Culture device, culture method and substance production of hydrogen bacteria with non-explosive mixed gas substrate

Abstract

To provide a culture device and culture method of hydrogen bacteria with a non-explosive mixed gas substrate.SOLUTION: According to the present invention, there is provided a closed gas-phase circulating culture device for culturing hydrogen bacteria, in which the hydrogen concentration is maintained below the lower explosive limit, comprising: a hydrogen supply section for generating low-concentration hydrogen by water electrolysis; a carbon dioxide supply section; an air supply section; a cell culture reactor containing a culture solution for culturing hydrogen bacteria; and a circulation flow path for establishing gas-phase circulation. Furthermore, the present invention provides a method for culturing hydrogen bacteria, in which the hydrogen concentration is maintained below the lower explosive limit, based on the use of the closed gas-phase circulating culture device.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] The present invention relates to a culture device for hydrogen bacteria using a non-explosive mixed gas substrate for the purpose of efficient and stable carbon dioxide fixation in hydrogen bacteria, a culture method for hydrogen bacteria, and the production of useful substances from hydrogen bacteria. [Background technology]

[0002] Hydrogen bacteria are chemoautotrophic bacteria that can grow using hydrogen as an energy source and carbon dioxide as a carbon source. Compared to photosynthetic organisms, hydrogen bacteria have high carbon dioxide fixation and growth capacity, and do not require a large light-receiving area, and are not affected by seasonal or weather conditions such as sunlight conditions, allowing for efficient and stable carbon dioxide fixation. Reducing emissions of carbon dioxide, a greenhouse gas, is an urgent issue for preventing global warming and achieving sustainable development, and autotrophic cultivation of hydrogen bacteria is a promising technology for fixing carbon dioxide and converting it into useful substances.

[0003] Cupriavidus necator (Ralstonia eutropha) H16 is a type of hydrogen-producing bacterium, a facultative chemoautotrophic bacterium that can grow heterotrophically using carbohydrates, amino acids, and lipids as carbon and energy sources, and autotrophically using hydrogen as an energy source and carbon dioxide as a carbon source. This bacterium is also highly productive of polyhydroxyalkanoates (PHA), a biopolyester, and has been studied in many research projects as a producer of biodegradable, environmentally friendly polymeric materials that can be produced from biomass raw materials. Poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) copolymers [P(3HB-co-3HHx)], which are currently being produced by domestic companies and are being developed for various purposes, are produced from vegetable oils and fats by genetically modified strains of this bacterium.

[0004] By genetically engineering this bacterium for substance production and utilizing its autotrophic growth ability (carbon dioxide fixation ability) as a hydrogen bacterium, it is expected that it will be able to produce useful substances from carbon dioxide, including PHA, a biodegradable plastic, and alcohols that can be used as biofuels. On the other hand, this bacterium is aerobic and uses oxygen as an electron acceptor. That is, a mixed gas substrate of hydrogen, oxygen, and carbon dioxide is supplied for the autotrophic cultivation of this bacterium, but the major problem is that the mixed gas of hydrogen and oxygen is explosive. Since the explosive range of hydrogen is about 4-75% when mixed with air, in the cultivation of many aerobic hydrogen bacteria, the hydrogen concentration of the mixed gas substrate supplied is generally 80% or more (for example, hydrogen:oxygen:carbon dioxide = 80:10:10, etc.) to avoid the explosive range. However, in this case, if a high-concentration hydrogen mixed gas leaks from the cultivation device, the surrounding area may enter the explosive range, so strict management of the hydrogen concentration and prevention of gas leaks and explosions are required for large-volume cultivation.

