Culture medium for photosynthetic microorganisms and method for producing the same

Abstract

The present invention provides an acid-soluble culture medium for combustion ash, which is a culture medium for photosynthetic microorganisms in which deterioration during storage is suppressed, or a culture medium more suitable for the growth of photosynthetic microorganisms. [Solution] The culture medium is a culture medium for photosynthetic microorganisms comprising an acidic solution of combustion ash and a chelating agent. The combustion ash is preferably woody biomass combustion ash. The acid is preferably at least nitric acid and hydrochloric acid and / or sulfuric acid. The chelating agent is preferably a hydroxycarboxylic acid or a polycarboxylic acid.

Description

Technical Field

[0001] The present invention relates to a culture medium for photosynthetic microorganisms.

Background Art

[0002] The biomass power generation business is expanding as a carbon-neutral renewable energy. However, a large amount of combustion ash (about 3,000 tons / year) discharged from woody biomass power plants is currently partly reused as roadbed materials and the rest is disposed of as waste. Since the combustion ash is highly alkaline, it is difficult to directly use it as a fertilizer for agriculture and the like.

[0003] On the other hand, in the cultivation of microalgae (phytoplankton), which is expected for next-generation biofuels and the like, the production cost of the culture medium containing nutrients is high, so it has not been widely spread. The combustion ash contains inorganic nutrients (phosphorus, potassium, sodium, magnesium, sulfur, etc.) contained in firewood and wood chips. Therefore, if the inorganic nutrients contained in the combustion ash are used for the cultivation of microalgae, waste can be effectively utilized and the cultivation cost of microalgae can be reduced, and a contribution to the construction of a recycling society can be expected.

[0004] However, since the solubility of the inorganic nutrients contained in incineration ash (combustion ash) is low, it cannot be used as a nutrient source (culture medium) for microalgae by simply dissolving the incineration ash in water. Therefore, a method of acid-dissolving incineration ash and using it as a culture medium has been proposed (Patent Document ). Specifically, Patent Document proposes dissolving chicken manure incineration ash in an aqueous mixed acid solution containing nitric acid, sulfuric acid, and hydrochloric acid, removing the undissolved incineration ash by centrifugation, and diluting the obtained supernatant with distilled water to obtain a culture medium. It is also stated that the nitric acid contained in the aqueous mixed acid solution can be used as a nitrogen source necessary for the growth of photosynthetic organisms such as algae.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] However, when the culture medium described in Patent Document 1 was used after being stored at room temperature (or low temperature), a phenomenon was observed where the growth of microorganisms deteriorated. Furthermore, it is desirable to further improve the culture medium described in Patent Document 1 to make it more suitable for microbial growth. The present invention has been made in view of the above circumstances, and its purpose is to provide an acid-soluble culture medium for combustion ash, which is a culture medium for photosynthetic microorganisms in which deterioration during storage is suppressed, or a culture medium more suitable for the growth of photosynthetic microorganisms. A preferred purpose is to provide a culture medium for photosynthetic microorganisms in which deterioration during storage is suppressed and which is more suitable for the growth of photosynthetic microorganisms. In this specification, the term "combustion ash" is used in a sense that includes incinerated ash, regardless of whether it was previously considered waste or not. [Means for solving the problem]

[0007] The inventors of the present invention have diligently investigated the above problem and found that the combustion ash culture medium deteriorates over time because of the dissolved iron (Fe) contained in the combustion ash culture medium. 2+ We suspected that the oxidation of ) was the cause. Therefore, we dissolved the combustion ash in acid in the presence of a chelating agent, and the resulting Fe 2+ We found that immediately supplementing the ash with a chelating agent suppresses the deterioration of the combustion ash culture medium. Furthermore, we discovered that the ability of the culture medium to grow microorganisms is also enhanced, thus completing the present invention.

