As described above, the multi-nozzle of the present invention discharges biological culture liquid containing fine bubbles of useful gas in multiple directions at angles of 0° to 90° with respect to the direction of the central axis of the multi-nozzle toward the culture tank, thereby allowing the biological culture liquid in the culture tank to be sufficiently stirred without the use of an agitator, and the activity of microorganisms, etc. to be maintained.

Furthermore, when the multi-nozzle of the present invention is used in a biological reactor that uses fine bubbles containing gases useful for culturing microorganisms, etc., it becomes unnecessary to use an agitator as described above, and the internal structure of the culture tank of the biological reactor can be simplified, improving cleanability and preventing bacterial contamination.

By not using an agitator, it is possible to solve the following problems that were previously encountered: a) microorganisms are stressed and damaged, which reduces their activity or inhibits their growth; b) there are areas that are difficult to sterilize before a biological reaction (seal parts of the agitator's rotating shaft, bearing parts of the rotating shaft, agitator blades, baffles, baffles, etc.), making it difficult to prevent contamination; and c) the costs involved in installing, operating, maintaining, and managing the agitator.

In this way, by using the multi-nozzle of the present invention without using an agitator, the biological culture solution in the culture tank can be agitated economically and efficiently.

1 is a schematic cross-sectional view showing an overview of a multi-nozzle according to the present invention. FIG. 2 is a schematic cross-sectional view showing the X-X' cross section of FIG. FIG. 2 is a schematic external view showing the left end surface of FIG. 1 . 10 is a schematic diagram for explaining an acute angle formed between a conveying pipe and a discharge pipe in a multi-nozzle of the present invention. FIG. 10 is a schematic diagram for explaining an acute angle formed between a conveying pipe and a discharge pipe in a multi-nozzle of the present invention. FIG. FIG. 1 is a schematic diagram showing an overview of a first embodiment of a biological reaction apparatus to which a multi-nozzle of the present invention is applied. FIG. 1 is a schematic diagram showing an overview of a second embodiment of a biological reaction apparatus to which a multi-nozzle of the present invention is applied. FIG. 11 is a schematic diagram showing an overview of a third embodiment of a biological reaction apparatus to which the multi-nozzle of the present invention is applied. FIG. 2 is a schematic cross-sectional view showing an outline of a fine bubble generating device used in the multi-nozzle of the present invention. FIG. 1 is a schematic external view showing a first example of a fine bubble generating device suitably used in the multi-nozzle of the present invention. FIG. 11 is a schematic cross-sectional view showing the operating state of the micro-bubble generating device of FIG. 12 is a schematic diagram showing a cross section taken along line II in FIG. 11. FIG. 11 is a schematic diagram showing the appearance of the micro-bubble generating device of FIG. 10 provided with a gas supply unit. FIG. 2 is a schematic cross-sectional view showing a second example of a fine bubble generating device suitably used in the multi-nozzle of the present invention. FIG. 11 is a schematic cross-sectional view showing a third example of a fine bubble generating device that can be suitably used in the multi-nozzle of the present invention. FIG. 11 is a schematic cross-sectional view showing a fourth example of a fine bubble generating device that can be suitably used in the multi-nozzle of the present invention. FIG. 1 is a schematic diagram showing the appearance of a conventional fine bubble generating device. FIG. 1 is a schematic diagram showing a cross section of a conventional fine bubble generating device. 19 is a schematic diagram showing a cross section taken along line II-II in FIG. 18. FIG. 1 is a schematic diagram showing a biological reaction apparatus using a conventional agitator. FIG. 1 is a diagram showing the configuration of an adherent cell culture device described in Patent Document 7.

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

<General aspects of the multi-nozzle of the present invention>
First, general aspects of a biological reaction apparatus to which the multi-nozzle of the present invention is applied will be described.

The biological reaction device to which the multi-nozzle of the present invention is applied 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, fermentation, etc., 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.