[0005] Professor Tanaka of Kinki University (then Kyushu University) and his colleagues reported in 1995 that high-density cultivation of C. necator and highly efficient production of poly(3-hydroxybutanoic acid) [P(3HB)], a representative PHA, were possible using a closed circulation fermenter (with sufficient safety measures taken into consideration) supplied with a mixed gas of high-concentration hydrogen (Non-Patent Document 1). Research into the production of substances by autotrophic growth of C. necator has been intensified worldwide, and biosynthesis of acetoin and other substances from carbon dioxide has been reported (Non-Patent Document 2), but these are cultures that were supplied with high-concentration hydrogen. Cultivation and substance production of recombinant strains of C. necator using hydrogen generated by electrolysis with electrodes immersed in the medium have also been reported, and although it is said to be highly efficient (Non-Patent Document 3), there are difficulties in scaling up. Associate Professor Tsuge of the Tokyo Institute of Technology has reported the biosynthesis of P(3HB) by flask culture of C. necator strain supplied with a mixed gas substrate containing low concentration of hydrogen (Non-Patent Document 4), and Professor Tanaka of Kinki University has reported the biosynthesis of P(3HB-co-3HHx) by flask culture of recombinant C. necator strain supplied with a mixed gas substrate containing high concentration of hydrogen (collaboration with the present inventor) (Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] K. Tanaka, et al.,Biotechnol.Bioeng.,45(3),268-275,1995 [Non-Patent Document 2] C. Windhorst and J. Gescher, Biothechnol. Biofuels, 12, 163, 2019 [Non-Patent Document 3] C.LIU,et al.,1210-1213,352,2016 [Non-Patent Document 4] Y. Miyahara,et al.,Biotechnol.Lett.,49,1655-1662,2020 [Non-Patent Document 5] K. Tanaka,et al.,Bioengineering,8, 179, 2021 Summary of the Invention [Problem to be solved by the invention]

[0007] Hydrogen is a next-generation energy source, and research and development is being actively conducted on its production, transportation, and storage, and it is expected that its use will spread to society in the future. However, considering the production cost of hydrogen, the risk of explosion, and the problems of handling it, one of the solutions to cultivating hydrogen bacteria is thought to be to efficiently convert hydrogen generated on-site at low concentrations into hydrogen bacteria. [Means for solving the problem]

[0008] The present inventors have discovered that, as a typical example, hydrogen produced by highly efficient water electrolysis using a polymer electrolyte membrane (PEM) type water electrolysis reactor with a manganese-iridium composite oxide (Mn-IrOx) as an anode catalyst can be supplied at a low concentration to a closed circulation bioreactor, and that the hydrogen can be efficiently utilized by C. necator to grow the bacteria from carbon dioxide, thereby producing biopolyester, thus completing the present invention.

[0009] That is, the present invention is as follows. [1] A closed gas-phase circulation culture apparatus for culturing hydrogen bacteria, in which the hydrogen concentration is maintained below the lower explosion limit, A hydrogen supply for generating low-concentration hydrogen by water electrolysis; Carbon dioxide supply section; Air supply section; A cell culture reactor containing a culture medium for culturing hydrogen bacteria; and Circulation flow path for establishing gas phase circulation A closed gas-phase circulating culture device equipped with a [2] The closed gas-phase circulating culture apparatus according to the above [1], wherein the hydrogen supply unit is a solid polymer water electrolysis reactor. [3] The closed gas phase circulation culture apparatus according to [1] or [2] above, which is provided with a pump for gas phase circulation. [4] The closed gas-phase circulating culture apparatus according to any one of the above [1] to [3], wherein the mixed gas in the circulation flow path is composed of hydrogen, carbon dioxide, and the oxygen and nitrogen in the air being discharged, and the composition ratio of hydrogen, carbon dioxide, oxygen and nitrogen is 1:3 to 5:3 to 5:20 to 30. [5] The closed gas-phase circulating culture apparatus according to any one of the above [1] to [4], wherein the hydrogen bacteria is a strain belonging to any one of the genera Cupriavidus, Ralstonia, Alcaligenes, Wautersia, Pseudomonas, Bacillus, Paracoccus, Nocardia, Hydrogenobacter, Hydrogenophilus, Hydrogenovibrio, or Hydrogenophaga, or a recombinant strain of said strain imparted with poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid)-producing ability. [6] The closed gas-phase circulating culture apparatus according to [5], wherein the strain is a Cupriavidus necator strain or a recombinant Cupriavidus necator strain imparted with poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) producing ability. [7] The closed gas-phase circulating culture apparatus described in [6] above, wherein the C. necator strain is the H16 strain (DSM428). [8] The closed gas-phase circulating culture apparatus according to [6] above, wherein the recombinant Cupriavidus necator strain is MF01 strain, MF02 strain, MF03 strain, MF04 strain, NSDG strain, NSDGΔA strain, MF01ΔB1 strain, MF01ΔB1B3 strain, NSDG2-GG strain, or NSDG-GGΔB1 strain, which are modified strains of the H16 strain. [9] A method for culturing hydrogen bacteria using a closed gas-phase circulating culture apparatus defined in any one of [1] to [8] above.