[0008] In other words, the present invention is defined by the following configuration. [1] A culture medium for photosynthetic microorganisms, comprising an acidic solution of combustion ash and a chelating agent. [2] The culture medium according to [1], wherein the combustion ash is combustion ash of woody biomass. [3] The culture medium according to [1] or [2], wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid. [4] The culture medium according to any one of [1] to [3], wherein the chelating agent is a hydroxycarboxylic acid or a polycarboxylic acid. [5] The culture medium according to [4], wherein the hydroxycarboxylic acid is gluconic acid, and the polycarboxylic acid is at least one selected from hydroxyl group-containing polycarboxylic acids selected from citric acid; and nitrogen atom-containing polycarboxylic acids selected from ethylenediaminetetracarboxylic acid, diethylenetriaminepentacarboxylic acid, ethylenebisiminobis[(2-hydroxyphenyl)acetic acid], and dicarboxymethyl aspartate. [6] The culture medium according to any one of [1] to [5], wherein the photosynthetic microorganism is an alga. [7] A culture medium according to any of [1] to [6], wherein the pH before culturing is 4 to 8. [8] A method for producing a culture medium for photosynthetic microorganisms, comprising dissolving combustion ash with acid in the presence of a chelating agent. [9] The method for producing the product according to [8], wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid. In this specification, the term "culture medium" is not limited to a medium used directly for culturing microorganisms, but also includes media on which other components are added or which are diluted before being used for culturing microorganisms. [Effects of the Invention]

[0009] According to the present invention, the deterioration of microbial culture media during storage can be suppressed. Furthermore, it is possible to create a culture medium that is more suitable for microbial growth. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a graph showing the culture results using the culture medium of the present invention. [Figure 2] Figure 2 is a graph showing the culture results using the culture medium of the comparative example. [Figure 3]Figure 3 is a graph showing other culture results using the culture medium of the present invention. [Figure 4] Figure 4 is a graph showing another culture result using the culture medium of the present invention.

Mode for Carrying Out the Invention

[0011] (1) Combustion ash In the present invention, a culture medium is prepared from combustion ash. By using combustion ash as an inorganic nutrient component, a mineral component, etc. of the medium, waste can be effectively utilized and resources can be saved. The combustion ash may be combustion ash of factory emissions (including incineration ash), ash discharged when solid fuels such as coal are burned in a thermal power plant, a combustion furnace, etc., but livestock wastes such as feces, pig manure, and cow manure; food wastes such as garbage; forestry wastes such as waste wood and thinned wood; sewage treatment plant sludge; etc. The ash obtained by burning (including incineration) biomass resources is preferred.

[0012] Among them, as the combustion ash, combustion ash of lignocellulosic biomass is preferred. Lignocellulosic biomass includes wood and plants, and is a fuel that can be used as a fuel for thermal power generation (lignocellulosic biomass power generation) and contribute to carbon neutrality. In addition to the above forestry wastes, it may be wood grown for power generation purposes, or may be charcoal, wood chips, bark, etc. By effectively using such lignocellulosic biomass in the medium, the contribution to a low-carbon society can be increased.

[0013] The proportion of lignocellulosic biomass combustion ash in the combustion ash is, for example, 50 to 100% by mass, preferably 60 to 100% by mass, more preferably 80 to 100% by mass.

