A method for cultivating anaerobic bacteria using an anaerobic bacteria cultivator equipped with a mechanism for adding fine and ultra-fine bubbles of gas, mainly composed of nitrogen gas, to the culture solution.

Abstract

To provide an anaerobic bacterium culture device and an anaerobic bacterium culture method, with which dissolved oxygen concentration of a culture solution may be efficiently lowered before start of the culture, the dissolved oxygen concentration of the anaerobic bacterium culture solution may be continuously kept low even during the culture, and culturing of anaerobic bacterium may be executed economically and efficiently.SOLUTION: There is provided an anaerobic bacterium culture device in which a culture solution 1 and an anaerobic bacterium are stored in a culture tank 2 to cultivate anaerobic bacteria. The anaerobic bacterium culture device comprises a mechanism (I) for containing fine bubbles and ultrafine bubbles of a gas containing 90% by volume or more of nitrogen gas in the culture solution.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to an anaerobic bacteria culturing apparatus for culturing anaerobic bacteria, and an anaerobic bacteria culturing method using the anaerobic bacteria culturing apparatus. [Background technology]

[0002] Anaerobic bacteria are bacteria that grow in environments with low oxygen concentrations such as the hydrosphere, underground, and animal intestines where convection is poor. There are facultative anaerobes such as lactic acid bacteria and Escherichia coli that can grow even in the presence of oxygen, and obligate anaerobes such as bifidobacteria whose growth is inhibited by exposure to oxygen at atmospheric levels. In order to culture such anaerobic bacteria, it is necessary to provide an anaerobic bacteria culture device and an anaerobic bacteria culture method that can efficiently reduce the dissolved oxygen concentration in a culture solution (hereinafter also referred to as "culture solution") before inoculation of anaerobic bacteria before initiation of anaerobic bacteria culture (hereinafter also referred to as "before initiation of culture"), and can continuously maintain a low dissolved oxygen concentration in a culture solution (hereinafter also referred to as "anaerobic bacteria culture solution") after inoculation of anaerobic bacteria even during initiation of anaerobic bacteria (hereinafter also referred to as "during culture"), thereby economically and efficiently culturing anaerobic bacteria.

[0003] The dissolved oxygen concentration in the culture medium and the anaerobic culture medium must be kept low enough not to cause stress or damage to the anaerobic bacteria, and is preferably substantially 0 ppm.

[0004] Anaerobic bacteria are generally cultured by the following steps. 1) Cleaning the inside of the culture tank 2) Supplying culture medium to the culture tank 3) A process to sterilize the inside of the culture tank, etc. using high-temperature, high-pressure steam. 4) A process in which nitrogen gas is supplied into the culture tank to compensate for the reduced pressure caused by the liquefaction of water vapor, maintain a positive pressure inside the culture tank, and reduce the dissolved oxygen concentration in the culture solution. 5) A process in which anaerobic bacteria are inoculated into the culture solution and cultured while stirring the anaerobic culture solution with a stirrer.

[0005] Non-Patent Document 1 discloses that in the production of yogurt using lactic acid bacteria, which are facultative anaerobic bacteria, the fermentation time can be shortened by using a "deoxygenated fermentation method" in which nitrogen is bubbled through a culture solution (10% skim milk powder) in advance to reduce the dissolved oxygen concentration to 0 ppm before fermentation is performed.

[0006] Furthermore, Patent Document 1 discloses that in culturing lactic acid bacteria, which are facultative anaerobes, the culture solution is deoxygenated before the start of culturing by injecting nitrogen gas into the culture tank to maintain a positive pressure state in the cooling process after high-temperature sterilization, and that it is preferable to continue injecting nitrogen gas during culturing or to keep the inside of the culture tank in an anaerobic state by sealing the culture tank.

[0007] Furthermore, Patent Document 2 discloses that in culturing lactic acid bacteria, which are facultative anaerobic bacteria, the culture is carried out under anaerobic conditions maintained by aeration with carbon dioxide gas and inert gases (nitrogen gas, argon gas, etc.) during culture.

[0008] Furthermore, Patent Document 3 discloses that propionic acid bacteria may be cultured anaerobically by bubbling in carbon dioxide or nitrogen gas during the culture.