[10] A method for producing poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) from a recombinant strain imparted with poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid)-producing ability, using the culture method defined in [9] above. Effect of the Invention

[0010] According to the present invention, low-concentration hydrogen can be efficiently taken up and used for substance production by closed gas-phase circulating culture of hydrogen bacteria, and by combining this with water electrolysis using a PEM reactor, highly efficient substance conversion using electrical energy can be made possible. [Brief description of the drawings]

[0011] [Figure 1] The P(3HH-co-3HHx) biosynthetic pathway in C. necator MF01ΔB1 carrying pBPP-ccrMeJAc-emd is shown. PhaJAc: enoyl-CoA hydratase (from Aeromonas caviae); PhaB3: acetoacetyl-CoA reductase; Ccrme: crotonyl-CoA carboxylase / reductase (from Methylorubrum extorquens); EmdMm: ethylmalonyl-CoA decarboxylase (from mouse); PhaCNSDG: engineered PHA polymerase with broad substrate specificity (from Aeromonas caviae); BktB: β-ketothiolase. [Figure 2A] A typical closed gas-phase circulation culture apparatus for culturing hydrogen bacteria is shown. Medium: MB, total volume: 1 L; total gas circulation flow rate: 600 mL / min; agitation: 400 rpm; circulation gas composition: H2:O2:CO2:N2 (approximately 3:10:10:77). [Figure 2B] A polymer electrolyte membrane (PEM) type water electrolysis reactor is shown. [Figure 3A] The graph shows the time course of various parameters during the growth and PHA production process of the MF01ΔB1 / pBPP-ccrMeJAc-emd strain using a closed gas-phase circulation culture apparatus. OD600: turbidity at a wavelength of 600 nm; DO: dissolved oxygen concentration. [Figure 3B] The results of PHA analysis of the MF01ΔB1 / pBPP-ccrMeJAc-emd strain harvested 72 and 168 hours after the start of culture are shown. RCM: residual cell weight (total weight of dry cells minus PHA weight). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] For the purposes of illustrating the present invention, a detailed description of a preferred embodiment will now be given. The inventors have succeeded in producing biopolyester by supplying hydrogen at low concentration to a closed circulation bioreactor by highly efficient water electrolysis using a polymer electrolyte membrane (PEM) type water electrolysis reactor (see FIG. 2B) and allowing C. necator to efficiently utilize the hydrogen to grow bacteria from carbon dioxide. In other words, the present invention provides a culture device, a culture method, and a substance production method for hydrogen bacteria using a non-explosive mixed gas substrate.

[0013] (1) Hydrogen Bacteria Although hydrogen bacteria are used in the culture apparatus, culture method, and substance production of the present invention, the type of hydrogen bacteria that can be used is not limited. Hydrogen bacteria are generally bacteria that can perform chemosynthesis using the oxidation energy of hydrogen and grow by fixing carbon dioxide autotrophically. Examples of hydrogen bacteria include, but are not limited to, strains belonging to any of the genera Cupriavidus, Ralstonia, Alcaligenes, Wautersia, Pseudomonas, Bacillus, Paracoccus, Nocardia, Hydrogenobacter, Hydrogenophilus, Hydrogenovibrio, and Hydrogenophaga. For example, a typical example of a strain belonging to the genus Cupriavidus is Cupriavidus necator H16 strain (DSM428).

[0014] In addition, according to the present invention, it is possible to produce substances from carbon dioxide depending on the capabilities of the strain. For example, the C. necator H16 strain and recombinant and mutant strains derived from the H16 strain have the ability to synthesize P(3HB) during autotrophic growth, and can produce P(3HB) from carbon dioxide. The recombinant C. necator strain may be the MF01 strain, the MF02 strain, the MF03 strain, the MF04 strain, the NSDG strain, the NSDGΔA strain (see Patent 5807878), the NSDG-GG strain, or the NSDG-GGΔB1 strain (Zhang, M., et al. Microb. Cell Fact., 18, 147, 2019).

[0015] Furthermore, according to the present invention, a recombinant strain in which an artificial metabolic pathway is constructed by genetic engineering to give a new substance production ability to a hydrogen bacteria strain can also be used. For example, the present inventors have modified Cupriavidus necator H16 strain, which has the ability to synthesize P(3HB), to create a recombinant strain capable of synthesizing P(3HB-co-3HHx) from carbohydrate raw materials. One such recombinant strain is the C. necator MF01ΔB1 / pBPP-ccrMeJ4a-emd strain (sometimes simply referred to as "MF01ΔB1 strain"; see Patent 6755515) and related strains, which are used in substance production tests in the examples described below.