[0014] The combustion ash to be subjected to acid dissolution preferably contains each element in the following ranges. In the following, except for carbon and halogen atoms, it is shown as the mass of the oxide. Ferric oxide was calculated as Fe2O3 without distinguishing between divalent and trivalent. And it is expressed as the amount when the total of oxides of elements from carbon, halogen atoms, and 5B to 92U (however, excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass. Carbon (C): 1 to 15 parts by mass, preferably 3 to 10 parts by mass Cl atom: 0.1 to 3 parts by mass, preferably 0.5 to 2 parts by mass MgO: 0.5 to 10 parts by mass, preferably 3 to 7 parts by mass Na2O: 0.1 to 8 parts by mass, preferably 1 to 5 parts by mass Al2O3: 0.5 to 10 parts by mass, preferably 1 to 5 parts by mass SiO2: 5 to 25% by mass, preferably 10 to 20 parts by mass P2O5: 0.5 to 10 parts by mass, preferably 1 to 5 parts by mass SO3: 1 to 20 parts by mass, preferably 5 to 15 parts by mass K2O: 10 to 30 parts by mass, preferably 15 to 25 parts by mass CaO: 10 to 40 parts by mass, preferably 20 to 30 parts by mass TiO2: 0.01 to 3 parts by mass, preferably 0.01 to 1 part by mass MnO: 1 to 5 parts by mass, preferably 2 to 4 parts by mass Fe2O3: 1 to 5 parts by mass, preferably 2 to 4 parts by mass ZnO: 0.01 to 1 part by mass, preferably 0.01 to 0.5 part by mass Br atom, Cr2O3, NiO, CuO, As2O3, Rb2O, SrO, ZrO, Y2O3, ZrO2, Nb2O5, PbO, BaO, etc.: Each is 1 part by mass or less, preferably 0.1 part by mass or less

[0015] (2) Acid By dissolving the combustion ash in an aqueous solution of an acid, inorganic nutrient components and mineral components can be dissolved as ions. The acid is not particularly limited as long as it does not include an acid corresponding to the chelating agent described later, and various acids such as mineral acids, organic acids, and carbonic acid can be mentioned. It is preferable to contain at least a mineral acid (such as nitric acid, hydrochloric acid, sulfuric acid, etc.), and it is particularly preferable to contain at least nitric acid. Since the combustion ash often lacks a nitrogen component, the nitrogen component can be supplemented by including nitric acid in the dissolving acid. In addition to nitric acid, it is also preferable for the acid to contain hydrochloric acid and / or sulfuric acid. When using hydrochloric acid, sulfuric acid, etc., FeO contained in the combustion ash is converted to Fe 2+This allows for efficient dissolution. By dissolving Fe in its divalent state, it contributes to the growth of microorganisms.

[0016] The proportion of mineral acid in the acid is, for example, 50 to 100% by mass, preferably 70 to 100% by mass, and more preferably 90 to 100% by mass. When the acid contains nitric acid, hydrochloric acid, and / or sulfuric acid, the amount of nitric acid is, for example, 40 to 95 moles, preferably 50 to 90 moles, and more preferably 60 to 85 moles, per 100 moles of the total of hydrochloric acid, nitric acid, and sulfuric acid. The amount of hydrochloric acid is, for example, 5 to 70 moles, preferably 15 to 60 moles, and more preferably 25 to 50 moles, per 100 moles of the total of hydrochloric acid and sulfuric acid.

[0017] When dissolving combustion ash, the acid concentration in the aqueous solution is, for example, 0.005 to 5 mol / L, preferably 0.01 to 1 mol / L, and more preferably 0.05 to 0.3 mol / L. Furthermore, the amount of combustion ash to be added is 1 m³ of acid solution. 3 The amount per unit is, for example, 1 to 100 kg, preferably 5 to 50 kg, and more preferably 10 to 30 kg. The amount of combustion ash that dissolves in the acid is 1 m³ of the acid aqueous solution. 3 For example, the amount per unit is 0.5 to 50 kg, preferably 2 to 25 kg, and more preferably 5 to 15 kg.

[0018] (3) Chelating agents The acid solution of the combustion ash preferably contains a chelating agent. By including a chelating agent, Fe can be maintained in its divalent state during storage, thereby suppressing the deterioration of the culture medium during storage. Furthermore, because the recovery efficiency of Fe in the combustion ash as divalent can be increased, the growth rate of microorganisms can be enhanced even before storage.