[0009] However, it is difficult to efficiently reduce the dissolved oxygen concentration of the culture solution by aerating nitrogen into the culture solution in advance to set the dissolved oxygen concentration to 0 ppm as disclosed in Non-Patent Document 1. In addition, in the method disclosed in Patent Documents 1 to 3, in which nitrogen gas is injected into the culture tank in the cooling step after sterilization to reduce the dissolved oxygen concentration of the culture solution, and nitrogen gas is continued to be injected during the culture to keep the dissolved oxygen concentration of the anaerobic culture solution low, it is difficult to efficiently reduce the dissolved oxygen concentration of the culture solution before the start of the culture, and it is also difficult to continuously maintain the dissolved oxygen concentration of the anaerobic culture solution low during the culture. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Hirofumi Horiuchi, "Development of yogurt deoxygenation fermentation technology", Journal of Biotechnology and Life Sciences, The Society of Biotechnology, Japan, November 25, 2010, Vol. 88, No. 11, pp. 594-600 [Patent documents]

[0011] [Patent Document 1] Patent No. 4512265 [Patent Document 2] Japanese Patent Application Publication No. 9-121844 [Patent Document 3] JP 2012-201599 A Summary of the Invention [Problem to be solved by the invention]

[0012] The objective of the anaerobic bacteria culture apparatus of the present invention and the anaerobic bacteria culture method using this anaerobic bacteria culture apparatus (hereinafter referred to as the "culture apparatus of the present invention" and the "culture method of the present invention", respectively, and collectively referred to as the "culture apparatus and culture method of the present invention") is to provide an anaerobic bacteria culture apparatus and an anaerobic bacteria culture method that can efficiently reduce the dissolved oxygen concentration in the culture solution before the start of culture, and can continuously maintain the dissolved oxygen concentration in the anaerobic bacteria culture solution low even during culture, thereby enabling anaerobic bacteria to be cultured economically and efficiently. [Means for solving the problem]

[0013] The present inventors have surprisingly found that by adding nitrogen gas in the form of fine or ultrafine bubbles (hereinafter also referred to as "fine bubbles") to an anaerobic culture solution, it is possible to efficiently reduce the dissolved oxygen concentration in the culture solution before the start of culture, and to continuously maintain the dissolved oxygen concentration in the anaerobic culture solution low during culture, and have thus completed the present invention.

[0014] The culture apparatus and culture method of the present invention are characterized by being equipped with a mechanism (I) for blowing fine bubbles of a gas containing 90% by volume or more of nitrogen gas with an oxygen gas concentration of substantially 0 ppm (hereinafter also referred to as "nitrogen gas-based gas") into the culture solution before the start of culture and into the anaerobic bacteria culture solution during culture, thereby causing the culture solution to contain the bubbles.

[0015] In addition, preferably, in addition to the mechanism (I), a mechanism (II) is provided for blowing fine bubbles of nitrogen gas as the main component into the culture solution supplied to the culture tank, thereby making the culture solution contain the gas. This further solves the above problems. Effect of the Invention

[0016] The culture apparatus and culture method of the present invention, by being equipped with the above-mentioned mechanism (I), can efficiently reduce the dissolved oxygen concentration in the culture solution before the start of culture, and can continuously maintain the dissolved oxygen concentration in the anaerobic culture solution low during culture.

[0017] Furthermore, by providing the culture apparatus and culture method of the present invention with the mechanism (II) in addition to the mechanism (I), the dissolved oxygen concentration in the culture solution before the start of culture can be reduced in an even shorter time, and even when additional culture solution is added to the anaerobic culture solution during culture, the dissolved oxygen concentration in the anaerobic culture solution can be prevented from fluctuating significantly. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing a first reference embodiment of a culture apparatus according to the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing a second reference embodiment of the culture apparatus of the present invention. [Diagram 3] FIG. 1 is a schematic diagram showing a first embodiment of a culture device of the present invention. [Figure 4] FIG. 2 is a schematic diagram showing a second embodiment of the culture device of the present invention. [Diagram 5] FIG. 1 is a cross-sectional view showing an example of a fine bubble generating nozzle used to generate fine bubbles of nitrogen gas as a main component gas according to the present invention. [Figure 6] FIG. 1 is a schematic diagram showing a conventional anaerobic bacteria culturing device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

[0020] <Conventional Anaerobic Bacteria Cultivation Apparatus and Method> First, a conventional anaerobic bacteria culturing apparatus and method will be described with reference to FIG.