[Aerobic or facultative anaerobic microorganisms, etc.] include aerobic and facultative anaerobic microorganisms such as Aspergillus and other koji molds, natto bacteria, acetic acid bacteria, yeast bacteria, and lactic acid bacteria that are conventionally used in technical fields such as brewing and fermentation, as well as various aerobic and facultative anaerobic microorganisms created by genetic engineering. In addition, examples of cells include animal cells, particularly genetically modified animal cells, for producing physiologically active peptides or proteins used as antibody drugs.

Furthermore, the bioreactor to which the multi-nozzle of the present invention is applied can also be used to cultivate [obligate anaerobic microorganisms, etc.] whose growth is inhibited by exposure to oxygen at atmospheric concentrations, such as bifidobacteria. When culturing [obligate anaerobic microorganisms, etc.], nitrogen is used as the useful gas.

Furthermore, the biological reactor to which the multi-nozzle of the present invention is applied can also be used to cultivate [organic matter synthesis 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 synthesis microorganisms, etc.], useful gases are used, such as carbon dioxide gases derived from natural gas and carbon dioxide gas discharged from thermal power plants.

<Fine bubbles used in a biological reaction device to which the multi-nozzle of the present invention is applied>
Next, the multi-nozzle of the present invention and the fine bubbles used in the biological reaction device to which the multi-nozzle of the present invention is applied will be described.

The term "fine bubbles/ultra-fine bubbles" (microbubbles) used in the multi-nozzle of the present invention and in the biological reaction device to which the multi-nozzle of the present invention is applied means "fine bubbles" and/or "ultra-fine bubbles". While "normal bubbles" rise rapidly in the water and burst at the surface and disappear, "fine bubbles", microscopic bubbles with a diameter of 100 μm or less, shrink in the water and disappear, and at that time, together with free radicals, generate "ultra-fine bubbles", which are extremely small bubbles with a diameter of 1 μm or less, and these "ultra-fine bubbles" remain in the water for a relatively long time.

In the multi-nozzle of the present invention and in the biological reaction device to which the multi-nozzle of the present invention is applied, 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.

<Characteristics of a biological reactor to which the multi-nozzle of the present invention is applied>
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. 20, 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 the first embodiment of FIG. 6, the biological reaction apparatus to which the multi-nozzle of the present invention is applied includes a culture tank 2 that contains a biological culture solution 1, and an extraction pipeline 4 that extracts the biological culture solution 1 from the culture tank 2 using a culture tank pump 3 or the like. The multi-nozzle 5 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 includes a micro-bubble generator 5-1 that causes the biological culture solution 1 to contain micro-bubbles of gas A that contains a useful gas, and a plurality of discharge ports 5-2 provided at the outlet on the culture tank 2 side, and is characterized in that the plurality of discharge ports 5-2 are provided to discharge the micro-bubble-containing biological culture solution in multiple directions that are 0° to 90° with respect to the direction of the culture tank 2 side of the central axis B of the multi-nozzle 5.

<Installation of the multi-nozzle of the present invention>
The multi-nozzle of the present invention is installed outside the culture tank 2, but the number of multi-nozzles, the installation positions of the multi-nozzles, etc. can be set by experiments, simulations, etc. so that the biological culture solution 1 contained in the culture tank 2 can be appropriately stirred. For example, one multi-nozzle 5 can be installed at the bottom of the vertical culture tank 2 as in the first embodiment of Fig. 6, one multi-nozzle 5 can be installed at the side of the vertical culture tank 2 as in the second embodiment of Fig. 7, or multiple multi-nozzles 5 can be installed at the bottom of the horizontal culture tank 2 as in the third embodiment of Fig. 8.

The shape of the culture tank in which the multi-nozzle of the present invention is installed can be any commonly used cylindrical, cubic, or rectangular shape, but from the perspective of performing uniform and homogeneous stirring, a cylindrical shape is preferred.

