Method and apparatus for culturing chemosynthetic autotrophic bacteria

Abstract

This invention provides a method and apparatus for culturing chemosynthetic autotrophic bacteria that enable safe operation while maintaining high culture efficiency. [Solution] The culture apparatus 1 comprises a plurality of culture regions 10, a first supply line 21 for supplying hydrogen to the culture medium 11a contained in the first culture region 10a, a first recovery line 22 for recovering gas released from the culture medium 11a contained in the first culture region 10a, a second supply line 31 for supplying oxygen to the culture medium 11b contained in the second culture region 10b, a second recovery line 32 for recovering gas released from the culture medium 11b contained in the second culture region 10b, and a connecting channel 60 for circulating the culture medium 11a contained in the first culture region 10a and the culture medium 11b contained in the second culture region 10b so as to mix.

Description

[Technical Field]

[0001] This invention relates to a method and apparatus for culturing chemosynthetic autotrophic bacteria. More specifically, it relates to a method and apparatus for culturing chemosynthetic autotrophic bacteria that can convert carbon dioxide into organic matter using hydrogen and oxygen. [Background technology]

[0002] In recent years, culturing chemosynthetic autotrophic bacteria, such as hydrogen-oxidizing bacteria, which have attracted attention from the perspective of CO2 fixation, requires the simultaneous supply of hydrogen and oxygen to the culture medium. However, the resulting gas mixture is explosive and requires careful handling. Until now, this explosiveness has not been a major issue in laboratory-level cultivation, but it becomes apparent in industrial cultivation. The following patent documents are known regarding this technology. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 03-127983 [Patent Document 2] Japanese Patent Application Publication No. 04-271778 [Overview of the project] [Problems that the invention aims to solve]

[0004] Patent Document 1 discloses a method for maintaining a gas mixture outside the explosion range by reducing the amount of oxygen supplied to the culture medium. In this regard, chemosynthetic autotrophic bacteria are inherently capable of extremely efficient growth and are therefore attracting attention as a means of CO2 fixation. However, when attempting to achieve highly efficient growth, it is expected that oxygen will become depleted as growth progresses. In the method of Patent Document 1, it is necessary to reduce the amount of oxygen supplied to the culture medium. This directly leads to a reduction in the culture rate, and consequently, a decrease in the CO2 fixation rate. Patent Document 2 discloses a method of using fluorocarbons with excellent oxygen solubility as a culture medium. However, there are concerns that fluorocarbons may inhibit or reduce the growth of chemosynthetic autotrophic bacteria, leading to a decrease in the CO2 fixation rate.

[0005] This invention has been made in view of the above circumstances, and aims to provide a method and apparatus for culturing chemosynthetic autotrophic bacteria that enable safe operation while maintaining high culture efficiency of chemosynthetic autotrophic bacteria. [Means for solving the problem]

[0006] This invention includes the following inventions. [1] A method for culturing chemosynthetic autotrophic bacteria using hydrogen and oxygen, A method for culturing chemosynthetic autotrophic bacteria, characterized by supplying hydrogen to a first culture medium and oxygen to a second culture medium, from a culture medium containing chemosynthetic autotrophic bacteria divided into multiple portions, separately recovering the gas released from the first culture medium and the gas released from the second culture medium, and circulating the culture mediums so that they are mixed with each other. [2] The method for culturing chemosynthetic autotrophic bacteria according to [1] above, wherein carbon dioxide is supplied to the second culture medium. [3] A method for culturing chemosynthetic autotrophic bacteria according to [1] or [2] above, wherein the proportion of oxygen in the gas released from the first culture medium is controlled to less than 5% by volume. [4] A method for culturing chemosynthetic autotrophic bacteria according to any one of [1] to [3] above, wherein the proportion of hydrogen in the gas released from the second culture medium is controlled to less than 4% by volume. [5] A method for culturing chemosynthetic autotrophic bacteria according to any one of [1] to [4] above, wherein the ratio of each component to the total amount of hydrogen, oxygen, and carbon dioxide supplied to the entire plurality of culture media is 50-90% by volume for hydrogen, 5-25% by volume for oxygen, and 5-25% by volume for carbon dioxide. [6] A method for culturing chemosynthetic autotrophic bacteria according to any one of [1] to [5] above, wherein hydrogen contained in the gas recovered from the first culture medium is refluxed as hydrogen supplied to the first culture medium. [7] A method for culturing chemosynthetic autotrophic bacteria according to any one of [1] to [6] above, wherein the oxygen contained in the gas recovered from the second culture medium is refluxed as oxygen supplied to the second culture medium. [8] A culture apparatus for chemosynthetic autotrophic bacteria using hydrogen and oxygen, Equipped with multiple culture regions, A first supply line for supplying hydrogen to the culture medium contained in the first culture region among the aforementioned multiple culture regions, A first recovery line for recovering gas released from the culture medium contained in the first culture region, A second supply line for supplying oxygen to the culture medium contained in the second culture region among the aforementioned multiple culture regions, A second recovery line for recovering gas released from the culture medium contained in the second culture region, A connecting channel for circulating the culture medium contained in the first culture region and the culture medium contained in the second culture region so as to mix them, A culture apparatus for chemosynthetic autotrophic bacteria, characterized by being equipped with [a specific feature / feature]. [9] The apparatus for culturing chemosynthetic autotrophic bacteria according to [8], further comprising a third supply line for supplying carbon dioxide to the culture medium contained in the second culture region.

[10] A culture apparatus for chemosynthetic autotrophic bacteria as described in [8] or [9] above, wherein the second supply line and the third supply line are common.

[11] The connecting channel is provided in the depth between the first culture region and the second culture region, and the apparatus for culturing chemosynthetic autotrophic bacteria according to any one of [8] to

[10] above.

[12] A culture apparatus for chemosynthetic autotrophic bacteria according to any one of [8] to

[11] above, comprising a culture medium transfer means for transferring the culture medium contained in the first culture area to the second culture area via the connecting channel. 〔13〕The culture apparatus for chemosynthetic autotrophic bacteria according to any one of the above [8] to

[12] , wherein the first culture region includes oxygen concentration measuring means for measuring the concentration of oxygen contained in the gas released from the culture solution contained in the first culture region. 〔14〕The culture apparatus for chemosynthetic autotrophic bacteria according to any one of the above [8] to

[13] , wherein the second culture region includes hydrogen concentration measuring means for measuring the concentration of hydrogen contained in the gas released from the culture solution contained in the second culture region. 〔15〕A method for culturing chemosynthetic autotrophic bacteria using the culture apparatus for chemosynthetic autotrophic bacteria according to [8] above, supplying hydrogen to the culture solution contained in the first culture region via the first supply line, collecting the gas released from the culture solution contained in the first culture region separately from the gas released from the culture solution contained in the second culture region via the first recovery line, supplying oxygen to the culture solution contained in the second culture region via the second supply line, collecting the gas released from the culture solution contained in the second culture region separately from the gas released from the culture solution contained in the first culture region via the second recovery line, A method for culturing chemosynthetic autotrophic bacteria, characterized by mixing the culture solution contained in the first culture region and the culture solution contained in the second culture region by flowing them through the connection channel. 〔16〕 The method for culturing chemosynthetic autotrophic bacteria according to

[15] above, wherein the ratio of each component to the total of hydrogen, oxygen and carbon dioxide supplied to the entire mixed culture solution is 50 to 90% by volume of hydrogen, 5 to 25% by volume of oxygen, and 5 to 25% by volume of carbon dioxide. 〔17〕The method for culturing chemosynthetic autotrophic bacteria according to

[15] or

[16] above, wherein hydrogen in the gas recovered from the first recovery line is refluxed to the first supply line. 〔18〕The method for culturing chemosynthetic autotrophic bacteria according to any one of

[15] to

[17] above, wherein oxygen in the gas recovered from the second recovery line is refluxed to the second supply line.

Advantages of the Invention

[0007] According to the method for culturing chemosynthetic autotrophic bacteria of the present invention, safe operation is enabled while maintaining a high culture efficiency of chemosynthetic autotrophic bacteria. According to the culture apparatus for chemosynthetic autotrophic bacteria of the present invention, safe operation is enabled while maintaining a high culture efficiency of chemosynthetic autotrophic bacteria.

Brief Description of the Drawings

[0008] [Figure 1] It is an explanatory diagram for explaining an example (1A) of the culture apparatus. [Figure 2] It is an explanatory diagram for explaining another example (1B) of the culture apparatus. [Figure 3] It is an explanatory diagram for explaining another example (1C) of the culture apparatus. [Figure 4] It is an explanatory diagram for explaining another example (1D) of the culture apparatus. [Figure 5] It is an explanatory diagram for explaining another example (1E) of the culture apparatus.

Modes for Carrying Out the Invention

[0009] Hereinafter, the present invention will be described with reference to the drawings. The matters shown here are exemplary and for exemplarily explaining the embodiments of the present invention, and are described for the purpose of providing an explanation that can most effectively and easily understand the principle and conceptual features of the present invention. In this regard, it is necessary for a fundamental understanding of the present invention and does not intend to show the structural details of the present invention more than a certain degree, and it is to clarify to those skilled in the art how some forms of the present invention are actually embodied by the explanation combined with the drawings.

[0010] [1] Method for Culturing Chemosynthetic Autotrophic Bacteria The present invention relates to a method for culturing chemosynthetic autotrophic bacteria using hydrogen and oxygen, characterized in that, of a culture medium containing chemosynthetic autotrophic bacteria divided into multiple portions, hydrogen is supplied to a first culture medium and oxygen is supplied to a second culture medium, the gas released from the first culture medium and the gas released from the second culture medium are collected separately, and each culture medium is circulated so as to be mixed with the others.

[0011] Chemosynthetic autotrophic bacteria are bacteria that grow through chemosynthetic autotrophic metabolism, utilizing energy from the oxidation of inorganic substances to reduce carbon dioxide (CO2) and synthesize organic matter. Examples of such bacteria include hydrogen-oxidizing bacteria, methanogens, methane-oxidizing bacteria, nitrate bacteria, nitrite bacteria, acetic acid bacteria, sulfur-oxidizing bacteria, iron-oxidizing bacteria, CO-utilizing bacteria, and Anammox bacteria. These can be used individually or in combination of two or more species.

[0012] Among these, the chemosynthetic autotrophic bacteria used in this method are preferably those that can utilize the oxidation reaction of at least hydrogen (free hydrogen, an inorganic substance) as an energy source and carbon dioxide as a carbon source. For this reason, hydrogen-oxidizing bacteria are preferred as the chemosynthetic autotrophic bacteria used in this method. That is, hydrogen-oxidizing bacteria are bacteria that reduce carbon dioxide and synthesize organic matter by utilizing the energy from the oxidation reaction of free hydrogen. Since hydrogen-oxidizing bacteria have a fast growth rate among chemosynthetic autotrophic bacteria, selecting hydrogen-oxidizing bacteria allows for a high bacterial concentration in a short time. In addition, unlike photosynthetic bacteria, hydrogen-oxidizing bacteria do not require light supply, so selecting hydrogen-oxidizing bacteria allows for cultivation in a more space-saving manner. Furthermore, the organic substances synthesized by chemosynthetic autotrophic bacteria are not limited and include carbohydrates, amino acids, and proteins. Among these, carbohydrates include, for example, various organic acids such as hydroxybutyric acid (hydroxybutanoic acid) and hydroxyvaleric acid (hydroxypentanoic acid), and polymers thereof (polyhydroxyalkanoic acid, etc.). These may be used individually or in combination of two or more.

