Method for producing macroalgae seedling clusters

Abstract

The present invention addresses the problem of providing a technique for simply and efficiently producing macroalgae seedling clusters. The present invention solves the problem by providing a method for producing macroalgae seedling clusters that comprises: a step for obtaining a parent alga body that is substantially free from microalgae adhesion; and a step for culturing the parent alga body in which spore formation has induced to such an amount that the spore deposition density with respect to the inner wall area of a culture vessel is 103 spores / cm2 or more.

Description

Method for producing macroalgae agglomerate seedlings

[0001] The present invention relates to a method for producing agglomerated seedlings of macroalgae.

[0002] As a method for cultivating macroalgae such as seaweed on land, the onshore tank production of seaweed using agglomerated spores, as described in Patent Document 1, is widely practiced. This seaweed cultivation method is characterized by first seeding seaweed spores at high density on a flat plate and culturing them to form spore agglomerates in which multiple spores are linked together, or germinated spores in which the germinated spores are entangled, and then culturing the resulting agglomerates. This method allows the seaweed to grow in a suspended state in a culture solution, eliminating the need for a step of removing the seaweed from the solid phase during harvesting, making harvesting easier than conventional methods.

[0003] However, this method requires a large amount of spore purification and collection processes using spore phototaxis and centrifugation to obtain a high-density spore suspension, which is labor-intensive and time-consuming, and results in spore loss.

[0004] Patent No. 3828359

[0005] An object of the present invention is to provide a technique for easily and efficiently producing agglomerate seedlings of macroalgae.

[0006] In view of the above problems, the present inventors have conducted extensive research and found that by isolating mother algae bodies that are substantially free of attached microalgae and adjusting the culture density of the mother algae bodies before releasing spores, it is possible to omit the cumbersome process of purifying and collecting large amounts of spores and simply and efficiently produce macroalgae agglomerate seedlings.Further research based on this finding led to the completion of the present invention.

[0007] That is, the present invention includes the following aspects.

[0008] Item 1. A method for producing agglomerated seedlings of macroalgae, comprising: (1) a step of isolating and growing mother algae bodies; (2) a step of inducing spore formation in the isolated and grown mother algae bodies; and (3) culturing the mother algae bodies after spore formation in a culture vessel such that the spore settlement density relative to the inner wall area of ​​the vessel is 10 ３ pieces / cm ２Item 2. A method for producing agglomerate seedlings of macroalgae, comprising (a) a step of adjusting the density to an amount equal to or greater than 1000 kJ / cm, and (b) a step of forming agglomerates of spores and / or germinated bodies obtained in the culture vessel. Item 2. The production method according to Item 1, further comprising one or more steps selected from the group consisting of a step of culturing fine fragments of the mother algae, a step of culturing the mother algae at 20°C or higher, and a step of culturing under dark conditions. Item 3. The production method according to Item 1 or 2, wherein the macroalgae is a seaweed belonging to any of the orders Hilocatorales, Ulvales, Laminariales, Paleophytales, and Bangiocarpus. Item 4. The production method according to any of Items 1 to 3, wherein the macroalgae is a seaweed belonging to any of the genus Metastases, Ulva, Laminariales, Undaria pinnatifida, Cladophora, Porphyra, or Porphyra. Item 5. Item 6. A method for culturing macroalgae, comprising a step of agitating and culturing clumped seedlings obtained by the method according to any one of Items 1 to 4. Item 7. A method for culturing macroalgae, comprising a step of agitating and culturing clumped seedlings obtained by the method according to any one of Items 1 to 4.

[0009] According to the present invention, the step of purifying and collecting a large amount of spores in the spore agglomeration method can be omitted, thereby making it possible to produce agglomerated seedlings of macroalgae more simply and efficiently.

[0010] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

[0011] In one aspect, the present invention provides a method for producing a clump seedling of macroalgae, comprising: (1) a step of isolating and growing mother algae; (2) a step of inducing spore formation in the isolated and grown mother algae; and (3) a step of cultivating the mother algae in which spore formation has been induced in a culture vessel such that the spore settlement density relative to the inner wall area is 10 ３ pieces / cm ２ The present invention relates to a method for producing macroalgae clump seedlings (sometimes referred to as the "production method of the present invention" in this specification), which comprises (1) a step of adjusting the density to the amount above, and (2) a step of forming clumps of spores and / or germinated bodies obtained in the culture vessel. This method will be described below.