[0019] The timing of adding the chelating agent is not particularly limited; it may be added after dissolving the combustion ash in acid, or the combustion ash may be dissolved in acid in the presence of the chelating agent. When dissolving the combustion ash in acid in the presence of the chelating agent, the chelating agent and combustion ash may be mixed first, and then this chelating agent / combustion ash mixture may be mixed with the acid, or the chelating agent and acid may be mixed first, and then this chelating agent / acid mixture may be mixed with the combustion ash, or the combustion ash and combustion ash may be added to the acid at the same time. It is preferable to mix the chelating agent and acid first, and then mix this chelating agent / acid mixture with the combustion ash.

[0020] Examples of chelating agents include hydroxycarboxylic acids and polycarboxylic acids, and one or more of these can be used. Examples of hydroxycarboxylic acids include gluconic acid. Examples of polycarboxylic acids include hydroxyl group-containing polycarboxylic acids such as citric acid (e.g., mono-, dihydroxy-di-, or tricarboxylic acids); nitrogen atom-containing polycarboxylic acids selected from one or more such as ethylenediaminetetracarboxylic acid (EDTA), diethylenetriaminepentacarboxylic acid (DTPA), ethylenebisiminobis[(2-hydroxyphenyl)acetic acid] (EDDHA), and dicarboxymethyl aspartate (ASDA) (e.g., nitrogen atom-containing polycarboxylic acids having 2 to 6, preferably 2 to 4, carboxylic acid groups), and one or more of these can be used.

[0021] The amount of chelating agent is, for example, 0.1 to 20 parts by mass, preferably 0.3 to 10 parts by mass, and more preferably 0.5 to 5 parts by mass, per 100 parts by mass of combustion ash.

[0022] (4) pH adjustment, filtration When combustion ash is dissolved in acid, the pH increases as the amount dissolved increases. It is preferable to stop the dissolution of combustion ash with acid once the pH reaches 4 or higher. By continuing the dissolution until the pH reaches 4 or higher, the combustion ash can be sufficiently dissolved. The pH range after dissolution is preferably 4 to 8, more preferably 5.0 to 7.5, and even more preferably 6.0 to 7.0. By preventing the pH from becoming too high, it is possible to prevent the chelating agent from dissociating from divalent iron, and to prevent the divalent iron from becoming trivalent during storage of the culture medium. If the pH becomes too high, acid may be added to lower the pH to the above range. It is also preferable for storage conditions to have the pH within the above range.

[0023] The acid solution of combustion ash, whose pH has been adjusted to a predetermined range, may be separated from insoluble matter by solid-liquid separation. Solid-liquid separation includes centrifugation and filtration. The composition of the culture medium after solid-liquid separation is preferably as follows. In the following, carbon is specified as the amount of chelating agent component, so the amounts of components other than carbon are shown. Also, in the following, except for halogen atoms, the amounts are shown as the mass of oxides. Iron oxide was calculated as Fe2O3 without distinguishing between divalent and trivalent iron oxides. The amounts are expressed as the total of halogen atoms and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) when the total is set to 100 parts by mass.

[0024] Cl atoms: 0.1 to 5 parts by mass, preferably 1.0 to 3.0 parts by mass Na2O: 1 to 10 parts by mass, preferably 2 to 6 parts by mass MgO: 0.05 to 8 parts by mass, preferably 1 to 5 parts by mass SO3: 10-40 parts by mass, preferably 15-30 parts by mass K2O: 20-60 parts by mass, preferably 30-50 parts by mass CaO: 20-50 parts by mass, preferably 30-50 parts by mass MnO: 0.01 to 3 parts by mass, preferably 0.1 to 1 part by mass Fe2O3: 0.01 to 3 parts by mass, preferably 0.1 to 1 part by mass Br atoms, Al2O3, SiO2, P2O5, TiO2, Cr2O3, NiO, CuO, ZnO, As2O3, Rb2O, SrO, Y2O3, ZrO2, Nb2O5, PbO, BaO: 0.3 parts by mass or less each, preferably 0.03 parts by mass or less

[0025] PO4 3- The amount of solution is, for example, 0.01 to 1000 mg / L, preferably 0.1 to 100 mg / L, and more preferably 1 to 30 mg / L.

