Method and device for culturing hydrogen bacteria

Abstract

Provided is a method in which bacteria belonging to the genus Hydrogenophilus is cultured in a culture solution while hydrogen gas, oxygen gas, and carbon dioxide gas are supplied, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are directly supplied to the culture solution, and if the oxygen gas is supplied to the culture solution without being previously mixed with hydrogen gas, the danger of a gas explosion caused by mixing the oxygen gas and the hydrogen gas can be simply avoided, and it is thereby possible to easily culture the bacteria belonging to the genus Hydrogenophilus.

Description

Method and apparatus for culturing hydrogen bacteria

[0001] The present invention relates to a culturing method and apparatus capable of safely culturing hydrogen bacteria.

[0002] The Paris Agreement adopted in 2015 stipulates the rapid reduction of greenhouse gas emissions worldwide. In accordance with this, Japan aims to reduce greenhouse gas emissions such as carbon dioxide and methane by 46% by 2030 compared to 2013.

[0003] As a technology for fixing and effectively using carbon dioxide, interest has been focused on technologies for producing useful substances using microorganisms that utilize carbon dioxide. Hydrogen bacteria are chemosynthetic autotrophic bacteria that can grow using hydrogen as an energy source and carbon dioxide as a carbon source. Biomass and chemical products obtained by culturing hydrogen bacteria autotrophically have attracted attention as sustainable substances because carbon dioxide is the raw material.

[0004] Here, with the increase in the world population, food demand is increasing, and there is concern that food shortages may become a serious problem in the future. Among them, meat consumption is feared to disrupt the balance between meat production and demand due to the limitations of the current livestock system, and microbial protein is expected as a sustainable alternative protein that does not depend on livestock. Therefore, the biomass obtained by the growth of hydrogen bacteria is being considered for use as an alternative protein in food and feed.

[0005] In addition, most of the world's chemical production depends on petroleum raw materials, and there are problems such as an increase in greenhouse gas emissions. The method of producing organic substances by the original metabolism of hydrogen bacteria or genetic recombination of hydrogen bacteria has attracted attention as a method that does not depend on petroleum because it uses carbon dioxide as a raw material. Also, there is high expectation for producing useful substances from carbon dioxide using hydrogen bacteria in order to reduce carbon dioxide, which is one of the greenhouse gases.

[0006] However, the technology for culturing hydrogen bacteria on a large scale has not been sufficiently developed. Hydrogen bacteria are aerobic and utilize oxygen as an electron acceptor. Hydrogen bacteria grow using the energy generated by the oxidation of hydrogen and carbon dioxide as a carbon source, so cultivation requires the supply of hydrogen, oxygen, and carbon dioxide. Conventionally, a method of supplying a mixed gas of hydrogen, oxygen, and carbon dioxide to the culture vessel has been adopted. This is because hydrogen bacteria belong to a group of bacteria that, although aerobic, exhibit the highest sensitivity to oxygen (Non-Patent Literature 1), and also because molecular oxygen strongly inhibits the growth of hydrogen bacteria in the early stages of growth, i.e., when the bacterial concentration is low (Non-Patent Literature 2). If oxygen gas, i.e., molecular oxygen, is supplied directly to the culture medium, hydrogen bacteria are exposed to high concentrations of oxygen gas, inhibiting their growth. Therefore, conventionally, when supplying oxygen during the cultivation of hydrogen bacteria, it has been supplied not as molecular oxygen, but as a mixed gas of hydrogen and carbon dioxide.

[0007] In the gas mixture used for culturing hydrogen bacteria, the volume ratio of hydrogen:oxygen:carbon dioxide is often set to around 8:1:1 to 7:1:1, which is optimal for the efficient growth of hydrogen bacteria. The lower limit concentration at which a mixture of hydrogen, oxygen, and carbon dioxide will explode depends on the culture conditions, but it is generally considered to be around 4 v / v% for hydrogen and around 6 v / v% for oxygen. Therefore, if a gas ratio is adopted that provides a sufficiently fast growth rate for practical use, there is a risk of explosion depending on other conditions. For example, Patent Documents 1 and 2 indicate that hydrogen bacteria cannot grow well unless they are in an explosive gas mixture containing hydrogen, oxygen, and carbon dioxide. Consequently, it is necessary to establish a safe culture process.

