Method and apparatus for culturing hydrogen-oxidizing bacterium

Abstract

The present invention provides a method for culturing a hydrogen-oxidizing bacterium that utilizes a gas including a combustible gas, with which it becomes possible to improve both culturing efficiency and safety in the proliferation of the hydrogen-oxidizing bacterium and / or the production of a useful substance by the hydrogen-oxidizing bacterium.　Provided are: a method for culturing a hydrogen-oxidizing bacterium, the method being characterized by comprising (i) a step for supplying an oxygen-containing gas and carbon dioxide gas to a fermentation tank that contains the hydrogen-oxidizing bacterium and a liquid culture medium and (ii) a step for supplying hydrogen gas to the fermentation tank separately from the oxygen-containing gas, wherein the hydrogen gas supply step includes a step (a) for controlling the supplying of the hydrogen gas to the fermentation tank in such a manner that the hydrogen gas concentration in the gas in a head space of the fermentation tank after the supply of the gases becomes less than 4.0 vol%; and a culture apparatus for the method.

Description

Method and apparatus for culturing hydrogen-oxidizing bacteria

[0001] The present invention relates to a method for culturing hydrogen-oxidizing bacteria and an apparatus for culturing hydrogen-oxidizing bacteria.

[0002] Hydrogen-oxidizing bacteria are autotrophic microorganisms that grow using carbon dioxide as their sole carbon source, utilizing the energy generated by hydrogen oxidation. Their cells contain high concentrations of proteins and nucleic acids, and have a wide range of uses, including as food and biodegradable bioplastics.

[0003] However, under autotrophic conditions, hydrogen-oxidizing bacteria grow well only in an explosive gas mixture containing hydrogen and oxygen. Furthermore, because this combustible feed gas has extremely low solubility in the culture solution, the utilization efficiency of the feed gas is extremely low. While it is possible to reuse unused feed gas by circulating it without discharging it, circulating a gas mixture with a composition within the explosive range still poses a great risk. Therefore, it is currently difficult to mass-cultivate hydrogen-oxidizing bacteria industrially.

[0004] Various culture methods have been reported to solve this problem. In Patent Document 1, hydrogen gas and oxygen gas are dissolved separately in culture solutions introduced into a hydrogen gas adsorption device and an oxygen gas adsorption device, respectively, and the culture solutions are circulated between a fermenter, a hydrogen tank, and an oxygen tank. However, in the method of Patent Document 1, the bacteria in the culture solutions introduced into the hydrogen gas adsorption device and the oxygen gas adsorption device are exposed to hydrogen-rich and oxygen-rich conditions that are unsuitable for growth, resulting in a slow growth rate. Therefore, it is difficult to expect sufficient and stable productivity for practical use.

[0005] In Patent Document 2, the supply of oxygen contained in the raw material gas is made independently controllable, the dissolved oxygen concentration in the culture solution is measured, and the amount of oxygen supplied to the fermenter is limited based on the measured dissolved oxygen concentration. However, the method of Patent Document 2 does not take any measures regarding the composition ratio of the mixed gas released from the culture solution, which raises concerns from the viewpoint of preventing explosion.

[0006] Incidentally, the explosive concentration of hydrogen gas in a fermenter is known to be 4.0 to 75.0 vol%. On the other hand, even if the concentration exceeds 75.0 vol%, the high concentration always poses a risk of explosion during cultivation, and there is also the risk of explosion due to gas leakage from the fermenter. Therefore, Non-Patent Document 1 discloses a method for producing polyhydroxyalkanoic acid using hydrogen-oxidizing bacteria, in which hydrogen gas and air are mixed immediately before being supplied to the fermenter (the hydrogen gas concentration in the mixed gas is 3.8 vol%) and then continuously supplied to the fermenter. However, in the method described in Non-Patent Document 1, the hydrogen concentration in the supply gas is always adjusted to less than 4.0 vol% to ensure safety, which results in an insufficient amount of hydrogen gas being supplied, and good bacterial growth may not be achieved.

[0007] Also reported is a microbial culture device that includes a fermenter that contains a culture solution, a raw material gas supply unit that supplies raw material gas to the fermenter, a composition ratio measurement unit that measures the composition ratio of the mixed gas in a space that exists above the liquid surface of the culture solution in the fermenter, and a composition ratio control mechanism that maintains the composition ratio of the mixed gas in the space at a predetermined target value outside the explosion range (oxygen concentration less than 5% or hydrogen concentration less than 4%) by supplying an adjustment gas into the space in accordance with the measured composition ratio of the mixed gas (Patent Document 3).

[0008] Japanese Patent Publication No. 51-38480 Japanese Patent Publication No. 2564008 International Publication No. 2024 / 048796

[0009] Y. Miyahara, et al., Bioengineering 2022, 9, 586. https: / / doi.org / 10.3390 / bioengineering9100586

[0010] Therefore, there has been a demand for a method for culturing hydrogen-oxidizing bacteria that can improve both the culture efficiency and safety in the growth of hydrogen-oxidizing bacteria and / or the production of useful substances using gases including combustible gases.

[0011] In view of the above circumstances, the present inventors have conducted extensive research and have found that hydrogen (H ２ ), oxygen (O ２ ) and carbon dioxide (CO ２The inventors have discovered a method for culturing hydrogen-oxidizing bacteria that can maximize the amount of hydrogen gas supplied while maintaining the hydrogen concentration in the headspace gas at less than 4.0 vol% by adjusting the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the headspace gas in the fermenter after each gas supply is less than 4.0 vol%, thereby completing the present invention. The inventors have also discovered an apparatus for culturing hydrogen-oxidizing bacteria that can achieve such culturing, and have completed the present invention.