[0026] Furthermore, the amount of chelating agent dissolved is, for example, 0.1 to 20 parts by mass, preferably 0.3 to 10 parts by mass, and more preferably 0.5 to 5 parts by mass, per 19 parts by mass of K2O. Also, per 100 parts by mass of the total of halogen atoms and oxides of elements from 5B to 92U (excluding nitrogen oxides and oxygen), the amount of chelating agent dissolved is, for example, 0.1 to 20 parts by mass, preferably 0.3 to 10 parts by mass, and more preferably 0.5 to 5 parts by mass.

[0027] (5) Concentration and component adjustment The acidic solution of combustion ash obtained as described above can be used as a culture medium. The pH of the culture medium is, for example, 4 to 8, preferably 5.0 to 7.5, and more preferably 6.0 to 7.0, before storage and before culturing. In this specification, the culture medium includes not only the acidic solution of combustion ash, but also concentrated or diluted solutions as needed, and also includes solutions with additional necessary components.

[0028] Water is used for the aforementioned dilution. This dilution water may be naturally derived water containing appropriate components. For example, when culturing freshwater microorganisms, water or water derived from freshwater is preferred as the dilution water. When culturing marine microorganisms, water derived from seawater, such as seawater or artificial seawater, is preferred as the dilution water.

[0029] Furthermore, whether or not dilution is performed, the culture medium may be appropriately supplemented with active ingredients. Active ingredients include NaCl, Fe salts such as FeSO4·7H2O, and Cu salts such as CuSO4·7H2O. Adding active ingredients can promote cultivation. In this invention, since the combustion ash is improved with a chelating agent, cultivation can be performed even without the addition of active ingredients.

[0030] (6) Photosynthetic microorganisms The culture medium described above can be used for culturing photosynthetic microorganisms. By using it for culturing photosynthetic microorganisms, it can contribute to the fixation of carbon dioxide from the air. Furthermore, by culturing it as feed for other organisms, it can contribute to aquaculture.

[0031] The aforementioned photosynthetic microorganisms include algae. The algae may be species that grow in freshwater, brackish water, or saltwater. Examples of such algae include cyanobacteria, green algae, glaucophytes, Euglena algae, dinoflagellates, brown algae, diatoms, chlorarachnion algae, red algae, haptophytes, cryptophytes, prasinophytes, and euspot algae, with green algae, diatoms, haptophytes, prasinophytes, and euspot algae being preferred. Green algae are useful for carbon dioxide fixation, while diatoms, haptophytes, prasinophytes, and euspot algae are useful as feed.

[0032] Green algae include microalgae such as Chlorella, Chlorella vulgaris, and Chlorella sp., with Chlorella being preferred. Chlorella includes the Chlorella genus, such as Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella kessleri, and Chlorella sp.

[0033] Diatoms include genera such as Chaetoseros gracilis and Chaetoseros calcitrans, and Phaeodactylum tricornutum. Haptophytes include genera such as Pavlova lutheri and Isochrysis galbana. Prasinoales include species of the genus Tetraselmis (such as Tetraselmis tetrathele). The true eyespot algae include species of the genus Nannochloropsis (such as Nannochloropsis oculata).

[0034] The culture conditions should preferably involve culturing the microorganisms under light irradiation and aeration conditions, supplying carbon dioxide and air, at salinity levels appropriate to the specific environment of the microorganism, such as freshwater, brackish water, or seawater. During cultivation, the pH and other components may be adjusted as needed. Photosynthetic microorganisms may be directly seeded into the culture medium of the present invention, or they may be pre-cultured in a known algal medium before being added to the culture medium of the present invention. [Examples]

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by the following examples, and it is certainly possible to implement it with appropriate modifications within the scope that is consistent with the spirit of the preceding and following descriptions, and all such modifications are included within the technical scope of the present invention.