[0021] Anaerobic bacteria are generally cultured using a culture apparatus as shown diagrammatically in FIG.

[0022] The culture tank 2 is airtight to prevent oxygen from entering from the outside air, and a seal portion 4c such as a mechanical seal is provided at the location where the rotating shaft 4b of the agitator 4 penetrates the culture tank 2.

[0023] Anaerobic bacteria are usually cultured by batch culture. In order to prevent contamination, when the previous batch culture is finished and the next batch culture is started, the inside of the culture tank 2 is washed, and then the culture solution 1 is supplied to the culture tank 2 through the culture solution supply path C. The culture solution 1, the agitator 4, the culture solution supply path C, etc. are sterilized with high-temperature, high-pressure steam.

[0024] Thereafter, in order to compensate for the reduced pressure caused by the liquefaction of the water vapor and to maintain a positive pressure inside the culture tank 2, and to reduce the dissolved oxygen concentration of the culture solution 1 supplied to the culture tank 2, nitrogen gas is supplied into the culture tank 2 in a cooling process to create a pressurized state, anaerobic bacteria are inoculated into the culture solution 1, and the stirring blades 4d are rotated by the electric motor 4a of the stirrer 4 to stir the anaerobic bacteria culture solution 3 while culturing.

[0025] In Non-Patent Document 1, the above-mentioned anaerobic bacteria culture device and method are used to culture lactic acid bacteria (Lactobacillus bulgaricus strain 2038 and Lactobacillus thermophilus strain 1131), which are facultative anaerobic bacteria. However, since it takes time to reduce the dissolved oxygen concentration in the culture solution 1 (10% skim milk powder) simply by pressurizing the inside of the culture tank 2 with nitrogen as described above, in order to improve this, nitrogen is aerated into the culture solution 1 before it is supplied to the culture tank 2 to set the dissolved oxygen concentration to 0 ppm.

[0026] In Patent Document 1, the anaerobic bacteria culture device and method described above are used to culture lactic acid bacteria, which are facultative anaerobes. In the cooling process after sterilization, nitrogen gas is injected into the culture tank 2 to reduce the dissolved oxygen concentration in the culture solution 1, and nitrogen gas is continued to be injected into the anaerobic bacteria culture solution 3 during culture to maintain an anaerobic state.

[0027] Furthermore, in culturing obligate anaerobes, which are more susceptible to stress and damage from oxygen than facultative anaerobes such as lactic acid bacteria, it is said that the dissolved oxygen concentrations in culture solution 1 before the start of culturing and in anaerobic culture solution 3 during culturing (hereinafter referred to as "culture solutions 1 & 3") must be kept as close to 0 ppm as possible.

[0028] <Characteristics of the culture device and culture method of the present invention> The culture apparatus and culture method of the present invention are mainly characterized by the use of mechanism (I) (hereinafter simply referred to as "mechanism (I)") for blowing fine bubbles of nitrogen gas as the main component into culture solution 1&3 to cause it to be contained therein.

[0029] Before the start of cultivation, the dissolved oxygen concentration in the culture solution can be efficiently reduced by adding nitrogen gas as the main component gas in the form of fine bubbles to the culture solution 1. That is, by making the gas in the form of fine bubbles, the contact area (interface) between the culture solution 1 and the nitrogen gas as the main component gas can be increased, and the dissolved oxygen (O2) in the culture solution 1 can be efficiently replaced with nitrogen (N2).

[0030] Furthermore, the fine bubbles of the gas mainly composed of nitrogen gas blown into the culture solution 1 before the start of the culture remain in the anaerobic culture solution 3 for a long period of time even during the culture, so that the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained low. Furthermore, by continuously blowing fine bubbles of the gas mainly composed of nitrogen gas into the anaerobic culture solution 3 during the culture, the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained even lower.

[0031] Furthermore, in the culture apparatus and culture method of the present invention, in addition to the above-mentioned mechanism (I), a mechanism (II) (hereinafter simply referred to as "mechanism (II)") is used in which fine bubbles of nitrogen gas as the main component are blown into the culture solution 1 supplied to the culture tank to cause it to contain the gas. This makes it possible to quickly reduce the dissolved oxygen concentration in the culture solution before the start of culture, and also to suppress fluctuations in the dissolved oxygen concentration in the anaerobic culture solution 3 even when additional culture solution 1 is added to the anaerobic culture solution 3 during culture.