[0013] Furthermore, from the viewpoint of CO2 fixation efficiency, bacteria with high efficiency in converting carbon dioxide into organic matter are preferable. For example, bacteria that use carbon dioxide as their sole carbon source (i.e., bacteria that synthesize all organic matter in their bodies from carbon dioxide) can be used. Furthermore, the chemosynthetic autotrophic bacteria used in this method can be any bacteria capable of using carbon dioxide as a carbon source, as described above. For example, it is not limited to bacteria capable of using sugars and / or lipids as carbon sources in addition to carbon dioxide.

[0014] The species of このようなchemically synthesized independent phytotrophic bacteria (special に, hydroacidifying bacteria) are limited to されるものではないが, example えば, Alcaligenes genus (A. hydrogenophilus, A.ruhlandii, A.latus, A.paradoxus, etc.), Aquaspirillum Genus (A.autotrophicum, etc.), Arthrobacter (A.globiformis, etc.), Azospirillum (A.lipoferum, etc.), Calderobacterium (C.hydrogenophilum, etc.), Cupriavid *C. us* (C. necator, C. oxalaticus, C. pauculus, C. respiraculi, C. taiwanensis, C. metallidurans, etc.), *Derxia* (D. gummosa, etc.), *Flavobacterium* (F. autothermophilum, etc.), *Hydrogenibacillus* (H. schlegelii, etc.), *Hydrogenobacter* (H. thermophilus, etc.), *Hydrogenophaga* (H. aquatica, H. atypica, H. bi... sanensis,H.caeni,H.crassostreae,H.defluvii,H.flava,H.laconesensis,H.luteola,H.intermedia,H.palleronii,H.pseudoflava,H.soli,H. taeniospiralis, etc.), Hydrogenophilus (H.thermoluteolus), Hydrogenovibrio (H.marinus, etc.), Kyrpidia (K.tusciae, etc.), Microcyclus (M.aquatic us, M.ebruneus, etc.), Mycobacterium (M.gordonae, M.llatzerense, etc.), Nocardia (N.autotrophica, N.opaca, etc.), Paracoccus (P.denitrificans, P.pa ntotrophus, etc.), Pseudomonas (P.facilis, P.flava, P.pseudoflava, P.hydrogenovora, P.hydrogenothermophila, P.palleronii, P.thermophila, P.Examples include the genera *Saccharophila*, *Renobacter* (e.g., *R. vacuolatum*), *Rhizobium* (e.g., *R. japonicuni*), *Rhodobacter* (e.g., *R. sphaeroides*, *R. capsulatus*), *Rhodococcus* (e.g., *R. opacus*, *R. erythropolis*), *Rhodopseudomonas* (e.g., *R. sphaeroides*, *R. palustris*), *Rhodospirillum* (e.g., *R. rubrum*), *Ralstonia* (e.g., *R. eutropha*), *Wautersia* (e.g., *W. eutropha*), and *Xanthobacter* (e.g., *X. autotrophicus*, *X. flavus*). These can be used individually or in combination of two or more species.

[0015] The origin of these bacteria is not limited, and those isolated from various environments (aquatic environments such as soil, ponds, rivers, lakes, swamps, and the sea) can be used. Furthermore, the chemosynthetic autotrophic bacteria (especially hydrogen-oxidizing bacteria) mentioned above may not only be strains that inherently possess organic matter synthesis genes (organic matter synthesis enzyme genes), but also recombinant strains (transformed organisms) into which acquired genes encoding organic matter (organic matter synthesis enzymes derived from other bacteria, etc.) have been introduced. When using recombinant strains, only one type may be used, or two or more types may be used in combination. In addition, either non-recombinant strains or recombinant strains may be used alone, or in combination. Among the chemosynthetic autotrophic bacteria (especially hydrogen-oxidizing bacteria) used in this method, the following species can be suitably used: Cupriavidus necator, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus respiraculi, Cupriavidus taiwanensis, and Cupriavidus metallidurans. Of these, Cupriavidus necator (e.g., strain H16) is particularly suitable.

[0016] This method has the following configuration: (1) Divide the culture medium into multiple portions and place them in the containers. (2) Of the divided culture media, hydrogen is supplied to the first culture media. (3) Supply oxygen to a second culture medium, which is different from the first culture medium. (4) Separately collect the gas released from the first culture medium (i.e., containing hydrogen) and the gas released from the second culture medium (i.e., containing oxygen). (5) Each culture medium is circulated so that it is mixed with the others.

[0017] A culture medium is a culture medium used for culturing chemosynthetic autotrophic bacteria. Culture mediums typically contain water as their primary liquid component. Using a liquid culture medium (i.e., a culture solution) instead of a solid medium facilitates the management of culture conditions (controlling substrate supply, measuring substrate mass, controlling pH, etc.) and improves culture efficiency. Furthermore, it facilitates the separation of the culture (chemosynthetic autotrophic bacteria) from the culture medium after cultivation.

[0018] As described below, hydrogen, oxygen, and carbon dioxide are supplied to the culture medium as substrates, but other substrates (inorganic nutrients) may also be included. Examples of other substrates include inorganic salts. The types of inorganic salts are not limited, but examples include nitrogen sources (ammonia, ammonium salts, nitrates, etc.), phosphorus sources (phosphates, etc.), and other necessary bioelements (alkaline earth metal salts such as MgSO4 and CaCl2, transition metal salts such as FeCl3 and NiCl2, etc.). These may be used individually or in combination of two or more. Furthermore, the culture medium may contain buffering components (Na2HPO4, KH2PO4, NaHCO3, etc.) to buffer the pH fluctuation (pH decrease) associated with the dissolved carbon dioxide. These components other than hydrogen, oxygen, and carbon dioxide may be supplied to the culture medium as needed, may be contained in the culture medium from the beginning, or may be used in combination.

[0019] Furthermore, in this invention, the culture medium is divided and contained in multiple portions. That is, the culture medium is divided and contained in two or more culture regions (see Figures 1 to 5). The number of divisions is not limited, and it is sufficient to have two or more (usually 1000 or less), for example, three or more, four or more, five or more, ten or more, twenty or more, etc. Also, the amount of culture medium contained in each division is not limited, and may be the same or different. More specifically, for example, it may be 10 L or more (usually 10,000 L or less), further 50 L or more, further 100 L or more, and further 500 L or more. Furthermore, when distinguishing between culture media contained in different culture regions, this specification will refer to them as the first culture medium, the second culture medium, etc. However, since these are distributed in a manner that allows them to be mixed with each other, there is no distinction between them as culture media. Therefore, although the types and concentrations of various components in each culture medium may differ, culture media that have been sufficiently distributed can usually be substantially the same.

[0020] By dividing the culture medium into separate containers, hydrogen and oxygen can be recovered individually. In other words, by dividing the culture medium into separate containers, hydrogen and oxygen can be supplied individually to different culture mediums. This prevents hydrogen and oxygen from coexisting during supply. Furthermore, by supplying hydrogen and oxygen individually to different culture mediums, hydrogen or oxygen that is not dissolved in each culture medium and is released from the culture medium can be recovered individually in each culture area without mixing them. Therefore, from gas supply to gas recovery, the coexistence of hydrogen and oxygen is prevented, and the reaction between hydrogen and oxygen is prevented, allowing for the safe cultivation of chemosynthetic autotrophic bacteria.

[0021] The culture medium can be divided in any way. For example, the culture medium can be divided by separating a single container into multiple culture regions using partitions (see Figures 1 and 3). Alternatively, the culture medium can be divided by placing the culture medium in separate, independent culture regions (see Figures 2, 4, and 5).

[0022] Furthermore, the culture media contained in each culture region are circulated so that they are mixed with each other. In other words, the culture media, which are separated from each other, can be mixed through circulation. By ensuring the flow between culture media, it is possible to mix a culture medium supplied with hydrogen with a culture medium supplied with oxygen. As a result, it becomes possible to dissolve both hydrogen and oxygen in the culture medium.

[0023] The culture medium can be circulated in any way, but for example, between culture regions separated by a partition wall, the culture medium can be circulated through an opening in the partition wall (see Figures 1 and 3). There may be only one opening, or there may be two or more. It is possible to circulate through only one opening, but from the viewpoint of more efficient circulation, two or more openings (for example, three or more, four or more, five or more, ten or more, twenty or more, etc., usually 1000 or less) may be provided. When there are two or more openings, the first opening can be used as a path for moving the culture medium from region A to region B, and the second opening can be used as a path for moving the culture medium from region B to region A (see Figures 1 and 3).

[0024] Furthermore, the flow of culture medium between culture regions separated by a partition wall may occur naturally (for example, by leaving the opening open), but it can also be controlled using a pump or the like. When using a pump, it can be installed in any way, but for example, as illustrated in Figures 1 and 3, the pump can be installed to block the opening, allowing the flow of culture medium only when the pump is running.

[0025] On the other hand, between separate culture regions, the culture medium can be circulated through channels connecting each culture region (see Figures 2, 4, and 5). For example, it is possible to do so through only one channel, but from the viewpoint of more efficient circulation, two or more channels (for example, three or more, four or more, five or more, ten or more, twenty or more, etc., usually 1000 or less) can be provided. When two or more channels are provided, the first channel can be used as a path to move the culture medium from region A to region B, and the second channel can be used as a path to move the culture medium from region B to region A.

[0026] Furthermore, the flow of culture medium between separate culture regions may occur naturally (for example, by leaving the flow path open), but it can also be controlled using a pump or the like. When using a pump, it can be installed in any way, but for example, as illustrated in Figures 2, 4, and 5, the pump can be installed within the flow path to block it, allowing the flow of culture medium only when the pump is running.

[0027] The flow of culture medium (whether between culture regions separated by partitions or between separate individual culture regions) can be performed to any extent without limitation. For example, if the total amount of culture medium (the total amount of culture medium contained in each culture region) is 10 to 100 L, the flow rate per minute (the volume of culture medium flowing between culture regions) can be set to 70 to 100% of that amount. Furthermore, the flow rate per minute can be set to 90 to 100%.

[0028] The hydrogen supplied to the first culture medium is a gas (free hydrogen), and hydrogen is a substrate for the chemosynthetic autotrophic bacteria being cultured. Hydrogen is a component that is oxidized, and it is a component that provides the energy necessary for carbon dioxide reduction during organic matter synthesis through its oxidation. The concentration of the supplied hydrogen is not limited; for example, a gas with a hydrogen partial pressure of 100% may be supplied, or a gas with a hydrogen partial pressure of less than 100% may be supplied. When using a gas with a hydrogen partial pressure of less than 100%, from the viewpoint of supply efficiency, it is preferable that the gas has a hydrogen partial pressure of 50% or more, and further, it can be 60% or more, further, 70% or more, and further, 80% or more. In addition, the hydrogen may be supplied discontinuously (i.e., in a batch-type supply as needed) or continuously. When gas is supplied continuously, it becomes a continuous aeration culture.

[0029] The source of hydrogen is not limited; manufactured hydrogen may be used, or hydrogen produced as a by-product in various facilities and equipment may be used. Among the above, examples of hydrogen produced include hydrogen produced by steam reforming of fossil fuels (natural gas, petroleum, etc.), hydrogen produced by partial oxidation reforming of fossil fuels (natural gas, petroleum, etc.), hydrogen produced by reforming alcohols (methanol, ethanol, etc.), and hydrogen produced by electrolysis of water. These may be used individually or in combination of two or more. Furthermore, examples of by-product hydrogen include hydrogen produced during the manufacturing process of caustic soda and hydrogen produced during the manufacturing process of chlorine. These may be used individually or in combination of two or more. In addition, if by-product hydrogen is contained in the mixed gas, the mixed gas may be used as is, or it may be used as a mixed gas with a higher hydrogen concentration after passing through a filter, concentrator, separator, etc., or it may be used as purified hydrogen after passing through a filter, concentrator, separator, etc.