[0008] For example, in the apparatus described in Non-Patent Documents 3-5, as a safety measure, a closed-system hydrogen gas culture apparatus is used to prevent the mixed gas from being released into the atmosphere. A gas chamber is attached to the culture vessel, and the gas supplied to the culture vessel is circulated between the culture vessel and the gas chamber, maintaining the oxygen concentration in that space below the explosion limit of the mixed gas. In addition, oxygen gas is supplied to the culture vessel via a separate line as needed. Furthermore, explosion-proof pumps and electrical systems are used for circulating or delivering the gas, and the generation of electrostatic sparks is prevented by using oil-free treated stainless steel pipes in the gas circulation line. Because the apparatus in Non-Patent Documents 3-5 has a gas chamber with a capacity of approximately 10 times that of the culture vessel, the apparatus becomes very large when used for industrial-scale cultivation. In addition, the cost is high because various equipment for explosion prevention is required.

[0009] Furthermore, Patent Document 1 discloses a method for mass-culturing hydrogen bacteria, in which a mixed gas of hydrogen gas and carbon dioxide is supplied to the upper space of a culture tank containing the hydrogen bacteria culture medium, while oxygen is supplied directly to the culture medium by itself. Unused gases accumulating in the space within the culture tank are circulated through a closed circulation system and supplied to the culture medium to cultivate hydrogen bacteria. During this process, the amount of oxygen gas supplied is controlled while monitoring the amount of dissolved oxygen in the culture medium, thereby maintaining the mixed gas composition of both the culture tank and the closed circulation system outside the explosion range. Unused gases include hydrogen gas, oxygen gas, and carbon dioxide gas. Patent Document 1 states that this method is safe and economical because it can maintain and control the mixed gas concentration of the raw materials outside the explosion range, and because the raw material gases can be utilized 100% without being discharged outside the culture system. Patent Document 1 also states that Alcaligenes eutrophas (currently known as Capriavidus necatol) was cultured using this method. However, the method described in Patent Document 1 requires a device equipped with a closed circulation system consisting of a mixed gas circulation pipeline and a pump, making it complicated and costly. Furthermore, since the closed circulation system contains not only hydrogen gas and carbon dioxide but also oxygen gas derived from unused gas, gas concentration adjustment is necessary to avoid explosions, which is also complicated.

[0010] Furthermore, Patent Document 2 discloses that by introducing the culture medium from a mixing tank containing the culture medium of hydrogen bacteria into a hydrogen gas absorber and an oxygen gas absorber, respectively, dissolving the hydrogen gas and oxygen gas into separate culture media, and then returning both culture media to the mixing tank, the culture can be cultivated while circulating the culture media, without forming a mixed gas with a composition within the explosive combustion range within the system, and without reducing the utilization efficiency of the hydrogen gas and oxygen gas. In this method, carbon dioxide gas can be dissolved in each culture media by the hydrogen gas absorber and the oxygen gas absorber, or it can be dissolved in the culture media in the mixing tank. Patent Document 2 describes culturing Alcaligenes eutrophas (Capriavidus necatol) using this method. In the method of Patent Document 2, culture media with a very high hydrogen gas ratio and culture media with a very high oxygen gas ratio are prepared in advance, and the main culture is started by mixing the two, so the method and the apparatus used therein are complex.

[0011] Japanese Patent No. 2564008, Japanese Unexamined Patent Publication No. 51-38480

[0012] E Wilde, et al., Antonie Van Leeuwenhoek, 1982, 48:131-43E Goto, et al., Agr. Biol. Chem., 1977, 41:521-5T Komada, et al., Agr. Biol. Chem., 1975, 39:77-82K Tanaka, et al., Biotechnol Bioeng. 1995, 45:268-75.K Tanaka, et al., Bioengineering 2023, 10, 1304.

