Culture medium for photosynthetic microorganism and method for producing same
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- HARIMA SHIPPING SERVICE CO LTD
- Filing Date
- 2025-10-30
- Publication Date
- 2026-08-06
Smart Images
Figure JP2025038153_06082026_PF_FP_ABST
Abstract
Description
Culture medium for photosynthetic microorganisms and method for producing the same
[0001] This disclosure relates to a culture medium for photosynthetic microorganisms.
[0002] Biomass power generation is expanding as a carbon-neutral renewable energy source. However, the large amount of combustion ash (approximately 3,000 tons per year) emitted from wood biomass power plants is currently partially reused as roadbed material, while the rest is disposed of as waste. Because the combustion ash is highly alkaline, it is difficult to use it directly as fertilizer in agriculture, etc.
[0003] On the other hand, the cultivation of microalgae (phytoplankton), which are attracting attention as next-generation biofuels, has not become widespread due to the high cost of manufacturing nutrient-rich culture media. Combustion ash contains inorganic nutrients (phosphorus, potassium, sodium, magnesium, sulfur, etc.) found in firewood and wood chips. Therefore, if the inorganic nutrients contained in combustion ash are used for cultivating microalgae, waste can be effectively utilized, and the cost of cultivating microalgae can be reduced, which is expected to contribute to the creation of a circular economy.
[0004] However, because the inorganic nutrients contained in incinerated ash (combustion ash) have low solubility, simply dissolving incinerated ash in water is insufficient for use as a nutrient source (culture medium) for microalgae. Therefore, a method has been proposed to use incinerated ash as a culture medium by dissolving it in acid (Patent Document 1). Specifically, Patent Document 1 proposes dissolving chicken manure incinerated ash in a mixed acid aqueous solution containing nitric acid, sulfuric acid, and hydrochloric acid, removing undissolved incinerated ash by centrifugation, and diluting the resulting supernatant with distilled water to use as a culture medium. It also states that the nitric acid contained in the mixed acid aqueous solution can be used as a nitrogen source necessary for the growth of photosynthetic organisms such as algae.
[0005] Japanese Patent Publication No. 2009-11197
[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 in which 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 the growth of microorganisms. This disclosure has been made in view of the above circumstances, and the problem to be solved is to provide a culture medium for photosynthetic microorganisms that is an acid-soluble culture medium for combustion ash and in which deterioration during storage is suppressed, or a culture medium that is more suitable for the growth of photosynthetic microorganisms. The preferred problem to be solved is to provide a culture medium for photosynthetic microorganisms that is both in which deterioration during storage is suppressed and more suitable for growth. In this specification, the term "combustion ash" is used in a sense that does not include whether or not it was conventionally considered waste, and includes incineration ash.
[0007] The inventors of the present invention have diligently investigated the above problem and found that the reason why the combustion ash culture medium deteriorates over time is 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, leading to the technology disclosed herein.
[0008] That is, the present disclosure is specified by the following configurations. [1] A culture medium for photosynthetic microorganisms, comprising an acid-dissolved solution of combustion ash and a chelating agent. [2] The culture medium according to [1], wherein the combustion ash is combustion ash of lignocellulosic biomass. [3] The culture medium according to [1] or [2], wherein the acid contains 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 hydroxy group-containing polycarboxylic acids selected from citric acid; and nitrogen atom-containing polycarboxylic acids selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, ethylenebisiminobis[(2-hydroxyphenyl)acetic acid], and aspartic acid dicarboxymethyl. [6] The culture medium according to any one of [1] to [5], containing each element within the following ranges. Cl atom: 0.1 to 5 parts by mass Na ２ O: 1 to 10 parts by mass MgO: 0.05 to 8 parts by mass SO ３ : 10 to 40 parts by mass K ２ O: 20 to 60 parts by mass CaO: 20 to 50 parts by mass MnO: 0.01 to 3 parts by mass Fe ２ O ３ : 0.01 to 3 parts by mass Br atom, Al ２ O ３ 、SiO ２ 、P ２ O ５ 、TiO ２ 、Cr ２ O ３ 、NiO, CuO, ZnO, As ２ O ３ 、Rb ２ O、SrO、Y ２ O ３ 、ZrO ２ 、Nb ２ O ５PbO, BaO: 0.3 parts by mass or less each, where the total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass. [7] The amount of chelating agent is K ２ [1] to [6] a culture medium according to any one of [1] to [6], wherein the amount of O is 0.1 to 20 parts by mass per 19 parts by mass. [8] A culture medium according to any one of [1] to [7], wherein the photosynthetic microorganism is an algae. [9] A culture medium according to any one of [1] to [8], wherein the pH before cultivation is 4 to 8.