[0030] Means and methods for separating or concentrating hydrogen include gas separation membranes, cryogenic separation, and pressure swing adsorption (PSA). These may be used individually or in combination of two or more. Among these, gas separation membranes include polyimide membranes, polyamide membranes, and palladium alloy membranes. These may be used individually or in combination of two or more.

[0031] Of the above, the permeation rates of various gases such as hydrogen, oxygen, carbon dioxide, and nitrogen differ for gas separation membranes (separators utilizing gas separation membranes). For example, when using a polyimide membrane as a gas separation membrane, hydrogen has the highest permeation rate, followed by carbon dioxide, then oxygen, and among these four gases, nitrogen has the lowest permeation rate. In other words, differences in permeation rates can be created for each gas by using a gas separation membrane. By utilizing these differences in permeation rates for gas separation membranes, it is possible to easily prepare gases that are substantially hydrogen only, mixed gases with a high concentration of hydrogen, gases that are substantially oxygen only, mixed gases with a high concentration of oxygen, gases that are substantially carbon dioxide only, mixed gases with a high concentration of carbon dioxide, gases that are substantially oxygen and carbon dioxide only, and mixed gases with high concentrations of oxygen and carbon dioxide.

[0032] Furthermore, when supplying hydrogen, if there are other gases to be supplied to the culture medium besides hydrogen and oxygen, they can be supplied together. In this invention, hydrogen can be supplied together with, for example, carbon dioxide. On the other hand, in the gas separation using the polyimide membrane described above, the separation of hydrogen and carbon dioxide tends to be less precise than the separation of hydrogen and oxygen. Therefore, from the perspective of using gas separation with a polyimide membrane, it is preferable to avoid coexistence with amounts of carbon dioxide that may hinder the reuse of hydrogen.

[0033] While hydrogen can be supplied to the culture medium in any way, from the viewpoint of efficiently dissolving hydrogen into the culture medium, it is preferable to supply gas into the culture medium. That is, it is preferable to supply gas into the culture medium within the area (culture tank) in which the culture medium is contained. Furthermore, from the viewpoint of more efficient dissolution of hydrogen into the culture medium, and from the viewpoint of more efficiently obtaining the stirring effect brought about by excess gas (gas that is not dissolved from the gas supplied to the culture medium) in the culture medium, it is preferable to supply gas to a deeper location in the culture area. That is, for example, as shown in Figures 1 to 5, it is preferable to discharge gas from the opening of a gas supply pipe inserted to near the bottom of the tank in the culture area.

[0034] Furthermore, in the culture method of the present invention, hydrogen contained in the gas recovered from the first culture medium can be refluxed as hydrogen supplied to the first culture medium. That is, the recovered gas can be reused as the supply gas. Since the amount of soluble hydrogen is small relative to the amount of culture medium, it is difficult to supply only the soluble amount to the culture medium. For this reason, hydrogen is usually supplied in an amount exceeding the soluble amount, resulting in a surplus of hydrogen (which replenishes the substrate consumed in the culture medium and substantially keeps the dissolved hydrogen saturating at all times). Consequently, when such gas is supplied, hydrogen that is not dissolved in the culture medium is released from the surface of the culture medium. On the other hand, the culture method of the present invention allows for the supply of only hydrogen to the first culture medium (Figures 1 to 6). When only hydrogen is supplied to the first culture medium, hydrogen will account for the majority of the gas recovered from the first culture medium. Therefore, the gas recovered from the first culture medium can be reused as hydrogen for supplying to the first culture medium. This makes it possible to reduce the amount of hydrogen required for operation and provides excellent cost benefits.

[0035] Furthermore, in the culture method of the present invention, hydrogen and other gases can also be supplied to the first culture medium. When hydrogen and other gases are supplied to the first culture medium, the gas recovered from the first culture medium will also contain the other gases. However, as described above, by separating or concentrating hydrogen from the recovered gas, a gas with a high hydrogen concentration can be obtained. This gas (containing hydrogen) can be reused as hydrogen to be supplied to the first culture medium. In this case as well, the amount of hydrogen required for operation can be reduced, and excellent cost benefits can be obtained.

[0036] The oxygen supplied to the second culture medium is gaseous (free oxygen), and oxygen is a substrate for the chemosynthetic autotrophic bacteria being cultured. Oxygen is an electron acceptor and typically constitutes the reaction [H2 + 1 / 2O2 → H2O]. While this method supplies at least oxygen as an electron acceptor, it does not prevent the supply of other electron acceptors besides oxygen. Other components that can function as electron acceptors include carbon dioxide and nitrate ions (NO2). 3- Examples include the following. These may be used individually or in combination of two or more.

[0037] The concentration of oxygen supplied is not limited; for example, gas with an oxygen partial pressure of 100% may be supplied, or gas with an oxygen partial pressure of less than 100% may be supplied. When using gas with an oxygen partial pressure of less than 100%, from the viewpoint of supply efficiency, it is preferable that the gas has an oxygen partial pressure of 50% or more, and furthermore, it can be 60% or more, furthermore 70% or more, and furthermore 80% or more. In addition, oxygen may be supplied discontinuously (i.e., in batches as needed) or continuously. When gas is supplied continuously, it becomes continuous aeration culture.

[0038] The source of oxygen is not limited; manufactured oxygen may be used, oxygen produced as a by-product in various facilities and equipment may be used, or air (atmosphere) may be used. Among the above, examples of manufactured oxygen include oxygen separated from air (such as high-oxygen-concentration gas obtained by reducing the nitrogen concentration in the air through physical adsorption) and oxygen produced by the electrolysis of water. These may be used individually or in combination of two or more. Other examples of by-product oxygen include combustion exhaust gas (combustion exhaust gas containing excess oxygen). These can be used individually or in combination of two or more. Since combustion exhaust gas contains carbon dioxide along with oxygen, these two can be used without separation. Furthermore, combustion exhaust gas containing excess oxygen can be used as is, or it can be used as a mixed gas with increased oxygen concentration after being filtered, concentrated, or separated.

[0039] Means and methods for separating or concentrating oxygen include gas separation membranes, zeolite adsorption methods, and cryogenic separation methods. These may be used individually or in combination of two or more. Among these, gas separation membranes include polyimide membranes, polyamide membranes, and ceramic membranes. These may be used individually or in combination of two or more. Furthermore, similar to the supply of hydrogen, oxygen may be supplied together with oxygen and / or carbon dioxide, but it is preferable to supply hydrogen separately from oxygen. Furthermore, since carbon monoxide (CO) can inhibit the growth of hydrogen bacteria, if CO is present in the combustion exhaust gas, it is preferable to remove the carbon monoxide or convert it to carbon dioxide by oxidation.

[0040] While oxygen can be supplied to the culture medium in any way, from the viewpoint of efficiently dissolving oxygen into the culture medium, it is preferable to supply gas into the culture medium. That is, it is preferable to supply gas into the culture medium within the area (culture tank) in which the culture medium is contained. Furthermore, from the viewpoint of more efficient dissolution of oxygen into the culture medium, and from the viewpoint of more efficiently obtaining the stirring effect brought about by excess gas (gas that is not dissolved from the gas supplied to the culture medium) in the culture medium, it is preferable to supply gas to a deeper location in the culture area. That is, for example, as shown in Figures 1 to 5, it is preferable to discharge gas from the opening of a gas supply pipe inserted to near the bottom of the tank in the culture area. When supplying oxygen, if there are other gases to be supplied to the culture medium besides oxygen and hydrogen, they can be supplied together. In this invention, oxygen can be supplied together with, for example, carbon dioxide.

[0041] Furthermore, in the culture method of the present invention, the oxygen contained in the gas recovered from the second culture medium can be refluxed as oxygen supplied to the second culture medium. That is, the recovered gas can be reused as the supply gas. Since the amount of soluble oxygen is small relative to the amount of culture medium, it is difficult to supply only the soluble amount to the culture medium. For this reason, oxygen is usually supplied in an amount exceeding the soluble amount, resulting in excess oxygen (which replenishes the substrate consumed in the culture medium and substantially keeps the dissolved oxygen saturating at all times). Consequently, when such gas is supplied, the oxygen that is not dissolved in the culture medium is released from the surface of the culture medium. On the other hand, the culture method of the present invention can supply only oxygen to the second culture medium. When only oxygen is supplied to the second culture medium (Figures 5 and 6), oxygen will account for the majority of the gas recovered from the second culture medium. Therefore, the gas recovered from the second culture medium can be reused as oxygen to be supplied to the second culture medium. This makes it possible to reduce the amount of oxygen required for operation and obtain excellent cost benefits.

[0042] Furthermore, in the culture method of the present invention, oxygen and other gases can also be supplied to the second culture medium (Figures 1 to 4). When oxygen and other gases are supplied to the first culture medium, the gas recovered from the second culture medium will also contain other gases. However, as described above, by separating or concentrating oxygen from the recovered gas, a gas with a high oxygen concentration can be obtained. This gas (containing oxygen) can be reused as oxygen to be supplied to the second culture medium. In this case as well, the amount of oxygen required for operation can be reduced, resulting in excellent cost benefits.

[0043] In the culture method of the present invention, carbon dioxide is supplied as a substrate in addition to the hydrogen and oxygen mentioned above. Carbon dioxide is a carbon source for chemosynthetic autotrophic bacteria. As described above, carbon dioxide may be supplied to the first culture medium, to the second culture medium, or to a third culture medium different from the first and second culture mediums (Figures 5 and 6). When carbon dioxide is supplied to the third culture medium, it can be supplied separately from hydrogen and oxygen, so that these three gases can be recovered without mixing them. Therefore, each recovered gas can be reused and circulated as a supply gas in each region (tank).

[0044] The concentration of carbon dioxide supplied is not limited; for example, gas with a partial pressure of 100% carbon dioxide may be supplied, or gas with a partial pressure of less than 100% may be supplied. When using gas with a partial pressure of less than 100% carbon dioxide, from the viewpoint of supply efficiency, it is preferable that the gas has a partial pressure of 50% or more, and further, it can be 60% or more, further, 70% or more, and further, 80% or more. In addition, carbon dioxide may be supplied discontinuously (i.e., in batches as needed) or continuously. When gas is supplied continuously, it becomes continuous aeration culture.

[0045] The source of carbon dioxide is not limited; manufactured carbon dioxide may be used, carbon dioxide produced as a by-product in various facilities and equipment may be used, or air (atmosphere) may be used. Typically, carbon dioxide can be supplied from either by-product carbon dioxide or the atmosphere. Examples of by-products of carbon dioxide include combustion exhaust gas, exhaust gas from steelmaking, and exhaust gas from petrochemical plants. These can be used individually or in combination of two or more. Since these exhaust gases contain oxygen along with carbon dioxide, they can be used without separating them. Furthermore, exhaust gas containing carbon dioxide can be used as is, or it can be used as a mixed gas with a higher carbon dioxide concentration after being filtered, concentrated, or separated.