[0012] That is, the present invention provides the following: [1] A culture device for hydrogen-oxidizing bacteria, comprising: (1) a fermenter containing hydrogen-oxidizing bacteria and a culture solution, (2) a supply unit that supplies an oxygen-containing gas to the fermenter, (3) a supply unit that supplies hydrogen gas to the fermenter separately from the oxygen-containing gas, (4) a supply unit that supplies carbon dioxide gas to the fermenter, and (5) a detection unit connected to the fermenter that detects the hydrogen gas concentration in the gas in the headspace in the fermenter, wherein the hydrogen gas supply unit includes a first supply unit that adjusts the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%. [2] The culture device according to [1], wherein the hydrogen gas supply unit further includes a second supply unit that constantly supplies hydrogen gas. [3] The culture device according to [1] or [2], wherein the first supply unit continues or stops the supply of hydrogen gas to the fermenter depending on the hydrogen gas concentration in the gas in the headspace in the fermenter. [4] The culture device according to any one of [1] to [3], which includes a supply unit that supplies hydrogen gas as a mixed gas with carbon dioxide gas. [5] The culture device according to any one of [1] to [4], wherein the carbon dioxide gas is derived from exhaust gas. [6] The culture device according to any one of [1] to [5], wherein the oxygen-containing gas is derived from air or exhaust gas. [7] The culture device according to any one of [1] to [6], wherein the hydrogen gas concentration in the gas in the headspace in the fermenter is in the range of 1.0 vol% to less than 4.0 vol%. [8] A method for culturing hydrogen-oxidizing bacteria, comprising: (i) a step of supplying an oxygen-containing gas and a carbon dioxide gas to a fermenter containing hydrogen-oxidizing bacteria and a culture solution, respectively; and (ii) a step of supplying hydrogen gas to the fermenter separately from the oxygen-containing gas, wherein the hydrogen gas supply step includes a step (a) of adjusting the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%. [9] The culture method according to [8], wherein the hydrogen gas supply step further includes a step (b) of constantly supplying hydrogen gas.

[10] The method according to [8] or [9], wherein in step (a), the supply of hydrogen gas to the fermenter is continued or stopped depending on the hydrogen gas concentration in the gas in the headspace of the fermenter.

[11] The method according to

[10] , wherein in step (a), the supply of hydrogen gas to the fermenter is continued when the hydrogen gas concentration in the gas in the headspace of the fermenter is less than 4.0 vol%, and the supply of hydrogen gas to the fermenter is stopped when the hydrogen gas concentration in the gas in the headspace of the fermenter is 4.0 vol% or higher.

[12] The method according to any one of [8] to

[11] , wherein the hydrogen gas concentration in the gas in the headspace of the fermenter is in the range of 1.0 vol% to less than 4.0 vol%.

[13] The method according to any one of [8] to

[12] , wherein trace elements are added sequentially during culture.

[14] The method according to any one of [8] to

[13] , wherein exhaust gas containing carbon dioxide is used as the carbon dioxide.

[15] The method according to any one of [8] to

[14] , wherein air or exhaust gas is used as the oxygen-containing gas.

[16] The method according to any one of [9] to

[15] , wherein in step (b), the hydrogen gas concentration in the gas in the headspace of the fermenter is maintained in the range of 1.0 vol% to less than 4.0 vol%.

[17] The method according to any one of [8] to

[16] , wherein the hydrogen-oxidizing bacterium is a hydrogen-oxidizing bacterium into which a gene encoding a polyhydroxyalkanoate (PHA) synthase has been introduced.

[18] The method according to any one of [8] to

[17] , wherein the hydrogen-oxidizing bacterium is Ralstonia eutropha.

[0013] The method of the present invention has made it possible to achieve both high culture efficiency and high safety in the growth of hydrogen-oxidizing bacteria that utilize gases containing flammable gases and / or the production of useful substances.

[0014] FIG. 1 is a diagram showing one embodiment of the culture apparatus of the present invention. The hydrogen gas supply unit includes only a first hydrogen gas supply unit. The open arrows indicate the gas flow. FIG. 2 is a diagram showing another embodiment of the culture apparatus of the present invention. The hydrogen gas supply unit includes only a first hydrogen gas supply unit and uses exhaust gas containing carbon dioxide as a carbon dioxide supply source. The open arrows indicate the gas flow. FIG. 3 is a diagram showing yet another embodiment of the culture apparatus of the present invention. The hydrogen gas supply unit includes a first hydrogen gas supply unit and a second hydrogen gas supply unit. The open arrows indicate the gas flow. FIG. 4 is a diagram showing the growth curve of hydrogen-oxidizing bacteria. The solid line indicates the growth of hydrogen-oxidizing bacteria in Example 1, and the dashed line indicates the growth without feedback control (Comparative Example 1). FIG. 5 is a diagram showing the growth curve and hydrogen gas concentration of hydrogen-oxidizing bacteria versus culture time. A indicates the growth of hydrogen-oxidizing bacteria and hydrogen gas concentration in Example 1, and B indicates the growth and hydrogen gas concentration in Example 2. The filled triangles indicate the hydrogen gas concentration in the headspace measured by TCD-GC, and the filled circles indicate the amount of bacterial growth.

[0015] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.

[0016] A first embodiment of the present invention relates to an apparatus for culturing hydrogen-oxidizing bacteria. Specifically, the culture apparatus 1 of the present invention includes: (1) a fermenter 12 containing hydrogen-oxidizing bacteria and a culture solution; (2) a supply unit 2 that supplies an oxygen-containing gas to the fermenter; (3) a supply unit 4 that supplies hydrogen gas to the fermenter separately from the oxygen-containing gas; (4) a supply unit 3 that supplies carbon dioxide gas to the fermenter; and (5) a detection unit 8 connected to the fermenter that detects the hydrogen gas concentration in the gas in the headspace in the fermenter, wherein the hydrogen gas supply unit includes a first supply unit that adjusts the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%.