[0032] <Fine bubbles / Ultra-fine bubbles> In the present invention, "fine bubbles / ultrafine bubbles" means "fine bubbles" and / or "ultrafine bubbles". "Normal bubbles" rise rapidly in water and burst at the surface, disappearing, whereas "fine bubbles", tiny bubbles with a diameter of less than 100 μm, shrink in water and disappear, generating "ultrafine bubbles", which are extremely tiny bubbles with a diameter of less than 1 μm, together with free radicals, and these "ultrafine bubbles" remain in water for a certain period of time. In the present invention, as standardized by the ISO (International Organization for Standardization), bubbles with a number-average diameter of less than 100 μm are called "fine bubbles", and bubbles with a number-average diameter of less than 1 μm are called "ultrafine bubbles". Methods commonly used to measure the diameter of fine bubbles include image analysis, laser diffraction scattering, electrical detection band method, resonance mass measurement method, and optical fiber probe method, while methods commonly used to measure the diameter of ultrafine bubbles include dynamic light scattering, Brownian motion tracking method, electrical detection band method, and resonance mass measurement method.

[0033] <Nitrogen gas as main component> The nitrogen gas-based gas used in the form of fine bubbles of the present invention is a high-purity gas with an oxygen gas content as close to 0% by volume as possible and a nitrogen gas content of 90% by volume or more, preferably 99% by volume or more, and more preferably as close to 100% by volume as possible. Nitrogen gas is an inert gas that can be obtained simply and economically from air, so from this viewpoint, it is preferable that the nitrogen gas content of the nitrogen gas-based gas be as close to 100% by volume as possible.

[0034] Examples of gas components other than nitrogen gas that can be contained in the nitrogen gas-based gas include those that are beneficial for culturing anaerobic bacteria, such as carbon dioxide gas (content: about 5 to 10% by volume), hydrogen gas (content: about 1 to 5% by volume), etc. In addition, any gas that has a favorable effect on the culturing of anaerobic bacteria depending on the type of anaerobic bacteria, such as high-purity ammonia gas (purity of 99.999% or more), can be blended as a gas component in the nitrogen gas-based gas.

[0035] <The first aspect of the culture device of the present invention reference Form> FIG. 1 shows a first embodiment of the culture device of the present invention. reference Show the form. No. 1 reference The culture apparatus of this embodiment is a conventional anaerobic culture apparatus shown in FIG. 6, and is equipped with a mechanism (I) for blowing and containing fine bubbles of the nitrogen gas as the main component gas into the culture solution 1&3 by increasing the pressure of the nitrogen gas as the main component gas supplied from a nitrogen gas supply source 5 to a porous body 7 such as sintered metal or porous glass placed in the culture solution 1&3 using a compressor 6 and supplying the gas through a nitrogen-containing gas supply path A.

[0036] No. 1 reference In the culture apparatus of this type, fine bubbles of nitrogen gas as the main component are blown into the culture solutions 1 & 3 to make them contain the gas in the following process. 1) The inside of the culture tank 2 is washed, the culture solution 1 is supplied into the culture tank 2 through the culture solution supply path C, and the inside of the culture tank etc. is sterilized using high-temperature, high-pressure steam. Then, fine bubbles of nitrogen gas, the main component gas, are blown into the culture solution 1 through the porous body 7 to contain it. 2) When the dissolved oxygen concentration in culture solution 1 has decreased sufficiently to the extent that it does not cause stress or damage to the anaerobic bacteria, anaerobic bacteria are inoculated into culture solution 1, and cultivation is started while stirring the anaerobic culture solution 3 with agitator 4. 4) During the culture, fine bubbles of nitrogen gas, the main component of which is gas, are blown into the anaerobic culture solution 3 through the porous body 7 to be contained therein.

[0037] No. 1 reference In the culture device having the above structure, the dissolved oxygen concentration in the culture solution can be efficiently reduced by the nitrogen gas main component gas blown into the culture solution 1 in the form of fine bubbles before the start of culture. That is, by using the fine bubbles, the contact area (interface) between the culture solution 1 and the nitrogen gas main component gas can be increased, and the dissolved oxygen (O2) in the culture solution 1 can be efficiently replaced with nitrogen (N2).