[0046] Methods and techniques for separating or concentrating carbon dioxide include gas separation membranes, cryogenic separation, pressure swing adsorption (PSA), and chemical absorption using alkaline solutions. These may be used individually or in combination of two or more. Examples of gas separation membranes include polyimide membranes, polyamide membranes, and ceramic membranes. These may be used individually or in combination of two or more.

[0047] In the culture method of the present invention, the carbon dioxide contained in the recovered gas can be refluxed as carbon dioxide supplied to the culture medium. That is, the recovered gas can be reused as the supply gas. Since the amount of soluble carbon dioxide is small relative to the amount of culture medium, it is difficult to supply only the soluble amount to the culture medium. For this reason, the amount of carbon dioxide supplied is usually greater than the soluble amount, resulting in excess carbon dioxide. On the other hand, the culture method of the present invention can supply only carbon dioxide to the third culture medium. When only carbon dioxide is supplied to the third culture medium (Figures 5 and 6), carbon dioxide will account for the majority of the gas recovered from the third culture medium. Therefore, the gas recovered from the third culture medium can be reused as carbon dioxide supplied to the third culture medium. This makes it possible to suppress the amount of carbon dioxide required for operation and obtain excellent cost benefits. That is, the equipment and man-hours required for the separation and concentration of carbon dioxide can be reduced.

[0048] Furthermore, in the culture method of the present invention, carbon dioxide and oxygen can also be supplied to the second culture medium (Figures 1 to 4). When carbon dioxide and oxygen are supplied to the second culture medium, the gas recovered from the second culture medium will contain oxygen and carbon dioxide. As described above, this gas (containing oxygen and carbon dioxide) can be reused as a gas to be supplied to the second culture medium. In this case as well, the amount of oxygen and carbon dioxide required for operation can be reduced, resulting in excellent cost benefits. That is, the equipment and man-hours required for the separation and concentration of oxygen and carbon dioxide can be reduced.

[0049] In the culture method of the present invention, the gas released from the first culture medium (i.e., containing hydrogen) and the gas released from the second culture medium (i.e., containing oxygen) are recovered separately. As mentioned above, hydrogen is supplied to the first culture medium, but oxygen is not. Therefore, the gas released from the first culture medium contains virtually no oxygen, or only a small amount. Consequently, the gas recovered from the first culture medium does not reach the explosive limits in the coexistence of hydrogen and oxygen, allowing for safe operation. In addition, the gas recovered from the first culture medium contains abundant hydrogen and can therefore be reused as a hydrogen source. That is, from the viewpoint of hydrogen reuse, it is preferable not to supply any gas other than hydrogen to the first culture medium (see Figures 1-5). Furthermore, when reusing the gas recovered from the first culture medium as a hydrogen source, the hydrogen concentration can be monitored by providing a hydrogen concentration measuring means, and hydrogen can be replenished as needed.

[0050] Similarly, since oxygen is supplied to the second culture medium but not hydrogen, the gas released from the second culture medium contains virtually no hydrogen, or only a small amount. Therefore, the gas recovered from the second culture medium does not reach the explosive limits in the coexistence of hydrogen and oxygen, and operations can be conducted safely. In addition, the gas recovered from the second culture medium contains abundant oxygen and can therefore be reused as an oxygen source. Furthermore, when reusing the recovered gas from the second culture medium as an oxygen source, the oxygen concentration can be monitored by providing an oxygen concentration measuring device, and oxygen can be replenished as needed.

[0051] Furthermore, for example, as shown in Figures 1 to 4, when oxygen and carbon dioxide are supplied to the second culture medium, the gas recovered from the second culture medium will contain oxygen and carbon dioxide. This gas can also be reused as an oxygen source and a carbon dioxide source. When reusing the gas, the concentration of each gas can be monitored by providing means for measuring the concentration of each gas (oxygen concentration meter, carbon dioxide concentration meter), and oxygen and / or carbon dioxide can be replenished as needed.

[0052] Furthermore, as shown in Figure 5, for example, if hydrogen is supplied to the first culture medium, oxygen to the second culture medium, and carbon dioxide to the third culture medium, mixing of substrate gases can be avoided. Therefore, the gas recovered from the first culture medium can be easily reused as a hydrogen source, the gas recovered from the second culture medium can be easily reused as an oxygen source, and the gas recovered from the second culture medium can be easily reused as a carbon dioxide source.

[0053] Each gas can be recovered in any way, but for example, a recovery space can be provided above each culture area, and the gas present in that space can be recovered. Alternatively, a duct can be provided above the culture area, and the gas released from the surface of the culture medium can be recovered through the duct. In this case, gas can be recovered without substantially providing a recovery space, or if a recovery space is provided, its volume can be reduced. That is, substrate (gas) that is not dissolved in the culture medium can be recovered without waste without being released outside the system.

[0054] Furthermore, through the circulation and mixing of the first and second culture media, the second culture media, which contains dissolved oxygen, is mixed with the first culture media. As oxygen is released from the second culture media, oxygen may be introduced into the first culture area containing the first culture media. However, the amount is small and does not usually reach the explosive limit due to the coexistence of hydrogen and oxygen. Similarly, through the circulation and mixing of the culture media, the first culture media, which contains dissolved hydrogen, is mixed with the second culture media. As hydrogen is released from the first culture media, hydrogen may be introduced into the second culture area containing the second culture media. However, the amount is small and does not usually reach the explosive limit due to the coexistence of hydrogen and oxygen. Therefore, according to the culture method of the present invention, chemosynthetic autotrophic bacteria can be cultured safely.

[0055] However, from the perspective of achieving a higher level of safety, the proportion of oxygen in the gas released from the first culture medium can be controlled to less than 5% by volume. This makes it more reliable to prevent the recovered gas from the first culture medium from reaching the explosive limit due to the coexistence of hydrogen and oxygen. This control is more effective when the recovered gas from the first culture medium is reused as a hydrogen source. Similarly, from the perspective of achieving a higher level of safety, the proportion of hydrogen in the gas released from the second culture medium can be controlled to less than 4% by volume. This makes it more reliable to prevent the recovered gas from the second culture medium from reaching the explosive limit due to the coexistence of hydrogen and oxygen. This control is more effective when the recovered gas from the second culture medium is reused as an oxygen source.

[0056] The recovered gas from each culture medium may contain hydrogen, oxygen, and carbon dioxide (and other gases may be present if other gases are supplied). When hydrogen and oxygen coexist, the resulting gas mixture may become explosive. Specifically, within a wide concentration range, the recovered gas (gas mixture) may become explosive if hydrogen accounts for 4 to 95 volume percent and oxygen for 5 volume percent or more, relative to a total of 100 volume percent of hydrogen and oxygen. Therefore, the explosiveness can be avoided by (A) controlling the concentration of each component gas in the recovered gas so that the proportion of oxygen in the recovered gas from the first culture medium is less than 5 volume percent, and / or (B) controlling the concentration of each component gas in the recovered gas so that the proportion of hydrogen in the recovered gas from the second culture medium is less than 4 volume percent.

[0057] The above (A) and (B) may be achieved in any way, for example, as follows (M A1 )~(M A3 ) any of the operations listed below, or any combination thereof, and the following (M B1 )~(M B3 This can be achieved by any of the operations listed above, or by a combination of these operations. (M A1): Hydrogen is mixed with the recovered gas of the first culture solution to achieve the above (A). (M A2 ): Part or all of the oxygen contained in the recovered gas of the first culture solution is removed to achieve the above (A). (M A3 ): Another gas is mixed with the recovered gas of the first culture solution to achieve the above (A). (M A3 ): Another gas is mixed with the recovered gas of the first culture solution to achieve the above (A). (M B1 ): Oxygen is mixed with the recovered gas of the second culture solution to achieve the above (B). (M B2 ): Part or all of the hydrogen contained in the recovered gas of the second culture solution is removed to achieve the above (B). (M B3 ): Another gas is mixed with the recovered gas of the second culture solution to achieve the above (B).

[0058] In the above operations (M A1 ) to (M A3 ), from the perspective of recycling the recovered gas, (M A1 ) and (M A2 ) are preferred, and (M A1 ) is more preferred. That is, the operation of the above (M A1 ) is to add hydrogen to the recovered gas. By mixing hydrogen, a mixed gas with an oxygen ratio of less than 5% by volume can be suitably recycled as a hydrogen source.

[0059] Also, the oxygen removal in the operation of the above (M [[ID=)43]] A2 ) can be carried out in any manner. For example, it can be carried out by removal using a gas separation membrane, a chemical adsorption method (amine-based, etc.), a physical adsorption method (activated carbon, zeolite, etc.), etc. These can be used alone or in combination of two or more. Among these, from the perspectives of energy efficiency and simplification of equipment, removal using a gas separation membrane can be suitably used.

[0060] When using a gas separation membrane, oxygen can be removed (the oxygen concentration reduced) by passing the recovered gas through the membrane. For example, when a polyimide membrane is used as a gas separation membrane, the permeation rate of oxygen through the polyimide membrane is lower than that of hydrogen and carbon dioxide. Therefore, the gas that permeates through the polyimide membrane (the gas on the permeation side of the polyimide membrane) results in a gas mixture with a low oxygen concentration (especially one where the oxygen content in the gas mixture is less than 5% by volume), i.e., a gas mixture with a high hydrogen concentration. This gas mixture can be reused as a hydrogen source. On the other hand, by recovering gases that have a low permeation rate through the polyimide membrane, and / or gases that do not permeate the polyimide membrane (gases on the non-permeable side of the polyimide membrane), a mixed gas with a high oxygen concentration can be obtained. This mixed gas can be reused as an oxygen source.

[0061] Furthermore, the above (M A3 When performing the operation described above, other gases that can be mixed with the recovered gas include noble gases (helium, neon, argon, krypton, xenon), nitrogen (free nitrogen), and carbon dioxide, from the viewpoint of being less reactive gases. These may be used individually or in combination of two or more. Among these, carbon dioxide is preferred from the viewpoint of maximizing the dissolved amounts of hydrogen and oxygen. That is, among the gases mentioned above, nitrogen and carbon dioxide can be used inexpensively, but of these, nitrogen, when dissolved in the culture medium, reduces the dissolved amounts of hydrogen and oxygen, which are substrates. For this reason, carbon dioxide is preferred from the viewpoint of maximizing the dissolved amount of substrates. Although carbon dioxide is more soluble in water than other gases and tends to lower the pH of the aqueous solution when dissolved, the pH of the culture medium generally changes to the acidic side as the culture progresses. For this reason, it is preferable to adjust the pH of the culture medium constantly or periodically, and the acidification caused by carbon dioxide can be corrected by such pH adjustment.

[0062] The above (M B1 )~(M B3In the operation described above (M), from the viewpoint of reusing the recovered gas, any of the operations can be suitably used, but from the viewpoint of cost, B1 Operation ) is preferable. That is, the above (M B1 The operation involves adding oxygen to the recovered gas. By mixing in oxygen, the hydrogen content of the resulting gas mixture is less than 4% by volume, making it suitable for reuse as an oxygen source.

[0063] Also, the above (M B2 Hydrogen removal in the operation of ) can be carried out in any way, but for example, it can be done by removal using a gas separation membrane, chemical adsorption (amine-based, etc.), physical adsorption (activated carbon, zeolite, etc.), etc. One of these may be used alone or two or more may be used in combination. Among these, removal using a gas separation membrane is preferably used from the viewpoint of energy efficiency and equipment simplification.