[0012] Step (1) Isolating and Growing Mother Algae Macroalgae are not particularly limited as long as they are algae, such as seaweed, whose algae grow into multicellular or polynuclear bodies. However, algae whose algae grow to about 1 cm or more are preferred, and seaweed that can be cultured in industrial quantities and that can be used as edible seaweed, useful substances such as pharmaceuticals, or raw materials for biofuels are particularly preferred. Examples of macroalgae include seaweeds such as green algae, red algae, and brown algae. Examples of green algae include seaweeds belonging to the Ulvales order, Ulva family, Ulva genus, and the Hibuliales order, Ulva family, Hibuliales genus. Examples of such seaweeds include Enteromorpha spp., Hibulia spp., Hibulia spp., Hibulia spp., Hibulia spp., Hibulia spp., Hibulia spp., and Hibulia spp. Examples of brown algae include seaweeds belonging to the Laminariales family, the Porphyrales family, the Porphyrales family, and the Sclerotiniales family, such as Laminaria japonica, Undaria pinnatifida, Kurome, Okinawan Cladosiphon okamuranus, Kishū-no-mozuku, Sclerotinia spp., and Habanori. Examples of red algae include seaweeds belonging to the Gelidaceae family, the Gigariniles family, the Fructaceae family, the Celastraceae family, the Bacillarioles family, the Porphyrales family, and the Gracilariales family, such as Porphyra genus, and examples of such seaweeds include Porphyra gracilaria, Porphyra porphyra, Gelidium nigricans, Tosakanori, Igisu, Gracilaria verrucosa, and Mafunori. Macroalgae may be used alone or in combination of two or more species. According to the production method of the present invention, agglomerated seedlings can be produced from any species of macroalgae or from algal bodies freshly collected from the field.

[0013] Spores are reproductive cells. Examples of spores include zoospores, gametes, zygotes, tetraspores, carpospores, uniospores, neutozoospores, and monospores. Spores may be of one type alone or a combination of two or more types. In this specification, germinated bodies refer to germinated spores.

[0014] By sowing spores at high density on a flat plate and allowing them to intertwine, a mat-like agglomeration of spores and / or germinated bodies, in which two or more spores are connected in a planar or three-dimensional manner, can be obtained (Patent Document 1). The spore agglomerations are peeled from the plate and, if necessary, finely crushed to a diameter of approximately 5 mm or less using a mixer or the like, and then transferred to a culture vessel and cultured with aeration to obtain agglomerated seedlings in which the germinated bodies intertwine and the algal bodies extend radially from the center. By culturing the agglomerated seedlings under agitation, the germinated bodies can be cultured while suspended in the culture solution without requiring adhesion to walls or carriers, eliminating the need for a process of removing the seaweed from the solid phase at harvest, making it easier to harvest the seaweed.

[0015] As used herein, microalgae refers to microscopic algae or microorganisms that live in water and perform photosynthesis, typically unicellular algae or microorganisms such as diatoms, dinoflagellates, and cyanobacteria. Microalgae are usually attached to macroalgae collected from the wild. Generally, the growth rate of microalgae is faster than that of multicellular macroalgae, and they compete with macroalgae for nutrients and light, reducing the growth efficiency of the macroalgae. Furthermore, in aquaculture, the commercial value of algae with attached microalgae is significantly reduced. After spore culture is initiated, it is difficult to physically separate microalgae from the spores or grown algae, or to selectively remove them using chemicals, etc. Therefore, in a method for cultivating macroalgae using spores as a starting material, it is preferable to take measures to prevent microalgae contamination before initiating spore culture.

[0016] The means for preventing the inclusion of microalgae in the spores is not particularly limited, and examples include chemically or physically removing the microalgae from the mother algae before spore formation, or recovering and washing the spores after they have been released from the mother algae, and then sowing them.The production method of the present invention has a high microalgae removal rate, is simple, does not affect spore growth due to the treatment, can be used regardless of the type or origin of microalgae or macroalgae, and further allows for the production of multiple agglomerate seedlings with a single treatment.From these viewpoints, it is preferable to obtain agglomerate seedlings by a method including a step of forming agglomerates of spores and / or germinated bodies obtained from isolated mother algae.

