Culture apparatus

Abstract

This culture apparatus is for culturing a microorganism or a cell that uses a source gas containing a combustible gas to grow itself or produce an organic substance, and includes: a culture tank that holds a culture solution for culturing the microorganism or the cell; a circulation path through which the culture solution flows out through an outlet port provided in the culture tank and the outflow culture solution flows back through a return port provided in the culture tank to the culture tank; a pump that generates a pressure for circulating the culture solution via the circulation path to generate, as the flow of the culture solution in the circulation path, a downflow in the vertical top-to-bottom direction; and a bubble generation device that generates bubbles having a diameter of 100 μm or more to supply the source gas in the form of bubbles to the circulation path. The outlet port is disposed above the return port in the vertical direction, and the bubble generation device is disposed upstream of the midpoint of the length from the outlet port to the return port.

Description

Culture device

[0001] The technology of the present disclosure relates to a culture device.

[0002] Research into the de-petroleumization of raw materials is active worldwide with the aim of reducing greenhouse gas emissions. As part of this research, technologies for utilizing carbon sources other than petroleum as raw materials have been developed in the fields of chemical and bioproduct manufacturing. For example, microorganisms are known that utilize carbon sources other than petroleum as raw materials for growth or for the production of organic substances. One example of such a microorganism is hydrogen-oxidizing bacteria. Hydrogen-oxidizing bacteria grow by consuming gases containing carbon dioxide, hydrogen, and oxygen as raw materials. Furthermore, genetic modification of hydrogen-oxidizing bacteria allows for the efficient production of organic substances such as alcohols and amino acids. Hydrogen-oxidizing bacteria is a collective term for chemosynthetic bacteria that utilize the energy generated by hydrogen oxidation to convert carbon from carbon dioxide into organic substances and incorporate them into the bacteria. Hydrogen-oxidizing bacteria are also attracting attention for their ability to effectively utilize carbon dioxide, a greenhouse gas, because carbon dioxide serves as a raw material for growth or the production of organic substances.

[0003] Japanese Patent Application Publication No. 51-038480 and U.S. Patent Application Publication No. 2019 / 0316072 disclose a culture device for culturing hydrogen-oxidizing bacteria. The culture device includes a culture tank containing a culture solution. A feed gas containing carbon dioxide, hydrogen, and oxygen is supplied into the culture tank, and some of the carbon dioxide, hydrogen, and oxygen contained in the feed gas are dissolved in the culture solution and consumed for the growth of hydrogen-oxidizing bacteria or the production of organic substances. The remaining feed gas is released into a space above the surface of the culture solution in the culture tank and then exhausted to the outside of the culture tank. A mixed gas containing flammable hydrogen and oxygen may pose an explosion risk depending on the composition ratio. Therefore, Japanese Patent Application Publication No. 51-038480 and U.S. Patent Application Publication No. 2019 / 0316072 disclose a technology for suppressing explosions.

[0004] In Japanese Patent Laid-Open No. 51-038480, the supply of oxygen contained in the feed gas is independently controlled, the dissolved oxygen concentration in the culture solution is measured, and the amount of oxygen supplied to the culture tank is limited based on the measured dissolved oxygen concentration. In U.S. Patent Application Publication No. 2019 / 0316072, safety is ensured by controlling the components of the feed gas.

[0005] Furthermore, the culture devices described in JP 2016-002040 A and JP 2018-117578 A use a bubble generator that generates microbubbles or nanobubbles, which are tiny bubbles with a diameter of less than 100 μm, and supply the raw material gas to the culture solution as microbubbles or nanobubbles.

[0006] The culture apparatuses described in JP 51-038480 A and U.S. Patent Application Publication No. 2019 / 0316072 A may limit the amount of raw material gas supplied to ensure safety, raising concerns about raw material shortages. On the other hand, the culture apparatuses described in JP 2016-002040 A and JP 2018-117578 A can improve the dissolution rate of the raw material gas by miniaturizing the bubbles, thereby resolving raw material shortages while ensuring the efficiency and safety of the raw material gas.

[0007] However, the culture devices described in JP 2016-002040 A and JP 2018-117578 A consume a lot of energy to miniaturize the bubbles, which raises concerns that this goes against the social trend toward reducing greenhouse gas emissions.

[0008] The technology of the present disclosure provides a culture apparatus that can improve the dissolution rate of a raw material gas while suppressing the energy consumption required for miniaturizing bubbles.

[0009] In order to achieve the above object, a culture apparatus according to a first aspect of the technology disclosed herein is a culture apparatus for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, and is equipped with a culture tank that contains a culture solution for culturing the microorganisms or cells, a circulation path that extracts the culture solution from an outlet provided in the culture tank and returns the extracted culture solution to the culture tank from a return port provided in the culture tank, a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a downward flow from above in the vertical direction as the flow of the culture solution in the circulation path, and a bubble generator that generates bubbles with a diameter of 100 μm or more, and supplies the raw material gas as bubbles to the circulation path, wherein the outlet is located vertically above the return port, and the bubble generator is located upstream of the midpoint of the length from the outlet to the return port.

[0010] In the first embodiment, the average flow velocity of the downward flow of the culture solution in the circulation path is further set to V ｐｉｐｅ , the bubble rising speed is V Ｂ In this case, V ｐｉｐｅ It is preferable that the condition of the following formula is satisfied.

[0011] In the first embodiment, the vertical height from the return port in the circulation path to the bubble generating device is set to H ｐｉｐｅ The height from the bottom of the culture tank to the liquid surface of the culture medium is H Ｌ The height from the bottom of the culture tank to the return port is H ｉｎ In this case, V ｐｉｐｅ It is preferable that the condition of the following formula is satisfied.

[0012] In the first aspect, the circulation path is preferably a path dedicated to circulation that is provided independently of a recovery path for recovering a portion of the microorganisms or cells from the culture solution.

[0013] In the first aspect, the return port is preferably provided in a range from the bottom of the culture tank to ¼ or less of the liquid level.

[0014] In the first embodiment, it is preferable that the microorganism is a hydrogen-oxidizing bacterium, and the raw material gas contains hydrogen as a combustible gas, and oxygen and carbon dioxide as other components.

[0015] In the first embodiment, the raw material gas preferably contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components, and the hydrogen, oxygen, and carbon dioxide components are supplied separately.

[0016] In the first aspect, it is preferable to provide a composition ratio control mechanism that measures the composition ratio of the mixed gas in the space present above the liquid surface of the culture solution in the culture tank and supplies an adjustment gas into the space in accordance with the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a preset target value outside the explosion range.

[0017] A second aspect of the culture apparatus according to the technology of the present disclosure is a culture apparatus for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, and is equipped with a culture tank that contains a culture solution for culturing the microorganisms or cells, a circulation path that removes the culture solution from an outlet provided in the culture tank and returns the removed culture solution to the culture tank from a return port provided in the culture tank, a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a downward flow from above in a vertical direction as the flow of the culture solution in the culture tank, and a bubble generator that generates bubbles with a diameter of 100 μm or more, and supplies the raw material gas as bubbles to the culture tank.

[0018] In the second embodiment, the liquid level of the culture solution in the culture tank is further set to H Ｌ , the average flow velocity of the downward flow of the culture solution in the culture tank is V ｔａｎｋ , the bubble rising speed is V Ｂ In this case, V ｔａｎｋ It is preferable that the condition of the following formula is satisfied.

[0019] In the second aspect, the circulation path is preferably a path dedicated to circulation that is provided independently of a recovery path for recovering a portion of the microorganisms or cells from the culture medium.

[0020] In the second aspect, the outlet is preferably located below the bubble generator.

[0021] In the second aspect, it is preferable that the microorganisms are hydrogen-oxidizing bacteria, and the raw material gas contains hydrogen as a combustible gas, and oxygen and carbon dioxide as other components.

[0022] In the second embodiment, the source gas preferably contains hydrogen as the combustible gas and oxygen and carbon dioxide as other components, and the source gas preferably contains the hydrogen, oxygen, and carbon dioxide components supplied separately.

[0023] In the second aspect, it is preferable to provide a composition ratio control mechanism that measures the composition ratio of the mixed gas in the space present above the liquid surface of the culture solution in the culture tank and supplies an adjustment gas into the space in accordance with the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a preset target value outside the explosion range.

[0024] According to the technology of the present disclosure, it is possible to improve the dissolution rate of the source gas while suppressing the energy consumption required for miniaturizing the bubbles.

[0025] FIG. 1 is a diagram explaining the function of the culture apparatus. FIG. 2 is a diagram illustrating the overall configuration of the culture apparatus. FIG. 3 is a diagram illustrating a configuration for supplying raw material gas for each component. FIG. 4 is a diagram illustrating a configuration for circulating a culture solution. FIG. 5 is a diagram illustrating an example in which a circulation path extends in the vertical direction. FIG. 6 is a diagram illustrating an example in which a circulation path is inclined with respect to the vertical direction. FIG. 7 is a graph showing the relationship between dissolved gas concentration and complete dissolution residence time. FIG. 8 is a diagram illustrating a configuration for controlling composition ratio. FIG. 9 is a diagram illustrating the overall configuration of a culture apparatus of a second embodiment. FIG. 10 is a diagram illustrating a configuration for circulating a culture solution of a second embodiment.