[0016] In order to grow and proliferate hydrogen bacteria using carbon dioxide (or carbon dioxide gas) as the only carbon source, a mixed gas containing (or consisting of) hydrogen, oxygen, and carbon dioxide is used, but this mixed gas can become an explosive gas depending on the composition. The danger of scaling up a culture reactor to fix a huge amount of carbon dioxide has been pointed out. As a conventional culture method, it has been reported that the bacteria are cultured for about 1 to 100 hours in a synthetic medium containing no carbon source at pH 5 to 9 and a culture temperature of 20 to 37°C while sealing or supplying a mixed gas with a composition ratio of hydrogen, oxygen, and carbon dioxide of about 7 to 8:1 to 2:1. This mixed gas composition is not an explosive gas because the hydrogen concentration is above the upper limit of the explosive range, but if the hydrogen concentration decreases due to the inclusion of air, there is a risk that the inside of the culture vessel will become an explosive range, or that the surrounding environment will become an explosive range due to hydrogen leakage from the culture vessel. Therefore, in the present invention, even if the hydrogen concentration in the supplied mixed gas is set to 5% or less, and more preferably to a low rate (low concentration) of about 2 to 4%, by maintaining dissolved hydrogen in the system, hydrogen bacteria can grow and proliferate well as in conventional methods.

[0017] (2) Closed gas-phase circulating culture device According to the present invention, there is provided a closed gas phase circulating culture apparatus for growing and multiplying the hydrogen bacteria, and the closed gas phase circulating culture apparatus typically comprises: A closed gas-phase circulation culture apparatus for culturing hydrogen bacteria, in which the hydrogen concentration is maintained below the lower explosion limit, A hydrogen supply for generating low-concentration hydrogen by water electrolysis; Carbon dioxide supply section; Air supply section; A cell culture reactor containing a culture medium for culturing hydrogen bacteria; and Circulation channel for establishing gas phase circulation It is a closed gas-phase circulating culture device equipped with

[0018] "Closed gas-phase circulating culture" refers to cell culture or a cell culture system in which gas is circulated in a closed system, and refers to cell culture in which the circulating culture system is prevented from being contaminated by foreign matter (various gases), bacteria, viruses, etc. In the present invention, the closed gas-phase circulating culture apparatus mainly comprises a hydrogen supply unit, a carbon dioxide supply unit, and a cell culture reactor, and includes a circulation flow path for connecting each supply unit and aerating the mixed gas (gas-phase circulation). By using a pump (e.g., a diaphragm pump or a peristaltic pump) for gas-phase circulation, hydrogen, carbon dioxide, and air sent from each supply unit can be circulated at a constant speed within the system as a mixed gas. The circulation speed of the mixed gas can be appropriately adjusted by the pump, and can be, for example, 80 to 2300 ml / min, preferably 100 to 2000 ml / min, and more preferably 200 to 1000 ml / min. The piping used for circulating the mixed gas is preferably made of a material that prevents gas leakage and deterioration due to gas, and typically, although not limited to, piping made of a multi-layer plastic tube including a gas barrier layer or a stainless steel pipe may be used.

[0019] Additionally, oxygen / carbon dioxide detectors and hydrogen detectors can be placed in the circulation flow path to measure the gas composition online, including, but not limited to, exhaust gas analyzer DEX-1562A (Biotto) and exhaust gas analyzer OFF-GAS Jr. DEX-2562A (Biotto).