[0013] The object of this invention is to provide a simple method and apparatus for safely culturing hydrogen bacteria.

[0014] The inventors have discovered that among hydrogen bacteria, bacteria of the genus Hydrogenophilus can grow well even when oxygen gas is supplied to the culture medium alone from the early stages of cultivation, i.e., when molecular oxygen is supplied.

[0015] The present invention has been completed based on the above findings and provides the following [1] to

[0015] . [1] A method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium, and oxygen gas is supplied to the culture medium without being pre-mixed with hydrogen gas. [2] The method according to [1], wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually. [3] The method according to [1] or [2], wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20: 1: 0.01 to 10. [4] The method according to any one of [1] to [3], wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.0001 to 1000 L / hour per L of culture medium. [5] The method according to any one of [1] to [4], wherein the hydrogen bacterium is Hydrogenophilus thermoreolus. [6] A method for producing a target substance by culturing bacteria of the genus Hydrogenophilus in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium, and the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas. [7] The method according to [6], wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually. [8] The method according to [6] or [7], wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20: 1: 0.01 to 10. [9] The method according to any one of [6] to [8], wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.0001 to 1000 L / hour per L of culture medium.

[0010] The method according to any one of [6] to [9], wherein the hydrogen bacterium is Hydrogenophilus thermoreolus.

[0011] A culture apparatus for Hydrogenophilus bacteria comprising a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device directly supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in the culture vessel, and supplies oxygen gas to the culture medium in the culture vessel without pre-mixing it with hydrogen gas.

[0012] The apparatus according to

[0011] , wherein the gas supply device is a device that supplies a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas to the culture medium individually; supplies a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas to the culture medium individually; or supplies a gas containing oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas to the culture medium individually.

[0013] The apparatus according to

[0011] or

[0012] , wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in a volume ratio of 0.1 to 20: 1: 0.01 to 10.

[0014] The apparatus according to any one of

[0011] to

[0013] , wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium such that the total supply rate of these gases is 0.0001 to 1000 L / hour per L of culture medium.

[0015] The apparatus according to any one of

[0011] to

[0014] , wherein the hydrogen bacteria is Hydrogenophilus thermoreolus.

[0016] It is generally known that hydrogen bacteria have their growth inhibited when they come into contact with molecular oxygen, and this inhibition is particularly pronounced in the early stages of culture. For this reason, hydrogen bacteria have traditionally been cultured while supplying a mixed gas of hydrogen, oxygen, and carbon dioxide to the culture vessel. However, this method carries the risk of explosion in the piping or reservoir of the mixed gas depending on the gas mixing ratio. For this reason, a culture apparatus equipped with a large and complex explosion-proof closed-type gas-phase circulation system, such as the apparatus described in Non-Patent Documents 3-5 and Patent Document 1, is required. Furthermore, there is the problem that it is not always possible to adopt the gas mixing ratio that is optimal for the growth of hydrogen bacteria.

[0017] This invention solves these problems by supplying oxygen gas to the culture medium without pre-mixing it with hydrogen gas when culturing Hydrogenophilus bacteria. Therefore, the risk of explosion that occurs when a mixed gas containing oxygen and hydrogen gas is supplied to the culture vessel through piping or a reservoir can be avoided. In the method and apparatus of this invention, some gas that is not consumed by the bacteria during cultivation may escape from the culture medium into the space inside the culture vessel, but normally only a small amount escapes, and there is no risk of explosion.

[0018] Furthermore, the method and apparatus of the present invention are simpler than the method described in Patent Document 2, which involves preparing in advance a culture medium containing a gas with a high oxygen gas ratio and a culture medium containing a gas with a high hydrogen gas ratio, and then mixing the two culture media to start the actual culture, because the three raw material gases are supplied directly to the culture medium in which the target culture is performed.