[0017] As used herein, "isolation" refers to obtaining macroalgal cells in which the number of organisms other than the target algae (e.g., microalgae) is reduced (preferably free of such organisms). Preferably, "isolation" refers to obtaining a spore-containing solution in which the number of organisms other than the target algae (e.g., microalgae) is reduced (preferably free of such organisms), and to seeding the spore-containing solution in a container and culturing the solution to obtain macroalgal cells in which the attachment of other organisms (e.g., microalgae) is reduced (preferably free of such organisms). By using isolated algal cells as mother algal cells to obtain spores, spores that are substantially free of microalgae contamination can be obtained. Spores that are substantially free of microalgae contamination can be determined, for example, by culturing the obtained spores for one week under the same conditions as those used to obtain clumped spores, and observing the culture medium microscopically, and preferably by culturing the obtained spores under the same conditions as those used to obtain clumped spores, and observing the culture medium microscopically, and

[0018] The macroalgae from which spores are isolated for growth into mother algae may be collected from the field or cultured strains, and either sporophytes, gametophytes, asexually reproducing strains, or sexually reproducing strains can be used. The state of the macroalgae used as the starting material is not particularly limited as long as it can form spores, and may be a portion of a macroalgae leaf or fragment, or a spore or germinated form. Spores obtained from the macroalgae or from the spores or germinated forms grown in a manner that minimizes contamination with microalgae can be isolated to obtain a spore-containing solution that does not contain organisms other than the target algae. A preferred isolation method is one that minimizes damage to the spores and does not affect their growth. For example, if the spores are phototactic, the microalgae adhering to the exterior can be washed by repeatedly irradiating a solution such as sterilized seawater or sterilized culture medium and a container containing the spores with light from one direction, allowing the spores to swim to the light-irradiated side or the opposite side of the container, and then replacing the sterilized seawater or the like with fresh water. If the spores are not phototactic, they can be collected under a microscope. They can be washed and isolated by suspending them in sterilized seawater, sucking up a small amount of spores with a pipette, and resuspending them in fresh sterilized seawater.

[0019] The number and duration of spore washing are not particularly limited as long as the microalgae can be substantially removed. For example, when washing by phototaxis, the spores may be allowed to swim in sterilized seawater by phototaxis two or more times. Here, "substantially removed" refers to the complete absence of microalgae contamination in the spores, and the removal to the extent that no increase in microalgae is observed during the period in which the washed spores are cultured and grown into mother algae. Whether microalgae have been substantially removed can be determined, for example, by culturing the washed spores and unwashed spores for one week under the same conditions as when obtaining clumped spores, and comparing them with unwashed spores, by observing the culture medium microscopically. Preferably, the washed spores and unwashed spores are cultured under the same conditions as when obtaining clumped spores, and comparing them with unwashed spores, by observing the culture medium microscopically, by observing the culture medium microscopically, by observing the culture medium microscopically.

[0020] By culturing and growing the isolated spores in sterilized seawater or a sterilized medium, isolated mother algae bodies substantially free of attached microalgae can be obtained. The culture conditions for the mother algae may be the same as those for the spores. The culture termination period can be appropriately selected from the period after the spores have grown into algae bodies, matured, and become capable of producing spores, until they have grown to a desired size. Furthermore, in the case of macroalgae (e.g., sexually regenerating strains of Hibiscus diffusus) in which spores released from the algae bodies develop into a microgeneration of approximately 0.01 to 1 cm, which then form spores that grow into large algae, the microgeneration may be used as the mother algae. In one embodiment, it is desirable to terminate the culture of the mother algae bodies after growing the algae bodies to 1 cm or more. Furthermore, in one embodiment, when the microgeneration is used as the mother algae, it is desirable to terminate the culture of the mother algae bodies after growing the microgeneration to 0.03 mm or more, preferably 0.05 mm or more. In one embodiment, the culture period from spores to mother algae bodies is, for example, 1 to several tens of days, 2 to 1 month, or 3 to 14 days. The mother algae obtained by growing the isolated spores may be referred to as mother algae body A. The isolated mother algae obtained can be stored for 30 days or more under conditions that prevent maturation (for example, low-temperature conditions below 15°C), and can be maintained by successively cultivating them under conditions that prevent contamination with microalgae.

