Method for rearing shellfish larvae and / or shellfish fry

Abstract

Provided is a method for rearing shellfish larvae by: culturing photosynthetic microorganisms under aerobic conditions and light irradiation in a culture medium prepared by dissolving combustion ash in acid; and feeding the cultured photosynthetic microorganisms to shellfish larvae. This method enables the efficient growth of shellfish such as oysters without requiring the use of deep-sea water.　The combustion ash is preferably derived from woody biomass and preferably contains, in terms of oxides, 5-40 mass% of potassium, 1-10 mass% of phosphorus, and 0.1-10 mass% of iron, while containing substantially no nitrogen.

Description

Method for rearing shellfish larvae and / or juvenile shellfish

[0001] The present disclosure relates to a method for rearing shellfish larvae and / or juvenile shellfish.

[0002] Oysters have been conventionally cultivated in seawater. In recent years, "ocean acidification" has progressed along with the increase in carbon dioxide concentration, and seawater conditions have occasionally temporarily reached a level unsuitable for the growth of oysters. In addition, for Pacific oysters, there is a process called "suppression", where juvenile oysters are conditioned by exposing them to sunlight using the ebb and flow of the tide, causing the shellfish to open and close. If the seawater level changes abnormally at this stage due to influences such as typhoons, the production volume will decrease. Therefore, stable artificial seed production and the development of onshore aquaculture technology are desired.

[0003] For example, Patent Document 1 describes that microalgae are cultivated using deep ocean water to carry out onshore oyster aquaculture. However, this method requires large-scale equipment for taking and transporting water from depths of 200 m or deeper, leaving problems in terms of cost.

[0004] WO 2016 / 129703 A1 Japanese Patent Laid-Open No. 2009-11197

[0005] When carrying out aquaculture such as artificial seed production and onshore cultivation of oysters, if oysters can be grown efficiently without requiring the use of deep seawater, it becomes possible to reduce the amount or period of use of deep seawater, or even not use deep seawater at all. Accordingly, an object of the present disclosure is to efficiently grow shellfish such as oysters without requiring the use of deep seawater.

[0006] To efficiently grow shellfish such as oysters, it is possible to improve the growth conditions of the shellfish themselves. However, the inventors of this invention investigated whether it is possible to efficiently grow shellfish by devising a feed (microorganisms) to give them, and in that investigation, they focused on a culture medium made by dissolving combustion ash in acid. For example, Patent Document 2 proposes dissolving chicken manure incineration ash in a mixed acid aqueous solution containing nitric acid, sulfuric acid, and hydrochloric acid, removing undissolved incineration ash by centrifugation, and using the resulting supernatant diluted with distilled water as a culture medium. However, the microorganisms actually cultured in Patent Document 2 are Chlorella, Spirulina, etc., none of which are microorganisms that can be used as feed for oysters. Furthermore, even if it were possible to cultivate oyster feed (microorganisms) using the culture medium described in Patent Document 2, it is unclear whether that feed would be superior to feed cultured in other culture media (for example, commercially available culture media such as DAIGO-IMK medium manufactured by Nippon Pharmaceutical Co., Ltd.) in terms of oyster growth. Under these circumstances, the inventors conducted research and found that when oysters and other shellfish are fed feed cultured using a solution of combustion ash dissolved in acid as the culture medium, the larvae of the shellfish grow more efficiently compared to when they are fed feed cultured in commercially available culture media, for example, their shell height increases and the number of survivors increases, thus completing the content of this disclosure.

[0007] In other words, this disclosure is defined by the following components: [1] A method for raising mollusk larvae and / or juvenile mollusks by culturing photosynthetic microorganisms in a culture medium prepared by dissolving combustion ash with acid, irradiating them with light under aerobic conditions, and feeding them the cultured photosynthetic microorganisms. [2] The method for raising mollusks according to [1], wherein the culture medium contains a chelating agent. [3] The method for raising mollusks according to [1] or [2], wherein the NaCl concentration of the culture medium is lower than the NaCl concentration in natural seawater. [4] The method for raising mollusks according to any one of [1] to [3], wherein the photosynthetic microorganisms cultured in the culture medium contain triacylglycerol, and the concentration of said triacylglycerol is 150 mg / L or more. [5] The method for raising mollusks according to any one of [1] to [4], wherein in addition to the photosynthetic microorganisms, other microorganisms are also fed to the mollusk larvae and / or juvenile mollusks, and the amount and / or ratio of the photosynthetic microorganisms and other microorganisms is changed according to the growth stage of the mollusk larvae. [6] The cultivation method according to any one of [1] to [5], wherein the combustion ash is combustion ash of woody biomass. [7] The cultivation method according to any one of [1] to [6], wherein the combustion ash contains 5 to 40% by mass of potassium, 1 to 10% by mass of phosphorus, and 0.1 to 10% by mass of iron in terms of oxides, and substantially contains no nitrogen. [8] The cultivation method according to any one of [1] to [7], wherein the acid contains at least nitric acid and sulfuric acid and / or hydrochloric acid. [9] The cultivation method according to any one of [1] to [8], wherein the pH of the culture solution is 4 to 8.

