Method for preparing medium

Abstract

To provide a method for supplying medium nutrients required during microbial cultivation, particularly nitrate ions, to a medium by simple means.SOLUTION: The present invention provides a method for preparing a medium, whereby a plasma-containing gas generated from a source gas containing nitrogen and oxygen is applied to the medium during microbial cultivation.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for preparing a medium used for culturing microorganisms.

Background Art

[0002] In culturing microorganisms, a medium is used to supply nutrients and the like to the microorganisms. For example, Patent Document 1 describes a culturing method in which fermentable sake distillation waste liquid is diluted and used as a medium to culture microalgae as microorganisms. In the culturing method of Patent Document 1, the sake distillation waste liquid generated as a residue when producing sake is used as a medium in a fermentable state, thereby preventing the corruption of the organic substances contained in the sake distillation waste liquid and generating carbon dioxide gas necessary for culturing microalgae by the fermentation of the sake distillation waste liquid.

[0003] Patent Document 2 describes a continuous culturing method in which microorganisms such as yeast are cultured in a culture solution, and the product produced by the microorganisms is separated and recovered by a membrane separation device. In the continuous culturing method of Patent Document 2, the membrane separation device is installed in the culture tank, and in the culture tank, the culture solution and a draw solution having an osmotic pressure higher than the osmotic pressure of the culture solution are in contact with each other through a separation membrane. Thereby, the product of the microorganisms and water in the culture solution migrate to the draw solution side through the separation membrane by forward osmosis. Therefore, the product can be recovered from the draw solution, and the clogging of the separation membrane is less likely to occur. However, Patent Documents 1 and 2 do not describe or suggest any means for supplying medium nutrients (medium nutrient components) necessary for culturing to the medium used for culturing microorganisms.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] One of the problems to be solved by the present invention is to provide a method for supplying medium nutrients required for culturing microorganisms, particularly nitrate ions, to the medium by a simple means of applying a plasma-containing gas to the medium. Another problem to be solved by the present invention is to provide a method for preparing a medium in which a plasma-containing gas is applied to the medium.

Means for Solving the Problems

[0006] As a result of intensive studies to solve the above problems, the present inventors have completed the following invention. [Item 1] A method for preparing a medium, in which a plasma-containing gas obtained by using a source gas containing nitrogen and oxygen is applied to the medium when culturing microorganisms. [Item 2] The step of applying the plasma-containing gas to the medium is the following (a) and / or (b); (a) A step of applying a plasma-containing gas to the medium, (b) A step of using a material to which a plasma-containing gas has been applied in the medium, The method for preparing a medium according to Item 1, including the above. [Item 3] The method for preparing a medium according to Item 1 or 2, which is for supplying culture nutrients. [Item 4] The method for preparing a medium according to Item 3, wherein the culture nutrients contain nitrate ions. [Item 5] The method for preparing a medium according to any one of Items 1 to 4, wherein the microorganism includes algae. [Item 6] The method for preparing a medium according to any one of Items 1 to 5, wherein the medium is a liquid medium.

Effects of the Invention

[0007] According to the present invention, it becomes possible to provide a method for supplying medium nutrients required for culturing microorganisms, particularly nitrate ions, to the medium by a simple means of applying a plasma-containing gas to the medium. In addition, according to the present invention, there is provided a method for preparing a culture medium by applying a plasma-containing gas to the culture medium. In the method for preparing a culture medium of the present invention, by applying a plasma-containing gas, a culture nutrient containing nitrate ions is supplied, and the content of the culture nutrient containing nitrate ions in the culture medium can be increased. Therefore, it is not necessary to add a culture nutrient such as nitrate, and it is possible to reduce carbon dioxide generated during the production of a culture nutrient such as nitrate. Thus, it is an environmentally advantageous method. Further, the present invention can be easily incorporated without significantly modifying an existing culture system, and is also advantageous in terms of costs such as capital investment.

Mode for Carrying Out the Invention

[0008] The present invention relates to a method for preparing a culture medium, which applies a plasma-containing gas obtained using a nitrogen- and oxygen-containing gas to the culture medium during the culture of microorganisms. Hereinafter, the present invention will be described in detail.

[0009] [Microorganisms] The microorganisms cultured using the culture medium prepared in the present invention are not particularly limited, but any type of microorganism can be cultured as long as it can be cultured in an artificially prepared culture medium such as various known culture methods, for example, a culture method using a liquid culture medium or a culture method using a solid culture medium. The microorganisms to be cultured as described above include not only eubacteria and archaea, but also, for example, algae (microalgae) as eukaryotes, yeasts (fission yeast, budding yeast, etc.), protists, fungi, slime molds, and the like can be used.