[0026] [First embodiment] As shown in Fig. 1, a culture device 10 according to a first embodiment of the technology of the present disclosure cultures hydrogen-oxidizing bacteria, which are microorganisms, as an example of a culture target. The raw material for growing the hydrogen-oxidizing bacteria is hydrogen (H ２ ), oxygen (O ２ ) and carbon dioxide (CO ２), as well as inorganic components such as ammonium sulfate. Hydrogen is an example of a flammable gas. The culture device 10 supplies a raw material gas to the culture solution to grow hydrogen-oxidizing bacteria in the culture solution. Furthermore, in the culture solution, the hydrogen-oxidizing bacteria efficiently produce organic substances such as alcohol and amino acids by modifying the genes of the hydrogen-oxidizing bacteria. The culture solution is purified to extract the organic substances. The extracted organic substances are used, for example, in the production of chemical products or bioproducts. Furthermore, hydrogen-oxidizing bacteria are separated from the culture solution, and the isolated hydrogen-oxidizing bacteria are being considered for use as, for example, feed, food, and fuel.

[0027] As shown in FIG. 2 , the culture device 10 includes a culture tank 11, a raw material gas tank 12, a culture solution tank 13, an adjustment gas tank 14, a culture target tank 15, and a processor 17. The culture tank 11 contains a culture solution 18. Examples of the hydrogen-oxidizing bacteria 21 include bacteria of the genus Hydrogenophilus, as described in Japanese Patent No. 6528295. In this case, as described in Japanese Patent No. 6528295, for example, the culture solution 18 may contain ammonium sulfate ((NH ４ ) ２ SO ４ ), potassium dihydrogen phosphate (KH ２ P.O. ４ ), dipotassium hydrogen phosphate (K ２ HPO ４ ), sodium chloride (NaCl), or the like is dissolved in water. The culture tank 11 is an example of a "culture tank" according to the technology of the present disclosure. Note that, instead of or in addition to the raw material gas tank 12, a gas generator that generates raw material gas 26 may be provided.

[0028] The culture tank 11 is connected to supply paths 12A, 13A, 14A, and 15A, respectively, for a raw material gas tank 12, a culture solution tank 13, an adjustment gas tank 14, and a culture target tank 15. Each of the supply paths 12A, 13A, 14A, and 15A is composed of piping, valves, etc. A pump 22 and a flow meter 23 for measuring the flow rate of the fluid flowing through each supply path are disposed in each of the supply paths 12A, 13A, 14A, and 15A.

[0029] In the first embodiment, the supply path 12A of the raw material gas tank 12 is connected to the fermenter tank 11 via a culture solution circulation path (hereinafter simply referred to as the circulation path) 54 that circulates the culture solution 18. The raw material gas 26 is supplied from the supply path 12A to the culture solution 18 in the circulation path 54, and is supplied to the fermenter tank 11 as the culture solution 18 flows into the fermenter tank 11 via the circulation path 54.

[0030] The culture solution tank 13 contains a culture solution 18. The culture solution 18 is supplied from the culture solution tank 13 to the culture tank 11 through the supply path 13A by driving a pump 22 on the supply path 13A. The supply timing and supply amount of the culture solution 18 are controlled by a processor 17 that controls the driving of the pump 22.

[0031] The culture target tank 15 contains hydrogen-oxidizing bacteria 21, which are an example of a culture target. The hydrogen-oxidizing bacteria 21 are supplied from the culture target tank 15 to the culture tank 11 through the supply path 15A by driving a pump 22 on the supply path 15A. The supply timing and supply amount of the hydrogen-oxidizing bacteria 21 are controlled by a processor 17 that controls the driving of the pump 22.

[0032] The source gas tank 12 contains a source gas 26. As described above, the source gas 26 may be, for example, hydrogen (H ２ ), oxygen (O ２ ) and carbon dioxide (CO ２ The raw material gas 26 is a gas containing various components such as ammonium nitrate, ammonium nitrate, ammonium nitrate, and ammonium nitrate. The raw material gas 26 is supplied from the raw material gas tank 12 to the culture tank 11 through the supply path 12A by driving a pump 22 on the supply path 12A. The supply timing and supply amount of the raw material gas 26 are controlled by a processor 17 that controls the driving of the pump 22.

[0033] The downstream end of the supply path 12A is connected to the circulation path 54. More specifically, a sparger 28 is disposed in the circulation path 54, and the downstream end of the supply path 12A is connected to the sparger 28. The sparger 28 is used to disperse and supply the raw material gas 26 into the culture solution 18. As an example of the sparger 28, an aeration pipe made of a porous material is used. The porous material of the sparger 28 is preferably made of sintered metal or ceramics. The sparger 28 is a bubble generating device that generates the raw material gas 26 as bubbles in the culture solution 18. The sparger 28 generates bubbles with a diameter of 100 μm or more. In other words, the bubble diameter, which is the diameter of the bubbles of the raw material gas 26, is defined as D Ｂ Then, the sparger 28 has a D of 100 μm or more. Ｂ (i.e., D Ｂ ≧100 μm) (see FIG. 4). The sparger 28 is an example of a “bubble generator” that supplies the raw material gas 26 to the circulation path 54 as bubbles according to the technology of the present disclosure.

[0034] There are various types of bubble generators, such as a micropore type like the sparger 28, a swirling flow liquid type that generates bubbles by a high-speed swirling flow, an ejector type (also called a Venturi type) that generates bubbles by a sudden pressure change in the gas-liquid flow path, and a static mixer type that generates bubbles by the shear force of an obstacle in the gas-liquid flow path. Types other than the sparger 28 may also be used as the bubble generator.

[0035] The circulation path 54 is provided on the outer peripheral surface of the culture tank 11 (see also FIG. 4 ). The circulation path 54 circulates the culture solution 18 in the culture tank 11 by removing the culture solution 18 from an outlet 54A provided in the culture tank 11 and returning the removed culture solution 18 to the culture tank 11 from a return port 54B provided in the culture tank. In the circulation path 54 of the first embodiment, the outlet 54A is located vertically above the return port 54B (corresponding to the Z direction in FIG. 4 ). The circulation path 54 is also provided with a pump 22. The pump 22 generates pressure to circulate the culture solution 18 through the circulation path 54, and generates a downward flow of the culture solution 18 in the circulation path 54, moving vertically from top to bottom.

[0036] In addition, a sparger 28 is disposed in the circulation path 54 upstream of a midpoint Mp (see FIG. 4 ) of the length from the outlet 54A to the return port 54B of the circulation path 54. The pump 22 is disposed upstream of the sparger 28. Bubbles generated in the sparger 28 float upward in the vertical direction when the culture solution 18 is stationary, and are therefore swept toward the return port 54B by the downward flow of the culture solution 18 generated by the pump 22. As a result, bubbles of the raw material gas 26 flow into the culture tank 11.

[0037] A portion of the raw material gas 26 supplied into the culture solution 18 is dissolved in the culture solution 18. The dissolved raw material gas 26 is taken up by the hydrogen-oxidizing bacteria 21 and consumed for the growth of the hydrogen-oxidizing bacteria 21 or the production of organic substances 29. The organic substances 29 are, for example, alcohols or amino acids. The raw material gas 26 that is not dissolved in the culture solution 18 is released into a space 11A that exists above a liquid surface 18A of the culture solution 18. The space 11A is filled with a mixed gas 31 that is the raw material gas 26 that is released without being dissolved in the culture solution 18.

[0038] The adjustment gas tank 14 contains an adjustment gas 33. The adjustment gas 33 is supplied from the adjustment gas tank 14 to the space 11A of the culture tank 11 through the supply path 14A by driving a pump 22 on the supply path 14A. The supply timing and supply amount of the adjustment gas 33 are controlled by a processor 17 that controls the operation of the pump 22. The mixed gas 31 in the space 11A contains hydrogen and oxygen, which are components of the raw material gas 26. Hydrogen is a flammable gas. The adjustment gas 33 is used to maintain the composition ratio of the mixed gas 31 in the space 11A at a preset target value outside the explosive range. Composition ratio control will be described later (see FIG. 8 ).

[0039] Furthermore, the mixed gas 31 contains components of the raw material gas 26. Therefore, a portion of the mixed gas 31 can be returned to the culture tank 11 via a gas circulation path 39 and reused as the raw material gas 26. The gas circulation path 39 is connected to a supply path 12A for the raw material gas 26. The mixed gas 31 reused as the raw material gas 26 is also supplied to the culture tank 11 via a sparger 28 provided in the circulation path 54. The mixed gas 31 that is not reused is discharged. Although not shown in the figure, when discharging the mixed gas 31 that is not reused, the mixed gas 31 is diluted with an inert gas such as carbon dioxide and then released to the atmosphere.