[10] The cultivation method according to any one of [1] to [9], wherein the nitrogen atom concentration of the culture solution is 10 to 50 mg / L, the phosphorus atom concentration is 0.1 to 10 mg / L, and the potassium concentration is 10 to 200 mg / L.

[11] The cultivation method according to any one of [1] to

[10] , wherein the shellfish is oysters or clams.

[12] A culture medium for the growth of shellfish larvae or juvenile shellfish, which is an acidic solution of combustion ash.

[13] The culture medium according to

[12] , wherein the nutrients in the acidic solution are a nitrogen atom concentration of 10 to 50 mg / L, a phosphorus atom concentration of 0.1 to 10 mg / L, and a potassium concentration of 10 to 200 mg / L.

[14] The culture medium according to

[12] or

[13] , further containing a chelating agent.

[15] The medium according to any one of

[12] to

[14] , wherein the NaCl concentration is lower than the NaCl concentration in natural seawater.

[16] Triacylglycerol is 18×10 per cell on average. －１２ A feed for growing shellfish larvae or juvenile shellfish, which is a photosynthetic microorganism containing g / cells or more of triacylglycerol.

[17] The feed according to

[16] , which is an alga.

[18] The feed according to

[16] or

[17] , which is cultured by irradiation with light under aerobic conditions in a culture solution prepared by dissolving combustion ash with an acid. In the present specification, the term "combustion ash" is used regardless of whether it has been conventionally regarded as waste, and includes incineration ash. The term "larva" refers to shellfish in the planktonic stage after hatching, and the term "juvenile shellfish" refers to shellfish in a state where larvae have metamorphosed and settled.

[0008] According to the present disclosure, shellfish larvae and juvenile shellfish can be grown efficiently, and for example, the shell height can be increased and the number of surviving individuals can be increased. Further, since a culture solution derived from combustion ash is used, effective utilization of waste is possible, and an aquaculture method that is friendly to the global environment can be provided.

[0009] FIG. 1 is a conceptual diagram showing an example of the shellfish rearing method of the present disclosure. FIG. 2 is a graph showing an example of the results of culturing the feed according to the present disclosure. FIG. 3 is a graph showing an example of a feeding program during the breeding of shellfish according to the present disclosure. FIG. 4 is a graph showing an example of a change in feed density during the breeding of shellfish according to the present disclosure. FIG. 5 is a graph showing an example of the results of breeding shellfish according to the present disclosure. FIG. 6 is a graph showing another example of the results of breeding shellfish according to the present disclosure. FIG. 7 is a graph showing another example of the results of culturing the feed according to the present disclosure. FIG. 8 is a graph showing still another example of the results of culturing the feed according to the present disclosure. FIG. 9 is a graph showing another example of the results of culturing the feed according to the present disclosure. FIG. 10 is a graph showing still another example of the results of culturing the feed according to the present disclosure. FIG. 11 is a schematic perspective view showing an example of an apparatus used for culturing the feed according to the present disclosure. FIG. 12 is a graph showing an example of the results of culturing the feed according to the present disclosure. FIG. 13 is a graph showing still another example of the results of breeding shellfish according to the present disclosure.

[0010] (1) Culture medium for feed (1.1) Combustion ash In this disclosure, a culture medium is prepared by dissolving combustion ash in acid, and feed is cultured using this culture medium (also called a combustion ash medium), and the obtained feed is used to raise shellfish larvae and juvenile shellfish. When feed is cultured using a combustion ash medium, the feed can be cultured with the same efficiency as with a normal medium. When shellfish larvae and juvenile shellfish are raised using the obtained feed, the shellfish larvae and juvenile shellfish can be raised more efficiently than when feed obtained from a normal medium is used. Furthermore, by using combustion ash as inorganic nutrient and mineral components in the culture medium, waste can be effectively utilized and contribute to resource conservation.

[0011] The combustion ash may be combustion ash from factory emissions (including incineration ash), ash discharged when burning solid fuels such as coal 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] Among the various types of combustion ash, woody biomass combustion ash is preferred. 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. Woody biomass combustion ash has a high potassium concentration, and the resulting culture medium and the feed (algae) cultivated in this medium also have a high potassium concentration. It is presumed that feeding shellfish with this high-potassium feed (algae) is particularly suitable for raising shellfish. Furthermore, by effectively utilizing woody biomass as a culture medium, it is possible to increase the contribution to a low-carbon society.