[0020] "Hydrogen supply unit" refers to a device connected to the circulation flow path for generating hydrogen and supplying hydrogen to the circulation flow path. Examples of hydrogen supply include, but are not limited to, a PEM-type water electrolysis reactor that can perform water electrolysis with high efficiency to provide low-concentration hydrogen. Such a PEM-type reactor may be one developed in the art or a commercially available product. For example, a PEM-type reactor (manganese-iridium composite oxide for water splitting catalyst, manganese-iridium composite oxide electrode material and their manufacturing method (see WO2021 / 193467)) composed of a Pt-Ti mesh (cathode) and a manganese-iridium composite oxide (Mn-IrOx) membrane catalyst (anode) developed by the co-inventor (ELSI Professor Nakamura) can be used. By using this, hydrogen can be generated under constant current conditions with about 2 V applied from a power supply device, and can be introduced into the circulation flow path at a maximum of 10 mL / min in a PEM-type reactor using a 3 cm2 membrane catalyst (see FIG. 2B). The flow rate (or flow speed) of the hydrogen supplied can be adjusted by adjusting the magnitude of the constant current, and in the above device, it can be adjusted in the range of 1 to 10 mL / min. The amount of hydrogen supplied to such a circulation path may be controlled using a current control unit (e.g., a potentiostat / galvanostat, etc.), and the supply amount and the hydrogen in the mixed gas present in the circulation path can be maintained at a predetermined concentration (e.g., 3% to 5%) by appropriately adjusting the current value.

[0021] The "carbon dioxide supply unit" is a device for supplying carbon dioxide into the circulation flow path, and may be, for example, a carbon dioxide cylinder filled with carbon dioxide (hereinafter, may be simply referred to as a "CO2 cylinder"). The flow rate (or flow speed) of the carbon dioxide supplied can be controlled by opening and closing the valve of a flow meter installed in the flow path, or by a gas mass flow controller (for example, 1 to 10 mL / min), and by appropriately adjusting it, the carbon dioxide in the mixed gas present in the circulation flow path can be maintained at a predetermined concentration (for example, 10% to 15%).

[0022] The term "air supply unit" refers to a device for supplying air (mainly oxygen and nitrogen) into the circulation flow path. In the present invention, for example, a general-purpose air compressor can be used as the air supply unit. The flow rate (or flow speed) of the supplied air can be controlled by opening and closing the valve of a flow meter installed in the flow path or by a gas mass flow controller (for example, 1 to 10 mL / min), and by appropriately adjusting it, the oxygen concentration in the mixed gas present in the circulation flow path can be maintained at a predetermined concentration (for example, 5% to 10%).

[0023] According to the present invention, in a mixed gas containing hydrogen, carbon dioxide, oxygen, and nitrogen, it is necessary to maintain the hydrogen concentration at or below the lower explosion limit. Specifically, the amount or concentration of each gas supplied from each supply unit is appropriately adjusted by a valve, a mass flow controller, or the like, and the composition ratio of hydrogen, carbon dioxide, oxygen, and nitrogen in the mixed gas is preferably 1:3-5:3-5:20-30. In this specification, a mixed gas having such a composition may be particularly referred to as a "non-explosive mixed gas substrate."

[0024] As mentioned above, the explosive range of hydrogen is about 4-75% when mixed with air, but it varies depending on the gas composition, for example, the lower limit is about 5% when the oxygen concentration is 10%. When used in the present invention, "low concentration hydrogen" means that the hydrogen concentration of the gas in the circulation flow path is below the lower explosive limit, for example, 0.01-5%, preferably 0.1-5%, more preferably 1-5%, and may also be any value or range included in these ranges.

[0025] The term "cell culture reactor" refers to a culture tank widely recognized by those skilled in the art, which is used for growing and multiplying bacteria and for substance production or fermentation. In the present invention, there is no particular limitation as long as it is capable of growing and culturing hydrogen bacteria and for substance production, and the cell culture reactor may be filled with a culture medium suitable for the type of hydrogen bacteria to be cultured. In addition, since the cell culture reactor is used by being incorporated into a closed gas-phase circulating culture device, it is necessary to ensure a closed environment by installing a lid on the cell culture reactor that has an inlet and an exhaust port for connecting to piping that forms a circulation flow path.

[0026] The mixed gas from the circulation path is supplied to the culture solution through a pipe, and can be aerated into the culture solution by immersing the pipe or a glass tube connected to the pipe in the culture solution.

[0027] The hydrogen bacteria are preferably cultured in suspension in the cell culture reactor, which is equipped with a culture medium suitable for culturing the hydrogen bacteria, a stirrer for stirring the culture medium to keep the cells suspended, and may also be equipped with a thermometer for monitoring the temperature of the culture medium. It is preferable that the cell culture medium also has a composition suitable for culturing the hydrogen bacteria.

[0028] In addition, in order to avoid excessive pressure rise in a situation where the amount of mixed gas supplied exceeds the amount consumed by the hydrogen bacteria, a pressure adjustment bag (e.g., a general-purpose gas collection bag made of aluminum, polyester, or fluororesin) may be branched off from the circulation flow path.