[0036] Example 1: Chlorella genus (photosynthetic organism) 100 μL of commercially available plant fertilizer (Hyponex Japan, Hyponex® concentrate) was diluted with 100 mL of distilled water, sterilized in an autoclave, and prepared as a culture medium (hereinafter referred to as Hyponex® solution medium). Chlorella vulgaris NIES-2170, obtained from the National Institute for Environmental Studies Microbial System Preservation Facility, was inoculated into this culture medium, and aerated culture was performed for 14 days under LED lighting (3000 Lux, continuous light) at a temperature of 25°C or lower to obtain a pre-culture solution (pre-culture solution 1).

[0037] Reference example 2: Diatom, genus Chaetoceros As shown in Table 1, 25.2 mg of IMK standard medium (manufactured by Nippon Pharmaceutical Co., Ltd., DAIGO-IMK medium), 5.684 mg of sodium metasilicate pentahydrate, and 3.6 g of artificial seawater (Osaka Yakken Marine Art SF-1) were diluted in 100 mL of distilled water, sterilized by autoclaving, and prepared as a pre-culture medium. Natural Chaetoseros gracilis isolated from the Seto Inland Sea was inoculated into this pre-culture medium, and aerated culture was performed for 14 to 28 days under conditions of 25°C or below with LED lighting (3000 Lux, continuous light) to obtain a pre-culture solution (pre-culture solution 2). [Table 1]

[0038] Example 1 Preparation of culture media from combustion ash: A mixed acid (50 mL) prepared by mixing 0.1 mol / L HNO3, 0.1 mol / L H2SO4, and 0.1 mol / L HCl in a volume ratio of 25:3:5 was placed in a beaker. 0.019 g of anhydrous citric acid was added as a chelating agent, and the mixture was stirred with a magnetic stirrer for 5 minutes. 0.8 g of woody biomass combustion ash (fly ash discharged from a woody biomass power plant) was placed in another beaker, and the entire contents of the stirred mixed acid were added to this beaker and stirred with a magnetic stirrer. During the stirring operation, the pH of the mixture rose from around 2 over time. When the pH reached 6.7, stirring was stopped, and the mixture was filtered using quantitative filter paper (Global Life Science Technologies Japan, Whatman 1004-125) to remove undissolved combustion ash (referred to as mixed acid residue) and obtain the supernatant (supernatant 1). The obtained supernatant 1 (containing citric acid) was diluted 30 times with distilled water to serve as the culture medium (combustion ash medium 1). Approximately 51 parts by weight of the original 100g of woody biomass combustion ash was removed, and approximately 49 parts by weight was carried over to the supernatant 1 as dissolved components. The composition of the woody biomass combustion ash and the mixed acid residue used to dissolve the mixed acid, expressed as the amount of oxides (excluding carbon and halogen atoms), are shown in Table 2. The amounts in Table 2 are the amounts (mass%) when the total of only the detected elements was taken as 100% by mass using a wavelength-dispersive X-ray fluorescence analyzer (ZSX Primus IV) manufactured by Rigaku Corporation, targeting elements in the range of 5B to 92U.