[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, Fe２ O ３ was calculated as follows. The amount is expressed based on 100 parts by mass in total of carbon, halogen atoms, and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides and oxygen). 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 SiO ２ : 5 to 25 mass%, preferably 10 to 20 parts by mass P ２ O ５ : 1 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: 5 to 40 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 10 parts by mass, preferably 2 to 5 parts by mass ZnO: 0.01 to 1 part by mass, preferably 0.01 to 0.5 part 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.: each 1 part by mass or less, preferably 0.1 part by mass or less. Substantially no nitrogen is contained.

[0015] As described above, phosphorus (P ２ O ５ ) and iron (Fe ２ O ３By including a predetermined amount of ), the conditions become particularly suitable for cultivating shellfish feed (algae). However, since combustion ash does not contain substantially any nitrogen, it is preferable to supplement the nitrogen with nitrogen-containing components such as nitrate, as will be described later.

[0016] (1.2) Acid The combustion ash is dissolved in an acid to make a culture medium. Dissolving in an acid allows inorganic nutrients and mineral components to be dissolved as ions. The acid is not particularly limited and can be any of the following: mineral acids, organic acids, carbonates, etc. 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. Combustion ash is often deficient in nitrogen, so by including nitric acid in the dissolving acid, the nitrogen component can be supplemented. 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, the FeO contained in the combustion ash is dissolved by Fe ２＋ This allows for efficient dissolution. By dissolving Fe in its divalent state, it contributes to the growth of feed (microorganisms).

[0017] 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 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.

[0018] When dissolving combustion ash, the acid concentration in the aqueous acid 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 aqueous 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 serving is 0.5 to 50 kg, preferably 2 to 25 kg, and more preferably 5 to 15 kg.

[0019] (1.3) The acid solution of the combustion ash may contain a chelating agent. By including a chelating agent, the efficiency of recovering Fe as divalent in the combustion ash can be increased, and the growth rate of the feed can be increased.

[0020] 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.

[0021] 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. Chelating agents with particularly excellent safety are preferred, and examples include hydroxyl group-containing polycarboxylic acids such as citric acid.

[0022] 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.

[0023] (1.4) 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 5.5 to 6.5. By not raising the pH too high, it is possible to prevent divalent iron from becoming trivalent. If the pH becomes too high, acid may be added to lower the pH to the above range.

[0024] 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, the amounts of components other than carbon are shown. Also, in the following, halogen atoms are excluded and the amounts are shown as the mass of oxides. Iron oxide is expressed without distinguishing between divalent and trivalent iron oxides. ２ O ３ The calculation was performed as follows. The amount was then expressed as the sum of halogen atoms and oxides of elements from 5B to 92U (excluding carbon oxides, nitrogen oxides, and oxygen) with a total of 100 parts by mass.

[0025] Cl atoms: 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 atoms, Al ２ O ３ SiO ２ , P ２ O ５ , TiO ２ , Cr ２ O３ , NiO, CuO, ZnO, As ２ O ３ , Rb ２ O, SrO, Y ２ O ３ , ZrO ２ Nb ２ O ５ PbO, BaO: 0.3 parts by mass or less each, preferably 0.03 parts by mass or less.

[0026] The nitrogen atom concentration in the culture medium is, for example, 10 to 50 mg / L, the phosphorus atom concentration is, for example, 0.1 to 10 mg / L, and the potassium concentration is, for example, 10 to 200 mg / L. ４ 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.

[0027] The NaCl concentration in the culture medium can be appropriately set depending on the microorganism being cultured, and may be freshwater, brackish water, or seawater. Furthermore, microorganisms that grow in seawater may be cultured at a lower NaCl concentration than that of natural seawater, for example, 0-2.2% by mass, 0.1-1.5% by mass, or 0.5-1.0% by mass. Note that when cultivating microorganisms that grow in seawater in water with a lower NaCl concentration than that of natural seawater, other components (for example, the above-mentioned MgO, SO4) may be present. ３ _K ２ O, CaO, MnO, Fe ２ O ３ It is preferable that the content of these components is equivalent to that of natural seawater (for example, within ±30% of the content of each component in natural seawater).

[0028] Furthermore, if the culture medium contains a chelating agent, the amount of chelating agent dissolved is K ２For 19 parts by mass of O, the amount 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. Also, for 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 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. The concentration during cultivation is, for example, 13 to 38 mg / 1000 mL, preferably 18 to 33 mg / 1000 mL, and more preferably 23 to 28 mg / 1000 mL.

[0029] (5) Adjustment of concentration and components The acid solution of combustion ash obtained as described above can be used as a culture medium for feed. The pH of the culture medium is, for example, 4 to 8, preferably 5.0 to 7.5, and more preferably 5.5 to 6.5. In this specification, the culture medium includes not only the acid solution of combustion ash, but also concentrated or diluted solutions as needed, and also includes solutions with additional necessary components.