In addition, examples of the culture tank in which the multi-nozzle of the present invention is installed include a vertical culture tank as in the first embodiment of FIG. 6 and the second embodiment of FIG. 7, or a horizontal culture tank as in the third embodiment of FIG. 8, but a horizontal culture tank is preferred. By installing the multi-nozzle in a horizontal culture tank as in the third embodiment of FIG. 8, the number of multi-nozzles 5 and the installation position of the multi-nozzle 5 can be increased by experiments, simulations, etc., so that the biological culture solution 1 contained in the culture tank 2 can be appropriately stirred, making it easier to achieve a desired stirring state. In addition, if the amount of fine bubbles supplied per unit volume of the biological culture solution 1 is constant, when a horizontal culture tank is used, the amount of fine bubbles per surface of the biological culture solution 1 can be reduced, so that the thickness of the bubbles formed on the surface of the biological culture solution 1 can be reduced.

<Number of multi-nozzles installed>
It is preferable that a plurality of multi-nozzles 5 of the present invention are provided in the culture tank 2. By providing a plurality of multi-nozzles 5, the number of multi-nozzles, the installation position of the multi-nozzles, etc. can be increased by experiments, simulations, etc., so that the biological culture solution 1 contained in the culture tank 2 can be appropriately stirred, and a desired stirring state can be easily realized. Furthermore, by independently adjusting the discharge amount of the microbubble-containing biological culture solution discharged from the plurality of multi-nozzles, the number of options when designing to realize a desired stirring state can be further increased.

In this way, in the bioreactor to which the multi-nozzle of the present invention is applied, when the microbubble-containing biological culture liquid of gas A containing useful gas is returned to the culture tank 2, the biological reaction apparatus and biological reaction method can be provided in which the activity of microorganisms and the like can be maintained without using an agitator by discharging in multiple directions at 0° to 90° with respect to the direction of the central axis B of the multi-nozzle 5 toward the culture tank 2. And, by not using an agitator, it is possible to solve the conventional problems such as a) the microorganisms and the like being stressed and damaged, the activity of the microorganisms and the like being reduced or their growth being inhibited, b) the occurrence of places where it is difficult to sterilize before the biological reaction (seal part of the rotating shaft of the agitator, the bearing part of the rotating shaft, the agitator blades, baffles, baffles, etc.), making it difficult to prevent contamination, and c) the installation, operation, maintenance, management, etc. of the agitator. Furthermore, since there is no need to provide members 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 this way, by using a multi-nozzle without using a stirrer, the biological culture solution in the culture tank can be stirred economically and efficiently.

In a biological reaction apparatus to which the multi-nozzle of the present invention is applied, 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.

<Preferred embodiment of the multi-nozzle of the present invention>
Next, preferred embodiments of the multi-nozzle of the present invention will be described with reference to FIGS.

As a preferred 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 nozzle is characterized in that the nozzle 5 has a plurality of discharge ports 5-2 that are arranged 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 of the multi-nozzle 5 toward the culture tank 2.

As described above, the multiple outlets 5-2 of the multi-nozzle 5 are usually provided so as 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 of the multi-nozzle 5. However, when the multiple outlets 5-2 are installed so as 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 the 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.

Figure 2 is a schematic cross-sectional view showing the X-X' cross section of Figure 1, and in this way, the multiple transport pipes 5-5 are arranged along the central axis B of the multi-nozzle 5. Also, Figure 3 is a schematic external view showing the left end face of Figure 1, and in this way, the multiple discharge ports 5-2 are preferably arranged symmetrically with respect to the central axis B of the multi-nozzle 5 and along the periphery of the upper end 5-4.

Although the fine bubble generating device 5-1 is not shown in FIG. 1, 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 present invention, the multiple outlets are provided to discharge the microbubble-containing biological culture solution in multiple directions at angles of 0° to 90° with respect to the direction of the central axis of the multi-nozzle toward the culture tank. This allows the biological culture solution in the culture tank to be sufficiently stirred by the microbubble-containing biological culture solution discharged from the multiple outlets without using a stirrer, thereby maintaining the activity of microorganisms, etc.

In order to discharge the microbubble-containing biological culture liquid in multiple directions at angles of 0° to 90° with respect to the direction of the central axis B of the multi-nozzle 5 toward the culture tank 2, 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 4 and 5, 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 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 discharge port 5-2 to be in multiple directions away from the central axis B of the multi-nozzle 5, 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 Figure 5, 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.