[0021] Step (2): Inducing spore formation in the isolated and grown mother algae. Many macroalgae, such as seaweed, undergo vegetative growth at low water temperatures and mature and release spores at high water temperatures. By culturing the macroalgae at a temperature suitable for their maturation, spore formation can be induced in the macroalgae, resulting in the release of a large number of spores in a short period of time. The temperature during the temperature-raising treatment can be appropriately selected taking into account the known maturation temperature of the target macroalgae, and is typically approximately 20°C to 30°C. In the case of seaweeds, such as the Hibiscales and Bangiales, spore formation can also be induced by dark treatment, which involves culturing the algae under dark conditions for 1 to 10 days, preferably 1 to 7 days. After the dark treatment, it is preferable to agitate the container with a stirrer or the like to prevent the spores from concentrating and attaching to a part of the container. The dark treatment may be performed simultaneously with the temperature-raising treatment. In seaweeds such as those of the Ulvales order, spore formation can also be induced by physically wounding the algal cells and causing the sporulation inhibitors inside the algal cells to leak out of the algal cells. The wounds need only reach the interior of the algal cells, and fragmenting the cells to a length of approximately 1 to 20 mm, preferably approximately 2 to 10 mm, can more efficiently form spores. Culture can be initiated by directly adding the finely fragmented wound or fragmented algal cells to the medium, but it is preferable to wash the algal cells with sterilized seawater or distilled water before adding them to the medium. Wounding the algal cells may be performed simultaneously with the temperature increase treatment.

[0022] Step (3) Incubate the mother algae in which spore formation has been induced at a spore density of 10 to 10% relative to the inner wall area of ​​the culture vessel. ３ pieces / cm ２ By inducing spore formation in the obtained mother algae bodies using the method described in step (2) or the like, it is possible to obtain mother algae bodies that have formed a large amount of spores that are free from contamination by microalgae and do not require purification. The amount of spores released from algae bodies of a certain weight or area is determined in advance using any spore formation induction method, and the amount of spores released is determined based on the amount of spores released from the algae bodies of a certain weight or area, and the amount of spores released is determined based on the amount of spores released from the algae of a certain weight or area within 1 cm of the inner wall area of ​​the culture vessel. ２ 10 per ３ 10 or more, preferably 10 ４By placing an amount of algal cells that will release at least 100 spores into the culture vessel simultaneously with sporulation induction or before sporulation is completed and the spores are released, the spore settlement density in the culture vessel after spore release can be adjusted. This procedure eliminates the conventional process of purifying and collecting spores after spore release, eliminating the spore loss and labor required in this process, and enabling efficient production of clump seedlings.

[0023] After the spores are released from the mother algae, the inner wall area of ​​the culture vessel is 1 cm ２ The spore density per 10 ３ 10 or more, preferably 10 ４ The period for releasing spores into the mother algae is not particularly limited, but it is desirable that the spore formation induction conditions be such that spore release is completed within 10 days, 7 days, 5 days, 3 days, 2 days, or 1 day, for example.

[0024] Step (4): A step of forming clumps of spores and / or germinated bodies obtained in the culture vessel. The culture temperature is not particularly limited as long as it is a temperature suitable for the growth of spores or algal bodies, or for spore formation, and is, for example, 5 to 35°C, preferably 10 to 30°C, and more preferably 15 to 25°C.

[0025] The light conditions during cultivation are not particularly limited as long as they allow the growth of macroalgae or their spores, and examples thereof include natural light light-dark cycle conditions, artificial light conditions, and artificial light-dark cycle conditions.

[0026] The culture period from the start of culture of spores released from the mother algae bodies to the collection of clumped spores or germinated bodies is not particularly limited, but is, for example, 1 to several tens of days, 2 to 21 days, or 3 to 14 days.

[0027] The culture of the present invention is usually carried out in a container. The culture of the present invention is usually agitated culture using aeration or an agitator, but static culture is also possible. The container is not particularly limited as long as it can be used for algae culture, and a suitable capacity can be selected depending on the scale of culture. For example, glass or plastic flasks, beakers, and water tanks can be used. From the viewpoint of peeling off spores attached in a mat-like manner to the wall surface, materials with a smooth surface, such as glass or plastic, are preferred. A thin film made of agar, gelatin, or the like may be attached to the culture container in advance to make it easier to peel off the clumped spores.