[0019] Furthermore, compared to supplying a mixed gas to the culture vessel, the method and apparatus of the present invention only increase the number of lines for supplying gas to the culture vessel, without increasing the size or complexity of the apparatus. Moreover, because the method and apparatus of the present invention do not pose an explosion risk, it is easy to set the supply ratio of each gas to a ratio suitable for the growth of the bacteria being cultured. Also, because there is no explosion risk, a sufficient amount of gas can be supplied to enable high-density culture of the bacterial cells. Thus, the present invention makes it possible to efficiently culture hydrogen bacteria at high density without requiring large or complex culture equipment and without the risk of explosion.

[0020] This is a schematic diagram of the culture apparatus used in the examples to culture bacteria of the genus Hydrogenophilus.

[0021] The present invention will be described in detail below. (1) Method for culturing Hydrogenophilus bacteria and method for producing the target substance The present invention is a method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium, and oxygen gas is supplied to the culture medium without being mixed with hydrogen gas beforehand.

[0022] Examples of bacteria belonging to the genus Hydrogenophilus include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidance, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Bacteria of the genus Hydrogenophilus can be easily isolated from almost anywhere on Earth. Among them, Hydrogenophilus thermortheorus is preferred because it has a top-level growth rate and, consequently, carbon fixation capacity as a carbon-fixing microorganism. A preferred strain of Hydrogenophilus thermortheorus is strain TH-1 (NBRC 14978). Hydrogenophilus thermortheorus strain TH-1 (NBRC 14978) exhibits the highest growth rate among carbon-fixing microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubles in one hour). Hydrogenophilus thermortheorus strain NBRC 14978 is internationally deposited under the Budapest Convention and is publicly available. Hydrogenophilus bacteria may be bacteria isolated from nature, or bacteria that have been genetically modified from bacteria isolated from nature. For example, genetically modified strains can be created by constructing artificial metabolic pathways through genetic manipulation to confer new substance production capabilities.

[0023] Culture Method: Gases containing hydrogen gas, gases containing oxygen gas, and gases containing carbon dioxide gas are supplied to the culture medium. When supplying each gas to the culture medium, oxygen gas and hydrogen gas are supplied independently. "Supplying independently" means supplying them individually without mixing. For example, gases containing hydrogen gas, gases containing oxygen gas, and gases containing carbon dioxide gas can be supplied individually to the culture medium; gases containing hydrogen gas and mixed gases containing oxygen gas and carbon dioxide gas can be supplied individually to the culture medium; or gases containing oxygen gas and mixed gases containing hydrogen gas and carbon dioxide gas can be supplied individually to the culture medium. Each supplied gas may contain different gases to the extent that it does not inhibit the growth of Hydrogenophilus bacteria or the production of the target substance. Gases containing hydrogen gas, gases containing oxygen gas, gases containing carbon dioxide gas, mixed gases containing oxygen gas and carbon dioxide gas, and mixed gases containing hydrogen gas and carbon dioxide gas may be gases consisting of hydrogen gas, gases consisting of oxygen gas, gases consisting of carbon dioxide gas, mixed gases consisting of oxygen gas and carbon dioxide gas, and mixed gases consisting of hydrogen gas and carbon dioxide gas, respectively. In this case, it implies that each gas contains an unavoidable amount of a different gas.

[0024] Furthermore, in this invention, hydrogen gas, oxygen gas, and carbon dioxide gas are directly supplied to the culture medium in which Hydrogenophilus bacteria are cultured. In this invention, "directly" supply means supplying to the culture medium in which the target culture is performed. That is, this invention excludes cases in which a culture medium containing hydrogen gas, oxygen gas, and carbon dioxide gas is obtained by mixing multiple culture media supplied with one or two of the following gases, and Hydrogenophilus bacteria are cultured in this culture medium. For example, this excludes cases in which Hydrogenophilus bacteria are cultured in a culture medium in which hydrogen gas and carbon dioxide gas are supplied, after which a culture medium supplied with oxygen gas and carbon dioxide gas is mixed, or in which Hydrogenophilus bacteria are cultured in a culture medium in which hydrogen gas is supplied, after which a culture medium supplied with oxygen gas and carbon dioxide gas is mixed. In this invention, hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium in a ratio suitable for culturing bacteria of the genus Hydrogenophilus.