[0017] A typical culture apparatus of the present invention is shown in Figure 1. Hydrogen gas is supplied to the fermenter by a hydrogen gas supply unit provided separately from the carbon dioxide gas supply unit and the oxygen-containing gas supply unit. Preferably, the hydrogen gas supply unit further includes a second hydrogen gas supply unit (described below) in addition to the first hydrogen gas supply unit, but the culture apparatus shown in Figure 1 only includes the first supply unit.

[0018] As used herein, the term "hydrogen-oxidizing bacteria" refers to bacteria that oxidize free hydrogen and use the energy generated by the reaction to perform carbon dioxide assimilation. Examples of hydrogen-oxidizing bacteria include bacteria of the genus Ralstonia, such as Ralstonia eutropha; bacteria of the genus Alcaligenes, such as Alcaligenes latus; bacteria of the genus Hydrogenovibrio, such as Hydrogenovibrio marinus; bacteria of the genus Hydrogenophilus, such as Hydrogenophilus thermoluteolus; and bacteria of the genus Hydrogenobacter, such as Hydrogenobacter thermophilus. Among these, Ralstonia eutropha is preferably used because it can grow under conditions where gaseous carbon dioxide or carbonate is the sole carbon source, its entire genome information has been analyzed, and a genetic recombination method has been established (Cramm, R. et al., J. Mol. Microbiol. Biotechnol., 16, 38-52 (2009)). One example is the Ralstonia eutropha H16 strain (ATCC 17699). Ralstonia eutropha is also known as Cupriavidus necator.

[0019] As used herein, the "headspace" in a fermenter refers to the space present above the liquid surface of the culture medium in the fermenter. Typically, about 50% to about 60% of the total volume of the culture medium (shown by the dashed line in FIG. 1 ) is charged into the fermenter, and the remaining volume of about 40% to about 50% constitutes the headspace.

[0020] As used herein, "separately from the oxygen-containing gas" means that the supply unit and supply flow path for supplying hydrogen gas are provided independently from the supply unit and supply flow path for the oxygen-containing gas, and the hydrogen gas and oxygen gas are supplied to the fermenter via separate fermenter inlets.

[0021] The hydrogen gas supply unit includes a first supply unit that adjusts the supply of hydrogen gas to the fermenter, and preferably further includes a second supply unit, described below, in addition to the first supply unit. The "adjustment" in this first supply unit includes not only supplying or stopping the supply of hydrogen gas according to the hydrogen gas concentration in the gas in the headspace in the fermenter, but also adjusting the amount of hydrogen gas supplied. In this specification, such adjustment is sometimes referred to as "feedback control." Therefore, the culture apparatus of the present invention is characterized by including a hydrogen gas supply unit with a "feedback control" function.

[0022] For example, when a detection unit that detects the hydrogen gas concentration in the gas in the headspace in the fermenter detects that the hydrogen gas concentration is less than 4.0 vol% (appropriately referred to as "non-explosive range concentration"), hydrogen gas is continuously supplied at the same flow rate (e.g., about 0.001 to 0.08 vvm) as before detection. On the other hand, when the hydrogen gas concentration is detected to be 4.0 vol% or higher, for example, 4.0 vol%, the supply of hydrogen gas to the fermenter is automatically stopped. The supply rate when hydrogen gas supply is resumed may be set higher than about 0.001 to 0.08 vvm, and may be increased, for example, by about 1.1 to 2.5 times. By this feedback control, the hydrogen gas concentration in the gas in the headspace in the fermenter is maintained at less than 4.0 vol%, preferably in the range of 1.0 vol% to less than 4.0 vol%, more preferably in the range of 2.0 vol% to less than 4.0 vol%, and more preferably in the range of 3.0 vol% to less than 4.0 vol%.

[0023] Incidentally, when the hydrogen gas concentration in the gas in the headspace in the fermenter is 4.0 vol% or more, an explosion does not necessarily occur. Although it depends on the equipment used and the gas control conditions, when the water vapor derived from the culture medium in the headspace in the fermenter has a specific concentration, for example, 7 vol% or more, an explosion does not occur or the risk of explosion is very low.

[0024] Feedback control can be performed by a detector that detects the hydrogen gas concentration in the gas in the headspace in the fermenter, a gas flow control unit 5, a gas flow rate adjustment converter 7, and a valve opening / closing unit 6 that opens and closes the gas supply valve using an electrical signal. A mass flow controller can be used as the gas flow rate control unit, and a solenoid valve can be used as the valve opening / closing unit that opens and closes the gas supply valve. Specifically, the culture apparatus of FIG. 1 is equipped with a mass flow controller and a solenoid valve, and this solenoid valve opens when hydrogen gas is supplied and closes when the hydrogen gas supply is stopped. Hereinafter, the "first hydrogen gas supply unit" refers to a supply unit having a feedback control function. The first supply unit includes a device that controls the gas flow rate and a device that opens and closes the gas supply valve using an electrical signal, and may further include a detector and a gas flow rate adjustment converter.

[0025] In contrast, in a culture apparatus without a feedback control function, hydrogen gas is simply supplied to the fermenter at a constant flow rate, regardless of the hydrogen gas concentration in the headspace of the fermenter. Therefore, when the hydrogen gas consumption exceeds the hydrogen gas supply due to the proliferation of hydrogen-oxidizing bacteria, the hydrogen gas supply becomes insufficient. This reduces the cell growth rate or the production rate of the target substance, resulting in a decrease in the productivity of the target substance.