[0030] 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, artificial seawater, or low-salinity seawater, is preferred as the dilution water. Low-salinity seawater refers to water from which some or all of the NaCl has been removed, while other components are substantially maintained. Since the feed for shellfish such as oysters, whose habitat is brackish or seawater, is often cultured in brackish or seawater, the dilution water should be seawater derived from seawater, such as seawater, artificial seawater, or low-salinity seawater, in order to adapt the culture of the feed to brackish or seawater. When diluting with water derived from seawater, it is preferable to use low-salinity seawater. Diluting with low-salinity seawater allows for the reduction of reagents and the effective utilization of wastewater compared to using seawater or artificial seawater. Using a culture medium derived from combustion ash, it is possible to cultivate marine microorganisms for use as feed at a cultivation rate comparable to that of conventional culture media, even if the chlorine atom concentration is lower than that of seawater.

[0031] Furthermore, whether or not dilution is performed, the culture medium may be appropriately supplemented with active ingredients. Examples of active ingredients include NaCl and FeSO4. ４ 7H ２ Fe salts such as O, CuSO ４ 7H ２ It contains Cu salts such as O. Adding active ingredients can promote cultivation. In this disclosure, since the culture medium is made by dissolving combustion ash with acid, the feed can be cultivated even without adding active ingredients.

[0032] (2) Feed (photosynthetic microorganisms) The culture medium is used to cultivate feed, which is photosynthetic microorganisms, under aerobic conditions by irradiating it with light. Feed cultivated in the culture medium can promote the growth of shellfish compared to feed cultivated in conventional feed. Furthermore, cultivating the feed can also contribute to the fixation of carbon dioxide from the air.

[0033] The aforementioned feed (photosynthetic microorganisms) includes algae. The algae may be species that grow in freshwater, brackish water, or saltwater, but species that grow in brackish water or saltwater are preferred. Many shellfish targeted for aquaculture inhabit brackish water or saltwater, and using feed that grows in brackish water or saltwater allows the feed and the shellfish's habitat to be matched, and the shellfish can be fed algae, which are their primary food source.

[0034] Examples of the aforementioned algae include diatoms, haptophytes, euspot algae, and prasinophytes. Diatoms include the genus Chaetoceros (diatoms, centroidal order), such as Chaetoceros calcitrans and Chaetoceros gracilis, and the genus Pheodaguchilum (diatoms, pennate order), such as Pheodaguchilum tricornutum. Haptophytes include the genus Pavlova, such as Pavlova lutheri, and the genus Isochrysis, such as Isochrysis sp. and Isochrysis galbana. The true eyespot algae include the genus Nannochloropsis, such as Nannochloropsis sp. The Prasinoal algae include the genus Tetraselmis, such as Tetraselmis sp.

[0035] Diatoms are useful as feed for oysters (such as Pacific oysters and rock oysters), clams, pearl oysters, surf clams, cockles, and ark clams. Haptophytes are useful as feed for pearl oysters, ark clams, and scallops. Eyespot algae are useful as feed for mussels. Prasinophytes are useful as feed for pearl oysters. The preferred species to cultivate are bivalves, preferably oysters and clams, and especially preferably oysters.

[0036] The concentration of triacylglycerol contained in the feed (algae, etc.) cultured in the culture medium is, for example, 18 (×10 －１２ g / cells) or more, preferably 19 to 200 (x 10 －１２ g / cells), more preferably 20 to 100 (×10 －１２ The triacylglycerol concentration is g / cells. The feed solution is, for example, 150 mg / L or more, preferably 180 to 700 mg / L, and more preferably 200 to 500 mg / L. The higher the triacylglycerol concentration, the better the nutritional value of the feed. The cell density of the feed is, for example, 3 to 30 (×10 ６ cells / mL), 5-20 (×10６ cells / mL), 7-15 (×10 ６ The concentration is (cells / mL). The turbidity (OD730) of the feed is, for example, 0.5 to 3.0, preferably 0.8 to 2.0, and more preferably 1.0 to 1.5. However, in order to reduce transportation costs and to facilitate the evaluation of the number of cells being fed, these feed concentrations are set to 1 (×10). ９ It may be used after being concentrated to approximately cells / mL. Furthermore, the feed is preferably from the culture period from the logarithmic growth phase to the stationary phase, and more preferably from the logarithmic growth phase.

[0037] (3) Rearing of shellfish The feed cultured in the culture medium can be used to rear shellfish by feeding it to shellfish larvae and / or juvenile shellfish. For rearing shellfish whose habitat is the sea, it is preferable to use seawater, for example. During rearing, aeration may be performed and the water may be changed as needed. The feed may be given together with the culture medium in which the feed was cultured. The feeding frequency is, for example, 0.5 to 3 times / day, preferably 0.8 to 2 times / day. Alternatively, it may be added over a period of several hours (for example, 1 to 5 hours). Furthermore, multiple types of cultured feed (photosynthetic microorganisms) may be given in combination, or the cultured feed (photosynthetic microorganisms) may be given in combination with other microorganisms. When giving two or more types of microorganisms, the amount and / or ratio of them may be changed according to the growth stage of the shellfish larvae and / or juvenile shellfish.