[0028] The culture of the present invention is carried out in water. The liquid used for culture is not particularly limited as long as it is suitable for the growth of seaweed, but is typically seawater or a seawater medium supplemented with nutrients for algae culture, such as nitrates and phosphates. Commercially available nutrients can be used. The seawater is not particularly limited as long as it is suitable for the growth of seaweed, but is a liquid with a salt concentration of 0% to 5%. Either natural seawater or artificial seawater can be used. Artificial seawater refers to freshwater artificially adjusted to mimic the composition of natural seawater by adding salts such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and magnesium sulfate, as well as pH buffers and other trace components. It also includes seawater from which salt and other components have been removed and / or active ingredients have been added. Seawater may be sterilized or filtered before use.

[0029] If necessary, development and growth-promoting factors that promote the development and growth of seaweed, etc., may be added to the seawater or culture medium. In the production method of the present invention, it is preferable to co-culture bacteria that promote the development and growth of algae, or to add a seaweed morphogenesis inducer, in the step of isolating and growing mother algae bodies. The seaweed morphogenesis inducer refers to a substance produced by microorganisms such as bacteria present in seawater, and various previously reported substances can be used, without particular limitation. By culturing spores of seaweed such as green algae in the presence of a seaweed morphogenesis inducer or co-culturing them with bacteria that produce a seaweed morphogenesis inducer, it is possible to form the algae into multicellular bodies or maintain the multicellular morphology, even under culture conditions that do not contain the factor present in natural seawater (WO 2004 / 007510, JP 2003-189845, etc.). Examples of the bacteria include strains belonging to the Cytophaga-Flavobacterium-Bacteriodes complex, such as those of the genera Flavobacterium, Zobelia, and Tenacibaculum, as well as mutant strains derived from these strains. Examples of seaweed morphogenetic factors include salusin.

[0030] Conventionally, obtaining agglomerate seedlings required purifying and collecting large amounts of spores to prevent contamination with microalgae and adjust the density at sowing, which required considerable labor and time each time, and posed the problem of spore loss during the purification and collection process. The production method of the present invention allows for the initial purification of only a small amount of spores sufficient to form mother algae, making it possible to produce agglomerate seedlings more simply and efficiently. Once the mother algae are isolated and maintained, it is possible to obtain agglomerate seedlings without the need for repeated purification processes. Furthermore, in conventional agglomerate seedling cultivation methods, spores are obtained by inducing spore formation in algae with heterogeneous properties collected from the field, except for a very small number of algae for which cultivated strains have been established. Therefore, the timing and number of spores released after the spore formation induction treatment were not uniform. The method of the present invention uses isolated mother algae with uniform properties derived from any algae, making it possible to predict the timing and number of spores released by the spore formation induction treatment and adjust the density before spore release.

[0031] Macroalgae Cultivation Method: The spore and / or germinated clumps obtained by the production method of the present invention can be finely crushed to a diameter of approximately 5 mm or less as needed, cultured with aeration, and germinated to obtain clump seedlings. This allows for convenient and efficient production of macroalgae. The clump seedlings are preferably cultured under agitation. Cultivation under agitation is not particularly limited as long as the culture medium is partially or entirely agitated. Examples of culturing under agitation include aeration of the culture medium using an aeration pump (aeration culture), passing a liquid (e.g., culture medium) through the culture medium using a pump, moving a stirrer (e.g., rotation), and shaking the culture vessel. During culture, the macroalgae may grow, for example, in a floating state or partially fixed to a wall or suitable support. Cultivation under agitation removes oxygen bubbles adhering to the surface of the algae during photosynthesis, allowing for efficient photosynthesis. Furthermore, fluidizing the algae clumps allows each algae to be uniformly exposed to light, resulting in uniform growth. The obtained algal cells of the macroalgae can be used, for example, as food, biofuel, or as raw materials for active pharmaceutical ingredients.