[0010] The microorganism to be cultured is preferably an alga. The alga is not particularly limited and may be either a prokaryote or a eukaryote, and can be appropriately selected according to the purpose and the like. For example, examples of algae include Cyanophyta, Glaucophyta, Rhodophyta, Chlorophyta, Cryptophyta, Haptophyta, Heterokontophyta, Dinophyta, Euglenophyta, Chlorarachniophyta, and the like. These may be used alone or in combination of two or more. More specifically, examples of the algae include Phaeodactylum (e.g., Phaeodactylum tricornutum (microbial strain preservation number: NIES-4392, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Chaetoceros (e.g., Chaetoceros calcitrans (microbial strain preservation number: CCAP 1085 / 3, microbial strain preservation facility: Culture Collection of Algae and Protozoa (Algae and Protozoa Collection, CCAP))), Dunaliella (e.g., Dunaliella salina (microbial strain preservation number: NIES-2257, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Nannochloropsis (e.g., Nannochloropsis oceanica (microbial strain preservation number: NIES-2145, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Chlorella (e.g., Chlorella vulgaris (microbial strain preservation number: NIES-2170, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Haematococcus (e.g., Haematococcus lacustris (microbial strain preservation number: NIES-144, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Euglena (e.g., Euglena gracilis (microbial strain preservation number: NIES-48, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), Arthrospira (e.g., Arthrospira platensis (microbial strain preservation number: NIES-39, microbial strain preservation facility: Microbial Strain Preservation Facility of the National Institute for Environmental Studies, National Institute for Environmental Studies (NIES))), and the like. These may be used alone or in combination of two or more species.

[0011] The microorganism to be cultured may be yeast. The yeast is not particularly limited, and may be selected from the genera Endomyces, Eremascus, Schizosaccharomyces, Nadsonia, Saccharomycodes, Hanseniaspora, Wickerhamia, Saccharomyces, Kluyveromyces, Lodderomyces, Wingea, Endomycopsis, Pichia, Hansenula, Pachysolepis, and the like. Examples of yeasts include yeasts belonging to the genera Saccharomycopsis, Spermophthora, Eremothecium, Crebrothecium, Ashbya, Nematospora, Metschnikowia, Coccidiascus, and Candida. These may be used alone or in combination of two or more.

[0012] The method for obtaining microorganisms is not particularly limited and can be appropriately selected depending on the purpose. For example, a method of collecting from nature, a method of using a commercially available product, a method of obtaining from a preservation institution or a depository institution, etc. can be used. When the microorganism is an algae, it may be used after a purification process. The purification process is a process for the purpose of dividing microalgae into a single species, and does not necessarily mean dividing them into a completely single algae.

[0013] [Culture medium] The medium prepared in the present invention can use any known medium capable of culturing microorganisms and is not particularly limited, but it is preferably selected according to the type of microorganism to be cultured. The medium may be either a liquid medium or a solid medium, but a liquid medium is preferred. As the liquid medium, either a seawater medium or a freshwater medium may be used.

[0014] Examples of the medium include one or more selected from the group consisting of AF-6 medium, Allen medium, BBM medium, BG11 medium, C medium, CA medium, CAM medium, CB medium, CC medium, CHU medium, CM medium, CSi medium, CT medium, CYT medium, D medium, ESM medium, f / 2 medium, HUT medium, HSM medium, IMK medium, JM medium, M-11 medium, MA medium, MAF-6 medium, MF medium, MDM medium, MG medium, MGM medium, MKM medium, MNK medium, MW medium, PES medium, P35 medium, URO medium, VT medium, VTAC medium, VTYT medium, W medium, WESM medium, SW medium, SOT medium, and mixtures of two or more of these media. Among these, as the freshwater medium, C medium and / or SOT medium are preferred, and as the seawater medium, f / 2 medium is preferred.

[0015] The liquid medium contains a liquid medium and is not particularly limited as long as it is not harmful to the microorganism to be cultured. Examples of the liquid medium include one or more selected from the group consisting of tap water, industrial water, ultrapure water, ion-exchanged water, distilled water, seawater, and organic solvents. Nutrient sources such as proteins and minerals, energy sources, and components such as phosphorus sources may be added to the liquid medium as needed. Note that the components added to the liquid medium are not particularly limited. Also, the liquid medium may contain microorganisms such as bacteria.