[0038] The oysters targeted for cultivation in this disclosure include domestic Pacific oysters, domestic rock oysters, and oysters from overseas, regardless of species, and can be used if natural seedlings of these species are available. Furthermore, artificial seedlings obtained by fertilizing the eggs of these adult oysters can also be used.

[0039] This application claims the benefit of priority based on Japanese Patent Application No. 2025-040241, filed on 13 March 2025. The entire specification of Japanese Patent Application No. 2025-040241, filed on 13 March 2025, is incorporated herein by reference.

[0040] 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.

[0041] In the following examples, artificial seawater (Osaka Yakken Marine Art SF-1) was used as a substitute for natural seawater, as it has the same function as natural seawater. Synthetic desalination seawater was also used to simulate brackish water. Several types of synthetic desalination seawater with different NaCl concentrations (synthetic desalination seawater 1 to 5) were used. Specifically, five main components other than NaCl from the artificial seawater preparation reagent were mixed with water, and a predetermined amount of NaCl was added to prepare the seawater. The components of the synthetic desalination seawater are shown in Table 1.

[0042]

[0043] In addition, the following culture media and feed were used in the example. Culture Media 1-1: The components of the F / 2 medium of Gillard are shown in Table 2 below.

[0044] Culture medium 1-2: F medium - the nutrient concentration of the F / 2 medium has been doubled.

[0045] Culture medium 1-3: IMK seawater medium. 50.4 mg of IMK standard medium (manufactured by Nippon Pharmaceutical Co., Ltd., DAIGO-IMK medium) shown in Table 3, 11.368 mg of sodium metasilicate pentahydrate, and 7.2 g of artificial seawater (Osaka Yakken Marine Art SF-1) were diluted in 200 mL of distilled water, sterilized by autoclaving, and prepared as IMK seawater medium.

[0046] Culture medium 1-4: IMK seawater medium with citric acid. In the IMK seawater medium shown in Table 3, citric acid was added to a concentration of 25.3 mg / 1000 mL to prepare IMK seawater medium with citric acid.

[0047] Culture medium 2-1: Combustion ash seawater medium with 0.1 mol / L HNO ３ , 0.1 mol / L H ２ SO ４A mixed acid (33.3 mL) prepared by mixing HCl and 0.1 mol / L HCl in a volume ratio of 25.2:3.0:5.1 was placed in a beaker and stirred with a magnetic stirrer for 5 minutes. 0.53 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 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 6.0 to 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 was diluted 30 times with distilled water (to a volume of approximately 1 L after dilution), and 3.6 g of artificial seawater (Osaka Yakken Marine Art SF-1) was added to prepare a culture medium (combustion ash seawater medium, salinity 3.5% by mass, NaCl concentration 2.2% by mass). Of the original 100 parts by mass of woody biomass combustion ash, approximately 51 parts by mass were removed, and approximately 49 parts by mass were 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 as shown in Table 4. The amounts in Table 4 are the amounts (by 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.

[0048] 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 C 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 is maintained in the ratio of each component in the mixed acid residue. Then, by subtracting the value of Conversion 1 from the combustion ash analytical value, the amount of each component in the supernatant was determined (Conversion 2). Based on the result of Conversion 2, the component composition of supernatant 1 (total of all components in 100 mass%) was determined (Conversion 3). In the calculation result of Conversion 3, considering that there was an error of less than 4% in the amount of combustion ash removed during the process, unreliable digits were removed, 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 4. According to Table 4, phosphorus (P), a nutrient, is... 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.

[0049]

[0050] Culture media 2-2 to 2-6: Citric acid-containing desalted seawater media 1 to 5 The same procedure as for culture media 2-1 was followed, except that 25.3 mg of citric acid was added to the mixed acid (33.3 mL) (resulting in 4.8 parts by mass of citric acid per 100 parts by mass of incineration ash), and the 3.6 g of artificial seawater used for dilution was replaced with 3.6 g of synthetic desalted seawater 1 to 5. The citric acid concentration in citric acid-containing desalted seawater media 1 to 5 was 25.3 mg / 1000 mL. Furthermore, the NaCl concentration in citric acid-containing desalination seawater medium 1 was 2.2% by mass, the NaCl concentration in citric acid-containing desalination seawater medium 2 was 1.5% by mass, the NaCl concentration in citric acid-containing desalination seawater medium 3 was 1.0% by mass, the NaCl concentration in citric acid-containing desalination seawater medium 4 was 0.5% by mass, and the NaCl concentration in citric acid-containing desalination seawater medium 5 was 0.0% by mass.

[0051] Pre-culture medium 1: Pre-culture medium for Chaetoceros gracilis. Natural Chaetoceros gracilis isolated from the Seto Inland Sea was inoculated into IMK seawater medium (medium 1-3), and aerated culture was performed for 14 to 28 days under conditions of 25°C or below with fluorescent lighting (16000 Lux, continuous light) to obtain pre-culture medium (pre-culture medium 1).