[0032] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0033] Example 1. Preparation of agglomerate seedlings of asexually reproduced strains of Hibiscus tularensis 1. Isolating and growing mother algae bodies A single asexually reproduced strain of Hibiscus tularensis was collected off the coast of Kochi Prefecture, and asexual spores with two flagella were released into a glass petri dish filled with sterilized seawater. The spores were isolated by swimming in sterilized seawater using their negative phototaxis. The isolated spores were seeded in a 9 cm diameter glass petri dish containing sterilized seawater supplemented with 0.05% Porphyran Conco (manufactured by Daiichi Seimo Co., Ltd.) (hereinafter referred to as Porphyra medium). At this time, bacteria belonging to the Cytophaga-Flavobacterium-Bacteriodes complex, which induces the development of Hibiscus spores into multicellular bodies, were added, and the water temperature was 15°C and the light intensity was 100-200 μmol m －２ s －１The algae were statically cultured under a 12-hour light / 12-hour dark photoperiod (white fluorescent light source). The multicellular algae that had grown to a length of 5 mm through static culture were then aerated under the same light, temperature, and medium conditions, and grown to a length of 10 cm or more. These isolated algae were used as mother algae. Step 2. Inducing sporulation in the mother algae: Circular algae fragments with a diameter of 15 mm were excised from the mother algae and aerated cultured at a water temperature of 20°C under the same light and medium conditions as in Step 1. Due to this fragmentation and temperature increase, spores were formed in the cells of the algae fragments 2-3 days after the start of culture and released into the medium. Step 3. Adjusting the density of the sporulation-induced mother algae: At the start of the aerated culture in Step 2, four of the above algae fragments and 400 mL of Porphyra medium were placed in an 8 cm diameter glass beaker to adjust the algae density. Each algae fragment contained 1.2 x 10 ７ Therefore, 4.8 × 10 spores were formed from four algal fragments. ７ When spores are formed and all of them are released into the medium, the total inner wall area of ​​the glass beaker containing 400 mL of medium is 250 cm ２ Therefore, 1.9 × 10 ５ / cm ２ It was predicted that the spore settlement density would be 100%. Step 4. Step of forming clumps of spores and / or germinated bodies obtained in the culture vessel. By the third day after the start of aerobic culture in Step 2, all four algal fragments had formed spores and released them into the medium. After 10 days of aerobic culture, the spores that had grown on the inner wall of the glass beaker underwent cell division, growing into germinated bodies and forming clumps. No microalgae growth was observed. Seaweed could be produced by agitating the clumps (clump seedlings).

[0034] Example 2. Steps for Producing Aggregated Seedlings of Enteromorpha japonica 1. Isolation and Growth of Mother Algae A single asexually reproduced strain of Enteromorpha japonica was collected from the brackish waters of the Shimanto River in Kochi Prefecture, and asexual spores with two flagella were released into a glass petri dish filled with sterilized seawater. The spores were isolated by allowing them to swim in sterilized seawater using their negative phototaxis. The isolated spores were placed in a 9 cm diameter glass petri dish containing 30 mL of Porphyra medium, and exposed to a water temperature of 20°C and a light intensity of 100-200 μmol m －２ s －１The algae were statically cultured under a photoperiod of 12 hours light and 12 hours dark (white fluorescent light source). After two weeks of culture, the multicellular algae, which had grown to 1 cm in length, were aerated and cultured under the same light, temperature, and medium conditions, until they reached a length of 30 cm or more. These isolated algae were used as mother algae. Step 2: Inducing spore formation in mother algae. It was previously investigated that when the mother algae of Enteromorpha spp. were cultured under conditions involving fragmentation, washing, and elevated temperature, spores were formed within the cells of the algae fragments 2-3 days after the start of culture. Following this procedure, 200 algae fragments measuring 2-5 mm in length were cut with scissors from the 2 mm-wide tip of the mother algae. These fragments were washed with distilled water and sterilized seawater and then placed in a 1 L glass beaker containing 1 L of the same medium as in Step 1. This beaker was placed at a water temperature of 25°C under the same light conditions as in Step 1, and the algae fragments were aerated and cultured. Step 3: Step of adjusting the density of sporulated mother algal bodies At the start of the light period on the third day after aerobic culture, it was confirmed that sporulation had occurred in the algal body fragments. While non-sporulated algal bodies are bright green, sporulated fragments are yellow to brown and can be distinguished with the naked eye. Before spore release began, the algal body fragments that had sporulated entirely were transferred to another container to adjust the density. 20, 30, and 40 sporulated fragments were each placed in a 9 cm diameter glass petri dish together with 30 mL of the same medium as in step 2. In the genus Ulva, to which Enteromorpha belongs, sporulation was observed in 1 cm algal body fragments. ２ The number of spores formed per ７ ~10 ８ Based on this knowledge, 4 × 10 pieces of algal cells were obtained from a piece of algal body 2 mm in width and length. ５ ~10 ６ It was calculated that spores were produced. The total inner wall area of ​​the above petri dish containing 30 mL of medium was 77 cm ２ Therefore, if spores are released from 20, 30, and 40 fragments and all adhere, there will be at least 1 x 10 ４ ~10 ５ pieces, 1.5×10 ４ ~10 ５ pieces, 2×10 ４ ~10 ５ pieces / cm ２It was predicted that the spore settlement density would be above 100 μg / L. Step 4. Forming spore and / or germinated aggregates obtained in a culture vessel. Spores were released that day in the medium of the three Petri dishes in which the density of the algal fragments from Step 3 had been adjusted. Because spores are phototactic, they tend to gather in one area of ​​the Petri dish in a bright environment. To avoid this, the spores were thoroughly stirred before the dark period of the day and then placed in the dark to ensure uniform settlement within the Petri dish. The three Petri dishes were then placed under the same static culture conditions as in Step 2. The spores adhered to the inner walls of the Petri dish, repeatedly divided into cells within a few days, and formed germinated aggregates after two weeks of culture. Although the spore densities differed among the three Petri dishes, germinated aggregates were formed without any problems in all three. The germinated aggregates were peeled from the inner walls of the Petri dish, broken into small pieces using a commercially available blender, and cultured in a glass beaker with aeration, producing numerous germinated aggregate seedlings. Microalgae growth was not observed. Seaweed can be produced by culturing the clumps (clump seedlings) under stirring.