[0064] When using a gas separation membrane, hydrogen can be removed (the oxygen concentration can be reduced) by passing the recovered gas through the membrane. For example, when a polyimide membrane is used as a gas separation membrane, the permeation rate of hydrogen through the polyimide membrane is greater than that of oxygen and carbon dioxide. Therefore, by recovering gases that have a slow permeation rate through the polyimide membrane, and / or gases that do not permeate the polyimide membrane (gases on the non-permeable side of the polyimide membrane), a gas mixture with a low hydrogen concentration, i.e., a gas mixture with a high oxygen concentration, can be obtained. This gas mixture can be reused as an oxygen source. On the other hand, by recovering gases with a high permeation rate through the polyimide membrane, a gas mixture with a low oxygen concentration can be obtained. This gas mixture can then be reused as a hydrogen source.

[0065] Furthermore, the above (M B3When performing the operation, air (atmosphere) can be used as another gas that can be mixed with the recovered gas. Air contains no hydrogen at all, or if it does, the amount of hydrogen is very small (e.g., less than 1 ppm by volume). Therefore, when air is mixed with the recovered gas, the hydrogen concentration of the resulting gas mixture will decrease. More specifically, by monitoring the hydrogen concentration in the recovered gas, the amount of air required to reduce the hydrogen content in the gas mixture to less than 4% by volume can be calculated, and by mixing in that amount of air, (M B3 (B) can be achieved by the operation of ).

[0066] Similarly, the above (M B3 When performing the operation described above, gases other than air can be used. From the viewpoint of being less reactive, other gases include noble gases (helium, neon, argon, krypton, xenon), nitrogen (free nitrogen), and carbon dioxide. These may be used individually or in combination of two or more. Among these, carbon dioxide is preferred from the viewpoint of maximizing the dissolved amounts of hydrogen and oxygen. That is, among the gases mentioned above, nitrogen and carbon dioxide can be used inexpensively, but when nitrogen is dissolved in the culture medium, the dissolved amounts of hydrogen and oxygen, which are substrates, decrease. For this reason, carbon dioxide is preferred from the viewpoint of maximizing the dissolved amount of substrates. As mentioned above, the pH associated with the mixing of carbon dioxide can be adjusted.

[0067] The culture method of the present invention enables safe operation while maintaining high culture efficiency of chemosynthetic autotrophic bacteria. Specifically, increasing the culture volume for efficient bacterial cultivation requires larger amounts of hydrogen and oxygen. This results in a larger volume of recovered gas, increasing the risk of explosion. However, this method allows for maintaining the recovered gas outside the explosive range. Furthermore, this maintenance can be achieved at low cost and by a simple method.

[0068] In the culture method of the present invention, the amount of gas (gasic substrate) supplied is not limited, but for example, the ratio of each component to the total amount of hydrogen, oxygen, and carbon dioxide supplied to the entire culture medium can be set so that hydrogen is 50-90% by volume, oxygen is 5-25% by volume, and carbon dioxide is 5-25% by volume. Within this range, the growth efficiency of chemosynthetic autotrophic bacteria can be further improved. This ratio can be further set to 60-88% by volume for hydrogen, 6-20% by volume for oxygen, and 6-20% by volume for carbon dioxide; 70-86% by volume for hydrogen, 7-18% by volume for oxygen, and 7-18% by volume for carbon dioxide; or 75-85% by volume for hydrogen, 8-15% by volume for oxygen, and 8-15% by volume for carbon dioxide. Within these preferred ranges, chemosynthetic autotrophic bacteria can be produced even more efficiently.

[0069] In the culture method of the present invention, in addition to the operations described above, other operations may be added to the extent that the effects of the present invention are achieved. These other operations may be performed individually or in combination of two or more. Furthermore, these other operations may be performed, for example, before gas supply, before gas recovery, or between each operation; the order of these operations is not limited. Other operations include pH adjustment, temperature adjustment, culture medium recovery, and culture medium replenishment. These operations may be performed individually or in combination of two or more.

[0070] Of the above, the pH adjustment operation is the operation of adjusting the pH of the culture medium to a pH suitable for culturing chemosynthetic autotrophic bacteria. Generally, the pH of the culture medium tends to become acidic during cultivation, so an operation to neutralize it using ammonia (ammonia water, etc.) can be performed. Of the above, the temperature adjustment operation is the operation of adjusting the temperature of the culture medium to a temperature suitable for culturing chemosynthetic autotrophic bacteria. Of the above, the culture medium recovery operation is the operation of removing chemosynthetic autotrophic bacteria that have grown through cultivation from the culture medium. In other words, since the culture efficiency tends to decrease as bacteria grow in the culture medium, it is preferable to perform an operation to reduce the bacterial density continuously or periodically from the viewpoint of maintaining high culture efficiency. Of the above, the culture medium replenishment operation is the operation of replenishing the culture medium. In addition to the substrate supplied as a gas, the culture medium may contain various substrates as described above. These components are consumed during cultivation and can be supplied as needed, but it is also possible to replenish with a new culture medium that already contains these components.

[0071] The culture method of this invention allows for the safe and efficient cultivation of chemosynthetic autotrophic bacteria while maintaining high cultivation efficiency. Specifically, organic matter (carbohydrates, amino acids, proteins, etc.) produced by these bacteria can be obtained safely and efficiently through the cultivation of chemosynthetic autotrophic bacteria. This method can be widely applied to various fields such as resin (plastic) manufacturing, fiber manufacturing, fuel manufacturing, reagent manufacturing, industrial raw material manufacturing, food ingredient manufacturing, and feed manufacturing (fish and shellfish feed, poultry feed, pig feed, dairy cow feed, beef cattle feed).

[0072] Examples of organic matter produced by bacteria include polyhydroxyalkanoates (PHA), polyhydroxybutyric acid (PHB), 4-hydroxybenzoic acid (PHBA), hydroxyisobutyric acid, hydroxybutyric acid, methylcitric acid, lactic acid, carboxylic acids, acetic acid, etc.), polysaccharides, amino acids, peptides, proteins (e.g., lysine, arginine, tryptophan, methionine, cyanophycin, organophosphorus hydrolases, etc.), fatty acids (e.g., linear saturated fatty acids, etc.), amines, esters (e.g., lactones, phosphate esters, etc.), nucleic acids, enzymes, vitamins, physiologically active substances, and bioplastics (polyesters, etc.). These can be used individually or in combination of two or more.

[0073] [2] Culture apparatus for chemosynthetic autotrophic bacteria The culture apparatus 1 of the present invention is Equipped with multiple culture regions 10, A first supply line 21 for supplying hydrogen to the culture medium 11a contained in the first culture region 10a among multiple culture regions 10, A first recovery line 22 for recovering gas released from the culture medium 11a contained in the first culture region 10a, A second supply line 31 for supplying oxygen to the culture medium 11b contained in the second culture region 10b among the multiple culture regions 10, A second recovery line 32 for recovering gas released from the culture medium 11b contained in the second culture region 10b, A connecting channel 60 for circulating the culture medium 11a contained in the first culture region 10a and the culture medium 11b contained in the second culture region 10b so as to mix them. It is characterized by being equipped with [the following features].

[0074] The culture apparatus 1 of the present invention is a specific culture apparatus that can be used to perform the culture method of chemosynthetic autotrophic bacteria described above. The terminology used in culture apparatus 1 and the terminology used in the culture method described above are basically the same, and the explanation in the culture method described above applies similarly to the explanation of this culture apparatus.

[0075] The culture medium 11 may contain chemosynthetic autotrophic bacteria and, as mentioned above, inorganic salts, etc. The culture medium 11 is usually contained in the culture area 10. The culture area 10 may be a batch system or a continuous system. It may also be a single-chamber system or a multi-chamber system. In the case of a multi-chamber system, the processes may be arranged to proceed in parallel or in series. Furthermore, in this culture apparatus and the culture method described above, the bacteria in the culture medium may be cultured by being directly contained in the culture medium (e.g., with the bacteria suspended in the liquid), or they may be cultured by being supported on a carrier that is in contact with the culture medium.

[0076] The culture region 10 comprises multiple regions. Each culture region can be divided in any way; for example, it can be divided by partitioning with a septum (see Figures 1 and 3), or by separating the tank itself (see Figures 2, 4, and 5). Only one type of division may be used, or two or more types may be used in combination.

[0077] Furthermore, the culture area 10 may be an open-type tank that is open to the external environment (atmosphere, etc.), or a closed-type tank that is not open to the external environment (atmosphere, etc.). Of these, a closed-type culture area is preferred. By using a closed-type culture area, it is possible to easily control the internal environment of the culture area. That is, it is possible to make it less susceptible to the influence of the external environment outside the culture area. In addition, it is possible to easily recover the substrate (hydrogen, oxygen, carbon dioxide, etc.), and by circulating it as needed, the substrate can be utilized efficiently.

[0078] The culture area 10 may be composed of any material, but for example, metal (stainless steel, etc.), glass, etc. can be used. One of these materials may be used alone, or two or more may be used in combination. Using these materials makes the culture area less susceptible to external environmental influences (heat, pressure, humidity, etc.) compared to using other materials. Furthermore, excellent corrosion resistance can be obtained.

[0079] Furthermore, the culture area 10 does not necessarily have an insulating structure, but it may have one. By having an insulating structure, heat exchange between the inside and outside of the culture area can be suppressed compared to the case without an insulating structure. As the insulating structure, vacuum insulation, insulating materials, etc., can be used. These may be used alone or in combination of two or more. Vacuum insulation can be achieved, for example, by making the culture area itself (the outer shell of the culture tank) a double-walled structure or more, reducing the amount of gas between each layer or reducing the pressure to a state where there is virtually no gas, and maintaining that state. Also, the shape of the culture area is not limited, but the upper (top) and lower (bottom) can be dome-shaped. This can improve pressure resistance. It can also improve the circulation performance of the gas (substrate). Furthermore, the intermediate part (body) located between the upper and lower parts can be cylindrical, for example.

[0080] The size of the culture area 10 is not limited, but as mentioned above, problems associated with the mixing of hydrogen and oxygen are less likely to become apparent in small-scale cultures, while these problems are more likely to become apparent in large-scale cultures. From this perspective, although the amount of culture medium 11 is not limited, for example, the effects of using this culture apparatus and the culture method described above can be more easily obtained in cultures of 10 L or more. This amount of culture medium can be further set to 1,000 L or more, 5,000 L or more, or 10,000 L or more. On the other hand, the upper limit of the amount of culture medium is not limited, but for example, it can be set to 1,000,000 L or less. These upper and lower limits can be any combination of each other, and the numerical range specified by combining these upper and lower limits is also disclosed in the present invention. That is, for example, it can be 100 L or more and 1,000,000 L or less.

[0081] The culture region 10 may be equipped with control means for controlling its internal environment (temperature, pressure, humidity, etc.). That is, for example, it may be equipped with temperature control means, pressure control means, humidity control means, etc. Among these, the temperature control means may include a heating mechanism (such as a heater jacket) and / or a cooling mechanism (such as a cooling mechanism using refrigerant circulation or a cooling mechanism using forced air). For example, by providing a heating mechanism, it is possible to prevent the inactivation of chemosynthetic autotrophic bacteria due to a decrease in temperature within the culture area. Furthermore, by providing a cooling mechanism, it is possible to prevent the inactivation of chemosynthetic autotrophic bacteria due to a rise in temperature within the culture area. Furthermore, a pressurizing mechanism (such as a pressurizing pump) can be provided as a pressure control means. By providing a pressurizing mechanism, the amount of gaseous substrates such as hydrogen, oxygen, and carbon dioxide dissolved in the culture medium can be increased. Therefore, the culture efficiency of chemosynthetic autotrophic bacteria can be improved by pressurizing. A depressurization mechanism such as an exhaust valve or sub-tank can also be provided. A sub-tank refers to a culture area from which a portion of the gas contained in the culture area can be moved as the pressure rises. This movement allows for adjustment of the pressure within the culture area, but since the gas contained in the sub-tank is not exhausted to the outside, the gas in the sub-tank can also be reused. Furthermore, a dryer can be provided as a means of humidity control. In particular, when gas separation is used, a dryer can be provided in front of the gas separation mechanism.