[0052] Feed 1: Gracilis culture medium (combustion ash seawater medium) 2 L of combustion ash seawater medium (medium 2-1) was added to a screw-top bottle, and 50 mL of the above pre-culture solution 1 (Gracilis pre-culture solution) was inoculated. Culture (main culture) was carried out for 14 days under conditions of culture temperature 25°C, fluorescent light illumination (16000 Lux, continuous light), and air flow rate 0.8 L / min (continuous aeration). The cell density of the culture medium during culture was measured using a cell counter (LOGOS BIOSYSTEMS, LUNA-II), and it was confirmed that the period from 6 to 10 days from the start of culture corresponds to the logarithmic growth phase to the stationary phase. Cultured cells in this period from 6 to 10 days from the start of culture were used as Feed 1. When performing Example 1 and Comparative Example 1 described later, the cultivation of Gracilis using this combustion ash seawater medium was carried out in parallel with multiple lines with staggered start times, as shown in Figure 1, so that Feed 1 corresponding to the period from 6 to 10 days from the start of culture (logarithmic growth phase) could be used at the necessary timing.

[0053] Feed 2: Culture of Chaetoceros gracilis in IMK seawater medium Except for changing the culture medium used for the main culture from combustion ash seawater medium (medium 2-1) to IMK seawater medium (medium 1-3), Chaetoceros gracilis was cultured in the same manner as in Feed 1 to obtain Feed 2. The culture results of Feed 1 and Feed 2 are shown in Figure 2. As is clear from Figure 2, when using combustion ash seawater medium, Chaetoceros gracilis could be cultured in the same way as when using IMK seawater medium.

[0054] Feed 3: Chaetoceros calcitrans (500 million cells / mL) manufactured by Calcitrans ISC.

[0055] Example 1, Comparative Example 1 (Cultivation of Pacific Oysters) Two tanks (45 cm wide x 30 cm deep x 30 cm high) were prepared, each containing 20 L of artificial seawater (Marine Art SF-1 dissolved in distilled water at a concentration of 36 g / L) (Tank 1, Tank 2). Mature Pacific oysters (Magallana gigas) were harvested by dissection to collect eggs and sperm, and fertilized. 90,000 planktonic larvae (Type D larvae) 24 hours after fertilization were placed in Tank 1 and Tank 2 respectively, and reared in still water with aeration at 25°C room temperature and 0.1 L / min. However, 6 L of water was changed once a day. The day the larvae were placed in the tank was considered day 0, and rearing continued until day 15. Feeding was done once a day in the afternoon. In one tank (Example 1), feed 1 (Chaetoceros gracilis cultured in burnt ash seawater medium) and feed 3 were provided, while in the other tank (Comparative Example 1), feed 2 (Chaetoceros gracilis cultured in IMK seawater medium) and feed 3 were provided. The ratio of feed 1 or feed 2 to feed 3 was varied according to the number of days of growth. The relationship between the two is shown in Figure 3. The feeding volume was adjusted so that the number of cells was the same in tanks 1 and 2. The number of feeding cells was gradually increased in accordance with the growth of the larvae in the tanks. Specifically, the amount of feeding was adjusted while checking the turbidity of the seawater. The relationship between the density of feed cells (Chaetoceros gracilis, Chaetoceros calcitrans) in the tanks and the number of days of growth is shown in Figure 4.

[0056] Larval growth was assessed by randomly selecting 5 to 72 larvae from each of the 6 L of seawater (reservation water) collected during water changes in tanks 1 and 2. After fixing them in formalin, their shell heights were measured under a microscope, and the average value was calculated. In addition, the number of individuals was determined by taking 1 mL of four-fold concentrated rearing water on a framed border slide glass (Matsunami Glass, S6300) and counting the number of cells under a microscope. The relationship between growth days and shell height is shown in Figure 5, and the relationship between growth days and the number of individuals is shown in Figure 6.

[0057] Comparative Examples 2-5 and Examples 2-6 (Feed Preparation Stage) In Comparative Examples 2-5 and Examples 2-6, Chaetoceros gracilis was cultured in test tubes using the culture medium shown in Table 5 below, and its usefulness as a feed for Pacific oysters was evaluated.

[0058]

[0059] In each comparative example and example, the feed was cultured using two test tubes. The volume of the culture medium was 80 mL, and 2.4 mL of Chaetoceros gracilis pre-culture solution 1 was added. The culture was carried out in an incubator at a temperature of 25°C, with a light intensity of 14,000 Lux and an air supply rate of 0.035 L / min. To maintain a constant NaCl concentration in the test tubes, distilled water was added once a day to keep the culture water level constant.

[0060] During the culture period, 1.5 mL of culture medium was taken at a time, and its turbidity (OD730) was measured. The difference between the turbidity before and after culturing was used to evaluate cell growth. Cell density at the end of the culture period was also measured using a cell counter.