[0025] The volume ratio (hydrogen:oxygen:carbon dioxide) of hydrogen gas, oxygen gas, and carbon dioxide gas supplied to the culture medium can be 0.1 to 20: 1: 0.01 to 10, more specifically 1 to 20: 1: 0.1 to 5, and more specifically 1 to 10: 1: 0.1 to 3. Within this range, bacterial growth is good and the target substance can be produced efficiently. In other words, this ratio is suitable for culturing Hydrogenophilus bacteria. The volume ratio of the supplied gases may be constant or fluctuate during cultivation.

[0026] In this invention, the culture medium can be stirred while culturing. The gas can be supplied continuously or intermittently. The method of this invention can be carried out with the culture system in communication with the outside air. This can be done using an open system device, for example, a device equipped with an exhaust port on the culture vessel. In this method, oxygen gas is supplied to the culture medium in which the main culture is to be performed without pre-mixing it with hydrogen gas (i.e., supplied to the culture medium in which the target culture is to be performed, or supplied to the culture medium in the culture vessel), but it is preferable that there is substantially no time (particularly none) for supplying hydrogen gas and oxygen gas (especially hydrogen gas, oxygen gas, and carbon dioxide gas). That is, it is preferable to supply oxygen gas directly to the culture medium from the start to the end of the culture without pre-mixing it with hydrogen gas.

[0027] The total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas can be 0.0001–1000 L / hour, 0.001–100 L / hour, 0.01–60 L / hour, 0.05–30 L / hour, 0.1–10 L / hour, or 1–10 L / hour per liter of culture medium. The gas supply rate may be constant or variable during cultivation. By adjusting the supply rate of each gas to match the material balance shown in the literature (Kodama, Toru (1977). "Cultivation of hydrogen bacteria and production of SCP." Petroleum and Microorganisms, 18, 32-41) in accordance with the growth of the bacterial cells, bacterial growth will be good, the target substance can be produced efficiently, and gas waste will be minimized.

[0028] For example, the hydrogen gas supply rate can be 0.001 L / hour or more, 0.01 L / hour or more, 0.05 L / hour or more, 0.1 L / hour or more, 0.5 L / hour or more, or 1 L / hour or more per liter of culture medium, and can also be 100 L / hour or less, 50 L / hour or less, 30 L / hour or less, 10 L / hour or less, or 5 L / hour or less. The hydrogen gas supply rate per liter of culture medium is as follows: 0.001–100 L / hour, 0.001–50 L / hour, 0.001–30 L / hour, 0.001–10 L / hour, 0.001–5 L / hour, 0.01–100 L / hour, 0.01–50 L / hour, 0.01–30 L / hour, 0.01–10 L / hour, 0.01–5 L / hour, 0.05–100 L / hour, 0.05–50 L / hour, 0.05–30 L / hour, 0.05–10 L / hour, 0.05–5 Examples include L / hour, 0.1–100L / hour, 0.1–50L / hour, 0.1–30L / hour, 0.1–10L / hour, 0.1–5L / hour, 0.5–100L / hour, 0.5–50L / hour, 0.5–30L / hour, 0.5–10L / hour, 0.5–5L / hour, 1–100L / hour, 1–50L / hour, 1–30L / hour, 1–10L / hour, and 1–5L / hour. The supply rates of oxygen gas and carbon dioxide gas can be 0.0001 L / hour or more, 0.0005 L / hour or more, 0.001 L / hour or more, 0.01 L / hour or more, 0.05 L / hour or more, or 0.1 L / hour or more per liter of culture medium, respectively, and can also be 100 L / hour or less, 50 L / hour or less, 30 L / hour or less, 10 L / hour or less, or 5 L / hour or less.The supply rates of oxygen and carbon dioxide per liter of culture medium are as follows: 0.0001–100 L / hour, 0.0001–50 L / hour, 0.0001–30 L / hour, 0.0001–10 L / hour, 0.0001–5 L / hour, 0.0005–100 L / hour, 0.0005–50 L / hour, 0.0005–30 L / hour, 0.0005–10 L / hour, 0.0005–5 L / hour, 0.001–100 L / hour, 0.001–50 L / hour, and 0.001–3 L / hour. Examples include 0 L / hour, 0.001–10 L / hour, 0.001–5 L / hour, 0.01–100 L / hour, 0.01–50 L / hour, 0.01–30 L / hour, 0.01–10 L / hour, 0.01–5 L / hour, 0.05–100 L / hour, 0.05–50 L / hour, 0.05–30 L / hour, 0.05–10 L / hour, 0.05–5 L / hour, 0.1–100 L / hour, 0.1–50 L / hour, 0.1–30 L / hour, 0.1–10 L / hour, and 0.1–5 L / hour.