[0082] The culture apparatus 1 of the present invention is equipped with a gas supply line. The gas supply line is a means of supplying gas (hydrogen, oxygen, carbon dioxide, etc.) to the culture medium 11. That is, it is a means of supplying a gaseous substrate. The culture apparatus 1 includes, as a gas supply line, at least a first supply line 21 that supplies hydrogen to the first culture medium 11a contained in the first culture area 10a, and a second supply line 31 that supplies oxygen to the second culture medium 11b contained in the second culture area 10b. In addition, as necessary, a third supply line 33 that supplies carbon dioxide to the second culture medium 11b contained in the second culture area 10b, a fourth supply line 41 that supplies carbon dioxide to the third culture medium 11c contained in the third culture area 10c, and so on may be appropriately provided.

[0083] The configuration of the gas supply line (first supply line 21, second supply line 31, third supply line 33, etc.) is not limited, but can be composed of, for example, a supply pipe (flow pipe), flow control means (valve, etc.), storage tank, supply means, etc. One type of these may be used alone, or two or more types may be used in combination. Of the above, the supply pipe is a pipe through which gas can be circulated, and is used for supplying gas. Of the above, the flow rate control means is a means of controlling the amount of gas supplied to the culture area by being installed in the gas supply line. Valves and the like can be used as the flow rate control means. The flow rate control means can be used more efficiently by linking it with a concentration measuring means.

[0084] Of the above, the storage tank is a tank that stores the gas supplied to the culture area. Although it is possible to connect the gas source and the culture area directly via a gas supply line without using a storage tank, using a storage tank makes the gas supply more stable. In other words, it prevents the amount of gas to be supplied from falling below the required amount. This storage tank may be a separate tank for each gas, or it may be a tank in which, for example, oxygen and carbon dioxide coexist. On the other hand, if no storage tank is used, gas can be supplied by connecting the gas generation source and the culture area through a supply pipe.

[0085] Of the above, the supplying means is a means for supplying the gas to be supplied to the culture area, and can be, for example, a blower, a pump (compressor, gas pump), etc. This supplying means may be a separate supplying means for each gas, or it may be a supplying means common to, for example, oxygen and carbon dioxide. That is, for example, the gases can be supplied by a pump or blower. Alternatively, the gases can be supplied by liquefying and storing them, then gasifying them under reduced pressure.

[0086] The supply of gas via the gas supply line may be done directly or indirectly to the culture medium. One example of direct gas supply is bubbling the supply gas directly into the culture medium. On the other hand, one example of indirect gas supply is supplying gas to the water or other components of the culture medium (e.g., bubbling) to form a liquid in which the substrate is dissolved, and then mixing this liquid with the culture medium to supply gas to the culture medium. Another example is supplying gas to the gas phase space (a space within the culture area that is not filled with culture medium) when the culture region has a gas phase space, and dissolving the substrate into the culture medium from the phase boundary between the gas phase space and the culture medium. One of these methods may be used alone, or two or more may be used in combination.

[0087] The culture apparatus 1 of the present invention is equipped with a gas recovery line. The gas recovery line is a means for recovering gases (hydrogen, oxygen, carbon dioxide, etc.) released from the culture medium 11. In other words, it is a means for recovering excess gaseous substrate. The culture apparatus 1 includes, as a gas recovery line, at least a first recovery line 22 for recovering hydrogen-containing gas released from the first culture medium 11a contained in the first culture area 10a, and a second recovery line 32 for recovering oxygen-containing gas released from the second culture medium 11b contained in the second culture area 10b. In addition, as necessary, a third recovery line 34 for recovering carbon dioxide from the second culture medium 11b contained in the second culture area 10b, a fourth recovery line 42 for recovering carbon dioxide from the third culture medium 11c contained in the third culture area 10c, and so on may be appropriately provided.

[0088] The configuration of the gas recovery line (first recovery line 22, second circulation line 32, etc.) is not limited, but can be composed of, for example, a recovery pipe (flow pipe), flow control means (valve, etc.), recovery tank, supply means, etc. One type of these may be used alone, or two or more types may be used in combination. Of the above, the recovery pipe is a pipe through which gas can flow and is used for gas recovery. Of the above, the flow rate control means is a means of controlling the amount of gas recovered from the culture medium by being installed in the gas recovery line. Valves and the like can be used as the flow rate control means. The flow rate control means can be used more efficiently by linking it with the concentration measuring means.

[0089] Of the above, the recovery tank is a tank for recovering gases that are not dissolved in the culture medium. Specifically, if there is space above the culture area (for example, if there is an upper void within the culture area where no culture medium is stored), that space (above the culture area) can be used as a recovery tank. Also, if there is no space above the culture area (for example, if there is no upper void within the culture area where no culture medium is stored), a recovery tank can be provided separately from the culture area. This recovery tank may be a separate tank for each gas, or it may be a tank that contains both oxygen and carbon dioxide, for example. Alternatively, if no recovery tank is used, gas recovery can be performed by connecting the culture area and the recovery tank through a recovery pipe.

[0090] Of the above, the supplying means is a means for leading the gas to be recovered out of the culture area, and can be, for example, a blower, a pump (compressor, gas pump), etc. This supplying means may be a separate supplying means for each gas, or it may be a supplying means common to, for example, oxygen and carbon dioxide.

[0091] The culture apparatus 1 of the present invention is equipped with a connecting channel. The connecting channel is a means for circulating the culture media contained in each culture region in order to mix them together. The culture apparatus 1 includes a connecting channel 60, which is used as a connecting channel to allow at least the culture medium 11a contained in the first culture region 10a and the culture medium 11b contained in the second culture region 10b to flow so as to be mixed.

[0092] The configuration of the connecting channel (connecting channel 60, etc.) is not limited, but can be composed of, for example, an opening, a flow tube, a means for moving culture medium (pump, etc.), a flow rate control means (valve, etc.), etc. One of these may be used alone, or two or more may be used in combination. Of the above, the opening 131 can be used as a connecting channel 60 in a configuration in which the culture region 10 is divided by a partition wall 13, as illustrated in Figures 1 and 3. The opening 131 may be just one, or it may be two or more. Furthermore, if there are two or more openings 131, the flow direction can be changed using each opening.

[0093] Furthermore, while the installation location of the connecting channel 60 is not limited, it is preferable to install it in a location that makes it easier to suppress the entrainment of gas bubbles (hydrogen, oxygen, carbon dioxide, etc.) supplied to the culture medium 11. That is, for example, the connecting channel 60 can be provided in the depths of the culture region 10 (or the depths of the culture medium 11). Specifically, by providing an opening 131 in the depths between the first culture region 10a and the second culture region 10b, the connecting channels 61 and 62 can be provided in the depths. By providing the connecting channel 60 deep within the culture area 10, it is possible to suppress the entrainment of air bubbles in the culture medium 11 when moving the culture medium. As a result, it is possible to suppress the mixing of oxygen and hydrogen in a gaseous state. Specifically, for example, the connecting channel can be installed at a position deeper than the position of air bubbles released from the gas discharge section of the gas supply line. Typically, this position is lower than 1 / 2 of the height of the culture medium 11, can be 1 / 8 or less of the height of the culture medium 11, and can even be 1 / 12 or less of the height of the culture medium 11. Furthermore, when the opening 131 is used as a connecting channel, the opening 131 can be closed by a culture medium transport means (such as a pump), as illustrated in Figures 1 and 3. This allows for the flow of culture medium as needed.

[0094] The flow tube is a tube through which the culture medium can be circulated, and can essentially be the connecting channel 60 itself. Furthermore, as illustrated in Figures 2, 4, and 5, the flow tube can be blocked by a means of moving the culture medium (such as a pump), while allowing the culture medium to circulate as needed. Of the above, the culture medium transfer means is a means for moving the culture medium from a predetermined culture area to a different culture area, and for example, a pump (tubular pump, diaphragm pump, etc.) can be used. The culture medium transfer means may be installed individually for each flow pipe, or by installing it in a predetermined flow pipe, a flow of culture medium may be created, and the culture medium may be naturally distributed by that flow without installing the culture medium transfer means in other flow pipes. Furthermore, the flow rate control means is a means of controlling the flow rate of the culture medium. Valves and the like can be used as the flow rate control means. The flow rate control means can be used more efficiently by linking it with the flow rate measuring means.

[0095] The culture apparatus 1 of the present invention may include other configurations in addition to those described above. Other configurations include concentration measuring means. Examples of concentration measuring means include means for measuring the concentration of a predetermined component (hydrogen, oxygen, carbon dioxide, etc.) in a gas, and means for measuring the concentration of a predetermined component (hydrogen, oxygen, carbon dioxide, etc.) in a culture medium. These may be used individually or in combination of two or more. More specifically, the first culture region 10a may be equipped with an oxygen concentration measuring means for measuring the concentration of oxygen contained in the gas released from the first culture medium 11a contained in the first culture region 10a. The second culture region 10b may be equipped with a hydrogen concentration measuring means, etc., for measuring the concentration of hydrogen contained in the gas released from the culture medium contained in the second culture region 10b.

[0096] Furthermore, other components include gas separation means. Gas separation means can be installed in each culture region for the purpose of removing (partially or entirely) desired gases from the recovered gas, or picking up (partially or entirely) only desired gases. Specifically, this can be done, for example, by gas separation membranes (polyimide membranes, polyamide membranes, ceramic membranes, etc.), cryogenic separation, pressure swing adsorption (PSA), zeolite adsorption, or chemical absorption using alkaline solutions. These may be used individually or in combination of two or more. Among these, gas separation membranes are preferably used from the viewpoint of energy efficiency and simplicity of equipment.

[0097] Gas separation membranes can be used in various ways, but for example, they can be used to remove nitrogen from combustion exhaust gas to obtain a gas with a high concentration of carbon dioxide (and even oxygen). They can also be used to remove (partially or completely) oxygen from a gas mixture of hydrogen and oxygen. For example, when using a polyimide membrane as a gas separation membrane to remove nitrogen (e.g., reduce nitrogen concentration), the permeation rate of nitrogen through the polyimide membrane is lower than that of hydrogen, oxygen, and carbon dioxide. Therefore, the gas that permeates through the polyimide membrane (the gas on the permeation side of the polyimide membrane) can be obtained as a gas with a low nitrogen concentration. Similarly, when using a polyimide membrane as a gas separation membrane to remove oxygen (e.g., reduce oxygen concentration), the permeation rate of oxygen through the polyimide membrane is lower than that of hydrogen and carbon dioxide. Therefore, the gas that permeates through the polyimide membrane (the gas on the permeation side of the polyimide membrane) can be obtained as a gas with a low oxygen concentration (especially one where the oxygen content in the mixed gas is less than 5% by volume). On the other hand, gases with a low permeation rate through the polyimide membrane can be recovered as gases that have not permeated the polyimide membrane (gases on the non-permeation side of the polyimide membrane), resulting in a gas with a high oxygen concentration. Furthermore, other configurations include gas supply lines that supply gases other than hydrogen, oxygen, and carbon dioxide, in addition to those mentioned above.