[0040] An exhaust line 36 for extracting the mixed gas 31 is connected to the culture tank 11, and the exhaust line 36 is provided with a pump 22 and a three-way valve 38. The pump 22 generates pressure for discharging and circulating the mixed gas 31. The three-way valve 38 switches between a state in which the exhaust line 36 is connected to a gas circulation line 39 and a state in which the exhaust line 36 is disconnected from the gas circulation line 39. The three-way valve 38 switches between sending the mixed gas 31 to the gas circulation line 39 or exhausting it to the outside. The exhaust line 36 is also provided with a composition ratio measurement unit 16 that measures the composition ratio of the mixed gas 31. The composition ratio measurement unit 16 is used to control the composition ratio of the mixed gas 31, which will be described later.

[0041] (Supply of each component of raw material gas) In addition, in FIG. 2, for the sake of convenience in order to prevent the drawing from becoming too complicated, the supply path 12A of the raw material gas 26 and the gas circulation path 39 of the mixed gas 31 are shown as one line. However, in reality, as shown in FIG. 3, the supply path 12A and the gas circulation path 39 are separated for each component such as hydrogen, oxygen, and carbon dioxide, making it possible to supply a plurality of components individually. Each supply path 12A and gas circulation path 39 for each component is provided with a pump 22 and a flow meter 23. In addition, the sparger 28 also supplies hydrogen (H ２ ), oxygen (O ２ ) and carbon dioxide (CO ２ Each of the supply paths 12A and gas circulation paths 39 for each component is connected to a sparger 28 corresponding to each component.

[0042] (Removal of impurities from raw material gas) The connecting member 40 connects the supply channel 12A and the gas circulation path 39 upstream of the spargers 28 for each component. As a result, the supply channel 12A and the gas circulation path 39 are integrated into one and connected to the sparger 28. An impurity removal mechanism 51 is provided between each connecting member 40 and each sparger 28. The impurity removal mechanism 51 removes impurities from the raw material gas 26 supplied to the culture tank 11. The impurities include sulfur oxides or nitrogen oxides (NO and NO), ２ The impurity removal mechanism 51 is a mechanism that uses any one of membrane separation, cryogenic separation, physical adsorption, and chemical absorption as a method for separating impurities from the source gas 26.

[0043] (Separation of Components Contained in Mixed Gas) Furthermore, since the mixed gas 31 is a mixture of components such as hydrogen, oxygen, and carbon dioxide, the gas circulation path 39 is provided with a component separation mechanism 52 that separates the components contained in the mixed gas 31. The component separation mechanism 52 separates the components contained in the mixed gas 31, and the separated components are supplied to the gas circulation paths 39 provided for each component. The component separation mechanism 52 is a mechanism that uses any of membrane separation, cryogenic separation, physical adsorption, and chemical absorption as a method for separating the components from the mixed gas 31.

[0044] In this way, when the raw material gas 26 is supplied to the culture tank 11, hydrogen (H ２ ), oxygen (O ２ ) and carbon dioxide (CO ２ ) are supplied separately, which has advantages in terms of culture efficiency and safety. The advantage in terms of culture efficiency is that the raw material gas 26 supplied to the culture tank 11 can be easily adjusted to an appropriate composition from the viewpoint of culture efficiency. The advantage in terms of safety is as follows. That is, the three elements of an explosion are a flammable gas, oxygen, and an ignition source. However, before supplying the raw material gas 26 to the culture tank 11, it is possible to handle the flammable gases hydrogen and oxygen, which are among the three elements of an explosion, separately, thereby further reducing the risk of explosion.

[0045] In addition to hydrogen, oxygen, and carbon dioxide, the component separation mechanism 52 may separate components such as nitrogen and methane from the mixed gas 31. Nitrogen, methane, and the like have the effect of reducing the solubility of the source gas 26 in the culture solution 18, and therefore, by separating these components when reusing the mixed gas 31, the solubility of the source gas 26 can be improved.

[0046] (Culture Solution Recovery) As shown in FIG. 2 , a culture solution recovery path 56 is connected to the culture tank 11. One end of the culture solution recovery path 56 is connected to a recovery solution outlet 56A provided in the culture tank 11, and the other end is connected to a culture solution recovery mechanism 55. The culture solution recovery path 56 removes a portion of the culture solution 18 from the culture tank 11 as a recovery solution. The culture solution recovery mechanism 55 recovers the culture solution 18 containing organic substances 29 from the culture solution 18 removed from the culture tank 11 through the culture solution recovery path 56. The culture solution recovery mechanism 55 includes a recovery solution tank that stores the recovered culture solution 18, which is the recovered culture solution 18, and a separation mechanism that separates the target for purification from the recovered solution. The recovered solution is transferred to a purification device (not shown), where the organic substances 29 are extracted from the recovered solution. The remaining culture solution 18 after recovery by the culture solution recovery mechanism 55, which contains hydrogen-oxidizing bacteria 21, may be returned to the culture tank 11. When the grown hydrogen-oxidizing bacteria 21 are purified, they are recovered, for example, by a method different from that for recovering the organic matter 29 .

[0047] (Agitation of the culture tank, temperature adjustment, and sensing of various information) The culture tank 11 is also provided with an agitator 41 that agitates the culture solution 18. The agitator 41 is a support rod with a screw 41A attached, and is provided so that the screw 41A is disposed within the culture solution 18. The agitator 41 rotates around the support rod as a rotation axis by driving a motor 42 controlled by the processor 17. This agitates the culture solution 18. By agitating the culture solution 18, the agitator 41 diffuses the hydrogen-oxidizing bacteria 21 and raw material gas 26 in the culture solution 18 without causing localized imbalances.

[0048] In addition, the culture tank 11 is provided with a thermometer 46, a temperature regulator 47, a pressure gauge 48, a dissolved gas concentration measurement unit 49, and a bacterial cell density measurement unit 50. The thermometer 46 measures the temperature T in the culture solution 18. The temperature regulator 47 heats or cools the culture solution 18 in the culture tank 11. The pressure gauge 48 measures the pressure PS in the space 11A. The dissolved gas concentration measurement unit 49 measures the dissolved gas concentration Dr of the raw material gas 26 dissolved in the culture solution 18.

[0049] The culture solution 18 is maintained at a target temperature suitable for culturing the hydrogen-oxidizing bacteria 21. For example, the target temperature is between 40°C and 95°C. The target temperature is set for each microorganism. When the hydrogen-oxidizing bacteria 21 are of the genus Hydrogenophilus, the target temperature is preferably around 50°C. The culture solution 18 is heated to reach the target temperature, and may also need to be cooled. This is because the cultured microorganisms may generate heat during growth, causing the culture solution 18 to exceed the target temperature. Based on the temperature T measured by the thermometer 46, the processor 17 controls the operation of the temperature regulator 47 so that the temperature T of the culture solution 18 is maintained at the target temperature.

[0050] The processor 17 also controls the pressure of the mixed gas 31 in the space 11A based on the pressure PS measured by the pressure gauge 48. Setting the pressure PS of the mixed gas 31 to a target pressure improves the solubility of the source gas 26 in the culture solution 18. In this way, the processor 17 controls the pressure of the mixed gas 31 to ensure appropriate solubility in the culture solution 18, thereby improving the cultivation efficiency of the hydrogen-oxidizing bacteria 21. The pressure PS of the mixed gas 31 is adjusted to at least one of the supply amount of the source gas 26, the discharge amount of the mixed gas 31, and the supply amount of the adjustment gas 33. The processor 17 adjusts the supply amount or discharge amount of these gases by controlling the operation of each pump 22.

[0051] More specifically, the dissolved gas concentration measurement unit 49 measures the concentration of each component (hydrogen, oxygen, and carbon dioxide) contained in the source gas 26 dissolved in the culture solution 18. The dissolved gas concentration measurement unit 49 is, for example, composed of a dissolved hydrogen meter (e.g., model KM2100DH manufactured by Kyoei Electronics Laboratory) that measures the dissolved hydrogen concentration (Dr(H)), a dissolved oxygen meter (e.g., model InPro6860i manufactured by METTLE TOLEDO) that measures the dissolved oxygen concentration (Dr(O)), and a dissolved carbon dioxide meter (e.g., model InPro5000i manufactured by METTLE TOLEDO) that measures the dissolved carbon dioxide concentration (Dr(CO)). The dissolved gas concentration measurement unit 49 outputs the measurement values ​​to the processor 17.

[0052] 3, the source gas tank 12 accommodates each component (hydrogen, oxygen, carbon dioxide) of the source gas 26. A supply path 12A and a pump 22 are provided for each component.

[0053] The processor 17 acquires the measured value of the dissolved gas concentration for each component from the dissolved gas concentration measuring unit 49, compares the dissolved gas concentration for each component with a target value, and supplies any component that is lacking in the raw material gas 26 to the culture tank 11. By controlling the supply amount of each component of the raw material gas 26 in this way, the processor 17 can maintain the dissolved gas concentration of the raw material gas 26 in the culture solution 18 at an appropriate value.