[0039] Furthermore, the elemental composition of supernatant 1 was calculated based on the analytical values ​​of the combustion ash and mixed acid residue. In this calculation, it was assumed that all of the carbon component in the combustion ash remained in the mixed acid residue, and the converted component amount in the mixed acid residue (Conversion 1) was determined. The ratio of each component in Conversion 1 maintains the ratio of each component in the mixed acid residue. Then, the amount of each component in the supernatant was determined by subtracting the value of Conversion 1 from the analytical value of the combustion ash (Conversion 2). Based on the result of Conversion 2, the component composition of supernatant 1 (total 100 mass%) of all components was determined (Conversion 3). Note that in Conversion 2 and Conversion 3, carbon derived from the chelating agent is added, but as is clear from the conversion process, carbon derived from the chelating agent is not included in the total 100 mass% of all components. In addition, in the calculation result of Conversion 3, considering that there was an error of less than 4% in the amount of combustion ash removed due to the work, unreliable digits were deleted, and the calculation was corrected so that the sum of the remaining reliable values ​​equals 100 mass%. The process and results are shown in Table 2. According to Table 2, the amount of the nutrient phosphorus (P2O5) is 0% (i.e., less than 0.5%), and it is unclear whether or not it is present. However, by separately measuring with an ion analyzer, PO4 3- It has been confirmed that it contains 8.76 mg / L.

[0040] [Table 2]

[0041] Cultivation of the green alga Chlorella genus (photosynthetic organisms): 50 mL of combustion ash medium 1 (containing citric acid), prepared one day prior, was added to a test tube, and pre-culture solution 1 (Chlorella pre-culture solution) (1.5 mL) was inoculated. The culture was then incubated for 10 days at a temperature of 25°C, with fluorescent lighting (12000 Lux, continuous light) and an airflow rate of 0.05 L / min (continuous aeration). The absorbance (OD730) at a wavelength of 730 nm of the culture solution during incubation was measured using a UV-Vis spectrophotometer (JASCO, V-730), and this value was used as the growth rate.

[0042] For comparison, Chlorella vulgaris was cultured in the same manner as described above, except that combustion ash medium 1 (containing citric acid) was replaced with medium C (purchased from the National Institute for Environmental Studies) shown in Table 3, or with Hyponex® solution medium. The results for each medium are shown in Figure 1. The final growth rate is shown in Table 4. [Table 3]

[0043] Comparative Example 1 Except for not using anhydrous citric acid, a supernatant (comparative supernatant 1: citric acid-free) was prepared in the same manner as in Experimental Example 1, and then diluted 30-fold with distilled water to prepare a combustion ash medium (comparative combustion ash medium 1: citric acid-free). Using comparative combustion ash medium 1 (citric acid-free) that had been prepared for 1 day, Chlorella vulgaris was cultured in the same manner as in Experimental Example 1.

[0044] For comparison, Chlorella vulgaris was cultured in the same manner as described above, except that comparative combustion ash medium 1 (without citric acid) was replaced with medium C (purchased from the National Institute for Environmental Studies). The results for each medium are shown in Figure 2. The final growth rate is shown in Table 4.

[0045] [Table 4]

[0046] Due to the different culture periods, the final growth rate in medium C differed slightly between Example 1 and Comparative Example 1. However, as can be seen from Figures 1 and 2, comparative combustion ash medium 1 without added citric acid showed slightly inferior growth compared to medium C, and was roughly the same as the Hyponex® solution medium, while combustion ash medium 1 with added citric acid showed growth exceeding that of medium C. From this, it became clear that the addition of a chelating agent improves the growth rate of Chlorella vulgaris.

[0047] Example 2, Comparative Example 2 Supernatant 1 (containing citric acid) was obtained in the same manner as in Example 1. To confirm the preservation effect of the chelating agent (citric acid) on the culture medium, Supernatant 1 was placed in a screw-top bottle, sealed, and stored at room temperature for 1 month (Supernatant 2; containing citric acid, stored for 1 month). Comparative Supernatant 1, without the addition of a chelating agent (anhydrous citric acid), was prepared in the same manner as in Comparative Example 1 and stored at room temperature for 1 month in the same manner as Supernatant 1 (Comparative Supernatant 2; not containing citric acid, stored for 1 month). On the day of the start of cultivation, both Supernatant 2 and Comparative Supernatant 2 were diluted 30-fold with distilled water, and 3.6 g of artificial seawater was added to prepare the combustion ash medium (the medium obtained from Supernatant 2 was designated as Combustion Ash Medium 2, and the combustion ash medium obtained from Comparative Supernatant 2 was designated as Comparative Combustion Ash Medium 2).