[0016] [Raw material gas] The raw material gas containing nitrogen and oxygen used in the method for preparing the medium of the present invention is not particularly limited as long as it contains nitrogen atoms and oxygen atoms and can be plasmaized. Examples of the raw material gas containing nitrogen atoms and oxygen atoms and capable of being plasmaized include, for example, "a raw material gas containing a gas containing at least a nitrogen atom in the molecule and a gas containing at least an oxygen atom in the molecule" and / or "a raw material gas containing a gas containing at least a nitrogen atom and an oxygen atom in the molecule". In the "raw material gas containing a gas containing at least a nitrogen atom in the molecule and a gas containing at least an oxygen atom in the molecule", examples of the "gas containing at least a nitrogen atom in the molecule" include one or more selected from the group consisting of nitrogen gas, ammonia gas, dinitrogen monoxide, nitric oxide, nitrogen dioxide, and the like. Further, examples of the "gas containing at least an oxygen atom in the molecule" include one or more selected from the group consisting of oxygen gas, carbon dioxide gas, water vapor, dinitrogen monoxide, nitric oxide, nitrogen dioxide, and the like. Examples of the "raw material gas containing a gas containing at least a nitrogen atom in the molecule and a gas containing at least an oxygen atom in the molecule" include, for example, "air", "a gas containing nitrogen gas and oxygen gas in an arbitrary ratio", "a gas containing nitrogen gas and carbon dioxide in an arbitrary ratio", "a gas containing ammonia and oxygen gas in an arbitrary ratio", "a gas containing dinitrogen monoxide and nitric oxide in an arbitrary ratio", "other gases containing a gas containing at least a nitrogen atom in the molecule and a gas containing at least an oxygen atom in the molecule", and the like. One or more selected from the group are included. Examples of the "raw material gas containing a gas containing at least a nitrogen atom and an oxygen atom in the molecule" include a raw material gas containing one or more selected from the group consisting of "dinitrogen monoxide", "nitric oxide", "nitrogen dioxide", "other gases containing at least a nitrogen atom and an oxygen atom in the molecule", and the like. Further, the raw material gas containing nitrogen and oxygen may be, for example, a mixed gas containing one or more other gases such as noble gases (argon, neon, helium, etc.) and hydrocarbon gases. The total content ratio of nitrogen atoms and oxygen atoms in the raw material gas is not particularly limited, but is preferably, for example, 10 atom% or more, more preferably 30 atom% or more, still more preferably 70 atom% or more, even more preferably 90 atom% or more, and even more preferably 99 atom% or more.

[0017] The raw material gas is introduced into the plasma treatment space to obtain a plasma-containing gas, and at least a part of it is plasmaized. The raw material gas introduced into the plasma treatment space serves as both the raw material gas for plasma and the carrier gas. The raw material gas source may be a storage container (gas cylinder) for the raw material gas, or may be an outside air intake blower when air is used as the raw material gas.

[0018] [Plasma-containing gas] The plasma-containing gas is obtained by introducing the raw material gas into the plasma treatment space and plasmaizing at least a part of the raw material gas. The pressure at which plasma is generated in the plasma treatment space is not particularly limited. For example, it is 0.1 atm or more, preferably 0.7 atm or more, and for example, 10 atm or less, preferably 1.5 atm or less. By setting mild pressure conditions, there is no risk of the equipment becoming large-scale, which is advantageous in terms of cost.

[0019] The temperature of the plasma-containing gas is not particularly limited. Considering the handling properties of the medium and the influence on microorganisms, etc., it is, for example, 0°C or more, preferably 5°C or more, more preferably 10°C or more, and for example, 99°C or less, preferably 60°C or less, more preferably 40°C or less. When the temperature of the plasma-containing gas exceeds 99°C, (i) problems may occur in terms of safety, handling properties, etc. because the plasma-containing gas is at a high temperature, (ii) the microorganisms to be cultured are affected by heat and appropriate culturing cannot be carried out, (iii) liquid media such as water contained in the liquid medium may boil, etc. If the temperature of the plasma-containing gas is less than 0°C, there is a risk of (i) the microorganisms to be cultured being affected by the low temperature and unable to be properly cultured, (ii) the liquid medium such as water contained in the liquid medium freezing, and other effects.

[0020] The supply amount of the raw material gas when introducing the raw material gas into the plasma treatment space is not particularly limited. The supply amount of the raw material gas is, for example, 0.001 to 200,000 L / min, preferably 0.01 to 10,000 L / min, more preferably 0.1 to 1,000 L / min, and even more preferably 1 to 1,000 L / min. If the supply amount of the raw material gas is less than 0.001 L / min, the generation amount of the plasma-containing gas decreases, and thus the supply function of nutrients to the medium may decrease. Further, if the supply amount of the raw material gas exceeds 200,000 L / min, the apparatus may become large-scale, and the plasma concentration in the plasma-containing gas may decrease, resulting in a decrease in the supply function of nutrients to the medium.

[0021] The plasma treatment space into which the plasma raw material gas is introduced is provided between a pair of electrodes connected to a power source. When a high-frequency wave, a pulse wave, a microwave, etc. are applied from the power source to the pair of electrodes and the discharge start voltage is exceeded, an electric field is formed in the plasma treatment space. At least a part of the introduced raw material gas is plasmaized in the plasma treatment space and then discharged as a plasma-containing gas. Therefore, the plasma-containing gas includes all gases obtained by plasmaizing at least a part of the raw material gas.

[0022] The electric field strength generated in the plasma treatment space is not particularly limited. The electric field strength is, for example, 1 to 1,000 kV / cm, preferably 2 to 300 kV / cm. If the electric field strength exceeds 1,000 kV / cm, the apparatus may become large-scale and disadvantageous in terms of cost. If the electric field strength is less than 1 kV / cm, there is a risk that a sufficient amount of plasma cannot be obtained.