[0098] The culture apparatus 1 of the present invention enables safe operation while maintaining high culture efficiency for chemosynthetic autotrophic bacteria. Specifically, increasing the culture volume for efficient bacterial cultivation increases the amount of hydrogen and oxygen used. Consequently, the amount of gas recovered also increases, raising the risk of explosion. However, the culture apparatus 1 of the present invention allows for continuous control of the recovered gas to remain outside the explosive range. Furthermore, this control can be achieved at low cost and by simple means.

[0099] According to the culture apparatus 1 of the present invention, chemosynthetic autotrophic bacteria can be cultured safely while maintaining high culture efficiency. In other words, organic matter (carbohydrates, amino acids, proteins, etc.) produced by chemosynthetic autotrophic bacteria can be obtained safely and efficiently through the culture of these bacteria. The culture apparatus 1 of the present invention can be widely used in various fields such as resin (plastic) manufacturing, fiber manufacturing, fuel manufacturing, reagent manufacturing, industrial raw material manufacturing, food ingredient manufacturing, and feed (fish and shellfish feed, poultry feed, pig feed, dairy cow feed, beef cattle feed) manufacturing.

[0100] [3] Methods for culturing chemosynthetic autotrophic bacteria The culture method of the present invention involves supplying hydrogen to a first culture medium 11a contained in a first culture region 10a via a first supply line 21. The gas released from the first culture medium 11a contained in the first culture area 10a is recovered separately from the gas released from the second culture medium 11b contained in the second culture area 10b via the first recovery line 22. Oxygen is supplied to the second culture medium 11b contained in the second culture region 10b via the second supply line 31. The gas released from the second culture medium 11b contained in the second culture region 10b is recovered separately from the gas released from the first culture medium 11a contained in the first culture region 10a via the second recovery line 32. The method is characterized by mixing a first culture medium 11a contained in a first culture region 10a and a second culture medium 11b contained in a second culture region 10b by circulating them through a connecting channel 60.

[0101] That is, the above culture method is a method for culturing chemosynthetic autotrophic bacteria using the culture apparatus 1 described above, and corresponds to the culture method detailed in [1] above. With respect to the culture method in [3], the explanation of the culture method detailed in [1] above, and the explanation of the culture apparatus detailed in [2] above, apply similarly to the explanation of the culture method described in [3] of the present invention. [Examples]

[0102] The present invention will be described below by several embodiments. [Culture device 1A] The culture apparatus 1A (see Figure 1) includes a culture area that is divided into two culture areas, 10a and 10b, by partitioning one culture area 10 with a partition wall 13. The culture area 10 is constructed as a stainless steel pressure tank that can be sealed. The culture area 10 is also capable of containing 10 liters or more of culture medium 11, and has a gas containment space above the area containing the culture medium 11. In other words, the culture area 10 is divided into two culture areas, each containing a culture medium layer and a gas layer.

[0103] Furthermore, the culture region 10 includes a concentration measuring means 819 for measuring the concentration of dissolved gases (hydrogen, oxygen, and carbon dioxide) in the culture medium, a concentration measuring means 811 for measuring the oxygen concentration in the gas containment space of the first culture region 10a, and a concentration measuring means 812 for measuring the hydrogen concentration in the gas containment space of the second culture region 10b. The concentration measuring means 811 is used to keep the oxygen concentration of the gas retained in the gas containment space of the first culture region 10a below 5 volume percent. The concentration measuring means 812 is used to keep the hydrogen concentration of the gas retained in the gas containment space of the second culture region 10b below 4 volume percent.

[0104] Furthermore, the culture region 10 is equipped with stirring means (stirring blades) 90 (stirring means 90a for the first culture region and stirring means 90b for the second culture region) at its bottom, and is equipped with culture medium 11 (first culture medium 11a and second culture medium 11b), each of which is capable of being stirred. By stirring, the amount of dissolved substrate supplied in gaseous form can be increased and stabilized. The stirring blades can be rotated, for example, at 50 rpm to 1500 rpm.

[0105] The culture medium (100 vol%) contains hydrogen-oxidizing bacteria (Cupriavidus necator strain H16), disodium hydrogen phosphate dodecahydrate (0.56 vol%), potassium dihydrogen phosphate (0.06 vol%), magnesium sulfate heptahydrate (0.01 vol%), calcium chloride (0.0005 vol%), ammonium chloride (0.12 vol%), iron(III) chloride hexahydrate (0.0004 vol%), and nickel(II) chloride hexahydrate (0.009 vol%).

[0106] The culture apparatus 1A (see Figure 1) includes a first supply line 21 for supplying hydrogen to a first culture medium 11a contained in a first culture region 10a, and a first recovery line 22 for recovering gas released from the first culture medium 11a contained in the first culture region 10a. The first supply line 21 includes a supply pipe 211 and a valve 213, and the first recovery line 22 includes a recovery pipe 221.

[0107] The supply pipe 211 has one end connected to a hydrogen source (not shown) and the other end open into the culture medium 11a. The gaseous hydrogen supplied from the hydrogen source is configured to bubble in the depths of the culture medium 11a through the supply pipe 211. The amount of hydrogen supplied can be adjusted by a valve 213. A hydrogen tank filled with compressed purified hydrogen is used as the hydrogen source. Of the hydrogen released into the culture medium 11a from the other end of the supply pipe 211, any excess hydrogen that does not dissolve in the culture medium 11a is retained in a gas containment space above the culture area 10a.

[0108] One end of the recovery pipe 221 is connected to the gas containment space above the culture area 10a, and the other end is connected to the supply pipe 211. Therefore, the gas that remains in the gas containment space is returned to the supply pipe 211 via the recovery pipe 221. In other words, any excess hydrogen supplied to the culture medium 11a that does not dissolve in the culture medium 11a can be reused without being discarded.

[0109] The culture apparatus 1A includes a second supply line 31 for supplying oxygen to the second culture medium 11b contained in the second culture region 10b, and a third supply line 33 for supplying carbon dioxide. The second supply line 31 and the third supply line 33 are shared and essentially function as a single recovery line. Therefore, the second supply line 31 will be described below. Furthermore, the culture apparatus 1A includes a second recovery line 32 and a third recovery line 34 for recovering gas released from the second culture medium 11b contained in the second culture region 10b. The second recovery line 32 and the third recovery line 34 are shared and essentially function as a single recovery line. Therefore, the second recovery line 32 will be described below. The second supply line 31 includes a supply pipe 311, a valve 315, and a separator 313, while the second recovery line 32 includes a recovery pipe 321.

[0110] In this culture apparatus 1A, combustion exhaust gas is used as an oxygen source and a carbon dioxide source. Specifically, nitrogen contained in the combustion exhaust gas is removed by the separator 313 to form a mixed gas containing high concentrations of oxygen and carbon dioxide, and this mixed gas is supplied to the culture medium 11b via the second supply line 31. The separator 313 has a polyimide gas separation membrane (polyimide membrane). Since the rate of nitrogen permeation through the polyimide membrane is lower than the rate of oxygen and carbon dioxide permeation, the nitrogen concentration in the gas mixture that passes through the separator 313 is reduced, and a gas mixture consisting substantially of oxygen and carbon dioxide is obtained.

[0111] The supply pipe 311 has one end connected to the separator 313 and the other end open into the culture medium 11b, and is configured to bubble the aforementioned mixed gas (oxygen and carbon dioxide) that has passed through the separator from deep within the culture medium 11b through the supply pipe 311. The amount of this mixed gas supplied can be adjusted by valve 315. In addition, any excess gas from the mixed gas released into the culture medium 11b from the other end of the supply pipe 311 that does not dissolve in the culture medium 11b is retained in the gas containment space above the culture area 10b.

[0112] One end of the recovery pipe 321 is connected to the gas containment space above the culture area 10b, and the other end is connected to the supply pipe 311. Therefore, the gas that remains in the gas containment space is returned to the supply pipe 311 via the recovery pipe 321. In other words, any excess oxygen and carbon dioxide supplied to the culture medium 11b that does not dissolve in the culture medium 11b can be reused without being discarded.

[0113] This culture apparatus 1A is equipped with two openings 131 on the bottom side of the partition wall 13, and these openings 131 function as connecting channels 60 (connecting channels 61 and 62). That is, the first culture medium 11a contained in the first culture area 10a and the second culture medium 11b contained in the second culture area 10b are circulated through the openings 131 which are the connecting channels 60, so that the culture mediums of each other are mixed. Furthermore, a pump 71, which serves as a culture medium transfer means 70, is installed on one side of the opening 131, and the connecting channel 60 is blocked by the culture medium transfer means. This configuration ensures that the culture medium flows only when the pump 71 is in operation.

[0114] Furthermore, each opening 131 is positioned deeper than the open ends of the supply pipes 211 and 311 on the tank bottom side, and vertical walls 611 are provided on both sides of each opening 131. This prevents the gases bubbling into each culture region 10a and 10b from mixing with the flow of culture media 11a and 11b.

[0115] In this culture apparatus 1A, hydrogen and oxygen are supplied separately to each of the divided culture regions 10a and 10b, and any excess gases are also recovered separately. Therefore, hydrogen and oxygen are not mixed, enabling safe operation. In addition, since the supplied gases in each culture region 10a and 10b are not mixed with each other, the recovered gases can be reused. On the other hand, safety can be ensured by the gas supply and recovery described above, allowing for the maximum possible gas supply and maximizing the amount of substrate dissolved in the culture medium. Furthermore, since the culture media 11a and 11b contained in each culture region 10a and 10b are mixed through the connecting channel 60, the culture efficiency of chemosynthetic autotrophic bacteria can be maximized.

[0116] [Culture device 1B] Culture apparatus 1B (see Figure 2) differs from culture apparatus 1A in that its two culture regions 10a and 10b are composed of separate pressure-resistant tanks. These culture regions 10a and 10b are connected by connecting channels 60 (connecting channels 61 and 62). Culture medium transfer means 70 (pumps 71 and 72) are installed within each connecting channel 60 (connecting channels 61 and 62), and the connecting channels 60 are blocked by the culture medium transfer means 70. This configuration ensures that the culture medium flows only when pumps 71 and 72 are operated.

[0117] Furthermore, the culture apparatus 1B, like the culture apparatus 1A, is equipped with a first supply line 21, a first recovery line 22, a second supply line 31 (shared with the third supply line 33), and a second recovery line 32 (shared with the third recovery line 34). Of these, culture device 1B differs from culture device 1A in that it has a dedicated line in the second supply line 31 for supplying oxygen from an oxygen source. Specifically, culture device 1B has a supply pipe 311' connected to an oxygen source and a valve 312 for controlling the flow rate in the supply pipe 311' in the second supply line 31. This allows for the replenishment of oxygen from the oxygen source when the amount of oxygen in the combustion exhaust gas is low, thereby obtaining an appropriate oxygen concentration. In other words, it is possible to improve the culture efficiency of chemosynthetic autotrophic bacteria. As an oxygen source, an oxygen generator (such as an oxygen generator that removes nitrogen by physical adsorption), an oxygen tank (liquid oxygen storage tank), etc., can be used.