[10] A method for culturing photosynthetic microorganisms, comprising supplying carbon dioxide and / or air under light irradiation in a culture medium according to any one of [1] to [9].
[11] A method for producing a culture medium for photosynthetic microorganisms, comprising dissolving combustion ash with acid in the presence of a chelating agent.
[12] The method for producing a culture medium according to
[11] , comprising mixing the chelating agent and the acid, and mixing the chelating agent / acid mixture with the combustion ash.
[13] The method for producing a culture medium according to
[11] or
[12] , comprising mixing the combustion ash, the chelating agent and the acid, continuing the mixing until the pH is 4 or higher, and then removing insoluble matter by solid-liquid separation in the range of pH 4 to 8.
[14] The manufacturing method according to any one of
[11] to
[13] , wherein the amount of chelating agent is 0.1 to 20 parts by mass per 100 parts by mass of combustion ash.
[15] The manufacturing method according to any one of
[11] to
[14] , wherein the combustion ash includes combustion ash of woody biomass.
[16] The manufacturing method according to
[15] , wherein the proportion of woody biomass combustion ash in the combustion ash is 50 to 100% by mass.
[17] The manufacturing method according to any one of
[11] to
[16] , wherein each element in the combustion ash is within the following ranges: Carbon (C): 1 to 15 parts by mass Cl atoms: 0.1 to 3 parts by mass MgO: 0.5 to 10 parts by mass Na ２ O: 0.1 to 8 parts by mass Al ２ O ３ :0.5 to 10 parts by mass SiO ２ :5 to 25 parts by mass P ２ O ５ : 0.5 to 10 parts by mass SO ３ : 1 to 20 parts by mass K ２ O: 10 to 30 parts by mass CaO: 10 to 40 parts by mass TiO ２: 0.01 to 3 parts by mass MnO: 1 to 5 parts by mass Fe ２ O ３ : 1 to 5 parts by mass ZnO: 0.01 to 1 part by mass Br atom, Cr ２ O ３ , NiO, CuO, As ２ O ３ , Rb ２ O, SrO, ZrO, Y ２ O ３ , ZrO ２ , Nb ２ O ５ PbO, BaO: 1 part by mass or less each, where the total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass.
[18] The method for producing a culture medium according to any one of
[11] to
[17] , wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid.
[19] The method for producing a culture medium according to
[18] , wherein the nitric acid is 40 to 95 moles in proportion to 100 moles of the total of hydrochloric acid, nitric acid, and sulfuric acid. In this specification, the culture medium is not limited to a medium for culturing microorganisms as is, but also includes a medium in which other components are added or which is diluted before being used for culturing microorganisms.
[0009] According to this disclosure, it is possible to suppress the deterioration of microbial culture media during storage. Furthermore, it is possible to create a culture medium that is more suitable for growth.
[0010] Figure 1 is a graph showing the culture results using the culture medium of the present disclosure. Figure 2 is a graph showing the culture results using the culture medium of a comparative example. Figure 3 is a graph showing other culture results using the culture medium of the present disclosure. Figure 4 is a graph showing yet another culture result using the culture medium of the present disclosure.
[0011] (1) Combustion Ash In this disclosure, a culture medium is prepared from combustion ash. By using combustion ash as the inorganic nutrient and mineral component of the culture medium, waste can be effectively utilized and resource conservation can be achieved. The combustion ash may be combustion ash from factory emissions (including incineration ash), ash discharged when solid fuels such as coal are burned at thermal power plants or combustion furnaces, etc., but ash obtained by burning (including incineration) biomass resources such as manure, pig manure and cow manure; food waste such as kitchen waste; forestry waste such as scrap wood and thinned wood; and sewage treatment plant sludge.
[0012] In particular, woody biomass combustion ash is preferred as the combustion ash. Woody biomass includes wood and plants and is a fuel that can contribute to carbon neutrality when used as fuel for thermal power generation (woody biomass power generation). In addition to the aforementioned forest waste, it may also be timber grown for power generation purposes, or it may be charcoal, wood chips, or bark. By effectively utilizing such woody biomass as a growing medium, the contribution to a low-carbon society can be increased.