[0029] When supplying each gas into the culture medium in the culture vessel, it is sufficient to supply or blow the gas into the culture medium from the gas supply port at the end of the air supply piping that constitutes the gas supply line. The gas can be supplied as is or as fine bubbles. In either case, it is desirable that the supply ports for the oxygen gas and the hydrogen gas be separated, but they may be in contact.

[0030] The amount of culture medium should be sufficient to allow for adequate stirring by the stirring blades attached to the culture vessel, but it can be, for example, 0.1 to 250,000 L, 0.1 to 25,000 L, 0.5 to 2,500 L, 0.5 to 250 L, 1 to 25 L, or 1 to 2.5 L.

[0031] Since bacteria of the genus Hydrogenophilus can grow using hydrogen as an energy source and carbon dioxide as their sole carbon source, carbon dioxide can be efficiently fixed by culturing them using substantially only carbon dioxide as the carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic culture medium that does not contain carbon sources such as organic matter or carbonates, and to cultivate them using substantially only carbon dioxide as the carbon source (especially using only carbon dioxide as the carbon source). In the present invention, "using carbon dioxide as substantially the sole carbon source" includes cases where an unavoidable amount of other carbon sources are mixed in.

[0032] The pH of the culture medium is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and the solubility of each gas in the culture medium are high, resulting in good bacterial growth efficiency and substance production efficiency.

[0033] The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and most preferably 50 to 52°C. Within this range, bacterial growth is good, and the target substance can be produced efficiently.

[0034] The method for culturing Hydrogenophilus bacteria of the present invention described above can also be a method for producing a target substance when the target substance is to be produced by the Hydrogenophilus bacteria. The target substance is, for example, an organic compound. In addition to the culturing step, this production method may include a step of recovering the target substance from the culture medium.

[0035] (2) Culture apparatus for Hydrogenophilus bacteria The culture apparatus for Hydrogenophilus bacteria of the present invention comprises a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device directly supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in the culture vessel, and supplies oxygen gas directly to the culture medium in the culture vessel without pre-mixing it with hydrogen gas. This apparatus is for carrying out the culture method for Hydrogenophilus bacteria of the present invention as described above.

[0036] Furthermore, the gas supply device is a device that supplies oxygen gas and hydrogen gas independently. The gas supply device may, for example, supply hydrogen gas, oxygen gas, and carbon dioxide gas individually to the culture medium; supply hydrogen gas and a mixed gas containing oxygen and carbon dioxide gas individually to the culture vessel; or supply oxygen gas and a mixed gas containing hydrogen and carbon dioxide gas individually to the culture medium. Each supplied gas may contain different gases to the extent that it does not inhibit the growth of Hydrogenophilus bacteria or the production of the desired substance. The hydrogen gas, oxygen gas, carbon dioxide gas, mixed gas containing oxygen and carbon dioxide gas, and mixed gas containing hydrogen and carbon dioxide gas may be, respectively, a gas consisting of hydrogen gas, a gas consisting of oxygen gas, a gas consisting of carbon dioxide gas, a mixed gas consisting of oxygen and carbon dioxide gas, and a mixed gas consisting of hydrogen gas and carbon dioxide gas. In this case, each gas may contain an unavoidable amount of different gases.