[0026] The hydrogen gas supply unit preferably further includes a "second supply unit" that constantly supplies hydrogen gas at a low rate. The supply of hydrogen gas from the first hydrogen gas supply unit is regulated by the hydrogen gas concentration in the headspace gas in the fermenter. When the hydrogen gas concentration in the fermenter reaches 4 vol%, the supply of hydrogen gas is stopped. Therefore, although safety is ensured, immediately after the start of feedback control, there is a period when no hydrogen gas is supplied at all, and the bacteria do not grow well. For example, even if the hydrogen gas supply rate is increased by about 1.1 to 2.5 times at the start of feedback control, it takes several tens of seconds for hydrogen gas to be supplied to the fermenter. In other words, during this period, the supply of hydrogen gas to the fermenter is stopped (see A in Figure 5). The second hydrogen gas supply unit solves this problem and satisfies both safety and high growth efficiency (see B in Figure 5). The term "low supply rate" refers to a supply rate that maintains the hydrogen gas concentration in the headspace at preferably less than 4.0 vol%, more preferably from 1.0 vol% to less than 4.0 vol%, and more preferably from 2.0 vol% to less than 4.0 vol%. Specifically, the supply rate is about 0.001 to 0.08 vvm, preferably 0.005 to 0.06 vvm, and more preferably 0.01 to 0.04 vvm.

[0027] The hydrogen gas may be supplied to the fermenter separately from the oxygen gas, or may be mixed with, for example, carbon dioxide gas. Such a supply unit may be provided in the culture apparatus of FIGS.

[0028] Exhaust gas containing carbon dioxide can also be used as a carbon dioxide gas supply source (Fig. 2). The use of exhaust gas is preferable because it allows for effective use of the exhaust gas. Fig. 2 shows a typical culture apparatus of the present invention that uses exhaust gas. Since exhaust gas containing carbon dioxide is supplied from an exhaust gas supply unit 14, no dedicated supply unit is provided for supplying only carbon dioxide gas to the fermenter. Exhaust gases include those emitted from facilities that use fossil fuels, such as coal-fired power plants, waste incinerators, cement factories, and steel mills. Exhaust gases include carbon dioxide; oxygen; nitrogen; nitrogen dioxide (NO ２ nitrogen oxides (NOx) such as sulfur dioxide (SO ２In addition to these gas components, exhaust gas may also contain harmful components such as soot and dust, heavy metals, and the like.

[0029] In order to remove the above-mentioned harmful components, it is preferable to further provide a pretreatment device between the exhaust gas supply unit and the exhaust gas generation source. Examples of the pretreatment device include devices for membrane separation, cryogenic separation, physical absorption, chemical absorption, and adsorption separation. Furthermore, the concentration of carbon dioxide gas in the exhaust gas after pretreatment is preferably about 1 to 10% (v / v).

[0030] The amount of carbon dioxide gas supplied is, for example, about 0.001 to 0.2 vvm.

[0031] The oxygen-containing gas may be derived from air, or exhaust gas may be used. The supply amount of oxygen gas is, for example, about 0.005 to 0.4 vvm.

[0032] An example of the detection unit is a hydrogen gas sensor (hereinafter simply referred to as a "gas sensor"). The gas sensor is not particularly limited as long as it can measure the hydrogen gas concentration in the fermentation exhaust gas from the fermenter (herein referred to as "fermentation exhaust gas" to distinguish it from "exhaust gas" emitted from power plants, etc.). Examples of the gas sensor include semiconductor sensors, solid electrolyte sensors, insulator sensors, piezoelectric sensors, catalytic combustion sensors, optical sensors, electrochemical sensors, gas thermal conduction sensors, and hot wire semiconductor sensors. In addition, the concentration of each gas, including hydrogen, in the headspace can also be analyzed using a gas chromatograph equipped with a thermal conductivity detector. In the present invention, the supply of hydrogen gas to the fermenter is adjusted based on the hydrogen gas concentration in the headspace detected by the gas sensor.

[0033] The culture device of the present invention is provided with a detection unit and a CO ２ / O ２The gas analyzer 10 may be provided with a moisture removal unit 9 that can protect the gas analyzer 10 from moisture derived from the fermentation exhaust gas. Moisture removal is performed, for example, by a cooled water trap, a silica gel unit equipped with silica gel, a molecular sieve unit equipped with a molecular sieve, etc. In one embodiment, the fermentation exhaust gas from which moisture has been removed is returned to the fermenter or reused as a feed gas for pre-culture.

[0034] The fermenter may be equipped with an agitator for agitating the culture medium, a shaker for shaking the culture medium, or the like. This can further increase the cultivation rate of the hydrogen-oxidizing bacteria. FIG. 1 shows an example of a fermenter equipped with an agitator, and this agitator has a stirring rod equipped with a support screw. The fermenter is preferably equipped with a sparger for aerating gas in the form of bubbles into the culture solution in the fermenter. The sparger is preferably at least partially made of a porous material and is approximately spherical. Examples of spargers include ceramic spargers, sintered spargers, and metal spargers.

[0035] The culture apparatus of the present invention may be provided with a sterilizing filter 11 for the purpose of removing microorganisms and fine particles from the gas or the piping for supplying the gas. The sterilizing filter may be located, for example, in the supply flow path just before the gas is supplied to the fermenter, or just after the moisture removal section in the flow path that transports the fermentation exhaust gas from the fermenter. Examples of the sterilizing filter include an air vent filter, preferably a Teflon (registered trademark) (PTFE) filter, a polyvinylidene fluoride (PVDF) filter, etc.

[0036] A second embodiment of the present invention is a method for culturing hydrogen-oxidizing bacteria, comprising: (i) a step of supplying an oxygen-containing gas and a carbon dioxide gas to a fermenter containing hydrogen-oxidizing bacteria and a culture solution, respectively; and (ii) a step of supplying hydrogen gas to the fermenter separately from the oxygen-containing gas, wherein the hydrogen gas supply step comprises a step (a) of adjusting the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%.

[0037] The hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is preferably maintained in the range of 1.0 vol% to less than 4.0 vol%, more preferably in the range of 2.0 vol% to less than 4.0 vol%, and more preferably in the range of 3.0 vol% to less than 4.0 vol%.

[0038] In step (a), adjusting the supply of hydrogen gas to the fermentor includes, for example, continuing or stopping the supply depending on the concentration of hydrogen gas in the gas in the headspace in the fermentor.