[0054] The bacterial cell density measurement unit 50 is an example of a density measurement unit that measures the density of the culture target present in the culture solution 18 in the culture tank 11. The bacterial cell density measurement unit 50 is, for example, configured with a total cell density sensor (e.g., Dencytee RS485 manufactured by HAMILTON). In this example, the culture target is hydrogen-oxidizing bacteria 21, so the bacterial cell density measurement unit 50 measures bacterial cell density X, which is the density of the hydrogen-oxidizing bacteria 21 in the culture solution 18. The bacterial cell density X is the mass or number of bacteria (in this example, the hydrogen-oxidizing bacteria 21) contained per unit volume of the culture solution 18. The bacterial cell density X has a positive correlation with the consumption amount of the raw material gas 26 consumed by the hydrogen-oxidizing bacteria 21. Measuring the bacterial cell density X can be used, for example, to calculate the consumption amount of the raw material gas 26 consumed by the hydrogen-oxidizing bacteria 21, or to perform a process check to verify whether the consumption amount of the raw material gas 26 is appropriate for the amount of the hydrogen-oxidizing bacteria 21.

[0055] The water level meter 66 measures the water level of the culture solution 18 in the culture tank 11, i.e., the height of the liquid surface 18A. The amount of the culture solution 18 present in the culture tank 11 can be determined from the water level of the culture solution 18 measured by the water level meter 66. The water level meter 66 is used for process checks to determine whether the amount of the culture solution 18 is appropriate, and for calculating the necessary amount of replenishment of the culture solution 18.

[0056] The culture tank 11 is also provided with an explosion vent 67. The explosion vent 67 is configured to be destroyed before other parts in the event of an explosion inside the culture tank 11, thereby safely releasing the pressure inside the culture tank 11 and preventing the spread of damage. The vicinity of the explosion vent 67 is selected to be a location where no major damage will occur even if pressure is released, or a location where measures have been taken to prevent the damage from spreading.

[0057] The processor 17 is composed of, for example, a CPU (Central Processing Unit) and a RAM (Random Access Memory), and as described above, controls each part of the culture device 10. The memory 17A is a non-volatile memory, such as a flash memory, in which various setting information is stored.

[0058] The operation of the above configuration will be described with reference to FIG. 4. FIG. 4 shows how the bubble diameter of the raw material gas 26 supplied changes in the culture solution 18 circulating through the culture tank 11 and the circulation path 54. In FIG. 4, for the sake of simplicity and to avoid complication of the drawing, only the components necessary for explaining the circulation of the culture solution 18 are shown, and other components are omitted. As described above, the sparger 28 is disposed upstream of the midpoint Mp of the circulation path 54, and is configured to generate bubbles with a diameter of 100 μm or more (i.e., D Ｂ ≧100 μm) of the raw material gas 26 is generated.

[0059] Because the bubbles of the raw material gas 26 have a lower specific gravity than the culture solution 18, they tend to rise upward in the vertical direction (Z direction in FIG. 4 ) if there is no flow of the culture solution 18. However, the pump 22 generates a downward flow of the culture solution 18 in the circulation path 54. The bubbles of the raw material gas 26 are swept away by the downward flow and move through the circulation path 54 toward the return port 54B. The longer the residence time of the raw material gas 26 in the culture solution 18, the more it dissolves. As dissolution progresses, the bubble diameter gradually decreases, and the bubbles disappear when completely dissolved. In the example shown in FIG. 4 , the bubble diameter is 100 μm or more immediately after generation of the sparger 28. However, as the residence time of the culture solution 18 increases, the bubble diameter decreases, and the bubble diameter is, for example, less than 100 μm near the return port 54B of the circulation path 54. With the bubble diameter reduced in this manner, the raw material gas 26 flows into the culture tank 11 from the return port 54B.

[0060] The bubbles of the raw material gas 26 that have flowed into the culture tank 11 rise toward the liquid surface 18A in the culture tank 11. Even during this rising, the bubbles continue to dissolve in the culture solution 18, and the closer they are to the liquid surface 18A, the smaller the bubbles become. The smaller the bubble diameter, the slower the rising speed. Therefore, the smaller the bubble diameter at the time of flowing into the culture tank 11, the slower the rising speed to the liquid surface 18A in the culture tank 11, and the longer the residence time in the culture solution 18 compared to when the bubble diameter is large. Furthermore, the smaller the bubble diameter, the more rapidly the raw material gas 26 dissolves, so by flowing the raw material gas 26 with a small bubble diameter into the culture tank 11, the dissolution rate of the raw material gas 26 into the culture solution 18 is improved.

[0061] As described above, the culture apparatus 10 includes a circulation path 54 that extracts the culture solution 18 through an outlet 54A provided in the culture tank 11 and returns the extracted culture solution 18 to the culture tank 11 through a return port 54B provided in the culture tank 11; a pump 22 that generates pressure to circulate the culture solution 18 through the circulation path 54 and generates a downward flow from above to below in the vertical direction as the flow of the culture solution 18 within the circulation path 54; and a sparger 28, an example of a bubble generator. The sparger 28 generates bubbles with a diameter of 100 μm or more and supplies the raw material gas 26 as bubbles to the circulation path 54. The outlet 54A is located vertically above the return port 54B, and the sparger 28 is located upstream of a midpoint Mp along the length from the outlet 54A to the return port 54B. This reduces the energy consumption required for miniaturizing the bubbles while improving the dissolution rate of the raw material gas 26.

[0062] That is, the culture apparatus 10 generates bubbles of the raw material gas 26 upstream of the circulation path 54 and generates a downward flow of the culture solution 18 in the direction opposite to the bubble rising direction. This prevents the bubbles from flowing back toward the outlet 54A, while allowing the bubbles to flow into the culture tank 11 through the return port 54B. The bubbles are dissolved into the culture solution 18 from the upstream side of the circulation path 54 until they reach the return port 54B. Therefore, at the time of flowing into the culture tank 11 from the return port 54B, the bubbles are more miniaturized compared to, for example, a configuration in which a sparger 28 with the same performance is disposed at the bottom of the culture tank 11. The smaller the bubbles, the slower their rising speed, and therefore the residence time in the culture tank (i.e., the time it takes for the bubbles to rise to the liquid surface 18A in the culture tank 11) becomes longer. This improves the dissolution rate of the raw material gas.

[0063] Furthermore, one method for miniaturizing bubbles is to use a bubble generator that generates microbubbles or nanobubbles with a diameter of less than 100 μm, as described in the prior art. However, the culture device 10 does not use a bubble generator that generates microbubbles or nanobubbles, but instead uses a bubble generator with a diameter of 100 μm or more. In addition, the culture device 10 extends the residence time of bubbles by devising the arrangement of the circulation path and the bubble generator.

[0064] As a result, the culture device 10 achieves the above-mentioned effect of improving the dissolution rate of the raw material gas 26 while suppressing the energy consumption required to reduce the size of the bubbles, compared to when a bubble generating device that generates microbubbles or nanobubbles is used.

[0065] Improving the dissolution rate of the raw material gas not only improves the culture efficiency but also improves safety by suppressing the release of undissolved flammable raw material gas 26 into space 11A as mixed gas 31. Here, the dissolution rate of the raw material gas 26 is defined as follows: When the amount of raw material gas supplied to the culture tank is Qin, the amount of gas discharged from culture tank 11 is Qout, and the dissolution rate of the raw material gas is R (%), R (%) = (1 - Qout / Qin) x 100.

[0066] In such a culture device 10, it is preferable that the processor 17 controls the flow rate of the downward flow of the culture solution 18 flowing in the circulation path 54 as follows. That is, the average flow rate of the downward flow of the culture solution 18 in the circulation path 54 is V ｐｉｐｅ , the bubble rising speed is V Ｂ In this case, V is set to satisfy the condition of the following formula (1). ｐｉｐｅ It is preferable to control

[0067]

[0068]

[0069] Here, D in the above formula (2) Ｂ is the diameter of the bubbles of the source gas 26, ρ Ｌ is the density of the culture medium, g is the acceleration of gravity, and η is the viscosity of the culture medium 18. Ｌ is the mass of the culture solution 18 per unit volume, and is different from the above-mentioned bacterial cell density X. Ｌ is measured using a hydrometer (for example, the portable density hydrometer DA-130N manufactured by Kyoto Electronics Manufacturing Co., Ltd.). Ｌ does not fluctuate significantly even if the bacterial cell density X changes. Therefore, in the calculation of formula (2), the culture medium density ρ Ｌ The value measured in advance with a hydrometer is used. That is, the bubble rising speed V Ｂis the diameter D of bubbles generated by the culture medium 18 used and the sparger 28. Ｂ If the above equation is known, it can be calculated in advance before starting the culture using equation (2).

[0070] Furthermore, D in formula (2) Ｂ When there is a distribution of bubble diameters in the circulation path 54, the average volumetric bubble diameter D Ｂ is expressed by the following equation (3), where there are N bubbles and the diameter of the i-th bubble is di.