<Micro-bubble generating device installed in multi-nozzle>
As the fine bubble generating device used in the multi-nozzle of the present invention, 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 diameter of the pipe is narrowed to increase the flow rate. 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.

<Micro-bubble generating device suitable for use in the multi-nozzle of the present invention>
In the multi-nozzle of the present invention, a fine bubble generator 6 such as the first example shown in Figures 10 to 13, the second example shown in Figure 14, the third example shown in Figure 15, and the fourth example shown in Figure 16 can be suitably used.

In a conventional micro-bubble generator 6 as shown in Figures 17 and 18, gas is blown into the liquid flowing inside the cylindrical main body 6-6 from multiple air outlets 9 on the side of the main body 6-6 along a plane perpendicular to the central axis of the main body 6-6, forming multiple bands of gas G' that flow along the inner surface of the main body 6-6 and become micro-bubbles H' near the outlet 6-5. This results in poor efficiency in generating micro-bubbles, making it difficult to efficiently and sufficiently increase the micro-bubble content of the liquid.

On the other hand, in the microbubble generator 6 such as the first example shown in Fig. 10 to Fig. 13, the second example shown in Fig. 14, the third example shown in Fig. 15, and the fourth example shown in Fig. 16, as shown in Fig. 11 and Fig. 12, the gas blown into the liquid flowing in the main body 6-6 from the slits 7 provided continuously on the side along a plane perpendicular to the central axis of the main body 6-6 forms a continuous wide thin layer G of gas (hereinafter also referred to as "thin layer G") along the inner surface of the main body 6-6 and flows along the inner surface of the main body 6-6, and microbubbles H are gradually formed, and a large amount of microbubbles H is formed near the outlet portion 6-5, so that the generation efficiency of microbubbles is improved and the microbubble content of the liquid can be efficiently and sufficiently increased. Furthermore, the thin layer G acts as a cushion to prevent microorganisms, etc. from colliding with the inner surface of the microbubble generator 6, so that stress and damage to the microorganisms, etc. can be reduced.

The cross-sectional shape of the cylindrical body 6-6a of the main body 6-6 of the micro-bubble generator 6 can be circular or rectangular, but is preferably circular. By making the cross-sectional shape circular, the thickness of the thin layer G can be made uniform, which tends to efficiently increase the micro-bubble content of the liquid. The cross-sectional shape of the cylindrical body 6-6a of the main body 6-6 may be a perfect circle as shown in FIG. 12, or may be an approximately perfect circle or an ellipse.

In the micro-bubble generator 6, the slit 7 is "provided along a plane perpendicular to the central axis of the main body 6-6 (hereinafter also referred to as the "vertical plane")," which means that it is provided roughly along the vertical plane. Also, in the micro-bubble generator 6, the slit 7 is "provided continuously on the side of the main body 6-6," but as will be described later, in cases where the main body 6-6 is formed from a single cylindrical body 6-6a, it is possible to leave a connection in part so that the main body 6-6 does not separate by providing the slit 7.

In addition, it is preferable that the main body 6-6, which is made of a cylindrical body 6-6a, has a rod-shaped body, preferably a rod-shaped body with a circular cross-sectional shape, arranged along its central axis. This makes it possible to increase the flow rate of the liquid flowing through the main body 6-6 (hereinafter also referred to as the "liquid flow rate") even if the amount of liquid supplied to the main body 6-6 is the same, so that the thin layer G can be formed stably and liquid containing fine bubbles, such as fine bubbles or ultra-fine bubbles, can be efficiently produced.

In order to smoothly form the thin layer G in the fine bubble generating device 6,
1) the flow velocity of the liquid, and 2) the flow velocity of the gas blown through the slit 7 in a direction perpendicular to the flow of the liquid in the main body 6-6 (hereinafter also referred to as the "gas flow velocity").
It is necessary to strike an appropriate balance between the above. If the flow rate of the gas is too high compared to the flow rate of the liquid, the gas will be blown close to the central axis of the main body 6-6, making it difficult to form the thin layer G. On the other hand, if the flow rate of the gas is too low compared to the flow rate of the liquid, the liquid will leak out of the main body 6-6 through the slit 7.