[0013] The proportion of woody biomass combustion ash in the combustion ash is, for example, 50 to 100% by mass, preferably 60 to 100% by mass, and more preferably 80 to 100% by mass.
[0014] The combustion ash subjected to acid dissolution preferably contains each element within the following ranges, for example. Note that, excluding carbon and halogen atoms, the mass is given as the oxide mass. Iron oxide is given without distinction between divalent and trivalent forms, Fe ２ O ３ The calculation was performed as follows: The amount was expressed as the total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) being set at 100 parts by mass. Carbon (C): 1 to 15 parts by mass, preferably 3 to 10 parts by mass Cl atoms: 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 Na ２ O: 0.1 to 8 parts by mass, preferably 1 to 5 parts by mass Al ２ O ３ 0.5 to 10 parts by mass, preferably 1 to 5 parts by mass of SiO ２: 5 to 25 parts by mass, preferably 10 to 20 parts by mass P ２ O ５ : 0.5 to 10 parts by mass, preferably 1 to 5 parts by mass SO ３ : 1 to 20 parts by mass, preferably 5 to 15 parts by mass K ２ O: 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 TiO ２ : 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 Fe ２ O ３ : 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 parts by mass Br atoms, Cr ２ O ３ , NiO, CuO, As ２ O ３ , Rb ２ O, SrO, ZrO, Y ２ O ３ , ZrO ２ , Nb ２ O ５ PbO, BaO, etc.: 1 part by mass or less each, preferably 0.1 parts by mass or less (may be 0 parts by mass)
[0015] (2) Acid The combustion ash can be dissolved in an aqueous solution of acid, which allows the inorganic nutrients and mineral components to be dissolved as ions. The acid is not particularly limited as long as it does not contain an acid equivalent to the chelating agent described later, and various acids such as mineral acids, organic acids, and carbonic acid can be used, and it is preferable that it contains at least a mineral acid (nitric acid, hydrochloric acid, sulfuric acid, etc.), and it is particularly preferable that it contains at least nitric acid. Since combustion ash is often deficient in nitrogen components, the nitrogen components can be supplemented by including nitric acid in the dissolving acid. It is also preferable that the acid contains hydrochloric acid and / or sulfuric acid in addition to nitric acid. When hydrochloric acid, sulfuric acid, etc. are used, FeO contained in the combustion ash is dissolved. 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. The mass of hydrochloric acid is calculated as the amount of hydrogen chloride. When the acid contains nitric acid and 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 concentration of the acid 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. The amount of combustion ash added is 1 m³ of the acid aqueous 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 serving is 0.5 to 50 kg, preferably 2 to 25 kg, and more preferably 5 to 15 kg.
[0018] (3) Chelating agent The acid solution of the combustion ash preferably contains a chelating agent. By including a chelating agent, Fe can be maintained in the divalent state even during storage, and deterioration of the culture medium during storage can be suppressed. In addition, it is possible to increase the recovery efficiency of Fe in the combustion ash as divalent, and thus the growth rate of microorganisms can be increased 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-, dihydroxydi-, or tricarboxylic acids); nitrogen atom-containing polycarboxylic acids selected from one or more of 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 dissolving the combustion ash in acid when 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 not raising the pH too high, it is possible to prevent the chelating agent from dissociating from divalent iron, and to prevent 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 dissolution solution of combustion ash with the pH adjusted to a predetermined range may remove insoluble matters by solid-liquid separation. Solid-liquid separation includes centrifugation, filtration, etc. The composition of the medium after solid-liquid separation is preferably, for example, as follows. Hereinafter, since carbon is specified as the amount of the chelating agent component, the amounts of components other than carbon are shown. Also hereinafter, except for halogen atoms, it is shown as the mass of oxides. Iron oxide is calculated as Fe ２ O ３ And it is expressed as the amount when the total of halogen atoms and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass.