[0023] The rise time (and fall time) of the electric field is the time required for the voltage to continuously increase (or decrease) in the plasma processing space. The rise time of the electric field is not particularly limited and can be arbitrarily set based on the gas species of the source gas, pressure, source gas supply rate, electric field strength, processing voltage, processing current, etc. The rise time is, for example, 10 μs or less, preferably 50 ns to 5 μs. In order to make the time required for the rise of the electric field 10 μs or less, it is preferable to apply a pulse wave to the electrode.

[0024] The power in the plasma processing space is not particularly limited and can be arbitrarily set based on the gas species of the source gas, pressure, source gas supply rate, electric field strength, processing voltage, processing current, etc. The power can be, for example, 100 kW / h or less, preferably 10 kW / h or less. If the power exceeds 100 kW / h, the apparatus may become large-scale and disadvantageous in terms of cost. The processing voltage in the plasma processing space is not particularly limited and can be arbitrarily set based on the gas species of the source gas, pressure, source gas supply rate, electric field strength, processing current, etc. The processing voltage can be, for example, 10 to 1000 V, preferably 20 to 600 V, more preferably 40 to 500 V.

[0025] The processing current in the plasma processing space is not particularly limited and can be arbitrarily set based on the gas species of the source gas, pressure, source gas supply rate, electric field strength, processing voltage, etc. The processing current can be, for example, 0.001 to 1000 A, preferably 0.01 to 500 A, more preferably 0.1 to 100 A. The frequency when applying an electric field by a pulse wave in the plasma processing space is not particularly limited and can be arbitrarily set based on the gas species of the source gas, pressure, source gas supply rate, electric field strength, processing voltage, processing current, etc. The frequency can be, for example, 0.001 kHz or more, preferably 0.01 kHz to 300 MHz, more preferably 0.1 kHz to 150 MHz.

[0026] As the plasma used in the preparation method of the present invention, any scientifically defined plasma can be used without particular limitation. Plasma is a high-energy gaseous state containing charged particles generated by ionization, and as long as the number of ions and electrons is the same or approximately the same and it is in an electrically neutral or almost neutral state. Plasma can be generated by various methods such as discharge between electrodes spaced apart from each other.

[0027] The plasma in the plasma-containing gas is in a high-energy state with luminescence immediately after generation. Therefore, it emits light in a color corresponding to the type of plasma raw material gas and can induce various chemical reactions. The plasma in the plasma-containing gas becomes invisible by losing part of its energy. For example, when the plasma in the plasma-containing gas rides on an air current and is transported over a long distance, it gradually loses energy and extinguishes, eventually becoming invisible. Also, for example, by an operation of depriving energy from the luminescent plasma in the plasma-containing gas, it can be extinguished and made invisible. Even when the plasma in the plasma-containing gas has extinguished, by applying the plasma-containing gas to a medium, it becomes possible to supply nutrients to the medium.

[0028] [Means for producing a plasma-containing gas] The means for producing a plasma-containing gas is not particularly limited as long as it can produce a plasma-containing gas by subjecting at least a part of the raw material gas to a plasma treatment under the condition of a pressure of at least 0.1 to 10 atmospheres. The means for producing a plasma-containing gas has, for example, at least a raw material gas introduction part for introducing the raw material gas, a plasma treatment space for plasmaizing the raw material gas, a pair of electrodes for forming an electric field to form the plasma treatment space, a power source connected to the electrodes, and a plasma-containing gas discharge part for discharging the plasma-containing gas. The means for producing a plasma-containing gas preferably has a housing covering the plasma treatment space in order to facilitate control of pressure and the like.

[0029] The raw material gas introduction part connects the gas source of the raw material gas and the plasma treatment space. For example, the raw material gas introduction part can be connected to various raw material gas sources, and a switching valve or the like can be provided so that any raw material gas can be introduced at any timing as required. When the raw material gas is air, an air supply device such as a blower can also be used.

[0030] The housing covering the plasma treatment space can be made of a dielectric material such as glass or ceramic, for example. Also, a dielectric having a dielectric constant of 2000 or less, such as barium titanate, silicon oxide, aluminum nitride, silicon nitride, or silicon carbide, can be used. Further, by forming at least a part of the housing of a conductive material, the housing itself can be used as an electrode. The shape of the housing is not particularly limited and can be any shape such as cylindrical, spherical, box-shaped, etc. The housing covering the plasma treatment space may have a nozzle shape processed so as to become thinner as it approaches the plasma-containing gas discharge part.

[0031] The means for forming an electric field in the plasma treatment space to cause discharge is not particularly limited, and any means can be used. For example, a pair of electrodes with different polarities are formed on the outer surface or the inner surface of the housing so as to face each other with a gap therebetween, and each electrode is connected to a power source to form an electric field for discharging. The pair of electrodes can be provided to face each other inside the housing covering the plasma treatment space. Also, at least one of the pair of electrodes having a layer formed of an insulator or the like on the surface can be installed. The distance between the discharge electrodes is not particularly limited and can be appropriately optimized in consideration of the voltage, etc., and can be, for example, 0.5 to 50.0 mm, preferably about 0.5 to 5.0 mm. When discharging using electrodes, the plasma concentration can be increased.