[0029] (3)Culture method According to the present invention, a method for culturing hydrogen bacteria using a closed gas-phase circulation culture device is provided. Except for the condition that the mixed gas is adapted to the culture solution, the conditions for culturing hydrogen bacteria in the culture solution in the cell culture reactor can be the same as those of the conventional method. For example, the culture can be performed at a culture temperature of 30 to 37°C and an agitation speed of 100 to 500 rpm / min.

[0030] (4) Material production According to the present invention, by using the closed gas-phase circulating culture device of the present invention, carbon dioxide can be fixed efficiently and stably in the hydrogen bacteria, and useful substances can be produced by the hydrogen bacteria. Examples of useful substances include polyhydroxyalkanoic acid (PHAs) (e.g., P(3HB)), polyhydroxyalkanoic acid copolymers (e.g., P(3HB-co-3HHx), poly(3-hydroxybutanoic acid-co-4-hydroxybutanoic acid) copolymers [P(3HB-co-4HB)], poly(3-hydroxybutanoic acid-co-3-hydroxypropionic acid) copolymers [P(3HB-co-3HP)], poly(3-hydroxybutanoic acid-co-lactic acid) copolymers [P(3HB-co-LA)], poly(3-hydroxybutanoic acid-co-2-hydroxybutanoic acid) copolymers [P(3HB-co-2HB)], etc.), alcohols (e.g., isopropanol, butanol, isobutanol, isoamyl alcohol, etc.), organic acids (e.g., lactic acid, succinic acid, 3-hydroxypropionic acid, (R)-3-hydroxybutanoic acid, (S)-3-hydroxybutanoic acid, etc.), fatty acids, and oils and fats. EXAMPLES

[0031] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0032] (1)Stock used The C. necator MF01ΔB1 / pBPP-ccrMeJAc-emd strain constructed by the present inventors is a modified strain capable of biosynthesizing the flexible PHA copolymer P(3HB-co-3HHx) from fructose or CO2 (Fig. 1). The recombinant plasmid pBPP-ccrMeJAc-emd is a plasmid in which the (R)-enoyl-CoA hydratase gene phaJ4a (derived from C. necator) in the previous pBPP-ccrMeJ4a-emd (see Patent 6755515) was replaced with phaJAc (derived from Aeromonas caviae). Both phaJAc and phaJ4a are genes encoding (R)-enoyl-CoA hydratase, but the phaJAc expression product is known to be more active than the phaJ4a expression product and to have high specificity for short-chain substrates. It has been shown that a recombinant strain in which pBPP-ccrMeJAc-emd was introduced into the C. necator MF01ΔB1 strain efficiently synthesizes P(3HB-co-3HHx) with a 3HHx fraction of about 14 mol% from a fructose substrate, and P(3HB-co-3HHx) with a 3HHx fraction of about 9-14 mol% from a mixed gas substrate of carbon dioxide, high-concentration hydrogen, and oxygen (10:80:10) (Non-Patent Document 5). In the present invention, this recombinant strain was subjected to hydrogen culture.

[0033] (2) Supply of low-concentration hydrogen through gas-phase circulation culture and water electrolysis C. necator MF01ΔB1 / pBPP-ccrMeJAc-emd was cultured in a nitrogen source-limited inorganic salt medium (0.9% disodium hydrogen phosphate dodecahydrate, 0.15% potassium dihydrogen phosphate, 0.05% ammonium chloride, 0.02% magnesium sulfate heptahydrate, 1% trace metal solution, 100 mg / L kanamycin sulfate) (culture volume 1 L) without carbon source using a 2 L jar fermenter. A mixture of carbon dioxide and air supplied by a mass flow controller (Fcon C2000) and hydrogen generated from a PEM type reactor was pumped into the culture solution at 600 mL / min with a pump, and piping was provided to circulate the exhaust gas (Figure 2A). Multilayer plastic tubes containing a gas barrier layer were used for the piping, and O2 / CO2 and H2 detectors were placed in the gas phase flow path to measure the gas composition online. The culture temperature was 30°C, and the mixture was stirred at 400 rpm / min with a stirring blade. In order to prevent excessive pressure rise when the amount of gas supplied is greater than the amount consumed by the microorganisms, a pressure adjustment bag was placed branching off from the circulation flow path. The PEM reactor was composed of a Pt-Ti mesh (cathode) and a Mn-IrOx membrane catalyst (anode) developed by the inventors (Figure 2B). Hydrogen was generated under constant current conditions by applying approximately 2 V from a power supply device and introduced into the circulation flow path. The supply flow rates of carbon dioxide and air, and the hydrogen generation flow rate (current) were appropriately adjusted to maintain the composition of the circulation gas at approximately hydrogen:oxygen:carbon dioxide:nitrogen = 3:10:10:77.