[0024] Cl atom: 0.1 to 5 parts by mass, preferably 1.0 to 3.0 parts by mass Na ２ O: 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 SO ３ : 10 to 40 parts by mass, preferably 15 to 30 parts by mass K ２ O: 20 to 60 parts by mass, preferably 30 to 50 parts by mass CaO: 20 to 50 parts by mass, preferably 30 to 50 parts by mass MnO: 0.01 to 3 parts by mass, preferably 0.1 to 1 part by mass Fe ２ O ３ : 0.01 to 3 parts by mass, preferably 0.1 to 1 part by mass Br atom, Al ２ O ３ 、SiO ２ 、P ２ O ５ 、TiO ２ 、Cr ２ O ３ 、NiO, CuO, ZnO, As ２ O ３ Rb ２ O、SrO、Y ２ O ３ 、ZrO ２ 、Nb ２ O ５ 、PbO、BaO: Each is 0.3 parts by mass or less, preferably 0.03 parts by mass or less (it may be 0 parts by mass).
[0025] PO ４ ３－The dissolution amount is, for example, 0.01 to 1000 mg / L, preferably 0.1 to 100 mg / L, more preferably 1 to 30 mg / L.
[0026] Also, the dissolution amount of the chelating agent is, for example, 0.1 to 20 parts by mass, preferably 0.3 to 10 parts by mass, more preferably 0.5 to 5 parts by mass with respect to 19 parts by mass of KO. Also, with respect to a total of 100 parts by mass of oxides of elements from halogen atoms to 5B to 92U (excluding nitrogen oxides and oxygen), for example, it is 0.1 to 20 parts by mass, preferably 0.3 to 10 parts by mass, more preferably 0.5 to 5 parts by mass. ２ (5) Concentration and Component Adjustment The acid dissolution solution of the 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, more preferably 6.0 to 7.0 before storage, before culturing, etc. In the present specification, the culture medium includes not only the acid dissolution solution of the combustion ash, but also those concentrated or diluted as necessary, and those to which necessary components are added.
[0027] For the dilution, water is used. This dilution water may be natural - derived water containing appropriate components. For example, when culturing freshwater microorganisms, the dilution water used is preferably water or water derived from fresh water. When culturing marine microorganisms, the dilution water used is preferably seawater - derived water such as seawater or artificial seawater.
[0028] Further, whether or not the dilution is performed, effective components may be appropriately added to the culture medium. Examples of the effective components include Fe salts such as NaCl, FeSO
[0029] ４ ・7H ２ O, and Cu salts such as CuSO ４ ・7H ２ O. By adding the effective components, culturing can be promoted. In the present disclosure, since the combustion ash is improved with a chelating agent, culturing can be performed even without adding effective components.
[0030] (6) Photosynthetic Microorganisms The culture medium 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. It can also contribute to aquaculture by culturing them as feed for other organisms.
[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, Ipomoea quamoclit, and Ipomoea purpurea, with Chlorella being preferred. Chlorella includes species of 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 Pheodactylum tricornutum. Haptophytes include genera such as Pavlova lutheri and Isochrysis galbana. Prasinophytes include genera such as Tetraselmis tetrathele. Euophthalmophytes include genera such as Nannochloropsis oculata.
[0034] The culture conditions should preferably involve culturing the microorganisms under conditions of aeration, supplying carbon dioxide and air, under light irradiation (natural or artificial light), and at salinity levels appropriate to the specific environment of the microorganism, such as freshwater, brackish water, or seawater. During cultivation, the pH may be adjusted or the components may be adjusted as needed. Photosynthetic microorganisms may be directly seeded into the culture medium of this disclosure, or they may be pre-cultured in a known algal medium before being added to the culture medium of this disclosure.
[0035] This application claims the benefit of priority based on Japanese Patent Application No. 2025-016280, filed on 3 February 2025. The entire specification of Japanese Patent Application No. 2025-016280, filed on 3 February 2025, is incorporated herein by reference.
[0036] The present disclosure will be explained in more detail below with reference to examples, but the present disclosure is not limited by the examples below, and it is certainly possible to implement it with appropriate modifications to the extent that it is in line with the spirit of the preceding and following, and all such modifications are included within the technical scope of the present disclosure.
[0037] Reference Example 1: Chlorella (photosynthetic algae) 100 μL of commercially available plant fertilizer (Hyponex Japan, Hyponex® stock solution) 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 aeration 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).
[0038] Reference Example 2: Diatoms Chaetoceros genus 25.2 mg of IMK standard medium (DAIGO-IMK medium, manufactured by Nippon Pharmaceutical Co., Ltd.) shown in Table 1, 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 and sterilized by autoclaving to prepare the pre-culture medium. Natural Chaetoceros gracilis isolated from the Seto Inland Sea was inoculated into this pre-culture medium, and aeration 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).