[0032] Further, for example, a coil is provided on the outer or inner periphery of a housing that covers a plasma processing space, and an electrode core is provided in the plasma processing space. A means is provided in which the coil and the electrode core are connected to a power source to form an electric field and cause discharge. The interval, winding length, winding diameter, wire diameter of the coil, the interval between the electrode core and the coil, the shape of the electrode core, etc. are not particularly limited and are appropriately optimized in consideration of the voltage and the like. When the plasma-containing gas discharge section from the raw material gas introduction section is a housing having an elongated cylindrical shape, discharging using the coil provided on the outer or inner periphery of the housing and the corresponding electrode core, although the discharge density is relatively low, the discharge volume through which the plasma raw material gas passes can be increased, so that a large amount of plasma can be generated. The pair of electrodes or coils are preferably configured not to be in direct contact with the plasma raw material gas in order to obtain stable plasma discharge. Therefore, an insulating film such as a glassy material such as quartz or alumina or a ceramic material may be provided on the surface of the pair of electrodes or coils by known means such as coating.

[0033] [Preparation of Medium] In the method for preparing the medium of the present invention, the method for preparing the medium by applying a plasma-containing gas obtained using a raw material gas containing nitrogen and oxygen to the medium is not particularly limited. For example, the medium can be prepared by (a) a step of applying the plasma-containing gas to the medium and / or (b) a step of using a material to which the plasma-containing gas has been applied in the medium. As the application step (a) of applying the plasma-containing gas to the medium, there is a step of bringing the plasma-containing gas into direct contact with the liquid medium. For example, using a gas application means having at least a liquid tank in which the liquid medium is stored and a supply tube for supplying the plasma-containing gas, while holding the tip of the supply tube so as to be located in the liquid medium stored in the liquid tank, the plasma-containing gas can be applied to the medium by flowing out the plasma-containing gas from the tip of the supply tube to prepare the medium. The gas application means is preferably designed to increase the contact time of the plasma-containing gas with the medium, such as flowing the plasma-containing gas spirally in the medium in the liquid tank.

[0034] (b) As a step of using the material to which the plasma-containing gas is applied in the medium, steps such as preparing the medium using a liquid medium contacted with the plasma-containing gas can be mentioned. For example, by using the above-described gas application means to bring the plasma-containing gas flowing out from the supply tube into contact with a liquid medium such as water stored in a liquid tank or the like, the plasma-containing gas is applied to the liquid medium, and then the liquid medium to which the plasma-containing gas is applied is used to prepare a liquid medium, whereby the plasma-containing gas can be applied to the medium to prepare the medium. Examples of the material to which the plasma-containing gas is applied include a liquid medium such as water to which the plasma-containing gas is applied.

[0035] Between the above-described plasma-containing gas production means and the gas application means, a gas transport path having means such as heating, cooling, and heat preservation can be provided to set the temperature of the plasma-containing gas to 0°C to 99°C, preferably 5°C to 60°C, more preferably 10°C to 40°C. Thereby, when the plasma-containing gas production means and the gas application means are separated from each other, inactivation of the plasma during the transfer of the plasma-containing gas can be suppressed.

[0036] In the present invention, if necessary, one or more devices selected from the group consisting of pipelines and pumps for flowing liquids such as liquid media, nozzles for discharging and discharging liquids, control valves for adjusting flow rate, liquid pressure, etc., various sensors for flow rate, pressure, temperature, oxygen, carbon dioxide, humidity, water temperature, etc., imaging devices (cameras), stirring devices, air supply and exhaust devices, plasma-containing gas treatment devices, air conditioning devices, lighting devices, air circulation devices, raw material gas transport paths, etc. can be provided.

[0037] The above-described various devices may be configured to be monitored or operated from the outside. For example, by connecting various sensors and imaging devices (cameras) to a terminal such as a computer accessible via a network and performing data communication, it is possible to appropriately monitor the situation of applying the plasma-containing gas to the liquid medium. For example, by connecting devices such as pumps, valves, and various apparatuses to a terminal such as a computer that can be accessed via a network and performing data communication, the devices such as pumps, valves, and various apparatuses can be externally operated and controlled.

[0038] (a) A step of applying a plasma-containing gas to a medium and / or (b) a step of using a material to which the plasma-containing gas has been applied in a medium are performed, whereby culture nutrients can be supplied to the medium to be treated. In particular, the medium prepared in the present invention can supply culture nutrients containing nitrate ions without adding nutrient components such as nitrates by applying a plasma-containing gas, and can increase the content of nitrate ions in the medium. Further, by applying a plasma-containing gas to the medium or the material used in the medium, effects such as reusing the used medium can also be expected.