[0061] Furthermore, the usefulness of the culture medium as oyster feed was evaluated by measuring the amount of lipids (triacylglycerol, hereafter TAG) contained in the culture medium using the following method. 1.6 mL of the culture medium was taken into an Eppendorf tube and centrifuged at 10,000 rpm for 20 minutes in an Eppendorf 5425 centrifuge. 1.4 mL of the supernatant after separation was removed, and the remaining 0.2 mL was used as an 8-fold concentrated sample. A triglyceride kit (Fujifilm Wako Pure Chemical Industries) was used as the colorimetric analysis reagent. 1.5 mL of colorimetric reagent was added to the Eppendorf tube containing 0.2 mL of the sample, stirred with a vortex mixer for 10 seconds, and allowed to stand for 20 minutes. After standing, it was transferred to a cuvette, and the absorbance at a wavelength of 600 nm was measured using a spectrophotometer (JASCO V-730). A calibration curve (coefficient of determination R) prepared in advance according to the instructions was used. ２ The TAG concentration was calculated from (= 0.9997).

[0062] Figure 7 shows the culture results (turbidity and error bars) at the end of cultivation (360 hours) for standard media (Comparative Examples 2 and 3), commercially available media (Comparative Example 4), and its improved media (Comparative Example 5). Among these comparative examples, the growth in F medium (Comparative Example 3) was the best, while the growth rate in IMK medium (Comparative Example 4) was intermediate between that of F medium (Comparative Example 3) and F / 2 medium (Comparative Example 2). Furthermore, the example in which citric acid was added to IMK medium (Comparative Example 5) was inferior to that of IMK medium (Comparative Example 4).

[0063] Figure 7 also shows the culture results (turbidity and error bars) at the end of cultivation (360 hours) for the culture media using incinerated ash (Examples 2-6). The culture media of Examples 2-5 showed better growth than the F medium containing NaCl (Comparative Example 3).

[0064] Table 6 shows the turbidity, cell density, TAG concentration, and TAG amount per cell at the end of culture (360 hours) for Comparative Examples 2-5 and Examples 2-6. The values ​​shown in Table 6 are the average values ​​from two test tubes. Figures 8, 9, and 10 show the cell density and error bars, TAG concentration and error bars, and TAG amount per cell, respectively.

[0065]

[0066] In a comparison of standard media (Comparative Examples 2-3), commercially available media (Comparative Example 4), and its improved version (Comparative Example 5), Medium F (Comparative Example 3) showed the best results in terms of cell density and TAG concentration. Among the media using combustion ash (Examples 2-6), Examples 2-4 (NaCl concentration 2.2-1%) surpassed Medium F (Comparative Example 3) in both cell density and TAG concentration. Examples 5-6 (NaCl concentrations 0.5% and 0%) showed lower cell densities than Medium F (Comparative Example 3), but exhibited significantly higher TAG concentrations. Thus, the maximum TAG per cell was 56.0 × 10⁶ in Example 6 (NaCl concentration 0%). －１２ g / cells was obtained. These results indicate that the culture medium using combustion ash (Examples 2-6) can reduce the amount of NaCl contained in seawater due to the Na and Cl elements contained in the supernatant. It was also found that the environmental stress of low NaCl concentration increases the amount of TAG accumulated in cells. From this, it was found that the amount of NaCl in marine algae culture can be reduced by using a culture medium using combustion ash, thereby reducing culture costs.

[0067] In Example 7 and Comparative Example 6, two culture packs (Otsuka Techno, OT-algae) with a diameter of 120 mm and a height of approximately 1600 mm were used. One was filled with the IMK seawater medium (medium 1-3), and the other was filled with citric acid-containing incinerated ash desalted seawater medium 1 (NaCl 2.2%) (medium 2-2). Without autoclaving, Chaetoceros gracilis pre-culture solution 1 was inoculated into each, and the cultures were incubated for 14 days under conditions of fluorescent light illumination (15000 Lux, continuous light) and an airflow rate of 0.13 L / min (continuous aeration). A schematic perspective view of the culture apparatus is shown in Figure 11, and the culture results are shown in Figure 12.

[0068] Up to 50 hours, the turbidity of IMK seawater medium (medium 1-3) was higher, but at 100 hours, the turbidity of desalted seawater medium 1 with citric acid (medium 2-2) became higher. The results at the end of culture are shown in Table 7. Both turbidity and cell density were higher in desalted seawater medium 1 with citric acid. Furthermore, microscopic observation of the culture media after cultivation revealed no contamination in either IMK seawater medium or desalted seawater medium 1 with citric acid, confirming that only Gracilis cells were cultured. The obtained culture medium is useful as feed for Pacific oysters.

[0069]

[0070] Example 8: On September 1, 2025 (day 0), eggs and sperm were collected from Pacific oysters by dissection, and these were used for fertilization. Seawater sterilized with chlorine and filtered with sand (raw seawater) was placed in a 500L tank, and cultivation was started.