<Gas supply section>
13, in the fine-bubble generator 6 used in the present invention, a gas supply unit 8 that supplies pressurized gas (e.g., pressurized air and/or oxygen) is connected to the slit 7. Preferably, the gas supply unit 8 is provided airtightly outside the main body 6-6 so as to surround the slit 7.

The pressure of the gas supplied to the gas supply unit 8 (hereinafter also referred to as "gas pressure") is preferably higher than the pressure of the liquid flowing within the main body 6-6, in order to prevent the liquid from leaking out of the main body 6-6 through the slit 7.

On the other hand, increasing the gas pressure also increases the gas flow rate, so if the gas pressure is too high, the gas flow rate will be too high compared to the liquid flow rate, making it difficult to form the thin layer G.

The gas pressure can be set to an appropriate value taking these factors into consideration, but it is usually preferable to set the lower limit at a pressure that prevents liquid from leaking out of the main body 6-6 through the slit 7 (e.g., 1.5 atm) and the upper limit at 3.0 atm.

<Slit>
As shown in FIGS. 10 to 11 and 14 to 16, the slit 7 of the fine bubble generator 6 is a fine hole provided continuously on the side surface along a plane perpendicular to the central axis of the main body 6-6.

The gap of the slit 7 can be set to an appropriate value that allows the formation of a thin layer G, taking into consideration the liquid flow rate, gas flow rate, and gas pressure.

In addition, if the gap of the slit 7 is too narrow, the efficiency of generating fine bubbles H decreases, and if it is too wide, it becomes difficult for fine bubbles H to form from the thin layer G near the outlet portion 6-5, so an appropriate value can be set taking these factors into consideration.

The gap of the slit 7 can be set to an appropriate value taking these factors into consideration, but can usually be set in the range of 0.5 mm to 2.0 mm, preferably 0.5 mm to 1.5 mm, and more preferably 0.5 mm to 0.8 mm.

In order to efficiently increase the microbubble content of the liquid, it is preferable to increase the amount of gas injected by increasing the gas flow rate. However, as explained above in the section <Microbubble generating device installed in a multi-nozzle>, it becomes difficult to form a thin layer G as the gas flow rate is increased compared to the liquid flow rate.

In order to achieve both the above-mentioned "improvement of the microbubble content of the liquid" and the "stable formation of the thin layer G", it is preferable to incline the slit 7 upstream with respect to a plane perpendicular to the central axis of the main body 6-6, as in the microbubble generators of the second example shown in FIG. 14, the third example shown in FIG. 15, and the fourth example shown in FIG. 16. This reduces the gas flow rate in a direction perpendicular to the liquid flow in the main body 6-6, which prevents the formation of the thin layer G even if the gas flow rate itself is increased. The angle θ (hereinafter also referred to as the "inclination angle θ") of the inclination upstream with respect to the plane perpendicular to the central axis of the main body 6-6 can be set to an acute angle so that the thin layer G can be stably formed according to the liquid flow rate, gas flow rate, etc. In consideration of the above-mentioned action, the inclination angle θ is preferably 0° or more and 80° or less, more preferably 60° or more and 80° or less, and even more preferably 70° or more and 80° or less.

In order to achieve both the above-mentioned "improvement of the fine bubble content of the liquid" and the "stable formation of the thin layer G", it is also preferable to provide multiple stages of slits 7 in the main body 6-6. This allows the amount of gas blown in to be increased without increasing the gas flow rate itself. From the viewpoint of increasing the amount of gas blown in, it is preferable to have a large number of stages of slits 7, but if there are too many stages, the nozzle structure becomes complicated and manufacturing and maintenance costs increase, so these factors can be taken into consideration when setting the number of stages appropriately. It is usually preferable for the number of stages of slits 7 to be 1 to 3.

<Slit formation>
The slit 7 can be formed by cutting the peripheral surface of the cylindrical body while leaving a connection portion in part, as in the first example shown in Figures 10 to 13, in which the main body 6-6 is formed from a single cylindrical body 6-6a, or by forming the main body 6-6 from two or more cylindrical bodies 6-6b, 6-6c, etc., and forming the slit 7 at the connection portion of these cylindrical bodies 6-6b, 6-6c, etc., as in the second example shown in Figure 14, the third example shown in Figure 15, and the fourth example shown in Figure 16.