[0039] The application conditions for applying the plasma-containing gas to the medium or the material (for example, the flow rate and supply time of the plasma-containing gas, the size of the supply tube, etc.) are not particularly limited. The application conditions of the plasma-containing gas can be appropriately changed according to the type of medium to be prepared, the microorganism to be cultured, etc. However, the treatment time of the treatment of applying the plasma-containing gas to the medium is preferably 1 second or more. By setting the treatment time to 1 second or more, the nutrient supply function to the medium can be appropriately exerted. The setting of the treatment time can be arbitrarily determined based on the balance between the application effect and the cost. Also, the ratio of the application amount of the plasma-containing gas to the amount of the medium is not particularly limited and can be appropriately optimized. For example, it can be 0.1 vvm (volume per volume per minute) to 1000 vvm, preferably 0.1 vvm to 100 vvm. When the ratio of the application amount of the plasma-containing gas to the amount of the medium is increased, the application effect of the plasma-containing gas can be enhanced.

Examples

[0040] Hereinafter, specific examples will be given for more detailed explanation, but the present invention is not limited only to the following examples.

[0041] [Test Example 1] Four types of liquid media, namely f / 2 medium, C medium, CM medium, and SOT medium, were prepared as follows. The resulting liquid media were prepared by directly applying a plasma-containing gas to each of them. Also, in order to confirm the supply effect of nitrate ions by the application of the plasma-containing gas, the nitrate ion concentration in the liquid medium before the application of the plasma-containing gas and the nitrate ion concentration in the liquid medium after the application of the plasma-containing gas were measured.

[0042] [Preparation of Medium] (1) f / 2 Medium To prepare the f / 2 medium, first, the components shown in Table 1 below were mixed at the ratios shown in Table 1. Then, the resulting solution was sterilized in an autoclave at 121 °C for 20 minutes to prepare the f / 2 medium. Note that the "f / 2 metals" shown in Table 1 were prepared by adding the components shown in Table 2 below to purified water. Also, the "seawater" shown in Table 1 was prepared by dissolving Marine Art SF-1 (Osaka Yakken Co., Ltd.) in industrial purified water (MonotaRO Co., Ltd.) at a ratio of 38.2 g per liter.

[0043] [Table 1]

[0044] [Table 2]

[0045] (2) C Medium To prepare Medium C, first, the components shown in Table 3 below were added to purified water and mixed, and further, 1 M aqueous citric acid solution was added until the pH reached 7.5. Then, the obtained solution was sterilized in an autoclave at 121°C for 20 minutes to prepare Medium C. "P IV metals" shown in Table 3 was prepared by adding the components shown in Table 4 below to purified water. The purified water used for the preparation of Medium C is the same as the industrial purified water used for the preparation of artificial seawater used in f / 2 medium.

[0046]

Table 3

[0047]

Table 4

[0048] (3) CM Medium To prepare CM medium, first, the components shown in Table 5 below were added to purified water and mixed, and further, 1 M aqueous citric acid solution was added until the pH reached 3.5. Then, the obtained solution was sterilized in an autoclave at 121°C for 20 minutes to prepare CM medium. The purified water used for the preparation of CM medium is the same as the industrial purified water used for the preparation of artificial seawater used in f / 2 medium.

[0049]

Table 5

[0050] (4) SOT Medium To prepare the SOT medium, first, the components shown in Table 6 below were added to purified water and mixed. Then, the obtained solution was sterilized in an autoclave at 121 °C for 20 minutes to prepare the SOT medium. The "A5 solution" shown in Table 6 was prepared by adding the components shown in Table 7 below to purified water. The purified water used for the preparation of the SOT medium is the same as the industrial purified water used for the preparation of artificial seawater used in the f / 2 medium.

[0051]

Table 6

[0052]

Table 7

[0053] <Application of Plasma-Containing Gas> Using a plasma-containing gas production apparatus and a gas application apparatus, an application process of applying a plasma-containing gas was performed on the f / 2 medium, C medium, CM medium, and SOT medium. As the plasma-containing gas production apparatus used in Test Example 1, a cylindrical glass gas flow-through portion having dielectric properties, a raw material gas introduction portion for introducing a raw material gas into the gas flow-through portion, a central electrode portion disposed at the axial center portion of the gas flow-through portion, a counter electrode portion wound around the outer peripheral surface of the gas flow-through portion, a power source, and a plasma-containing gas discharge portion for discharging the plasma-containing gas was used. A plasma treatment space is provided in the gas flow-through portion. The central electrode portion is formed of a copper rod member and is connected to the cathode of the power source. The counter electrode portion is formed of a copper mesh member and is connected to the anode of the power source. When producing a plasma-containing gas using such a production apparatus, by supplying power from the power source, a discharge is generated in the direction of "central electrode portion → raw material gas → dielectric (gas flow-through portion) → counter electrode portion", and at least a part of the raw material gas in the gas flow-through portion can be made into plasma. Thereby, a plasma-containing gas can be generated and sent to the gas application apparatus.