[0039] As one example, in step (a), when a detection unit that detects the hydrogen gas concentration in the gas in the headspace in the fermenter detects that the hydrogen gas concentration is less than 4.0 vol%, the supply of hydrogen gas is continued at the same flow rate (e.g., about 0.001 to 0.08 vvm) as before the detection. On the other hand, when the hydrogen gas concentration is detected to be 4.0 vol% or higher, for example, 4.0 vol%, the supply of hydrogen gas to the fermenter is automatically stopped. The supply amount when hydrogen gas supply is resumed may be set higher than about 0.001 to 0.08 vvm, for example, may be increased by about 1.1 to 2.5 times. As another example, in step (a), when a detection unit that detects the hydrogen gas concentration in the gas in the headspace in the fermenter detects that the hydrogen gas concentration is less than 3.8 vol%, the supply of hydrogen gas is continued at the same flow rate (e.g., about 0.001 to 0.076 vvm) as before the detection. On the other hand, if the hydrogen gas concentration is detected to be 3.8 vol% or higher, the supply of hydrogen gas to the fermenter is automatically stopped. As another example, in step (a), if the detection unit that detects the hydrogen gas concentration in the gas in the headspace in the fermenter detects that the hydrogen gas concentration is less than 3.5 vol%, the supply of hydrogen gas is continued at the same flow rate (e.g., about 0.001 to 0.07 vvm) as before the detection. On the other hand, if the hydrogen gas concentration is detected to be 3.5 vol% or higher, the supply of hydrogen gas to the fermenter is automatically stopped.

[0040] Preferably, step (ii) further includes step (b) of constantly supplying hydrogen gas at a low rate. In step (a), the supply of hydrogen gas is regulated by the hydrogen gas concentration in the headspace of the fermenter. When the hydrogen gas concentration in the fermenter reaches 4 vol%, the supply of hydrogen gas is stopped. Therefore, although safety is ensured, there is a period of time after feedback control is activated during which no hydrogen gas is supplied at all, resulting in poor bacterial growth. Step (b) solves this problem and satisfies both safety and high growth efficiency. The term "low supply rate" refers to a supply rate that maintains the hydrogen gas concentration in the headspace at preferably less than 4.0 vol%, more preferably between 1.0 vol% and less than 4.0 vol%, and more preferably between 2.0 vol% and less than 4.0 vol%. Specifically, the supply rate is approximately 0.001 to 0.08 vvm, preferably between 0.005 and 0.06 vvm, and more preferably between 0.01 and 0.04 vvm.

[0041] The hydrogen gas may be supplied to the fermenter separately from the oxygen gas, or may be mixed with, for example, carbon dioxide gas.

[0042] Exhaust gas containing carbon dioxide can also be used as a carbon dioxide gas supply source (Figure 2). The use of exhaust gas is preferable because it allows for effective use of the exhaust gas. When exhaust gas is used, a dedicated supply unit for supplying carbon dioxide gas to the fermenter is not required. Examples of exhaust gas include exhaust gas emitted from facilities that use fossil fuels, such as coal-fired power plants, waste incineration plants, cement factories, and steel mills. Exhaust gas contains carbon dioxide, oxygen, nitrogen, nitrogen dioxide (NO ２ ), sulfur dioxide (SO ２ In addition to these gas components, exhaust gas may also contain harmful components such as soot and dust, heavy metals, and the like.

[0043] In order to remove the above-mentioned harmful components, it is preferable to pretreat the exhaust gas before supplying the exhaust gas from the exhaust gas discharge facility to an exhaust gas supply unit. Pretreatment methods include, for example, membrane separation, cryogenic separation, physical absorption, chemical absorption, and adsorption separation. Furthermore, the concentration of carbon dioxide gas in the exhaust gas after pretreatment is preferably about 1 to 10% (v / v).

[0044] The amount of carbon dioxide gas supplied is, for example, about 0.001 to 0.2 vvm.

[0045] The oxygen-containing gas may be derived from air, or exhaust gas may be used. The supply amount of oxygen gas is, for example, 0.005 to 0.4 vvm.

[0046] In the culture method of the present invention, trace elements can be added at any time after the start of culture while monitoring the growth of the bacterial cells and nutrient consumption after the start of culture. This is because trace elements added to the medium may be depleted as the culture progresses. Trace elements are required, particularly in autotrophic cultures using hydrogen gas, for the reaction that extracts energy from hydrogen (J. Ferment. Bioeng., 71, 254-257 (1991), https: / / doi.org / 10.1016 / 0922-338X(91)90277-N). Examples of trace elements include, but are not limited to, Fe (e.g., ferric chloride), Ca (e.g., calcium chloride), Ni (e.g., nickel chloride), Mn (e.g., manganese chloride), Zn (e.g., zinc sulfate), B (e.g., boric acid), Mo (e.g., sodium molybdate), Cu (e.g., copper sulfate), Co (e.g., cobalt chloride), and Mg (e.g., magnesium sulfate). The amount of trace elements added is determined based on the culture conditions, common technical knowledge in this field, etc., but is usually about 0.01 to 50 mg / L for each trace element. In the present invention, the amount of bacterial cell growth when trace elements are added is about 4.4 times or more compared to when no trace elements are added.

[0047] The culture method of the present invention can be widely used to produce useful substances such as food, feed, and biodegradable bioplastics. Such useful substances are not particularly limited as long as they can be produced by an autotrophic culture method using hydrogen-oxidizing bacteria, and an example thereof is polyhydroxyalkanoic acid (PHA) (e.g., Sudesh, K. et al., Prog. Polym. Sci. 2000, 25, 1503-1555). PHA is a biopolyester that accumulates intracellularly in microorganisms. In recent years, PHA has attracted attention not only as a biodegradable plastic material but also as a biomass-derived plastic material.