[0034] (3) Results By supplying low-concentration hydrogen from the PEM reactor, the bacteria grew well using carbon dioxide as a carbon source, and the final bacterial concentration reached 7.5 as turbidity at a wavelength of 600 nm (OD600) (Figure 3A). In the early stages of cultivation, the carbon dioxide and oxygen concentrations in the circulating gas phase decreased as the bacteria grew. After 48 hours of cultivation, the nitrogen source (NH4Cl) was consumed, and the bacteria stopped growing due to nitrogen source depletion, and the PHA biosynthesis phase began. In the PHA biosynthesis phase after 48 hours, the flow rates of each gas were set to 1.5 mL / min of oxygen (7.2 mL / min of air), 1 mL / min of carbon dioxide, and 3.1 mL / min of hydrogen (0.4 A, 1.9-2.0 V), and the oxygen concentration in the circulating gas phase was almost constant at 6.5-7.5%, 8.5%, and 1.9%, respectively, and the supply gas and the gas consumption by the bacteria were balanced. (Note that the above gas concentrations are based on detectors calibrated under atmospheric pressure and room temperature conditions, and are not accurate as absolute values ​​due to differences in pressure and temperature within a closed system.)

[0035] The PHA analysis was performed on the cells collected 72 hours and 168 hours after the start of the culture. After the culture was completed, the cells were collected by centrifugation, washed with 70% ethanol to remove the oily matter, and then washed with distilled water. The cells obtained were freeze-dried and the dry cell weight was measured. Furthermore, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added to 10 to 30 mg of the dried cells, the container was sealed, and heated at 100°C for 140 minutes to obtain the methyl ester of the intracellular polyester decomposition product. 1 ml of distilled water was added to this and vigorously stirred. After leaving it to stand and separating into two layers, about 0.5 mL of the lower organic layer was taken out and analyzed by gas chromatography. The gas chromatograph used was a Shimadzu GC-2014, the detector was a Shimadzu FID-2014 flame ionization detector, and the capillary column was a GL Science InertCap-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). Helium was used as the mobile phase, and the temperature was raised at a rate of 8°C / min from an initial temperature of 100°C. The structure of related genes on the chromosome of the recombinant C. necator strain and the results of PHA production are shown in Figure 3B and Table 1 below. After 168 hours, the dry cell weight was 1.74 g / L, the PHA production was 0.92 g / L (PHA accumulation rate 52.5 wt%), and the accumulated PHA was P(3HB-co-3HHx) copolymer with a 3HHx fraction of 6.5 mol%. It was shown that this strain can be used to biosynthesize PHA copolymer from low-concentration hydrogen and carbon dioxide.

[0036] [Table 1]

[0037] (4) Calculation of yield The concentrations of hydrogen, oxygen, and carbon dioxide were measured by gas chromatography for the mixed gas blown into the culture solution and the exhaust gas from the culture tank during the 96-hour PHA biosynthesis period. The gas chromatograph was a Shimadzu GC-2014, the detector was a Shimadzu thermal conductivity detector TCD-2014, and the column was a Shinwa Kako Shincarbon ST (column length 4 m, column inner diameter 3 mm). The mobile phase was argon, and the temperature was raised from the initial temperature of 40°C at a rate of 20°C / min. The detector current was set to 50 mA, and 500 μL of sampled gas was injected and analyzed. The change in concentration before and after the culture tank was calculated from the measured values, and the amount of each gas consumed in PHA biosynthesis without growth of this recombinant strain was calculated. The following stoichiometric formula was obtained from the consumption of each gas and the results of PHA biosynthesis during the period from 72 to 168 hours. 21.72H2+7.59O2+4.15CO2→P(3HB-co-7.4mol%3HHx)+nH2O (1)