[0071]

[0072] As shown in the above formula (1), the average velocity of the downward flow V ｐｉｐｅ is the bubble rising speed V Ｂ By setting the speed faster than the speed of the return port 54B, it is possible to more reliably prevent bubbles from flowing back in the direction of circulation of the culture solution 18. Therefore, bubbles that try to rise in the circulation path 54 are reliably pushed toward the return port 54B, and the amount of raw material gas supplied to the culture tank 11 can be kept appropriate.

[0073] The processor 17 controls the pump 22 that generates the downward flow (see also FIG. 4) in the circulation path 54, thereby controlling the V ｐｉｐｅ The memory 17A stores V that satisfies the formula (1) according to the characteristics of the culture solution 18 and the dimensions of the culture tank 11 and the circulation path 54. ｐｉｐｅ The output of the pump 22 is set so that the V of the downward flow is ｐｉｐｅ Control.

[0074] The bubble diameter D in the culture solution 18 ＢRegarding the above, the initial diameter of the bubbles generated by the sparger 28 is determined depending on the performance of the sparger 28. If the rate of change of the bubble diameter reduction depending on the dimensions of the circulation path 54 used is measured in advance, the average diameter of the bubbles in the culture solution 18 can be derived. The bubble diameter can be measured by, for example, laser diffraction / scattering method, dynamic image analysis method, or the like. Specific measuring devices that can be used include, for example, a focused beam reflectance measurement device ParticleTrack G400 manufactured by Mettler Toledo, or an in-line microscope EasyViewer 400.

[0075] In addition, the density of the culture medium ρ Ｌ fluctuates during the culture, but as mentioned above, the density of the culture medium ρ Ｌ is a previously measured value, and the previously measured culture medium density ρ Ｌ Based on V ｐｉｐｅ The density of the culture medium during the culture is controlled. Ｌ When considering the fluctuation of the density of the bacterial cells X and the density of the culture medium ρ, for example, Ｌ The processor 17 calculates the culture medium density ρ during the culture by using the correlation between the measured value of the bacterial cell density X measured by the bacterial cell density measuring unit 50 during the culture and the correlation stored in the memory 17A. Ｌ The processor 17 calculates the calculated culture medium density ρ Ｌ Using V ｐｉｐｅ Control.

[0076] As shown in Fig. 5, the circulation path 54 may extend parallel to the vertical direction, or may be inclined with respect to the vertical direction as shown in Fig. 6. As shown in Figs. 5 and 6, the average flow velocity V ｐｉｐｅ is the magnitude of the vertical component along the vertical direction.

[0077] In addition to a linear shape, the circulation path 54 may be entirely or partially curved (including a spiral). By using such a shape, the circulation path 54 can be flexibly arranged according to the surrounding space in which the culture tank 11 is installed. Furthermore, by using a spiral shape, a longer circulation path 54 can be arranged in a space of the same size. By lengthening the circulation path 54, the residence time of bubbles can be increased.

[0078] Furthermore, the average flow velocity V ｐｉｐｅ It is preferable that satisfies the condition of the following formula (4).

[0079]

[0080] In formula (4), H ｐｉｐｅ , is the vertical height from the center of the return port 54B in the circulation path 54 to the lower end of the sparger 28, as shown in FIGS. Ｌ is the height from the bottom of the culture tank 11 to the liquid level 18A. Ｌ is the average height of the liquid surface 18A of the culture medium 18 when the culture medium 18 is circulated during the culture. ｉｎ is the height from the bottom of the culture tank 11 to the center of the return port 54B. Ｂ is the bubble rising speed as described above.

[0081] Equation (4) expresses the average flow velocity V ｐｉｐｅ This is the condition that defines the upper limit of the mean flow velocity V ｐｉｐｅ If the velocity is too fast, the residence time of the bubbles will be short. ｐｉｐｅ By satisfying the condition of formula (4), it is possible to ensure the time required for practical use as the residence time of bubbles in the culture tank 11.

[0082] Equation (4) is derived as follows: The residence time of the bubbles supplied from the sparger 28 until they reach the liquid surface 18A is T, and the minimum time required for the bubbles to completely dissolve in the culture solution 18 is T ｍｉｎ Then, T is T ｍｉｎ That is, T > T ｍｉｎ The following conditions must be met.

[0083] T is the sum of the residence time of the bubbles in the culture tank 11 and the residence time in the circulation path 54. The residence time of the bubbles in the culture tank 11 is the value obtained by dividing the distance the bubbles rise in the culture tank 11 by the rising speed, and the residence time in the circulation path 54 is the value obtained by dividing the substantial flow velocity of the bubbles in the circulation path 54 by the vertical distance they travel in the circulation path 54. Therefore, T>T ｍｉｎ can be expressed by the following formula (5). By transforming formula (5), formula (6) is obtained. T ｍｉｎ Substituting 11.2 [s] into the above gives the above formula (4).

[0084]

[0085]

[0086] The time of 11.2 seconds in equation (4) is the time derived from the experimental results shown in Fig. 7. Fig. 7 is a graph showing the relationship between the dissolved gas concentration and the complete dissolution residence time. The horizontal axis represents the dissolved gas concentration, which is the concentration of the source gas 26 dissolved in the culture solution 18.

[0087] The experiment was carried out in the following manner. Degassed water was prepared in a cylindrical tank of sufficient height, and a mixed gas of hydrogen, oxygen, and carbon dioxide (hydrogen:oxygen:carbon dioxide = 55:15:12 ratio) was passed through a sparger installed at the bottom of the tank until a predetermined dissolved gas concentration was reached. The dissolved gas concentration was measured using a dissolved gas concentration meter (comprising a dissolved hydrogen meter (manufactured by Kyoei Electronics Laboratory: Model No. KM2100DH) for measuring the dissolved hydrogen concentration (Dr(H)), a dissolved oxygen meter (manufactured by METTLE TOLEDO: Model No. InPro6860i) for measuring the dissolved oxygen concentration (Dr(O)), and a dissolved carbon dioxide meter (manufactured by METTLE TOLEDO: Model No. InPro5000i) for measuring the dissolved carbon dioxide concentration (Dr(CO))) installed in the cylindrical layer. Then, a small amount of bubbles was supplied through the sparger, and the maximum height H reached by the bubbles without being completely dissolved was measured. ｍａｘ The bubble diameter D from the sparger was recorded. Ｂ is measured using a bubble diameter sensor (Mettler Toledo ParticleTrack G400, a focused beam reflectance measurement device) and calculated using the method for calculating the average volumetric bubble diameter described above.Ｂ At this time, the residence time of the bubbles was calculated as H ｍａｘ / V Ｂ The experiment was carried out using spargers with different bubble diameters, and the residence time of the shortest bubbles was determined as the residence time required for complete dissolution. The residence time required for complete dissolution was plotted against the dissolved gas concentration, and the experimental results shown in Figure 7 were obtained.

[0088] The complete dissolution residence time is the residence time required for the raw material gas 26 to completely dissolve in the culture solution 18 from the state of bubbles, and is expressed as T ｍｉｎ In the experimental results shown in FIG. 7, when the dissolved gas concentration is 10%, the residence time for complete dissolution is 11.2 seconds. As the dissolved gas concentration increases above 10%, the residence time for complete dissolution increases. Naturally, the dissolved gas concentration varies depending on various conditions, such as the amount of bacterial cells consumed, but if a residence time for complete dissolution of the dissolved gas is ensured when the dissolved gas concentration is 10%, the minimum dissolution rate of the raw material gas required in practice can be ensured. The time of 11.2 seconds is the minimum time required in practice to approach complete dissolution of the raw material gas 26.

[0089] Furthermore, the bubbles that flow into the culture tank 11 from the circulation path 54 become smaller as they rise in the culture tank 11. However, since it is complicated to take into account the change in the diameter of the bubbles, the bubble rising speed V Ｂ is calculated using the diameter of the bubbles at the time when they flow from the circulation path 54 into the culture tank 11 as a representative value. As described above, since the initial diameter of the bubbles generated by the sparger 28 is known, the diameter of the bubbles at the time when they flow into the culture tank 11 can be derived based on the initial diameter.

[0090] Furthermore, the return port 54B is preferably provided in a range from the bottom of the culture tank 11 to ¼ or less of the height of the liquid level 18A. As described above, the residence time of the bubbles of the raw material gas 26 in the culture tank 11 can be increased as the distance the bubbles travel to the liquid level 18A increases. By locating the height of the return port 54B, through which the bubbles of the raw material gas 26 flow into the culture tank 11, at a low position close to the bottom of the culture tank 11, the distance to the liquid level 18A can be increased.