[0039] Example 1 Preparation of culture medium from combustion ash: 0.1 mol / L HNO ３ , 0.1 mol / L H ２ SO ４A mixed acid (50 mL) prepared by mixing HCl and 0.1 mol / L HCl in a volume ratio of 25:3:5 was placed in a beaker, and 0.019 g of anhydrous citric acid was added as a chelating agent. 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 that beaker. The mixture was then 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 prepare the culture medium (combustion ash medium 1). Of the original 100g of wood biomass combustion ash, approximately 51 parts by weight were removed, and approximately 49 parts by weight were carried over to the supernatant liquid 1 as dissolved components. The composition of the wood 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 as shown in Table 2. The amounts in Table 2 are the amounts (mass%) when the total of only the detected elements is taken as 100 mass%, based on measurements of elements in the range of 5B to 92U using a wavelength-dispersive X-ray fluorescence analyzer (ZSX Primus IV) manufactured by Rigaku Corporation.
[0040] 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 (6.9410 mass%) in the combustion ash remained in the mixed acid residue (i.e., 6.9410 mass%), and the converted component amount in the mixed acid residue (Conversion 1) was determined. In Conversion 1, the ratio of each component to the amount of carbon component (6.9410 mass%) in the combustion ash is the same as the ratio of each component in the mixed acid residue before conversion. Then, the amount of each component in the supernatant was determined by subtracting the value of Conversion 1 from the combustion ash analytical value (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. Furthermore, considering that there was an error of less than 4% in the amount of combustion ash removed during the calculation of conversion 3, unreliable digits were removed, and the calculation was modified so that the sum of the remaining reliable values equals 100% by mass. The process and results are shown in Table 2. According to Table 2, the nutrient phosphorus (P ２ O ５ The amount of ) is 0% (i.e., less than 0.5%), and it is unclear whether it is present or not, but by measuring separately with an ion analyzer, PO ４ ３－ It has been confirmed that it contains 8.76 mg / L.
[0041]
[0042] Cultivation of the green alga Chlorella genus (photosynthetic organism): 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 carried out for 10 days under conditions of a culture temperature of 25°C, fluorescent light illumination (12000 Lux, continuous light), and an air flow rate of 0.05 L / min (continuous aeration). The absorbance (OD730) at a wavelength of 730 nm of the culture solution during cultivation was measured using a UV-Vis spectrophotometer (JASCO, V-730), and this value was used as the growth rate.
[0043] 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 Hyponex® solution medium. The results for each medium are shown in Figure 1. The final growth rate is shown in Table 4.
[0044] Comparative Example 1 A supernatant (comparative supernatant 1: citric acid-free) was prepared in the same manner as in Experimental Example 1, except that anhydrous citric acid was not used. Then, it was diluted 30 times 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 one day, Chlorella vulgaris was cultured in the same manner as in Experimental Example 1.
[0045] For comparison, Chlorella vulgaris was cultured in the same manner as described above, except that comparative combustion ash medium 1 (citric acid-free) 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.
[0046]
[0047] Due to the different culture periods, the final growth rate of 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.
[0048] 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-cap bottle, the lid was closed, and it was stored at room temperature for one month (supernatant 2; containing citric acid, stored for one 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 one month in the same manner as supernatant 1 (comparative supernatant 2; not containing citric acid, stored for one month). On the day of the start of cultivation, supernatant 2 and comparative supernatant 2 were each diluted 30 times with distilled water, and 3.6 g of artificial seawater was added to prepare combustion ash culture medium (the culture medium obtained from supernatant 2 was designated as combustion ash culture medium 2, and the combustion ash culture medium obtained from comparative supernatant 2 was designated as comparative combustion ash culture medium 2).
[0049] 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 of Chaetoceros gracilis (3 mL) 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.
[0050] 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.
[0051] In Example 3 and Comparative Example 3, combustion ash culture media were obtained by dilution and addition of artificial seawater in the same manner as in Example 2, except that supernatant 1 (containing citric acid) or comparative supernatant 1 (not containing citric acid) was used before storage (the media obtained from supernatant 1 was designated as combustion ash culture media 3, and the media 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 culture media 3 (containing citric acid and seawater; not stored), comparative combustion ash culture media 3 (not containing citric acid and containing seawater; not stored), or IMK medium, one day after preparation. The results for each medium are shown in Figure 4. The final growth rate is shown in Table 5.