[0035] Example 3. Process for Producing Asakusanori Agglomerated Seedlings 1. Isolation and Growth of Mother Algae A single Asakusanori alga was collected off the coast of Ehime Prefecture. Carpospores released from this strain were dispersed in sterilized seawater and observed under a microscope. They were then picked up one by one with a glass pipette and placed in wells of a microplate for static culture. After two months, microfilaments (sporophytes) measuring approximately 1 cm in size developed in some wells. A filamentous mass with a diameter of 5 mm was cut from one isolated microfilament and placed in a 200 mL glass beaker containing 100 mL of Porphyra medium. The water temperature was 20°C and the light intensity was 100-200 μmol m. －２ s －１The culture was aerated under a photoperiod of 12 hours light and 12 hours dark (white fluorescent light source). After one month of culture, multiple conchospores released from the microfilaments attached to the beaker wall, germinated, and grew into leaf-like multicellular algae (gametophytes). Of these multicellular algae, individuals with a length of 1 cm or more were designated as mother algae. Step 2: Inducing spore formation in mother algae The mother algae were placed in a 9 cm diameter glass petri dish filled with Porphyra medium, wrapped in aluminum foil to avoid exposure to light, and placed in a 25°C incubator for 24 hours. The culture was then aerated under the same water temperature and light conditions as in Step 1. Due to this dark treatment and high temperature treatment, the fresh weight of the algae increased from 1 g to 10 g within one week of the start of aerated culture. ８ It was previously confirmed that more than 5 × 10 single spores were released. Step 3. Step of adjusting the density of sporulation-induced mother algae For aerobic culture, 0.05 g of fresh weight of mother algae and 200 mL of the same medium as in step 1 were placed in a glass beaker with a diameter of 6 cm to adjust the density of the algae. From 0.05 g of mother algae, 5 × 10 single spores were released within one week. ６ When more than 100 single spores are released, the total inner wall area of ​​the beaker containing 200 mL of medium is 162 cm ２ Therefore, after one week of aeration culture, 3 × 10 ４ pieces / cm ２ It was estimated that the spore settlement density would be 10 or more. Step 4. Step of forming agglomerates of the obtained spores and / or germinated bodies in the culture vessel. On the second day after the start of aerobic culture, a small number of single spores were released, and on the fifth and sixth days, agglomerates of 10 or more were released. ６ The cumulative total number of single spores released into the medium up to the 7th day was 4.7 × 10 ６ After that, aerated culture was continued for one week while the Porphyra medium was replaced, and the spores that had grown on the inner wall of the glass beaker underwent cell division, growing into germlings and forming clumps. No microalgae growth was observed. The clumps were removed from the glass beaker, crushed into small clumps of 5 mm or less, and cultured with stirring to produce seaweed seedlings for floating culture.