[0037] The gas supply device directly supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in a culture vessel for culturing Hydrogenophilus bacteria. That is, the present invention excludes cases where multiple containers for supplying one or two of the following gases—a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas—to the raw material culture medium, and each gas supply device for supplying the gas to those containers are attached. For example, the present invention excludes cases where a container for supplying a gas containing hydrogen gas and carbon dioxide gas to the raw material culture medium, a container for supplying a gas containing oxygen gas and carbon dioxide gas to the raw material culture medium, and each gas supply device for supplying the gas to those containers are attached. Furthermore, the present invention excludes cases where a container for supplying a gas containing hydrogen gas to the raw material culture medium, a container for supplying a gas containing oxygen gas to the raw material culture medium, a container for supplying a gas containing carbon dioxide gas to the raw material culture medium, and each gas supply device for supplying the gas to those containers are attached. The gas supply device directly supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in the culture vessel where Hydrogenophilus bacteria are cultured, in a ratio suitable for culturing Hydrogenophilus bacteria.

[0038] The gas supply system comprises a gas tank or a gas generator, and a gas supply line connecting them to the culture medium. The gas tank may be a high-pressure gas tank, and the gas generator may be, for example, a device that generates gas by vaporizing liquefied gas.

[0039] The gas supply line may include a supply pipe that delivers gas from a gas tank or gas generator to the culture medium, and a gas reservoir that stores gas before supplying it to the culture medium. A sparger may be provided at the gas supply port at the end of the supply pipe to make the gas supplied to the culture medium into fine bubbles. A gas supply line may be provided for each gas that is supplied individually. It is desirable that the supply ports for oxygen-containing gas and hydrogen-containing gas be separated, but they may be in contact. Even if they are in contact, the gas supplied to the culture medium will diffuse quickly, so it will not explode in the culture medium.

[0040] The gas supply device may supply gas into the culture medium continuously or intermittently. Furthermore, the gas supply device shall not include a gas supply device that supplies a mixed gas containing hydrogen gas and oxygen gas, or in particular a mixed gas containing hydrogen gas, oxygen gas, and carbon dioxide gas.

[0041] A regulator for adjusting the gas supply rate or gas supply amount may be attached to the gas tank or gas generator, gas supply piping, or reservoir. The volume ratio of hydrogen gas, oxygen gas, and carbon dioxide gas supplied to the culture vessel is the same as that of the culture method of the present invention described above. The total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas, and the supply rates of each gas, are also the same as those of the culture method of the present invention.

[0042] The capacity of the culture vessel may be determined according to the culture purpose. For example, it can be 1 to 500,000 L, 1 to 50,000 L, 1 to 5,000 L, 1 to 500 L, 1 to 50 L, or 1 to 5 L. Also, the culture vessel may be equipped with a stirrer for stirring the culture solution. The culture vessel can be provided with a gas outlet, and the gas outlet may be connected to an exhaust pipe for gas discharge as needed. In this case, the device of the present invention is an open-system device (a device having an exhaust port at any location). The culture vessel may be equipped with a temperature regulator for adjusting the medium to the target temperature. The temperature regulator may be any device that adjusts the medium temperature to the temperature described for the method of the present invention.

[0043] When the device of the present invention is for continuous culture, it may be equipped with a device for supplying fresh medium to the culture vessel and a device for discharging old medium from the culture vessel. The device for supplying fresh medium to the culture vessel may be equipped with a medium tank, a liquid supply pipe connecting the medium tank and the culture vessel, and a pump for sending the medium, and the liquid supply pipe may be connected to the liquid supply port of the culture vessel. The device for discharging medium from the culture vessel may be equipped with a liquid discharge pipe connected to the liquid discharge port of the culture vessel and a pump for sending the medium. The device for supplying fresh medium to the culture vessel and the device for discharging medium from the culture vessel may cooperate to adjust the amount of the culture solution in the culture vessel to the amount described for the method of the present invention.

[0044] This culture device is a device for culturing bacteria of the genus Hydrogenophilus, and the bacteria of the genus Hydrogenophilus are as described for the method of the present invention.