[0091] Furthermore, in the above embodiment, the circulation path 54 is a path dedicated to circulation that is provided independently of the recovery path 56 that recovers a portion of the hydrogen-oxidizing bacteria 21, an example of microorganisms, from the culture solution 18. By providing the circulation path 54 independently of the recovery path 56, it is possible to reduce parameters that affect the average flow velocity of the culture solution 18, making it easier to control the average flow velocity of the culture solution 18. Furthermore, if a sparger 28 is provided in the recovery path 56, there is a risk that the raw material gas 26 that has been supplied with great care may be recovered before being supplied to the culture tank 11. By providing the circulation path 54 separately from the recovery path 56, it is possible to suppress such a decrease in the utilization efficiency of the raw material gas 26.

[0092] In the above embodiment, the microorganisms to be cultured in the culture device 10 are hydrogen-oxidizing bacteria 21, and the raw material gas 26 contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components. By making the raw material gas 26 have such a composition, the hydrogen-oxidizing bacteria 21 can be cultured.

[0093] Furthermore, in the above embodiment, the raw material gas 26 is supplied with each of the components hydrogen, oxygen, and carbon dioxide separately. Therefore, it is easy to adjust the supply amount of the necessary components as the raw material gas 26. Furthermore, when each component of the raw material gas 26 is supplied separately in this manner, it is necessary to provide multiple spargers 28 (an example of a bubble generator) for each component. If multiple devices that consume a lot of energy, such as bubble generators that generate microbubbles or nanobubbles, are provided, the energy consumption required to miniaturize the bubbles increases significantly as the number of bubble generators increases. Therefore, the technology disclosed herein is very effective when multiple bubble generators are provided.

[0094] (Control of Composition Ratio of Mixed Gas) In the above embodiment, the culture device 10 is equipped with a composition ratio control mechanism that measures the composition ratio of the mixed gas 31 in the space 11A present above the liquid surface 18A of the culture solution 18 in the culture tank 11 and supplies an adjustment gas 33 into the space 11A in accordance with the measured composition ratio of the mixed gas 31, thereby maintaining the composition ratio of the mixed gas 31 in the space 11A at a preset target value outside the explosion range. The provision of such a composition ratio control mechanism further improves safety.

[0095] An example of composition ratio control is shown in Fig. 8. The memory 17A of the processor 17 stores, as target values ​​outside the explosive range used for composition ratio control, examples of which include a target value of hydrogen concentration (TD(H)), a target value of oxygen concentration (TD(O)), a water vapor concentration (not shown), and a target temperature.

[0096] As described above, hydrogen is a flammable gas, and the adjustment gas 33 is used to maintain the composition ratio of the mixed gas 31 in the space 11A at a predetermined target value outside the explosive range. In the mixed gas 31, hydrogen, which is a flammable gas, is mixed with oxygen. In this case, an explosion may occur depending on the composition ratio of the mixed gas 31. The composition ratio of the mixed gas at which an explosion occurs is called the explosive range. As described above, the memory 17A of the processor 17 stores target values ​​outside the explosive range, such as the target hydrogen concentration (TD(H)), the target oxygen concentration (TD(O)), and the water vapor concentration. The target value outside the explosive range varies depending on the components of the mixed gas 31. In the present example, when the mixed gas 31 contains hydrogen and oxygen, the target value outside the explosive range is generally an oxygen concentration of less than 5% or a hydrogen concentration of less than 4% in the mixed gas 31.

[0097] Furthermore, the applicant has experimentally verified that the composition ratio of the mixed gas can be maintained outside the explosive range even when the oxygen concentration is 5% or more, as long as the water vapor concentration is 7% or more. Therefore, in this example, the target value outside the explosive range is set, for example, to a range of 5% to 9% for the oxygen concentration when the water vapor concentration is 7% to 90%.

[0098] The experiment was conducted as follows. A stainless steel chamber with an inner diameter of 200 mm, a depth of 200 mm, and a volume of 6 L was used as the explosion vessel. Hydrogen, oxygen, carbon dioxide, and water vapor were filled into the explosion vessel. (Water vapor was produced by pouring water into the explosion vessel and evacuating it to the saturated water vapor pressure at the temperature inside the vessel, thereby vaporizing the water. Hydrogen, oxygen, and carbon dioxide were filled from gas cylinders through gas inlet pipes attached to the explosion vessel.) After homogenizing the gas by turning on the stirring fan inside the explosion vessel, ignition of the gas was attempted using a thin wire explosion device attached to the explosion vessel. Ignition or non-ignition was determined based on the presence or absence of pressure increase and the presence or absence of negative pressure inside the explosion vessel due to the generation of water. Pressure was measured using a pressure sensor (Kyowa Electric Industry, PHS-B-10MP) attached to the upper valve of the gas exhaust pipe attached to the explosion vessel.

[0099] Table 1 shows the experimental results of whether or not ignition occurs depending on the gas concentration of the mixed gas. Example 1 and Comparative Example 1 in Table 1 are both examples where the water vapor concentration is 0%, and the concentrations other than the oxygen concentration are almost the same. In Example 1, where ignition did not occur, the oxygen concentration was 4%, less than 5%, while in Comparative Example 1, where ignition occurred, the oxygen concentration was 9%, greater than 5%. Example 1 and Comparative Example 1 show experimental results that, as is generally known, when the water vapor concentration is 0%, ignition will not occur if the oxygen concentration is less than 5%.

[0100] Furthermore, Examples 2, 3, and Comparative Example 2 all have a water vapor concentration of 7% or higher, with only the oxygen concentration being significantly different. Examples 2 and 3 have an oxygen concentration of 9%, while Comparative Example 2 has an oxygen concentration of 16%, which is higher than 9%. These experimental results show that when the water vapor concentration is 7% or higher, ignition does not occur as long as the oxygen concentration is 5% or higher but 9% or lower.

[0101]

[0102] Although Table 1 does not include experimental results for hydrogen concentrations less than 4%, it is known that if the hydrogen concentration is less than 4% instead of the oxygen concentration being less than 5%, the mixed gas will not ignite even when the water vapor concentration is 0%. Therefore, as a target value outside the explosive range, the hydrogen concentration of mixed gas 31 may be less than 4% instead of the oxygen concentration of mixed gas 31 being less than 5%.

[0103] The adjustment gas 33 is, for example, an inert gas. More specifically, in this example, carbon dioxide (CO ２ ) is used. By supplying the adjustment gas 33 to the space 11A, the oxygen concentration or hydrogen concentration decreases, and the composition ratio of the mixed gas 31 is maintained at a target value outside the explosive range. Note that any gas other than an inert gas can be used as the adjustment gas 33. For example, any gas may be used as the adjustment gas 33 to set the oxygen concentration of the mixed gas 31 to less than 5%, thereby setting the composition ratio of the mixed gas 31 to a target value outside the explosive range. In this way, any gas can be used as the adjustment gas 33. However, as described above, when the mixed gas 31 is reused, it is preferable to use the components of the raw material gas 26 as the adjustment gas 33.

[0104] As shown in FIG. 8 , the composition ratio measurement unit 16 is disposed on an exhaust path 36 that exhausts the mixed gas 31 from the space 11A of the culture tank 11. The composition ratio measurement unit 16 measures, for example, the hydrogen concentration (D(H ２ A gas analyzer (e.g., Horiba Ltd. Model TCA-51d) measuring the oxygen concentration (D(O ２ A gas analyzer (e.g., Horiba Ltd. Model No. VA-5113) measuring the carbon dioxide concentration (D(CO ２ )), and a flow meter (for example, model number CMFS007M manufactured by Emerson) that measures the total flow rate (Qout(total)) of the mixed gas 31. This enables the composition ratio measurement unit 16 to measure the concentration of each component of the mixed gas 31 and the total flow rate (Qout(total)). The composition ratio measurement unit 16 outputs each measurement value to the processor 17 as a measurement signal.

[0105] The processor 17 determines the supply amount of the adjustment gas 33 based on, for example, the total flow rate of the mixed gas 31 and the measured value of the oxygen concentration so that the oxygen concentration of the mixed gas 31 is maintained below a preset upper limit value. Then, the processor 17 controls the pump 22 to supply the determined supply amount of the adjustment gas 33 from the adjustment gas tank 14 to the space 11A. As a result, the supply amount of carbon dioxide (Qin(CO ２ )) increases, and the oxygen concentration (D(O ２ )) decreases, and the composition ratio of the mixed gas 31 in the space 11A reaches a preset target value (TD(O ２ By increasing the supply amount of the adjustment gas 33, the hydrogen concentration (D(H ２ )) also decreases, so the hydrogen concentration also reaches the target value outside the explosive range (TD(H ２ The processor 17, the adjustment gas tank 14, and the pump 22 on the supply line 14A are an example of a "composition ratio control mechanism" according to the technology of the present disclosure.

[0106] Furthermore, since both the culture solution 18 and the space 11A are present in the culture tank 11, by maintaining the temperature T of the culture solution 18 at a target temperature, the temperature in the space 11A can be maintained at approximately the same temperature. As will be described later, the composition ratio of the mixed gas 31 in the space 11A is maintained at a predetermined target value outside the explosion range. The target value also includes the water vapor concentration of the mixed gas 31. By controlling the temperature of the culture solution 18 to 40°C or higher, the temperature of the mixed gas 31 can be increased to 40°C or higher. This makes it possible to adjust the water vapor concentration of the mixed gas 31 to 7% or higher.