[0054] As the gas application device in Test Example 1, eight 250 mL synthetic resin storage containers were used, and each storage container was provided with a lid attached to the storage container and having two holes, a gas supply tube inserted through one hole of the lid, and an exhaust tube inserted through the other hole of the lid. In each storage container, 160 mL of a treatment liquid (medium) to which a plasma-containing gas is applied is stored. The gas supply tube is held such that the tip (supply port) of the gas supply tube is positioned in the treatment liquid in the storage container. The plasma-containing gas production device and the gas application device are connected by a gas transport path equipped with means for appropriately maintaining the temperature of the plasma-containing gas. This gas transport path branches into eight and is connected to the supply tubes of the gas application device.

[0055] In the application process of the plasma-containing gas in Test Example 1, first, the power supply of the plasma-containing gas production device is turned on to generate the plasma-containing gas. In Test Example 1, air was used as the raw material gas. The plasma-containing gas produced by the production device is supplied to each of the eight storage containers of the gas application device through the gas transport path, flows out from the tip of the supply tube arranged in the treatment liquid, and contacts the treatment liquid.

[0056] In the gas transport path, the temperature of the plasma-containing gas is maintained at 10 to 40 °C. The plasma-containing gas is supplied to the gas application device at a flow rate of 64 L / min. As a result, in each storage container, the plasma-containing gas can flow out from each supply tube at a flow rate of 8 L / min. In this application process, after 4 hours have elapsed since the supply of the plasma-containing gas to the storage container of the gas application device was started, the application process is stopped (ended) by turning off the power supply of the production device.

[0057] In Test Example 1, for each of the f / 2 medium, C medium, CM medium, and SOT medium, the nitrate ion concentration of the medium before the application treatment of the plasma-containing gas and the nitrate ion concentration of the medium obtained after the application treatment of the plasma-containing gas were measured. For the nitrate ion concentration of the f / 2 medium, measurement was performed using a nitrate ion concentration quantification kit for seawater (HI 781, Hanna Instruments Japan Co., Ltd.). For the nitrate ion concentrations of the C medium, CM medium, and SOT medium, measurement was performed using a nitrate ion meter (LAQUAtwin NO3-11, Horiba, Ltd.). The measurement results of the nitrate ion concentrations for each medium are shown in Table 8 below.

[0058] [Table 8]

[0059] In Test Example 1, as shown in Table 8, by preparing the medium by applying the plasma-containing gas to the f / 2 medium, C medium, CM medium, and SOT medium respectively, it was confirmed that in any of the media, the concentration of nitrate ions (nitrate nitrogen) contained in the medium was increased compared to before the application of the plasma-containing gas.

[0060] [Test Example 2] For each of the f / 2 medium, C medium, CM medium, and SOT medium prepared in Test Example 1 above, before preparing the medium, an application treatment of applying the plasma-containing gas was performed on the water, which is the liquid medium used for preparing each medium. For the f / 2 medium, the plasma-containing gas was applied to the purified water before seawater conversion, and then, the Marine Art SF-1 (Osaka Pharmaceutical Research Co., Ltd.) was dissolved in the obtained purified water to prepare artificial seawater. The application treatment of the plasma-containing gas in Test Example 2 was performed in the same manner as the application treatment performed in Test Example 1, except that the treatment liquid was changed from the medium to water. Also, the nitrate ion concentration in the water before the application treatment of the plasma-containing gas and the nitrate ion concentration in the water obtained after the application treatment of the plasma-containing gas were measured in the same manner as in Test Example 1. The measurement results of the nitrate ion concentration for each water are shown in Table 9 below.

[0061]

Table 9

[0062] In Test Example 2, as shown in Table 9, it was confirmed that the concentration of nitrate ions contained in the water increased by applying the plasma-containing gas to the water used for preparing the medium. It can be seen that by applying the plasma-containing gas to a liquid medium such as the water used in the medium, the concentration of nitrate ions in the liquid medium can be increased in advance.

[0063] [Test Example 3] In Test Example 3, plasma-containing gases were produced using various raw material gases, and the relationship between the treatment time of the application treatment of the plasma-containing gas and the change in the concentration of nitrate ions was investigated.

[0064] <Preparation of Raw Material Gas> As the raw material gas for producing the plasma-containing gas, a mixed gas (Examples 1 to 5) in which nitrogen gas and oxygen gas are in the ratio (volume ratio) shown in Table 10, air (Example 6), nitrogen gas (Comparative Example 1), oxygen gas (Comparative Example 2), carbon dioxide gas (Comparative Example 3), and argon gas (Comparative Example 4) were used. Note that air mainly contains nitrogen gas, oxygen gas, argon gas, and carbon dioxide gas, and the content of nitrogen gas and oxygen gas in the air is about 99.1%.