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. Culture apparatus A culture apparatus schematic shown in Figure 1 was used. This culture apparatus comprises a culture vessel 1 and a gas supply device 2. The culture vessel 1 has an exhaust port 1a on its top surface and is equipped with a stirrer 3 for stirring the culture medium. The gas supply device has a hydrogen gas tank, an oxygen gas tank, and a carbon dioxide gas tank 2a, and three gas supply lines 2b. Each of the three gas supply lines 2b is equipped with an air supply pipe 2b1 extending from the gas tank into the culture vessel, and a gas pressure regulator 2b2 and a gas flow meter 2b3 are provided on the air supply pipe 2b1. The capacity of the culture vessel is 2L. Regarding the gas supply ports of the air supply pipe 2b1, the distance between adjacent ends of the oxygen gas supply port and the hydrogen gas supply port is 10cm. Although not shown, the culture vessel has an intake port and an outlet port for the culture medium and is equipped with a temperature controller for adjusting the temperature of the culture medium.

[0046] The culture apparatus shown in Figure 1 was used for culturing bacteria of the genus Hydrogenophilus. 1 L of liquid medium [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4・7H2O 0.014 g, MgSO4・7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4・7H2O 28 mg, CuSO4・5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4・5H2O 4.0 mg, CoCl2・6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0)] was placed in the culture tank. Hydrogenophilus thermoreolus TH-1 (NBRC 14978) strain was inoculated into the culture medium and incubated at 52°C for 8 hours with stirring using an impeller (1,000 rpm). Hydrogen gas, oxygen gas, and carbon dioxide gas were supplied to the culture medium through separate gas lines. Hydrogen gas was supplied at a rate of 100 mL / hour for the first 2 hours of incubation, 150 mL / hour from 2 to 4 hours, 250 mL / hour from 4 to 6 hours, and 1,000 mL / hour from 6 to 8 hours. Oxygen gas was supplied at a rate of 30 mL / hour for the first 2 hours of incubation, 50 mL / hour from 2 to 4 hours, 75 mL / hour from 4 to 6 hours, and 300 mL / hour from 6 to 8 hours. Carbon dioxide gas was supplied at a rate of 25 mL / hour for the first 2 hours of incubation, 40 mL / hour from 2 to 4 hours, 60 mL / hour from 4 to 6 hours, and 250 mL / hour from 6 to 8 hours. The cells of Hydrogenophilus thermoreolus TH-1 strain proliferated approximately 200-fold in 8 hours of culture. No gas explosions occurred before, during, or after culturing.

[0047] The culture method and apparatus of the present invention can easily avoid the explosion risk associated with mixed oxygen and hydrogen gases, and can culture Hydrogenophilus bacteria at high density, making it highly valuable for industrial applications.

Claims

1. A method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium, and the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas.

2. The method according to claim 1, wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually.

3. The method according to claim 1 or 2, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

4. The method according to claim 1 or 2, wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.0001 to 1000 L / hour per L of culture medium.

5. The method according to claim 1 or 2, wherein the hydrogen bacterium is Hydrogenophilus thermorteolus.

6. A method for producing a target substance by culturing bacteria of the genus Hydrogenophilus in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the hydrogen gas, oxygen gas, and carbon dioxide gas are supplied directly to the culture medium, and the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas.

7. The method according to claim 6, wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually.

8. The method according to claim 6 or 7, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

9. The method according to claim 6 or 7, wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.0001 to 1000 L / hour per L of culture medium.

10. The method according to claim 6 or 7, wherein the hydrogen bacterium is Hydrogenophilus thermorteolus.

11. A culture apparatus for Hydrogenophilus bacteria comprising a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device directly supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in the culture vessel, and supplies oxygen gas to the culture medium in the culture vessel without pre-mixing it with hydrogen gas.

12. The apparatus according to claim 11, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium individually; supplies hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas to the culture medium individually; or supplies oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas to the culture medium individually.

13. The apparatus according to claim 11 or 12, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

14. The apparatus according to claim 11 or 12, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium such that the total supply rate of these gases is 0.01 to 60 L / hour per L of culture medium.

15. The apparatus according to claim 11 or 12, wherein the hydrogen bacterium is Hydrogenophilus thermortheorus.

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