[0048] 100 mL of combustion ash medium (culture medium 2 or comparative culture medium 2) or IMK medium (however, the components were adjusted with artificial seawater, etc., in the same way as the medium for pre-culture medium 2) was added to a test tube, pre-culture medium 2 (3 mL) of Chaetoceros gracilis was inoculated, and the culture was carried out for 10 days under conditions of a culture temperature of 25°C, fluorescent light illumination (8500 Lux, continuous light), and an air flow rate of 0.05 L / min (continuous aeration). The results for each medium are shown in Figure 3.

[0049] Comparative combustion ash medium 2, which did not contain anhydrous citric acid, deteriorated during storage and showed inferior growth compared to IMK medium. However, combustion ash medium 2, which contained anhydrous citric acid, showed increased growth compared to IMK medium even after one month of storage.

[0050] Example 3, Comparative Example 3 Except for using supernatant 1 (containing citric acid) or comparative supernatant 1 (not containing citric acid) before storage, dilution and addition of artificial seawater were performed in the same manner as in Example 2 to obtain combustion ash culture media (the material obtained from supernatant 1 was designated as combustion ash culture media 3, and the material obtained from comparative supernatant 1 was designated as comparative combustion ash culture media 3). Chaetoceros gracilis was cultured in the same manner as in Example 2 using combustion ash medium 3 (containing citric acid and seawater; not preserved), comparative combustion ash medium 3 (not containing citric acid and containing seawater; not preserved), or IMK medium, one day after preparation. The results for each medium are shown in Figure 4. The final growth stage is shown in Table 5.

[0051] Due to the short storage period, there was no deterioration of the culture medium, and comparative combustion ash medium 3, without the addition of anhydrous citric acid, showed almost the same growth rate as IMK medium. On the other hand, combustion ash medium 3 with the addition of anhydrous citric acid showed increased growth rate compared to IMK medium.

[0052] Table 5 shows the final growth rates achieved in Example 3, Comparative Example 3, Example 4, and Comparative Example 4. The cell density measured at the end of culture using a cell counter (LOGOS Biosystems, LUNA-II) is also included. This clearly demonstrates that the addition of chelating agents prevents the deterioration of the culture medium. Furthermore, it was found that the addition of chelating agents improves the growth rate of the combustion ash medium even with a short storage period. [Table 5] [Industrial applicability]

[0053] The culture medium of the present invention can be used for culturing photosynthetic microorganisms. By using it for culturing photosynthetic microorganisms, it can be used for fixing carbon dioxide from the air. It can also be used for cultivating other organisms that feed on photosynthetic microorganisms.

Claims

1. A culture medium for photosynthetic microorganisms containing an acidic solution of combustion ash and a chelating agent.

2. The culture medium according to claim 1, wherein the combustion ash is combustion ash of woody biomass.

3. The culture medium according to claim 1, wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid.

4. The culture medium according to claim 1, wherein the chelating agent is a hydroxycarboxylic acid or a polycarboxylic acid.

5. The culture medium according to claim 4, wherein the hydroxycarboxylic acid is gluconic acid, and the polycarboxylic acid is at least one selected from hydroxyl group-containing polycarboxylic acids selected from citric acid; and nitrogen atom-containing polycarboxylic acids selected from ethylenediaminetetracarboxylic acid, diethylenetriaminepentacarboxylic acid, ethylenebisiminobis[(2-hydroxyphenyl)acetic acid], and dicarboxymethyl aspartate.

6. The culture medium according to claim 1, wherein the photosynthetic microorganism is algae.

7. The culture medium according to claim 1, wherein the pH before culturing is 4 to 8.

8. A method for producing a culture medium for photosynthetic microorganisms, comprising dissolving combustion ash with acid in the presence of a chelating agent.

9. The production method according to claim 8, wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid.

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