[0065] <Preparation of Liquid Medium (Treatment Liquid)> As the material to which the plasma-containing gas is applied, a mixed solution prepared by mixing the stock solution of bacteria and purified water at a volume ratio of 1:9 was prepared and used as the treatment liquid. Regarding the stock solution of bacteria, first, 10 g of soil was collected from the premises of the Tokyo Factory of Sakata Inks Co., Ltd. and dispersed in 100 mL of purified water, and stirred at 300 rpm for 5 minutes using a magnetic stirrer. Then, suction filtration was performed on the obtained suspension, and the obtained soil extract was used as the stock solution of bacteria.

[0066] <Application of Plasma-Containing Gas> In Test Example 3, the nitrate ion concentration of the treatment liquid was measured, and then an application treatment for applying the plasma-containing gas was performed using a plasma-containing gas production apparatus and a gas application apparatus. In the application treatment of Test Example 3, the same production apparatus and gas transport path as those of the plasma-containing gas production apparatus and gas transport path used in Test Example 1 were used.

[0067] As the gas application apparatus of Test Example 3, the same apparatus as that used in Test Example 1 was used, except that it had four 250 mL synthetic resin storage containers. 80 mL of the treatment liquid was stored in each storage container. The gas supply tube was held such that the tip (supply port) of the gas supply tube was located in the treatment liquid in the storage container. The plasma-containing gas production apparatus and the gas application apparatus are connected by a gas transport path equipped with means for appropriately maintaining the temperature of the plasma-containing gas. This gas transport path branches into four and is connected to the supply tubes of the gas application apparatus.

[0068] In the application treatment of the plasma-containing gas in Test Example 3, the plasma-containing gas was produced using each of the raw material gases of the above-described Examples 1 to 6 and Comparative Examples 1 to 4, and the produced plasma-containing gas was supplied to the gas application apparatus at a flow rate of 32 L / min. In the gas application apparatus, the plasma-containing gas was applied to the treatment liquid in the storage container by flowing it out from each supply tube of the four storage containers at a flow rate of 8 L / min.

[0069] In Test Example 3, after starting the supply of the plasma-containing gas to the storage container of the gas application apparatus, the nitrate ion concentration and the conductivity of the treatment liquid were measured 1 minute, 3 minutes, 5 minutes, 10 minutes, and 15 minutes later. For the nitrate ion concentration of the treatment liquid, the same nitrate ion concentration meter as in Test Example 1 was used for measurement. For the conductivity of the treatment liquid, a conductivity meter (LAQUAtwin EC-33, manufactured by Horiba, Ltd.) was used for measurement. The conductivity of the treatment liquid shows a higher value as the nitrate ion concentration in the treatment liquid increases. The measurement results of the nitrate ion concentration and conductivity of the treatment liquid measured in Test Example 3 are shown in Table 10 below.

[0070]

Table 10

[0071] In Test Example 3, as shown in Table 10, by performing the application treatment of the plasma-containing gas using the source gases of Examples 1 to 6 containing nitrogen and oxygen, it was confirmed that as the treatment time became longer, the concentration of nitrate ions contained in the treatment liquid increased more, and the conductivity became higher. In particular, when using the source gases of Examples 1 to 5 containing only nitrogen and oxygen, it was found that both the nitrate ion concentration and the conductivity showed high values at a treatment time of 15 minutes or more compared to Example 6 using air as the source gas. On the other hand, when using the source gases of Comparative Examples 1 to 4 that do not contain at least one of nitrogen and oxygen, even when the application treatment of the plasma-containing gas was performed for 15 minutes, the nitrate ion concentration in the treatment liquid showed a low value of 21 ppm or less.

[0072] From the results of Test Example 3, it was confirmed that even when a plasma-containing gas is applied to a liquid medium such as water used in the medium and then the medium is prepared, it is possible to prepare a medium with an increased nitrate ion concentration.

[0073] As described above, each embodiment of the present invention has been described. However, each embodiment disclosed this time is illustrative in all respects and not restrictive. In particular, in each embodiment disclosed this time, matters not explicitly disclosed, such as operating conditions, operation conditions, various parameters, dimensions, weights, volumes, and other values of components, do not deviate from the scope normally implemented by those skilled in the art, and those skilled in the art can adopt values that can be easily assumed.

Claims

1. A method for preparing a culture medium, which comprises applying a plasma-containing gas obtained by using a raw material gas containing nitrogen and oxygen to the culture medium when culturing microorganisms.

2. The step of applying the plasma-containing gas to the culture medium is the following (a) and / or (b); (a) A step of applying a plasma-containing gas to the culture medium, (b) A step of using a material to which a plasma-containing gas has been applied in the culture medium, The method for preparing a culture medium according to claim 1, comprising the above.

3. The method for preparing a culture medium according to claim 1 or 2, which is for supplying a culture nutrient.

4. The method for preparing a culture medium according to claim 3, wherein the culture nutrient contains nitrate ions.

5. The method for preparing a culture medium according to claim 1 or 2, wherein the microorganism includes algae.

6. The method for preparing a culture medium according to claim 1 or 2, wherein the culture medium is a liquid culture medium.

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