[0107] In FIG. 8 , in order to avoid complication of the drawing, the circulation path 54 is omitted and the sparger 28 is illustrated inside the culture tank 11, but the culture apparatus 10 of the first embodiment actually has the circulation path 54, and the sparger 28 is arranged in the circulation path 54.

[0108] [Second Embodiment] The culture apparatus 100 of the second embodiment shown in Figures 9 and 10 differs from the culture apparatus 10 of the first embodiment in two points: first, a sparger 28, which is an example of a bubble generator, is disposed in the culture tank 11; and second, the direction in which the culture solution 18 circulates is reversed. Since the other components are the same as those of the first embodiment, their description will be omitted, and the following description will focus on the differences. In Figure 10, as with Figure 4 of the first embodiment, for the sake of simplicity, in order to avoid cluttering the drawing, only components necessary for describing the circulation of the culture solution 18 are shown, and other components are omitted.

[0109] As shown in Fig. 10, the sparger 28 is disposed on the lower side in the vertical direction (corresponding to the Z direction) of the culture tank 11, i.e., close to the bottom. As an example, the position of the sparger 28 is set at a height H Ｌ As described above, if the distance that the bubbles have to travel to reach the liquid surface 18A becomes longer, the residence time of the bubbles becomes longer, so it is preferable to position the sparger 28 as close to the bottom as possible.

[0110] In the culture apparatus 100 of the second embodiment, the sparger 28 is also a bubble generating device that generates bubbles having a diameter of 100 μm or more, and is similar to the first embodiment in that the raw material gas 26 is supplied to the culture tank 11 as bubbles.

[0111] 9, a sparger 28 is provided for each component of the source gas 26, and it is possible to control the supply amount of each component, such as hydrogen, carbon dioxide, and oxygen, as shown in FIG. 10. This is also the same as in the first embodiment, and the same effects as in the first embodiment can be obtained.

[0112] In the culture device 100 of the second embodiment, the circulation direction of the culture solution 18 is opposite to that of the first embodiment. That is, the outlet 54A of the circulation path 54 is provided near the bottom of the culture tank 11 and is positioned vertically lower than the return port 54B. In the culture device 100 of the second embodiment, the flow of the culture solution 18 in the circulation path 54 is an upward flow that flows vertically upward.

[0113] A pump 22 is provided near the return port 54B of the circulation path 54. The pump 22 generates pressure to circulate the culture solution 18 through the circulation path 54, and generates a downward flow in the vertical direction from above to below as the flow of the culture solution 18 in the culture tank 11.

[0114] That is, in the culture device 100 of the second embodiment, unlike the first embodiment, the flow of the culture solution 18 in the circulation path 54 is an upward flow, and the flow of the culture solution 18 in the culture tank 11 is a downward flow. Therefore, in the culture tank 11, the culture solution 18 flows in the opposite direction to the floating direction of the raw material gas 26. The downward flow of the culture solution 18 in the culture tank 11 acts as resistance to the bubbles trying to float up, and reduces the floating speed of the bubbles. As a result, the residence time of the bubbles until they reach the liquid surface 18A becomes longer, and the dissolution of the bubbles into the culture solution 18 progresses. In the culture device 100, the bubble diameter D is larger than when there is no downward flow of the culture solution 18 in the culture tank 11. Ｂ Since the residence time of the sparger 28 is longer, the bubble diameter D Ｂ Even if the bubble diameter D Ｂ tends to be less than 100 μm.

[0115] In this way, the culture device 100 of the second embodiment can improve the dissolution rate even when the diameter of the supplied bubbles is 100 μm or more. That is, in the second embodiment, as in the first embodiment, the dissolution rate of the source gas can be improved while suppressing the energy consumption required for miniaturizing the bubbles.

[0116] Furthermore, in the culture device 100 of the second embodiment, it is preferable that the processor 17 controls the flow rate of the downward flow of the culture solution 18 flowing in the culture tank 11 as follows. That is, the average flow rate of the downward flow of the culture solution 18 in the culture tank 11 is set to V ｔａｎｋ In this case, V is set to satisfy the condition of the following formula (7). ｔａｎｋ It is preferable to control

[0117]

[0118] Here, as in the first embodiment, H Ｌ is the liquid level of the culture medium in the culture tank, V Ｂ is the bubble rising speed.

[0119] Similar to the formula (4) in the first embodiment, the formula (7) is a condition necessary for ensuring the minimum time practically required from the viewpoint of ensuring the dissolution rate of bubbles as the residence time of bubbles in the culture tank 11. The basis for the value of 11.2 seconds is the same as in the first embodiment. ｔａｎｋ is controlled so that the residence time is greater than 11.2 seconds.

[0120] Furthermore, in the culture device 100 of the second embodiment, the outlet 54A of the circulation path 54 is preferably disposed vertically below the sparger 28, which is an example of a bubble generator, as shown in FIG. 10 . By disposing the outlet 54A below the sparger 28, it is possible to prevent bubbles generated from the sparger 28 from being drawn into the circulation path 54 immediately after generation. Because bubbles generated from the sparger 28 have a large diameter immediately after generation, if these bubbles are drawn into the circulation path 54, large-diameter bubbles will flow into the culture tank 11 through the return port 54B located above the outlet 54A. This shortens the residence time of the bubbles of the raw material gas 26. In addition, a large amount of the raw material gas 26 is released into the space 11A without being dissolved in the culture solution 18, which reduces the utilization efficiency of the raw material gas 26. Disposing the outlet 54A below the sparger 28 can prevent these problems.

[0121] Similarly to the first embodiment, the circulation path 54 of the second embodiment is a dedicated circulation path provided independently of the recovery path 56. Therefore, similar to the first embodiment, the effect of easily controlling the flow rate can be obtained. Also, in the second embodiment, the composition ratio control shown in FIG. 8 in the first embodiment may be performed.

[0122] 6 of the first embodiment, also in the second embodiment, the extension direction of the circulation path 54 may be inclined with respect to the vertical direction so that the positions of the outlet 54A and the return port 54B of the circulation path 54 are different in the width direction of the culture tank 11. In this way, a swirling flow of the culture solution 18 can be generated in the circumferential direction within the culture tank 11, and uneven concentration of the raw material gas 26 within the culture tank 11 can be suppressed.

[0123] As described in the first embodiment, the circulation path 54 may be entirely or partially curved (including spiral) in addition to being linear. By using such a shape, the circulation path 54 can be flexibly arranged depending on the surrounding space in which the culture tank 11 is installed. Furthermore, by using a spiral shape, a longer circulation path 54 can be arranged in a space of the same size. By lengthening the circulation path 54, the residence time of bubbles can be increased.

[0124] In each of the above embodiments, hydrogen-oxidizing bacteria 21 have been described as an example of the microorganisms, but the technology of the present disclosure can also be applied to microorganisms or cells other than the hydrogen-oxidizing bacteria 21. Examples of the microorganisms include acetogenic bacteria such as Clostridium autoethanogenum and methane-utilizing bacteria such as Methylococcus caprasulatus. In the case of acetogenic bacteria, the components of the raw material gas 26 include hydrogen (H ２ ), carbon monoxide (CO) and carbon dioxide (CO ２ In the case of methanotrophs, the raw material gas 26 contains methane (CH ４) and air. A cell is a morphological and / or functional unit that constitutes an organism, and the organism from which the cell originates is not particularly limited, but may be an animal or a plant. The cell may be an established cell line or a genetically modified cell, and may include artificial cells. Examples of the cell include CHO cells (Chinese hamster ovary cells), HEK293 cells (Human Embryonic Kidney cells 293), and Sf9 cells (Spodoptera afrugiperda cells 9).

[0125] The above embodiment also discloses the techniques described in the following first and second appended claims.