In the second example shown in FIG. 14, the main body 6-6 is formed from two cylindrical bodies 6-6b, 6-6c with a circular cross section, and a slit 7 with an inclination angle θ is formed at the connection between these cylindrical bodies 6-6b, 6-6c, etc. However, in this second example, as the slit 7 with a small cross section becomes longer, there are concerns that cleaning the slit 7 will become more difficult and that the pressure of the gas supplied to the main body 6-6 will decrease due to pressure loss.

The third example shown in Figure 15 addresses the above concerns by shortening the length of the slit 7 by cutting off the tip of the connection between the cylindrical body 6-6c, which has a circular cross-sectional shape, and the cylindrical body 6-6b, which also has a circular cross-sectional shape. This makes it easier to clean the slit 7 and reduces the pressure loss of the gas supplied into the main body 6-6.

<Exit>
As shown in FIG. 11, near the outlet 6-5, the thin layer G separates from the inner surface of the main body 6-6 to form fine bubbles H. In order to make it easier for the thin layer G to separate from the inner surface of the main body 6-6, the inner diameter of the main body 6-6 near the outlet 6-5 can be gradually expanded toward the downstream side.

Also, as in the fourth example shown in Figure 16, by forming irregularities on the inner surface of the main body 6-6 near the outlet 6-5 and generating turbulence in the liquid flow, it is possible to promote the formation of fine bubbles H. Methods for forming irregularities on the inner surface of the main body 6-6 include cutting the inner surface of the main body 6-6 to form recesses, joining a coil-shaped member to the inner surface of the main body 6-6 to form protrusions, etc.

＜Other＞
When oxygen is used as a gas useful for culturing microorganisms, 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 that the microorganisms, etc. receive 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, etc. 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, as a culture tank pump for extracting the biological culture solution from the culture tank, a positive displacement pump such as a tube pump, diaphragm pump, screw pump, or rotary pump, which causes relatively little stress or damage to microorganisms, etc., can be suitably used.

In addition, it is preferable to use metals such as stainless steel and titanium, which have excellent corrosion resistance, and synthetic resins such as Teflon (registered trademark), which have excellent heat and chemical resistance, as materials for the multi-nozzle of the present invention.

<Summary>
As described above, in the multi-nozzle of the present invention, the biological culture solution containing fine bubbles of a gas containing a useful gas is discharged in multiple directions at angles of 0° to 90° relative to the direction of the central axis of the multi-nozzle toward the culture tank. This allows the biological culture solution in the culture tank to be sufficiently stirred without the use of a stirrer, and the activity of microorganisms, etc. can be maintained.

Furthermore, in a biological reactor using the multi-nozzle of the present invention, by not using an agitator, the internal structure of the culture tank of the biological reactor can be simplified, and cleaning properties can be improved and contamination by bacteria can be prevented. By not using an agitator, problems such as a) stress and damage to microorganisms, which reduces their activity and inhibits their growth, b) difficulty in preventing contamination due to the presence of areas that are difficult to sterilize before biological reactions (sealing 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 in the culture tank can be stirred economically and efficiently.

1 Biological culture fluid (containing culture fluid, microorganisms, etc.) 2 Culture tank 3 Culture tank pump 4 Extraction pipe 5 Multi-nozzle 5-1 Fine bubble / ultra-fine bubble (fine bubble) generator 5-2 (Multiple) discharge port 5-3 Lower end 5-4 Upper end 5-5 (Multiple) transport pipes 5-6 (Multiple) discharge pipes 6 Fine bubble generator 6-1 Inlet section 6-2 Throat section 6-3 Gas inlet 6-4 Suction section 6-5 Outlet section 6-6 Main body 6-6a Cylindrical body 6-6b Cylindrical body 6-6c Cylindrical body 7 Slit 8 Gas supply section 9 Air outlet A Gas containing useful gas B (of multi-nozzle) Central axis C Each plane D including a pair of connected transport pipes and discharge pipes Plane E including the transport pipes and central axis B of the multi-nozzle included in each plane C Wide thin layer F Fine bubbles G A continuous wide thin layer G' of gas (formed along the inner surface of the main body) A plurality of bands H of gas (formed along the inner surface of the main body) Fine bubbles H' Fine bubbles α Angle β on the acute angle side formed by the transport pipe and the discharge pipe in each plane C Angle 101 on the acute angle side formed by each plane C and a plane D including the transport pipe and the central axis B of the multi-nozzle included in each plane C Culture tank pump 102 Culture tank 103 Biological culture liquid 104 Fine bubble generator 105 Agitator a Oxygen-containing gas 201 Carrier 202 Cell 203 Culture tank 204 Discharge port 205 Straightening plate 206 Discharge nozzle θ Tilt angle