[0071] On September 2nd (1 day old), 600,000 D-type larvae were confirmed. Feeding with Chaetoceros callistrans was started. On September 7th (6 days old), 389,000 larvae were confirmed. In addition to callistrans, feeding with commercially available gracilis (manufactured by Yanmar Co., Ltd.) was started from this day. In Example 1, it was confirmed that planktonic larvae could be raised until just before settling using feed 1 (gracilis cultured in burnt ash seawater medium), so in this example, commercially available gracilis was used until settling. On September 18th (17 days old), individuals with a shell height exceeding 300 μm appeared, so PET bottles were added to help them settle. From this day on, the feed was switched to feed 1 (gracilis cultured in burnt ash seawater medium), and from then on, only feed 1 was fed as gracilis. The feed 1 used in Example 8 is the same as the feed 1 used in Example 1, except that a 1 L culture bottle was used instead of a 2 L screw-top bottle. This feed 1 was cultured with staggered start dates, and the fact that it was used to feed Pacific oysters during the culture period from the logarithmic growth phase to the stationary phase is the same as that used in Example 1. The amount of feed was adjusted daily by observing the tank 24 hours after the previous day's feeding; if any Gracilis feed remained, the amount of feed was reduced, and if none remained, the amount of feed was increased. At 21 days old (September 22), there were 17,500 planktonic larvae. It is estimated that approximately 300,000 larvae settled between 18 and 22 days old. From 30 days old onward, artificial seawater (*1) was used instead of raw seawater. At 62 days old (November 22), individuals with a shell height exceeding 10 mm were observed. The above rearing conditions are summarized in Table 8. Figure 13 shows the changes in shell height and shell length as the age of the Pacific oyster larvae changes. Artificial seawater (*1): Marine Art SF-1 (manufactured by Osaka Yakken Co., Ltd.) is dissolved in tap water at a concentration of 36 g / L and neutralized with a sodium thiosulfate aqueous solution (concentration 0.1 g / L).

[0072]

[0073] According to this disclosure, useful shellfish can be cultivated.

Claims

1. A method for cultivating photosynthetic microorganisms in a culture medium prepared by dissolving combustion ash in acid, irradiating it with light under aerobic conditions, and feeding the cultured photosynthetic microorganisms to shellfish larvae and / or juvenile shellfish.

2. The cultivation method according to claim 1, wherein the culture medium contains a chelating agent.

3. The cultivation method according to claim 1, wherein the NaCl concentration of the culture medium is lower than the NaCl concentration in natural seawater.

4. The cultivation method according to claim 1, wherein the photosynthetic microorganisms cultured in the culture medium contain triacylglycerol, and the concentration of said triacylglycerol is 150 mg / L or more.

5. The cultivation method according to claim 1, wherein, in addition to the photosynthetic microorganisms, other microorganisms are also fed to the larvae and / or juvenile shellfish, and the amount and / or ratio of the photosynthetic microorganisms and other microorganisms is changed according to the growth stage of the larvae and / or juvenile shellfish.

6. The cultivation method according to claim 1, wherein the combustion ash is combustion ash of woody biomass.

7. The cultivation method according to claim 1, wherein the combustion ash contains 5 to 40% by mass of potassium, 1 to 10% by mass of phosphorus, and 0.1 to 10% by mass of iron, in terms of oxides, and substantially contains no nitrogen.

8. The growth method according to claim 1, wherein the acid comprises at least nitric acid and sulfuric acid and / or hydrochloric acid.

9. The cultivation method according to claim 1, wherein the pH of the culture medium is 4 to 8.

10. The cultivation method according to claim 1, wherein the nitrogen atom concentration of the culture medium is 10 to 50 mg / L, the phosphorus atom concentration is 0.1 to 10 mg / L, and the potassium concentration is 10 to 200 mg / L.

11. The cultivation method according to claim 1, wherein the shellfish is oyster or clam.

12. A culture medium for the growth of shellfish larvae or juvenile shellfish, which is an acidic solution of combustion ash.

13. The culture medium according to claim 12, wherein the nutrients in the acid solution have a nitrogen atom concentration of 10 to 50 mg / L, a phosphorus atom concentration of 0.1 to 10 mg / L, and a potassium concentration of 10 to 200 mg / L.

14. The culture medium according to claim 12, further comprising a chelating agent.

15. The culture medium according to claim 12, wherein the NaCl concentration is lower than the NaCl concentration in natural seawater.

16. Triacylglycerols per cell average 18 × 10⁶ －１２ Feed for the growth of shellfish larvae or juvenile shellfish, containing photosynthetic microorganisms at a concentration of g / cells or higher.

17. The feed according to claim 16, wherein the feed is algae.

18. The feed according to claim 16, wherein the feed is cultured in a culture medium prepared by dissolving combustion ash in acid, under aerobic conditions, and irradiated with light.

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