[0038] Furthermore, the fine bubbles of the gas mainly composed of nitrogen gas blown into the culture solution 1 before the start of the culture remain in the anaerobic culture solution 3 for a long period of time even during the culture, so that the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained low. Furthermore, by continuously containing fine bubbles of the gas mainly composed of nitrogen gas in the anaerobic culture solution 3 during the culture, the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained even lower.

[0039] In addition, if the nitrogen gas supply source 5 can provide high-pressure nitrogen gas that can convert the main component gas of nitrogen gas into fine bubbles in the porous body 7 such as sintered metal or porous glass, there is no need to use the compressor 6.

[0040] <The second aspect of the culture device of the present invention reference Form> FIG. 2 shows the second embodiment of the culture device of the present invention. reference Show the form. Second referenceThe culture apparatus of this embodiment is a conventional anaerobic culture apparatus shown in Fig. 6, and is provided with a mechanism (I) for extracting culture solutions 1&3 from the culture tank 2 using a circulation pump 8, supplying them to a fine bubble generator 9a through a culture solution circulation path B, and then blowing in and incorporating fine bubbles of nitrogen gas as the main component gas, and then returning the culture solution to the culture tank 2. The culture solutions 1&3 are supplied to the fine bubble generator 9a through the culture solution circulation path B, and the first mechanism shown in Fig. 1 is provided to the fine bubble generator 9a. reference As in the embodiment, nitrogen gas as the main component gas is supplied from a nitrogen gas supply source 5 through a nitrogen-containing gas supply path A and pressurized by a compressor 6, and fine bubbles of the nitrogen gas as the main component gas are formed.

[0041] Second reference In the culture apparatus of this type, fine bubbles of nitrogen gas as the main component are blown into the culture solutions 1 & 3 to make them contain the gas in the following process. 1) The inside of the culture tank 2 is washed, the culture solution 1 is supplied into the culture tank 2 through the culture solution supply path C, and the inside of the culture tank etc. is sterilized using high-temperature, high-pressure steam. Thereafter, the culture solution 1 is extracted from the culture tank 2 using the circulation pump 8 and supplied to the fine bubble generator 9a through the culture solution circulation path B, where fine bubbles of nitrogen gas as the main component are blown in to incorporate the culture solution, and then the culture solution is returned to the culture tank 2. 2) When the dissolved oxygen concentration in culture solution 1 has decreased sufficiently to the extent that it does not cause stress or damage to the anaerobic bacteria, anaerobic bacteria are inoculated into culture solution 1, and cultivation is started while stirring the anaerobic culture solution 3 with agitator 4. 3) During cultivation, the anaerobic culture solution 3 is extracted from the cultivation tank 2 using a circulation pump 8, and is supplied to a fine bubble generator 9a via the cultivation solution circulation path B, where fine bubbles of nitrogen gas as the main component are blown in to incorporate the solution, and then the solution is returned to the cultivation tank 2.

[0042] Second reference In the culture device of the first referenceAs with the culture apparatus having the above-mentioned configuration, the dissolved oxygen concentration in the culture solution can be efficiently reduced by blowing the nitrogen gas as a main component gas into the culture solution 1 in the form of fine bubbles before the start of culture. That is, by using the fine bubbles, the contact area (interface) between the culture solution 1 and the nitrogen gas as a main component gas can be increased, and the dissolved oxygen (O2) in the culture solution 1 can be efficiently replaced with nitrogen (N2).

[0043] Furthermore, the fine bubbles of the gas mainly composed of nitrogen gas blown into the culture solution 1 before the start of the culture remain in the anaerobic culture solution 3 for a long period of time even during the culture, so that the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained low. Furthermore, by continuously containing fine bubbles of the gas mainly composed of nitrogen gas in the anaerobic culture solution 3 during the culture, the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained even lower.

[0044] It should be noted that if the nitrogen gas supply source 5 can provide high-pressure nitrogen gas that can convert the main component gas of nitrogen gas into fine bubbles in the fine bubble generator 9a, there is no need to use the compressor 6.

[0045] <The first aspect of the culture device of the present invention> 1 Embodiment> FIG. 3 shows the first embodiment of the culture device of the present invention. 1 An embodiment is shown. No. 1 The culture device of the embodiment is a second embodiment having a mechanism (I) shown in FIG. reference The culture apparatus further includes a mechanism (II) for supplying the culture solution 1 to a fine bubble generator 9b through a culture solution supply path C, and then supplying the culture solution 1 to the culture tank 2 after blowing in fine bubbles of nitrogen gas as the main component gas.