[0048] Some hydrogen-oxidizing bacteria, for example, Ralstonia bacteria such as Ralstonia eutropha, are naturally capable of producing homopolymers (P(3HB)) primarily composed of (R)-3-hydroxybutanoic acid (3HB) as a structural unit. However, they are rarely able to synthesize copolymer PHAs containing additional HA units other than HB. Therefore, the main PHA polymerase naturally possessed by hydrogen-oxidizing bacteria is an enzyme that produces P(3HB). Therefore, to improve the hard and brittle physical properties of P(3HB), an external PHA polymerase (hereinafter referred to as a "broad substrate specificity PHA polymerase") can be added. Introducing a gene encoding a broad substrate specificity PHA polymerase (hereinafter referred to as a "PHA polymerase gene") into a host hydrogen-oxidizing bacterium enables the synthesis of copolymer PHAs containing additional HA units other than HB, thereby enabling the composition of the copolymer PHA to be controlled. The PHA polymerase gene may be introduced into the same plasmid vector as the one carrying the monomer supply gene, or into a separate plasmid vector. The plasmid vector is then introduced into a host microorganism, which is then used for cultivation. For details, see, for example, Y. Miyahara, et al., Bioengineering 2022, 9, 586. https: / / doi.org / 10.3390 / bioengineering9100586.

[0049] The culture temperature is a temperature at which the bacteria can grow, preferably 15 to 40°C, particularly preferably 20 to 40°C, and even more preferably 28 to 34°C. The culture time is not particularly limited, but for example, 1 to 7 days is preferred for batch culture, and continuous culture is also possible. The culture medium is not particularly limited as long as it can be used by the host of the present invention. A medium containing a nitrogen source, inorganic salts, other organic nutrient sources, etc. in addition to a carbon source can be used.

[0050] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and diammonium hydrogen phosphate, peptone, meat extract, and yeast extract. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium hydrogen phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, and vitamin C.

[0051] In the present invention, other medium components may be added. Examples include a pH adjuster (acid or alkali) 13 and / or an antifoaming agent 13. Examples of pH adjusters include organic acids such as sodium acetate, inorganic acids such as dipotassium hydrogen phosphate and calcium carbonate, organic bases such as ammonia, and inorganic bases such as sodium hydroxide and calcium hydroxide. Examples of antifoaming agents include polyglycerol fatty acid esters such as decaglycerol monooleate. The antifoaming agent is preferably added in a range of 0.4 to 0.6 vol% relative to the culture solution. The amount of antifoaming agent added to the culture solution affects the antifoaming effect, the amount of culture solution consumed by the bacterial cells, and the amount of useful substances produced by the bacterial cells. In other words, as the proportion of antifoaming agent increases, the antifoaming effect on the culture solution increases, bacterial growth is suppressed, and enzyme production tends to decrease.

[0052] Useful substances can be recovered from the bacterial cells, for example, by the following method. After the culture is completed, the bacterial cells are separated from the culture medium using a centrifuge or the like, washed with distilled water, methanol, or the like, and dried. The useful substances are then extracted from the dried bacterial cells using an organic solvent such as chloroform. Subsequently, bacterial components are removed from the organic solvent solution containing the useful substances by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the useful substances. The supernatant is removed from the precipitated useful substances by filtration or centrifugation, and the useful substances can be recovered by drying. Alternatively, useful substances can be recovered from the bacterial cells by the following method. The bacterial cells containing the useful substances are dispersed in an aqueous solvent containing a surfactant or the like, disrupted using an ultrasonic disrupter or high-pressure homogenizer, and the insoluble components are recovered by filtration or centrifugation. The recovered insoluble components are washed with an appropriate solvent such as water and dried, allowing the useful substances to be recovered. The obtained useful substances can be analyzed, for example, by gas chromatography, nuclear magnetic resonance, or the like.

[0053] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples in any way.

[0054] Materials 1. Culture media (1) NR medium: 2.0 g / L yeast extract, 10 g / L bactotryptone, 10 g / L bonito extract. (2) MSI medium: 9.0 g / L Na ２ HPO ４ ・12H ２ O, 1.5g / L KH ２ P.O. ４、 0.5g / L NH ４ Cl, 0.2g / L MgSO ４ ・7H ２ O, 1 mL trace elements solution. (3) Trace elements solution: 0.218g / L CoCl in 0.1N HCl ２ ・6H ２ O, 20.5g / L FeCl ３ ・6H ２ O, 7.8g / L CaCl ２, 0.118g / L NiCl ２ ・6H ２ O, 0.105g / L CrCl ３ ・6H ２ O, 0.156g / L CuSO ４ ・5H ２ (4) MSII medium (3.0 g / L Na ２ HPO ４ ・12H ２ O, 0.5g / L KH ２ P.O. ４、 1.0 g / L (NH ４ ) ２ SO ４ , 1.4g / L MgSO ４ ・7H ２ 0, 1 mL trace elements solution) 2. Strain: Ralstonia eutropha H16 strain (ATCC 17699)