[0038] 0.74 g of P(3HB-co-7.4 mol% 3HHx) synthesized during 72 to 168 hours of the PHA biosynthesis phase was 8.41 mmol as monomer units. The constant current applied to the PEM reactor was 0.4 A. Based on this and equation (1), the yield per electricity (Electricity-to-PHA yield) was calculated as follows: Number of electrons and maximum hydrogen generation (2H + +2e - →H2) 0.4(A)×60(min)×96(h) / 96485(sA / mol)×1000=1432.76mmol-electron 1432.76 / 2=716.38mmol-H2 ·yield 8.41(mmol)×21.72 / 716.38=25.5%

[0039] If this electricity is obtained by solar cell panels and the energy conversion efficiency of silicon solar cells is assumed to be 20%, the highest level currently available, then the equivalent solar-to-PHA yield in this invention is 25.5 x 0.2 = 5.1%. The biomass conversion efficiency by photosynthesis in general agricultural crops is said to be 1-2%, and it can be said that efficient material conversion from electrical energy is possible in cultivation using this system. [Industrial Applicability]

[0040] This invention has demonstrated that low-concentration hydrogen can be efficiently taken up and used for substance production by closed gas-phase circulating culture of hydrogen bacteria, and that highly efficient substance conversion using electrical energy is possible by combining with water electrolysis in a PEM reactor. Further improvements in conversion efficiency are expected through improvements such as preventing hydrogen leakage by improving the piping material and supplying oxygen generated by water electrolysis as the oxygen needed as an electron acceptor. As mentioned above, research and development into hydrogen bacteria and their use as a method of turning carbon dioxide into a resource to reduce greenhouse gas emissions has been active worldwide, but there have been no reports on closed circulating culture combined with a PEM reactor, which is also being actively researched and developed as a water electrolysis device, and this is considered to be an important fundamental technology.

[0041] All publications and patents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, those skilled in the art will readily understand that various modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A closed gas-phase circulation culture device for culturing hydrogen bacteria, which maintains the hydrogen concentration below the lower explosion limit, a hydrogen supply unit for generating low-concentration hydrogen by water electrolysis; Carbon dioxide supply unit; air supply; A cell culture reactor containing a culture medium for culturing hydrogen bacteria; and Circulation channel for establishing gas phase circulation A closed gas-phase circulating culture device equipped with a

2. 2. The closed gas-phase circulating culture apparatus according to claim 1, wherein the hydrogen supply unit is a solid polymer water electrolysis reactor.

3. 3. The closed gas phase circulation culture apparatus according to claim 1, further comprising a pump for gas phase circulation.

4. 3. The closed gas-phase circulating culture apparatus according to claim 1, wherein the mixed gas in the circulation flow path is composed of hydrogen, carbon dioxide, and oxygen and nitrogen in the air being sent out, and the composition ratio of hydrogen, carbon dioxide, oxygen, and nitrogen is 1:3 to 5:3 to 5:20 to 30.

5. The hydrogen bacteria include those of the genus Cupriavidus, Ralstonia, Alcaligenes, Watersia, Pseudomonas, Bacillus, Paracoccus, Nocardia, and Hydrogenobacter. The closed gas-phase circulating culture apparatus according to claim 1 or 2, wherein the bacterial strain is a strain belonging to any one of the genus Bacillus subtilis, Bacillus spp., Bacillus spp., Bacillus vibrio, and Bacillus spp., or a recombinant strain obtained by imparting poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid)-producing ability to said strain.

6. 6. The closed gas-phase circulating culture apparatus according to claim 5, wherein the strain is a Cupriavidus necator strain or a recombinant Cupriavidus necator strain imparted with poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid)-producing ability.

7. 7. The closed gas-phase circulating culture apparatus according to claim 6, wherein the Cupriavidus necator strain is the H16 strain (DSM428).

8. The closed gas-phase circulating culture apparatus according to claim 6, wherein the recombinant Cupriavidus necator strain is a modified strain of the H16 strain, namely, MF01 strain, MF02 strain, MF03 strain, MF04 strain, NSDG strain, NSDGΔA strain, MF01ΔB1 strain, MF01ΔB1B3 strain, NSDG-GG strain, or NSDG-GGΔB1 strain.

9. A method for culturing hydrogen bacteria using the closed gas-phase circulation culture device defined in claim 1 or 2.

10. A method for producing poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid) from a recombinant strain imparted with poly(3-hydroxybutanoic acid-co-3-hydroxyhexanoic acid)-producing ability, using the culture method defined in claim 9.

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