[0046] No. 1 In the culture device of the embodiment, by providing the mechanism (I), reference Form and second referenceAs with the culture apparatus having the above-mentioned configuration, the dissolved oxygen concentration in the culture solution can be efficiently reduced by blowing the nitrogen gas as a main component gas into the culture solution 1 in the form of fine bubbles before the start of culture. That is, by using the fine bubbles, the contact area (interface) between the culture solution 1 and the nitrogen gas as a main component gas can be increased, and the dissolved oxygen (O2) in the culture solution 1 can be efficiently replaced with nitrogen (N2).

[0047] Furthermore, the fine bubbles of the gas mainly composed of nitrogen gas blown into the culture solution 1 before the start of the culture remain in the anaerobic culture solution 3 for a long period of time even during the culture, so that the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained low. Furthermore, by continuously containing fine bubbles of the gas mainly composed of nitrogen gas in the anaerobic culture solution 3 during the culture, the dissolved oxygen concentration in the anaerobic culture solution 3 during the culture can be maintained even lower.

[0048] Furthermore, by providing the mechanism (II), the dissolved oxygen concentration of the culture solution 1 can be reduced in an even shorter time before the start of culture. That is, after the inside of the culture tank is washed, fine bubbles of nitrogen gas as the main component gas are blown into the culture solution 1 in the culture solution supply path C to make it contain the gas, and then the culture solution 1 is supplied to the culture tank 2. This makes it possible to further shorten the time required for the mechanism (I) to reduce the dissolved oxygen concentration before the start of culture ("instantaneous" when the amount of dissolved oxygen in the culture solution 1 is made close to 0 ppm by the mechanism (II)), and after the sterilization process using high-temperature and high-pressure steam and the cooling process, anaerobic bacteria can be inoculated to start culture.

[0049] Furthermore, by providing the mechanism (II), even when additional culture solution 1 is added to the anaerobic culture solution 3 during cultivation, it is possible to prevent the dissolved oxygen concentration in the anaerobic culture solution 3 from fluctuating significantly.

[0050] <The first aspect of the culture device of the present invention> 2 Embodiment> FIG. 4 shows the first embodiment of the culture device of the present invention. 2 An embodiment is shown. No. 2 The culture device of the embodiment is1 The structure of the culture device of the embodiment is simplified by integrating the mechanism (I) and the mechanism (II). 2 In the culture device of the embodiment, fine bubbles of a gas containing nitrogen as a main component are blown into the culture solutions 1 & 3 to cause them to contain the gas in the following process. 1) After cleaning the inside of the culture tank, with valve 10 open and valve 11 closed, culture solution 1 is supplied to fine bubble generator 9a through culture solution supply path C, fine bubbles of nitrogen gas as the main component are blown in to incorporate the culture solution 1 into the culture tank 2. 2) After the inside of the culture tank and the like are sterilized using high-temperature, high-pressure steam, valve 10 is closed and valve 11 is opened, and the culture solution 1 is extracted from the culture tank 2 using the circulation pump 8, supplied to the fine bubble generator 9a via the culture solution circulation path B, where fine bubbles of nitrogen gas as the main component are blown in to incorporate the culture solution, and then returned to the culture tank 2. 3) When the dissolved oxygen concentration in culture solution 1 has decreased sufficiently to the extent that it does not cause stress or damage to the anaerobic bacteria, anaerobic bacteria are inoculated into culture solution 1, and cultivation is started while stirring the anaerobic culture solution 3 with agitator 4. 4) During cultivation, with valve 10 closed and valve 11 open, the anaerobic culture solution 3 is extracted from the cultivation tank 2 using the circulation pump 8, and supplied to the fine bubble generator 9a via the culture solution circulation path B, where fine bubbles of nitrogen gas as the main component are blown in to incorporate the solution, and then the solution is returned to the cultivation tank 2.

[0051] <Microbubble generator> As the fine bubble generator (9a and 9b) used in the present invention, known or commercially available devices can be used, such as a "pressure dissolution type fine bubble generator" that creates a supersaturated condition for the dissolved gas by dissolving a sufficient amount of gas in water at a certain level of high pressure and then releasing the pressure, or a "loop flow type bubble generating nozzle" that uses the phenomenon in which bubbles are broken down by the turbulence generated by the water flow by stirring and mixing a mixed fluid consisting of liquid and gas as a loop-shaped flow by generating a water flow. In addition, as the ultra-fine bubble generator, known devices such as those described in JP-A-2007-312690, JP-A-2006-289183, JP-A-2005-245817, JP-A-2007-136255, and JP-A-2009-39600 can be used.