[0126] [First Supplementary Item 1] A culture device for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, the culture device comprising: a culture tank containing a culture solution for culturing the microorganisms or cells; a circulation path for extracting the culture solution from an outlet provided in the culture tank and returning the extracted culture solution to the culture tank from a return port provided in the culture tank; a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a vertical downward flow from above to below as the flow of the culture solution in the circulation path; and a bubble generator that generates bubbles with a diameter of 100 μm or more, the bubble generator supplying the raw material gas as bubbles to the circulation path, wherein the outlet is located vertically above the return port, and the bubble generator is located upstream of the midpoint of the length from the outlet to the return port. [First Supplementary Item 2] The culture device further comprises a device that controls the rising speed of the bubbles to V Ｂ , the average flow velocity of the downward flow of the culture solution in the circulation path is V ｐｉｐｅ In this case, V ｐｉｐｅ The culture device according to claim 1, wherein the condition of the following formula is satisfied: [First Supplementary Item 3] Furthermore, the vertical height from the return port in the circulation path to the bubble generator is H ｐｉｐｅ The height from the bottom of the culture tank to the liquid surface of the culture medium is H Ｌ The height from the bottom of the culture tank to the return port is H ｉｎ, the bubble rising speed is V Ｂ In this case, V ｐｉｐｅ is a culture device according to the first appended paragraph 2, which satisfies the following formula: [First Supplementary Item 4] The culture device according to any one of First Supplementary Item 1 to First Supplementary Item 3, wherein the circulation path is a path dedicated to circulation that is provided independently of a recovery path that recovers a portion of the microorganisms or cells from the culture solution. [First Supplementary Item 5] The culture device according to any one of First Supplementary Item 1 to First Supplementary Item 4, wherein the return port is provided in a range from the bottom of the culture tank to ¼ or less of the height of the liquid surface. [First Supplementary Item 6] The culture device according to any one of First Supplementary Item 1 to First Supplementary Item 4, wherein the microorganisms are hydrogen-oxidizing bacteria, and the feed gas contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components. [First Supplementary Item 7] The culture device according to any one of First Supplementary Item 1 to First Supplementary Item 6, wherein the feed gas contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components, and wherein the hydrogen, oxygen, and carbon dioxide components are supplied separately. [First Supplementary Item 8] The culture apparatus according to any one of First Supplementary Item 1 to First Supplementary Item 7, which includes a composition ratio control mechanism that measures the composition ratio of the mixed gas in a space present above the liquid surface of the culture solution in the culture tank and supplies an adjustment gas into the space according to the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a preset target value outside the explosion range.

[0127] [Second Supplementary Item 1] A culture device for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, the culture device comprising: a culture tank containing a culture solution for culturing the microorganisms or cells; a circulation path for removing the culture solution from an outlet provided in the culture tank and returning the removed culture solution to the culture tank from a return port provided in the culture tank; a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a vertical downward flow from above as the flow of the culture solution in the culture tank; and a bubble generator that generates bubbles with a diameter of 100 μm or more, the bubble generator supplying the raw material gas as bubbles to the culture tank. [Second Supplementary Item 2] Furthermore, the liquid level of the culture solution in the culture tank is controlled by a pressure of HＬ , the bubble rising speed is V Ｂ , the average flow velocity of the downward flow of the culture solution in the culture tank is V ｔａｎｋ In this case, V ｔａｎｋ The culture device according to claim 2, wherein the condition of the following formula is satisfied. [Second Supplementary Item 3] The culture device according to Second Supplementary Item 1 or Second Supplementary Item 2, wherein the circulation path is a path dedicated to circulation that is provided independently of a recovery path that recovers a portion of the microorganisms or cells from the culture solution. [Second Supplementary Item 4] The culture device according to any one of Second Supplementary Item 1 to Second Supplementary Item 3, wherein the outlet is located below the bubble generator. [Second Supplementary Item 5] The culture device according to any one of Second Supplementary Item 1 to Second Supplementary Item 4, wherein the microorganisms are hydrogen-oxidizing bacteria, and the feed gas contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components. [Second Supplementary Item 6] The culture device according to any one of Second Supplementary Item 1 to Second Supplementary Item 5, wherein the feed gas contains hydrogen as a combustible gas and oxygen and carbon dioxide as other components, and wherein the feed gas is supplied with the hydrogen, oxygen, and carbon dioxide components separately. [Second Supplementary Item 7] The culture apparatus according to any one of Second Supplementary Item 1 to Second Supplementary Item 6, which is equipped with a composition ratio control mechanism that measures the composition ratio of the mixed gas in a space present above the liquid surface of the culture solution in the culture tank and supplies an adjustment gas into the space according to the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a predetermined target value outside the explosion range.

[0128] The above-mentioned processors include general-purpose processors such as CPUs that execute software (programs) and function as various processing units, as well as programmable logic devices (PLDs) that are processors whose circuit configuration can be changed after manufacture, such as FPGAs (Field Programmable Gate Arrays), and dedicated electrical circuits that are processors with circuit configurations designed specifically for executing specific processes, such as ASICs (Application Specific Integrated Circuits).

[0129] Furthermore, more specifically, the hardware structure of these various processors can be an electric circuit that combines circuit elements such as semiconductor elements.

[0130] The above-described description and illustrations are a detailed explanation of the parts related to the technology of the present disclosure and are merely an example of the technology of the present disclosure. For example, the above description of the configuration, functions, actions, and effects is an explanation of an example of the configuration, functions, actions, and effects of the parts related to the technology of the present disclosure. Therefore, it goes without saying that unnecessary parts may be deleted, new elements may be added, or replacements may be made to the above-described description and illustrations within the scope of the gist of the technology of the present disclosure. Furthermore, to avoid confusion and facilitate understanding of the parts related to the technology of the present disclosure, the above-described description and illustrations omit explanations of common technical knowledge that do not require particular explanation to enable the implementation of the technology of the present disclosure.

[0131] The disclosure of Japanese Patent Application No. 2024-030603, filed on February 29, 2024, is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A culture apparatus for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, comprising: a culture tank that contains a culture solution for culturing the microorganisms or cells; a circulation path that extracts the culture solution from an outlet provided in the culture tank and returns the extracted culture solution to the culture tank from a return port provided in the culture tank; a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a vertical downward flow of the culture solution within the circulation path; and a bubble generator that generates bubbles with a diameter of 100 μm or more, the bubble generator supplying the raw material gas as bubbles to the circulation path, wherein the outlet is located vertically above the return port, and the bubble generator is located upstream of the midpoint of the length from the outlet to the return port.

2. Furthermore, the average flow velocity of the downward flow of the culture solution in the circulation path is V ｐｉｐｅ , the rising speed of the bubbles is V Ｂ In this case, V ｐｉｐｅ The culture device according to claim 1 , wherein the following formula is satisfied:

3. Furthermore, the vertical height from the return port to the bubble generator in the circulation path is H ｐｉｐｅ The height from the bottom of the culture tank to the liquid surface of the culture solution is H Ｌ The height from the bottom of the culture tank to the return port is H ｉｎ In this case, V ｐｉｐｅ The culture device according to claim 2 , wherein the following formula is satisfied:

4. The culture device according to claim 1, wherein the circulation path is a path dedicated to circulation that is provided independently of a recovery path for recovering a portion of the microorganisms or cells from the culture solution.

5. The culture apparatus according to claim 1, wherein the return port is provided within a range of 1 / 4 or less of the liquid level from the bottom of the culture tank.

6. The culture apparatus according to claim 1, wherein the microorganisms are hydrogen-oxidizing bacteria, and the raw material gas contains hydrogen as the combustible gas and oxygen and carbon dioxide as other components.

7. The culture apparatus according to claim 1, wherein the raw material gas contains hydrogen as the combustible gas and oxygen and carbon dioxide as other components, and the hydrogen, oxygen and carbon dioxide components are supplied separately.

8. The culture apparatus according to claim 1, further comprising a composition ratio control mechanism that measures the composition ratio of the mixed gas in the space above the surface of the culture solution in the culture tank and supplies an adjustment gas into the space in accordance with the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a preset target value outside the explosion range.

9. A culture apparatus for culturing microorganisms or cells that utilize a raw material gas containing a flammable gas for growth or production of organic substances, comprising: a culture tank that contains a culture solution for culturing the microorganisms or cells; a circulation path that removes the culture solution from an outlet provided in the culture tank and returns the removed culture solution to the culture tank from a return port provided in the culture tank; a pump that generates pressure to circulate the culture solution through the circulation path, the pump generating a vertical downward flow of the culture solution within the culture tank; and a bubble generator that generates bubbles with a diameter of 100 μm or more, the bubble generator supplying the raw material gas as bubbles to the culture tank.

10. Furthermore, the liquid level of the culture solution in the culture tank is H Ｌ , the average flow velocity of the downward flow of the culture solution in the culture tank is V ｔａｎｋ , the rising speed of the bubbles is V Ｂ In this case, V ｔａｎｋ The culture device according to claim 9 , wherein the following formula is satisfied:

11. The culture device according to claim 9, wherein the circulation path is a path dedicated to circulation that is provided independently of a recovery path for recovering a portion of the microorganisms or cells from the culture solution.

12. The culture device according to claim 1, wherein the outlet is located below the bubble generating device.

13. The culture apparatus according to claim 9, wherein the microorganisms are hydrogen-oxidizing bacteria, and the raw material gas contains hydrogen as the combustible gas and oxygen and carbon dioxide as other components.

14. The culture apparatus according to claim 1, wherein the raw material gas contains hydrogen as the combustible gas and oxygen and carbon dioxide as other components, and the raw material gas is supplied with each of the hydrogen, oxygen, and carbon dioxide components supplied separately.

15. The culture apparatus according to claim 9, further comprising a composition ratio control mechanism that measures the composition ratio of the mixed gas in the space above the surface of the culture medium in the culture tank and supplies an adjustment gas into the space in accordance with the measured composition ratio of the mixed gas, thereby maintaining the composition ratio of the mixed gas in the space at a preset target value outside the explosion range.

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