[0036] Example 4. Production of clump seedlings of Hibiscus punctatus (sexually reproduced strain) 1. Isolation and growth of mother algae. Female and male gametophytes were collected from a population of Hibiscus punctatus sexually reproducing along the coast of Uranouchi Bay in Kochi Prefecture, and the female and male gametes were released into glass petri dishes filled with sterilized seawater. The gametes were collected by swimming in sterilized seawater using positive phototaxis. The collected male and female gametes were mixed and placed in a glass petri dish to form zygotes. Furthermore, zygotes were isolated by swimming in sterilized seawater using negative phototaxis. The isolated, concentrated, and collected zygote suspension was placed on a glass slide and left at room temperature for two days. The zygotes attached to the slide and transformed into spherical sporophytes approximately 0.01 mm in diameter. The zygote suspension was inoculated with bacteria belonging to the Cytophaga-Flavobacterium-Bacteriodes complex, which promotes the development and growth of Hibiscus tularensis. The slide glass on which a large number of minute sporophytes had grown was placed in a glass dish containing a Porphyra medium, and the water temperature was 20°C and the light intensity was 100-200 μmol m －２ s －１ (white fluorescent light source) under a photoperiod of 12 hours light and 12 hours dark, after 3 to 4 weeks they grew into spherical to ellipsoidal algal bodies with a major axis of 0.05 to 0.06 mm. These grown sporophytes were used as mother algal bodies. Step 2. Step of inducing sporulation in the mother algal bodies The slide glass on which the mother algal bodies had grown was placed in a glass beaker containing Porphyra medium, and aerated culture was performed at a water temperature of 25°C under the same light conditions as in Step 1. It was previously investigated that this high-temperature treatment would release an average of 10 to 30 zoospores per minute mother algal body within about one week of culture. Step 3. Step of adjusting the density of sporulated mother algal bodies In the aerated culture in Step 2, 2 x 10 ５ The slide glass on which the microscopic mother algae bodies had grown was placed together with 200 mL of Porphyra medium to adjust the density of the mother algae bodies. ５ From 2 x 10 in a week ６ More than 100 zoospores were released, and the total inner wall area of ​​the beaker containing 200 mL of medium was 162 cm ２ Therefore, after one week of aeration culture, the number of bacteria was 1.2 × 10 ４pieces / cm ２ It was estimated that the spore settlement density would be above 100%. Step 4. Step of forming clumps of spores and / or germinated bodies obtained in the culture vessel. By the 10th day after the start of culture in Step 2, spores had been released from more than 80% of the mother algae on the glass slide, and it was confirmed that numerous spores had settled on the inner wall of the glass beaker. After that, when aerated culture was continued for one week while changing the Porphyra medium, the spores that had settled on the inner wall of the glass beaker underwent cell division, grew into germinated bodies, and formed clumps. No microalgae growth was confirmed. The clumps were peeled from the glass beaker, crushed into small clumps of 5 mm or less, and cultured with stirring to produce seaweed seedlings for floating culture.

Claims

1. A method for producing macroalgae agglomerate seedlings, comprising: (1) a step of isolating and growing mother algae; (2) a step of inducing spore formation in the isolated and grown mother algae; and (3) cultivating the mother algae in which spore formation has been induced to a density of 10 to 10 with respect to the inner wall area of ​​a culture vessel. ３ pieces / cm ２ (4) adjusting the density to a level above that of the culture vessel; and (5) forming a mass of spores and / or germinated bodies obtained in the culture vessel.

2. The production method according to claim 1, further comprising one or more steps selected from the group consisting of a step of culturing the fine fragments of the mother algae, a step of culturing the mother algae at 20°C or higher, and a step of culturing under dark conditions.

3. The production method according to claim 1, wherein the macroalgae are seaweeds belonging to any one of the orders Hibuliales, Ulvales, Laminariales, Paleophytales, and Bangiales.

4. The production method according to claim 1, wherein the macroalgae is seaweed belonging to any one of the genus Metastasis, Ulva, Laminaria, Undaria pinnatifida, Laminariales, or Porphyra.

5. A method for cultivating macroalgae, comprising a step of agitating and cultivating the agglomerated seedlings obtained by the method according to any one of claims 1 to 4.

6. A method for cultivating macroalgae, comprising the method according to any one of claims 1 to 4 and a step of agitating and cultivating the clumped seedlings.