[0052] The water flow type fine bubble generator (9a and 9b) used in the present invention is preferable because it can generate a large amount of fine bubbles economically. A water flow type fine bubble generator that can be suitably used in the present invention will be described with reference to FIG. 5. In this fine bubble generator 20, a fluid D (culture fluid 1 extracted from the culture tank 2 before the start of culture, anaerobic culture fluid 3 extracted from the culture tank 2 during culture, or culture fluid 1 supplied to the culture tank 2 before the start of culture) is supplied from the inlet 21 of the nozzle under pressure, and a turbulent flow is generated in the throat 22 while increasing the flow rate by narrowing the diameter of the pipe. In this state, nitrogen gas-based gas E is supplied from the gas inlet 24, mixed with the fluid D in the suction section 23, and turned into fine bubbles by the water flow, and a fluid F containing fine bubbles of the nitrogen gas-based gas is discharged from the outlet 25 and supplied to the culture tank 2.

[0053] <Culture method of the present invention> The culture method of the present invention is characterized in that, by culturing anaerobic bacteria using the culture apparatus of the present invention, The dissolved oxygen concentration in the culture medium before the start of cultivation can be efficiently reduced, and the dissolved oxygen concentration in the anaerobic culture medium during cultivation can be continuously maintained low, allowing anaerobic bacteria to be cultivated economically and efficiently. [Explanation of symbols]

[0054] 1 Culture solution 2 Culture tank 3 Anaerobic culture medium 4 Mixer 4a electric motor 4b Rotation axis 4c Seal part 4d Mixing blade 5. Nitrogen gas supply source 6. Compressor 7. Porous bodies such as sintered metals and porous glass 8 Circulation Pump 9a, 9b Fine bubble (ultra-fine bubble) generator 10 Valve 11 Valve A Nitrogen-containing gas supply route B. Culture medium circulation path C. Culture medium supply route 20. Microbubble (Fine and Ultrafine Bubble) Generator 21 Entrance 22 Throat 23 Suction part 24 Gas inlet 25 Exit section D Fluid (culture fluid 1 extracted from culture tank 2 before the start of culture, anaerobic culture fluid 3 extracted from culture tank 2 during culture, or culture fluid 1 supplied to culture tank 2 before the start of culture) E. Nitrogen gas as main component (gas with nitrogen gas as main component) F. Fluid containing fine bubbles of nitrogen gas as the main component

Claims

1. An anaerobic bacteria culture method using an anaerobic bacteria culture apparatus that contains a culture solution and anaerobic bacteria in a culture tank and cultures the anaerobic bacteria by batch culture, the method comprising the following steps 1) to 6): 1) A step of cleaning the inside of the culture tank 2) A step of supplying a culture solution containing fine or ultra-fine bubbles of gas containing 90% or more by volume of nitrogen gas into the culture tank. 3) A step of sterilizing the inside of the culture tank, etc. using high-temperature and high-pressure steam 4) A process of extracting the culture solution from the culture tank, blowing in fine bubbles or ultrafine bubbles of a gas containing 90% or more by volume of nitrogen gas to make the gas contain the fine bubbles, and then refluxing the gas into the culture tank. 5) A step of inoculating the culture solution with anaerobic bacteria and starting the culture while stirring the culture solution when the dissolved oxygen concentration in the culture solution has decreased sufficiently to prevent stress or damage to the anaerobic bacteria. 6) During the culture, the culture solution is extracted from the culture tank, fine bubbles or ultrafine bubbles containing 90% or more by volume of nitrogen gas are blown into the culture solution to contain the fine bubbles or ultrafine bubbles, and then the culture solution is returned to the culture tank.

2. 2. The method for cultivating anaerobic bacteria according to claim 1, wherein the gas containing 90% by volume or more of nitrogen gas consists of nitrogen gas alone.

3. 2. The method for cultivating anaerobic bacteria according to claim 1, wherein the gas containing 90% by volume or more of nitrogen gas is a mixed gas of nitrogen gas, hydrogen gas and / or carbon dioxide gas.

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