Claims (10)

A multi-nozzle used in a biological reaction device to which fine bubbles or ultra-fine bubbles containing a gas useful for culturing microorganisms or cells are applied,
A lower end portion to which the biological culture solution extracted from the culture tank containing the biological culture solution is supplied;
An upper end portion connected to the culture tank and returning the biological culture solution extracted from the culture tank to the culture tank;
A plurality of transport pipes provided parallel to the central axis, which are connected to the lower end and divide the biological culture solution supplied to the lower end and transport it to the upper end side;
A plurality of discharge pipes connected to each of the transport pipes; and a plurality of discharge ports provided at the outlets of the discharge pipes on the culture tank side,
Each of the discharge pipelines or each of the transport pipelines is provided with a fine bubble/ultra-fine bubble generator that causes fine bubbles/ultra-fine bubbles to be contained in the biological culture solution extracted from the culture tank,
A multi-nozzle characterized in that the biological culture solution contained in the culture tank is stirred by discharging the biological culture solution extracted from the culture tank, containing fine bubbles and ultrafine bubbles, from the multiple discharge ports in multiple directions at angles of 0° to 90° with respect to the direction of the central axis of the multi-nozzle toward the culture tank. The multi-nozzle according to claim 1, characterized in that in each plane including the pair of connected conveying pipes and the discharge pipes, the acute angle formed by the conveying pipes and the discharge pipes is greater than 90° and less than 180°. The multi-nozzle according to claim 2, characterized in that each of the planes is a plane that includes the central axis of the multi-nozzle. The multi-nozzle according to claim 2, characterized in that each of the planes 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 conveying pipes and the multi-nozzle is greater than 0° and less than 90°. The above fine bubble and ultra-fine bubble generator,
A main body consisting of a cylindrical body having an inlet portion on the upstream side through which the biological culture solution extracted from the culture tank flows in and an outlet portion on the downstream side through which the biological culture solution containing fine bubbles and ultrafine bubbles is discharged;
A slit is provided continuously on a side surface along a plane perpendicular to the central axis of the main body;
a gas supply unit connected to the slit and supplying a gas to the slit;
The multi-nozzle according to any one of claims 1 to 4 , characterized in that it comprises: The multi-nozzle according to claim 5 , wherein in the fine bubble/ultra-fine bubble generator, the angle θ of the slit inclined toward the upstream side with respect to a plane perpendicular to the central axis of the main body is an acute angle. The multi-nozzle according to claim 5 or 6 , in the fine bubble/ultra-fine bubble generator, characterized in that the slits are provided in multiple stages in the main body. A multi-nozzle according to any one of claims 5 to 7, characterized in that in the fine bubble/ultra - fine bubble generator, the main body is formed from a single cylindrical body, and the slits are provided by cutting the peripheral surface of the single cylindrical body while leaving a connection portion in a portion. A multi-nozzle as described in any one of claims 5 to 7, characterized in that in the fine bubble/ultra - fine bubble generator, the main body is formed of two or more cylindrical bodies, and the slit is formed at the connecting portion of the cylindrical bodies. A multi-nozzle according to any one of claims 5 to 9 , characterized in that in the fine bubble/ultra-fine bubble generator, the inner diameter of the main body in the vicinity of the outlet portion is gradually expanded toward the downstream side.

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