[0052] 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 compared to IMK medium.
[0053] Table 5 shows the final growth rate achieved in Example 3 and Comparative Example 3. The cell density at the end of culture, measured using a cell counter (LOGOS Biosystems, LUNA-II), is also included. This clearly demonstrates that the addition of a chelating agent prevents the deterioration of the culture medium. Furthermore, it was found that the addition of a chelating agent improves the growth rate of the combustion ash medium even with a short storage period.
[0054] The culture medium disclosed herein 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, containing each element within the following ranges. Cl atom: 0.1 to 5 parts by mass, Na ２ O: 1 to 10 parts by mass, MgO: 0.05 to 8 parts by mass, SO ３ : 10 to 40 parts by mass, K ２ O: 20 to 60 parts by mass, CaO: 20 to 50 parts by mass, MnO: 0.01 to 3 parts by mass, Fe ２ O ３ : 0.01 to 3 parts by mass, Br atom, Al ２ O ３ 、SiO ２ 、P ２ O ５ 、TiO ２ 、Cr ２ O ３ 、NiO, CuO, ZnO, As ２ O ３ 、Rb ２ O、SrO、Y ２ O ３ 、ZrO ２ 、Nb ２ O ５ 、PbO, BaO: Each 0.3 parts by mass or less. Here, the total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass.
7. The amount of chelating agent is K ２ The culture medium according to claim 1, wherein the amount is 0.1 to 20 parts by mass per 19 parts by mass of O.
8. The culture medium according to claim 1, wherein the photosynthetic microorganism is an alga.
9. The culture medium according to claim 1, wherein the pH before culturing is 4 to 8.
10. A method for culturing photosynthetic microorganisms, comprising supplying carbon dioxide and / or air under light irradiation in the culture medium according to claim 1.
11. A method for producing a culture medium for photosynthetic microorganisms, comprising dissolving combustion ash with acid in the presence of a chelating agent.
12. The manufacturing method according to claim 11, comprising mixing the chelating agent with an acid, and then mixing the chelating agent / acid mixture with the combustion ash.
13. The manufacturing method according to claim 11, comprising mixing the combustion ash, the chelating agent, and the acid, continuing the mixing until the pH reaches 4 or higher, and then removing insoluble matter by solid-liquid separation in a pH range of 4 to 8.
14. The manufacturing method according to claim 11, wherein the amount of chelating agent is 0.1 to 20 parts by mass per 100 parts by mass of combustion ash.
15. The manufacturing method according to claim 11, wherein the combustion ash includes combustion ash of woody biomass.
16. The manufacturing method according to claim 15, wherein the proportion of woody biomass combustion ash in the combustion ash is 50 to 100% by mass.
17. The manufacturing method according to claim 11, wherein each element in the combustion ash is within the following ranges: Carbon (C): 1 to 15 parts by mass Cl atoms: 0.1 to 3 parts by mass MgO: 0.5 to 10 parts by mass Na ２ O: 0.1 to 8 parts by mass Al ２ O ３ :0.5 to 10 parts by mass SiO ２ :5 to 25 parts by mass P ２ O ５ : 0.5 to 10 parts by mass SO ３ : 1 to 20 parts by mass K ２ O: 10 to 30 parts by mass CaO: 10 to 40 parts by mass TiO ２ : 0.01 to 3 parts by mass MnO: 1 to 5 parts by mass Fe ２ O ３ : 1 to 5 parts by mass ZnO: 0.01 to 1 part by mass Br atom, Cr ２ O ３ , NiO, CuO, As ２ O ３ , Rb ２ O, SrO, ZrO, Y ２ O ３ , ZrO ２ , Nb ２ O ５ PbO, BaO: 1 part by mass or less each, where the total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) is 100 parts by mass.
18. The method for producing a product according to claim 11, wherein the acid comprises at least nitric acid and hydrochloric acid and / or sulfuric acid.
19. The manufacturing method according to claim 18, wherein the nitric acid is in an amount of 40 to 95 moles per 100 moles of the total of hydrochloric acid, nitric acid, and sulfuric acid.