[0055] Example 1 1. Cultivation of Hydrogen-Oxidizing Bacteria (1) Overview of the Cultivation Apparatus Depicted in Figure 1 Substrate gases (oxygen, hydrogen, and carbon dioxide) required for the growth of hydrogen-oxidizing bacteria were supplied from an air compressor (PC3-5.5L, Yaezaki Air Pressure Co., Ltd.), a carbon dioxide gas cylinder (liquefied carbon dioxide, Toho Oxygen Industry Co., Ltd.), and a hydrogen generator (OPGU-2200, HORIBA STEC Co., Ltd.), respectively. Gas flow rates were controlled using mass flow controllers (HORIBA STEC Co., Ltd.: S48 32, MT-51, CU-2130, SEC-E40; ALICAT Co., Ltd.: MC-100SCCM-D). To prevent unexpected contamination, 0.2 μm or 0.45 μm air vent filters (ADVANTEC Co., Ltd.: 25JP020AN, and MILLIPORE Co., Ltd.: Millex-FG) were installed in the gas piping. A ceramic sparger (Cerapol Sparger NBS-M5, Noritake Co., Ltd.) was installed in the fermenter to dissolve the gas in the medium. (2) Analysis of gas concentration The gas concentration in the headspace was measured by measuring the fermentation exhaust gas from the fermenter with a gas sensor (BlueVary, BlueSense Co., Ltd.) and CO ２ / O ２Analysis was performed using a gas analyzer (Automatic System Research, Inc.). In addition, in Example 1 and the following Example 2, sampling of gas in the headspace was performed, and the gas concentration in the headspace in the fermenter was analyzed using a gas chromatography GC-2014 (Shimadzu Corporation) (TCD-GC) equipped with a thermal conductivity detector (TCD). GC-TCD conditions: Column: Shincarbon ST; Carrier gas: Argon; Detector current value: 65 mA; Injection amount: 0.5 mL of sampling gas; Column temperature: 40 ° C, 5 min analysis. (3) Hydrogen gas feedback control In order to efficiently supply hydrogen gas while avoiding the risk of explosion, hydrogen gas feedback control was performed using a gas sensor (BlueVary, BlueSense), a mass flow controller (MFC), a gas flow adjustment converter (Bioneer-Neo, Marubishi Bioengine Co., Ltd.) and a solenoid valve (STOP VALVE, SMC Corporation). When the gas sensor detected a hydrogen gas concentration of 4% in the headspace of the fermenter, the MFC and solenoid valve were automatically closed to temporarily stop the supply of hydrogen gas from the first hydrogen gas supply unit. When the gas sensor detected a hydrogen gas concentration of less than 4% in the headspace gas, the MFC and solenoid valve were opened to restart the supply of hydrogen gas from the first hydrogen gas supply unit.

[0056] (4) Overview of the culture apparatus shown in FIG. 3 The culture apparatus shown in FIG. 3 further includes a second hydrogen gas supply unit in addition to the culture apparatus shown in FIG.

[0057] (5) The pre-cultured strain was inoculated into 2 mL of NR medium and cultured overnight at 30°C with shaking to obtain a primary culture solution. Next, 1 mL of the primary culture solution was inoculated into a 250 mL fermenter (Bio Jr. 8, Able Co., Ltd.) containing 100 mL of MSI medium. ２ :O ２ :CO ２ :N ２A preculture solution was obtained by culturing the mixture of 1.0% ethanol (3.8:7.3:13.0:75.9% (v / v)) at a flow rate of 100 mL / min (1 vvm) at a stirring speed of 1200 rpm and a culture temperature of 30°C for 24 hours. 0.01 wt% of Antifoam 204 (Sigma Co., Ltd.) was added to the medium as an antifoaming agent to prevent foaming of the culture solution.

[0058] (6) Following the main culture, 100 mL of the preculture solution was centrifuged at 10,000 × g for 5 minutes to recover the bacterial pellet. Using the culture apparatus shown in Figure 1, the bacterial pellet was suspended in 25 mL of NR medium and inoculated into a 1 L fermenter (Bioneer-Neo, Marubishi Bioengineering Co., Ltd.) filled with 500 mL of MSII medium. Air at 470 mL / min (0.94 vvm), carbon dioxide at 10 mL / min (0.02 vvm), and hydrogen at 20 mL / min (0.04 vvm) were supplied to the fermenter, and the culture was carried out for 144 hours at a stirring speed of 600 rpm and a culture temperature of 30 °C. To prevent unexpected contamination, 0.2 μm or 0.45 μm air vent filters (ADVANTEC, 25JP020AN, and MILLIPORE, Millex-FG) were installed in the gas piping.

[0059] To prevent foaming of the culture solution, 0.01 wt% of Antifoam 204 (Sigma Co., Ltd.) was added to the medium. If foaming was severe during the culture, Antifoam was added appropriately to prevent the culture solution from leaking from the fermenter. In addition, to replenish the nutrients consumed as the culture progressed, phosphate, MgSO were added as the bacterial cells grew. ４ ・7H ２ O and trace elements solution were added appropriately.

[0060] During cultivation, 1 M HCl, 10% aqueous ammonia, and 1 M KOH were added to the medium as needed to maintain a pH of approximately 7. To minimize the risk of hydrogen gas explosion and ensure efficient gas supply, feedback control of hydrogen gas supply was initiated approximately 24 hours after the start of cultivation. To prevent carbon source depletion, the carbon dioxide gas flow rate was increased from 10 mL / min (0.02 vvm) to 20 mL / min (0.04 vvm) upon the initiation of feedback control. During feedback control, hydrogen gas was supplied at a flow rate of 20 to 50 mL / min (0.04 to 0.1 vvm). The hydrogen gas concentration contained in the fermentation exhaust gas was measured using a gas sensor, and the hydrogen gas concentration in the headspace was automatically controlled to be less than 4 vol%. During cultivation, the culture medium was periodically sampled, and the optical density (OD value) was measured to evaluate bacterial growth.

[0061] 2. Evaluation of P(3HB) Production After cultivation, the culture medium was collected by centrifugation (10,000 × g, 10 minutes). The cell pellet was washed three times with pure water and then lyophilized for three days to obtain dried cells. The PHA content was measured by gas chromatography (GC) using a Shimadzu GC-2014s instrument (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. GC samples were prepared as follows: 20 mg of dried cells was weighed into a screw-cap test tube, and 2 mL of sulfuric acid / methanol (15 vol% sulfuric acid) and 2 mL of chloroform were added. A methyl esterification reaction was carried out at 100°C for 140 minutes. After the reaction was completed, 1 mL of ultrapure water was added and the mixture was vigorously stirred. The chloroform layer was collected. An equal volume of chloroform solution containing 0.1% (w / v) methyl-n-octane as an internal standard was added to the collected solution to prepare the final sample for GC analysis. The sample was injected through a GC capillary column, InertCap 1 (30 m × 0.25 mm, GL Science). The column temperature was initially held at 90 °C for 2 min, then increased to 110 °C at a rate of 5 °C / min, and then increased to 280 °C at a rate of 20 °C / min. The P(3HB) content in the culture medium was calculated from the signal peak area. The results are shown in Table 1.