[0118] [Culture device 1C] Culture apparatus 1C (see Figure 3) differs from culture apparatus 1A in that it is equipped with a separator 223 capable of hydrogen separation in the first recovery line 22. In this configuration, the concentration measuring means 811 for measuring the oxygen concentration in the gas containment space of the first culture area 10a monitors the oxygen concentration, and if the oxygen concentration is less than 5 volume%, the recovered gas is returned to the first supply line 21 without passing through the separator 223. On the other hand, if the concentration measuring means 811 detects that the oxygen concentration in the gas containment space of the first culture area 10a has increased (i.e., the oxygen concentration is approaching 5% by volume), the flow path can be changed by the valve 222, allowing the recovered gas to be passed to the separator 223. This removes some or all of the oxygen contained in the recovered gas in the separator 223, and the gas with a reduced oxygen concentration can be returned to the first supply line 21. This allows for more effective reuse of hydrogen without discarding it, while maintaining safety.

[0119] Furthermore, the culture apparatus 1C is equipped with a dryer 225 and a compressor 227 in the first recovery line 22. Similarly, the second recovery line 32 is also equipped with a dryer 325 and a compressor 327. If the recovered gas contains moisture, passing it through separators 223 and 313 will also remove the moisture, which may reduce the separation efficiency. Therefore, some or all of the moisture can be removed from the recovered gas beforehand using dryers 225 and 325. Furthermore, by providing compressors 227 and 327, the recovered gas can be pressurized and supplied to the separator, thereby improving separation efficiency. These dryers and compressors can also be provided in common in culture devices 1A, 1B, 1D, and 1E.

[0120] [Culture device 1D] Culture device 1D (see Figure 4) differs from culture device 1B (see Figure 2) in that it has multiple second culture regions 10b. In other respects, it is the same as culture device 1B (see Figure 2). This configuration facilitates continuous operation of culturing chemosynthetic autotrophic bacteria. That is, both the second culture area 10b and the second culture area 10b' may be operated, but one of them can be stopped. For example, if the second culture area 10b is running and the operation of the second culture area 10b' is stopped, the culture medium 11b' in the second culture area 10b' can be recovered and replenished. Similarly, if the second culture area 10b' is running and the operation of the second culture area 10b is stopped, the culture medium 11b in the second culture area 10b can be recovered and replenished. Therefore, since the culture medium can be recovered and replenished while the system is in continuous operation, better production efficiency can be achieved.

[0121] [Culture device 1E] Culture apparatus 1E (see Figure 5) differs from culture apparatus 1B (see Figure 2) in that it includes a third culture region 10c. Specifically, culture apparatus 1B includes a second supply line 31 and a third supply line 33 that handle oxygen and carbon dioxide in common, as well as a second recovery line 32 and a third recovery line 34 that handle oxygen and carbon dioxide in common, while culture apparatus 1E includes these individually. More specifically, the culture apparatus 1E includes a first supply line 21 for supplying hydrogen to the first culture area 10a, and a first recovery line 22 for recovering hydrogen from the first culture area 10a. Furthermore, it includes a second supply line 31 for supplying oxygen to the second culture area 10b, and a second recovery line 32 for recovering oxygen from the second culture area 10b. Furthermore, it includes a fourth supply line 41 for supplying carbon dioxide to the third culture area 10c, and a fourth recovery line 42 for recovering carbon dioxide from the third culture area 10c. Therefore, since gases are handled individually in each culture region, each recovered gas can be easily reused. In addition, because the different dissolved amounts of each gas can be easily controlled, it becomes possible to obtain an effective gas concentration in the culture medium after mixing, thereby improving the culture efficiency. [Industrial applicability]

[0122] According to the present invention, chemosynthetic autotrophic bacteria can be cultured safely while maintaining high culture efficiency. In other words, organic matter (carbohydrates, amino acids, proteins, etc.) produced by chemosynthetic autotrophic bacteria can be obtained safely and efficiently through the culture of these bacteria. The culture apparatus 1 of the present invention can be widely used in various fields such as resin (plastic) manufacturing, fiber manufacturing, fuel manufacturing, reagent manufacturing, industrial raw material manufacturing, food ingredient manufacturing, and feed (fish and shellfish feed, poultry feed, pig feed, dairy cow feed, beef cattle feed). [Explanation of Symbols]

[0123] 1, 1A, 1B, 1C, 1D, 1E; Culture device, 10; Culture area (culture tank), 10a; First culture area (first culture tank), 10b; Second culture area (second culture tank), 10c; Third culture area (third culture tank), 11; Culture medium, 11a; First culture medium, 11b; Second culture medium, 11c; Third culture medium, 21; First supply line (hydrogen supply line), 211; Supply pipe, 213; Valve, 22; First recovery line (hydrogen recovery line), 221; Recovery pipe, 223; Separator, 225; Dryer, 227; Compressor, 31; Second supply line (oxygen supply line), 311; Supply pipe, 312; Valve, 313; Separator, 315; Valve, 32; Second recovery line (oxygen recovery line), 321; Recovery pipe, 323; Separator, 325; Dryer, 327; Compressor, 33; Third supply line (carbon dioxide supply line, common line), 34; Third recovery line (carbon dioxide recovery line, common line), 41; Fourth supply line (carbon dioxide supply line, independent line), 411; supply pipe, 413; valve, 42; Fourth recovery line (carbon dioxide recovery line, independent line), 421; Recovery pipe, 60; connecting channel, 61; Connecting channel (first culture region 10a → second culture region 10b), 611; Vertical wall 62; Connecting channel (second culture region 10b → first culture region 10a), 63; Connecting channel (first culture region 10a → third culture region 10c), 64; Connecting channel (third culture region 10c → first culture region 10a), 70; means for transferring culture medium, 71; means for transferring culture medium (means for transferring the first culture medium 11a to the second culture area 10b via the connecting channel 61), 72; means for transferring culture medium (means for transferring the second culture medium 11b to the first culture area 10a via the connecting channel 62), 73; means for transferring culture medium (means for transferring the first culture medium 11a to the second culture area 10b via the connecting channel 61), 74; means for transferring culture medium (means for transferring the second culture medium 11b to the first culture area 10a via the connecting channel 62), 80;Concentration measuring means 811; an oxygen concentration measuring means for measuring the oxygen concentration in the emitted gas of the first culture medium 11a; 812; a hydrogen concentration measuring means for measuring the hydrogen concentration in the emitted gas of the second culture medium 11b; 813; a hydrogen concentration measuring means for measuring the carbon dioxide concentration in the emitted gas of the third culture medium 11c; 819; a concentration measuring means for measuring the concentration of dissolved components in the culture medium. 90; stirring means, 90a; stirring means for stirring a first culture area, 90b; stirring means for stirring a second culture area, 90c; stirring means for stirring a third culture area.

Claims

1. A method for culturing chemosynthetic autotrophic bacteria using hydrogen and oxygen, A method for culturing chemosynthetic autotrophic bacteria, characterized by supplying hydrogen to a first culture medium and oxygen to a second culture medium, from a culture medium containing chemosynthetic autotrophic bacteria divided into multiple portions, separately recovering the gas released from the first culture medium and the gas released from the second culture medium, and circulating the culture mediums so that they are mixed with each other.

2. The method for culturing chemosynthetic autotrophic bacteria according to claim 1, wherein carbon dioxide is supplied to the second culture medium.

3. A method for culturing chemosynthetic autotrophic bacteria according to claim 1 or 2, wherein the proportion of oxygen in the gas released from the first culture medium is controlled to less than 5% by volume.

4. A method for culturing chemosynthetic autotrophic bacteria according to claim 1 or 2, wherein the proportion of hydrogen in the gas released from the second culture medium is controlled to less than 4% by volume.

5. A method for culturing chemosynthetic autotrophic bacteria according to claim 1 or 2, wherein the ratio of each component to the total amount of hydrogen, oxygen, and carbon dioxide supplied to the entire plurality of culture media is 50 to 90% by volume for hydrogen, 5 to 25% by volume for oxygen, and 5 to 25% by volume for carbon dioxide.

6. A method for culturing chemosynthetic autotrophic bacteria according to claim 1 or 2, wherein hydrogen contained in the gas recovered from the first culture medium is refluxed as hydrogen supplied to the first culture medium.

7. A method for culturing chemosynthetic autotrophic bacteria according to claim 1 or 2, wherein oxygen contained in the gas recovered from the second culture medium is refluxed as oxygen supplied to the second culture medium.

8. A culture apparatus for chemosynthetic autotrophic bacteria using hydrogen and oxygen, Equipped with multiple culture regions, A first supply line for supplying hydrogen to the culture medium contained in the first culture region among the plurality of culture regions, A first recovery line for recovering gas released from the culture medium contained in the first culture region, A second supply line for supplying oxygen to the culture medium contained in the second culture region among the aforementioned multiple culture regions, A second recovery line for recovering gas released from the culture medium contained in the second culture region, A connecting channel for circulating the culture medium contained in the first culture region and the culture medium contained in the second culture region so as to mix them, A culture apparatus for chemosynthetic autotrophic bacteria, characterized by being equipped with [a specific feature / feature].

9. The apparatus for culturing chemosynthetic autotrophic bacteria according to claim 8, further comprising a third supply line for supplying carbon dioxide to the culture medium contained in the second culture region.

10. The apparatus for culturing chemosynthetic autotrophic bacteria according to claim 8 or 9, wherein the second supply line and the third supply line are common.

11. The apparatus for culturing chemosynthetic autotrophic bacteria according to claim 8 or 9, wherein the connecting channel is provided deep between the first culture region and the second culture region.

12. A culture apparatus for chemosynthetic autotrophic bacteria according to claim 8 or 9, further comprising a culture medium transfer means for transferring the culture medium contained in the first culture region to the second culture region via the connecting channel.

13. The apparatus for culturing chemosynthetic autotrophic bacteria according to claim 8 or 9, wherein the first culture region is provided with an oxygen concentration measuring means for measuring the concentration of oxygen contained in the gas released from the culture medium contained in the first culture region.

14. The apparatus for culturing chemosynthetic autotrophic bacteria according to claim 8 or 9, wherein the second culture region is provided with a hydrogen concentration measuring means for measuring the concentration of hydrogen contained in the gas released from the culture medium contained in the second culture region.

15. A method for culturing chemosynthetic autotrophic bacteria using the chemosynthetic autotrophic bacteria culture apparatus described in claim 8, Hydrogen is supplied to the culture medium contained in the first culture region via the first supply line. The gas released from the culture medium contained in the first culture region is recovered separately from the gas released from the culture medium contained in the second culture region via the first recovery line. Oxygen is supplied to the culture medium contained in the second culture region via the second supply line. The gas released from the culture medium contained in the second culture region is recovered separately from the gas released from the culture medium contained in the first culture region via the second recovery line. A method for culturing chemosynthetic autotrophic bacteria, characterized by mixing a culture medium contained in the first culture region and a culture medium contained in the second culture region by circulating them through the connecting channel.

16. The method for culturing chemosynthetic autotrophic bacteria according to claim 15, wherein the ratio of each component to the total amount of hydrogen, oxygen, and carbon dioxide supplied to the entire mixed culture medium is 50 to 90% by volume for hydrogen, 5 to 25% by volume for oxygen, and 5 to 25% by volume for carbon dioxide.

17. A method for culturing chemosynthetic autotrophic bacteria according to claim 15 or 16, wherein hydrogen in the gas recovered from the first recovery line is refluxed to the first supply line.

18. A method for culturing chemosynthetic autotrophic bacteria according to claim 15 or 16, wherein oxygen in the gas recovered from the second recovery line is returned to the second supply line.

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