[0062] Comparative Example 1 P(3HB) was produced in the same manner as in Example 1, except that a culture apparatus not having the first hydrogen gas supply unit (i.e., no feedback control) was used.

[0063]

[0064] Example 2 Hydrogen-oxidizing bacteria were cultured and P(3HB) was produced in the same manner as in Example 1, except that the culture apparatus shown in Figure 3 was used. Specifically, hydrogen gas was supplied at 30 to 90 mL / min (0.06 to 0.18 vvm) from the first hydrogen gas supply unit, and hydrogen gas was continuously supplied at 10 mL / min (0.02 vvm) from the second hydrogen gas supply unit. When the gas sensor detected 4 vol% hydrogen gas, the hydrogen gas concentration in the headspace was controlled to be maintained between 2 vol% and less than 4 vol%.

[0065]

[0066] From Table 2, it can be seen that the amount of residual cells and the amount of PHA produced increased several times compared to Example 1, and increased several tens of times compared to Comparative Example 1.

[0067] The method for autotrophic cultivation of hydrogen-oxidizing bacteria of the present invention can produce useful substances with high cultivation efficiency and high safety, and is expected to be industrially applicable.

[0068] DESCRIPTION OF SYMBOLS 1 Cultivation device 2 Oxygen-containing gas supply unit 3 Carbon dioxide gas supply unit 4 Hydrogen gas supply unit 5 Gas flow rate control unit 6 Valve opening / closing unit 7 Gas flow rate adjusting converter 8 Detection unit 9 Moisture removal unit 10 CO ２ / O ２ Gas analyzer 11 Sterile filter 12 Fermenter 13 pH adjuster / antifoaming agent 14 Exhaust gas supply unit

Claims

1. A culture device for hydrogen-oxidizing bacteria, comprising: (1) a fermenter containing hydrogen-oxidizing bacteria and a culture medium; (2) a supply unit that supplies an oxygen-containing gas to the fermenter; (3) a supply unit that supplies hydrogen gas to the fermenter separately from the oxygen-containing gas; (4) a supply unit that supplies carbon dioxide gas to the fermenter; and (5) a detection unit connected to the fermenter that detects the hydrogen gas concentration in the gas in the headspace in the fermenter, wherein the hydrogen gas supply unit includes a first supply unit that adjusts the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%.

2. The culture device according to claim 1, wherein the hydrogen gas supply unit further includes a second supply unit that constantly supplies hydrogen gas.

3. A culture apparatus as described in claim 1 or 2, wherein the first supply unit continues or stops supplying hydrogen gas to the fermenter depending on the hydrogen gas concentration in the gas in the headspace in the fermenter.

4. The culture device according to claim 1 or 2, comprising a supply unit that supplies hydrogen gas as a mixed gas with carbon dioxide gas.

5. The culture device according to claim 1 or 2, wherein the carbon dioxide gas is derived from exhaust gas.

6. The culture device according to claim 1 or 2, wherein the oxygen-containing gas is derived from air or exhaust gas.

7. The culture apparatus according to claim 1 or 2, wherein the hydrogen gas concentration in the gas in the head space of the fermenter is in the range of 1.0 vol% to less than 4.0 vol%.

8. A method for culturing hydrogen-oxidizing bacteria, comprising: (i) a step of supplying an oxygen-containing gas and a carbon dioxide gas to a fermenter containing hydrogen-oxidizing bacteria and a culture solution, respectively; and (ii) a step of supplying hydrogen gas to the fermenter separately from the oxygen-containing gas, wherein the hydrogen gas supply step includes step (a) of adjusting the supply of hydrogen gas to the fermenter so that the hydrogen gas concentration in the gas in the headspace in the fermenter after each gas supply is less than 4.0 vol%.

9. The culture method according to claim 8, wherein the hydrogen gas supply step further comprises a step (b) of constantly supplying hydrogen gas.

10. The method according to claim 8 or 9, wherein in step (a), the supply of hydrogen gas to the fermenter is continued or stopped depending on the hydrogen gas concentration in the gas in the headspace in the fermenter.

11. The method according to claim 10, wherein in step (a), if the hydrogen gas concentration in the gas in the headspace in the fermenter is less than 4.0 vol%, the supply of hydrogen gas to the fermenter is continued, and if the hydrogen gas concentration in the gas in the headspace in the fermenter is 4.0 vol% or more, the supply of hydrogen gas to the fermenter is stopped.

12. The method of claim 8 or 9, wherein the hydrogen gas concentration in the gas in the headspace in the fermenter is in the range of 1.0 vol% to less than 4.0 vol%.

13. The method according to claim 8 or 9, wherein trace elements are added successively during the culture.

14. The method according to claim 8 or 9, wherein exhaust gas containing carbon dioxide is used as the carbon dioxide.

15. The method according to claim 8 or 9, wherein air or exhaust gas is used as the oxygen-containing gas.

16. The method according to claim 9, wherein in step (b), the hydrogen gas concentration in the gas in the headspace in the fermenter is maintained in the range of 1.0 vol% to less than 4.0 vol%.

17. The method according to claim 8 or 9, wherein the hydrogen-oxidizing bacterium is a hydrogen-oxidizing bacterium into which a gene encoding polyhydroxyalkanoate (PHA) synthase has been introduced.

18. The method of claim 8 or 9, wherein the hydrogen-oxidizing bacterium is Ralstonia eutropha.

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