Seaweed farming methods

Abstract

The present invention aims to provide an efficient method for cultivating seaweed. [Solution] The seaweed cultivation method of the present invention includes step A: shredding the seaweed leaf parts, step B: culturing the shredded leaf parts, and step C: culturing the zoospores produced from the cultured leaf parts, and is characterized in that step A and step B are repeated five or more times, and then step C is carried out.

Description

[Technical Field]

[0001] The present invention relates to an efficient method for cultivating seaweed. [Background technology]

[0002] Seaweed is a familiar food to the Japanese. For example, kelp, hijiki, mozuku, wakame, asakusanori, tengusa, sea lettuce, and green laver have been eaten since ancient times. However, in recent years, poor seaweed harvests have been reported throughout Japan. The cause of this is not entirely clear, but rising seawater temperatures due to global warming have been cited as one of the reasons. Therefore, various efficient seaweed cultivation methods have been investigated.

[0003] For example, Patent Document 1 describes a method for artificially harvesting green laver seedlings in which the mother algae of green laver is cut into pieces of approximately 5 mm and placed in salt water to release spores, and the released spores are then attached to a net for seedling cultivation.

[0004] Patent Document 2 describes a method for producing green algae seeds, in which pieces of green algae are cultured in a culture medium containing an extract of the green algae.

[0005] Patent Document 3 describes a method for producing seaweed cells by culturing seaweed spores or the like in a medium that does not contain a seaweed morphogenesis inducer. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 9-224511 [Patent Document 2] Japanese Patent Application Publication No. 2018-29492 [Patent Document 3] International Publication No. 2020 / 027002 Brochure Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, various efficient seaweed farming methods have been studied, but in recent years, poor seaweed harvests and shortages have become a hot topic across Japan, and further improvements in production efficiency are required. Therefore, an object of the present invention is to provide an efficient method for cultivating seaweed. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above problems. As a result, they found that although seaweed seedlings can be increased by repeatedly chopping and culturing seaweed, the efficiency of this process can be reduced by repeating the process. Therefore, they discovered that the seedling increase efficiency can be restored by culturing zoospores, which are produced from cultured thallus parts and have undergone sexual reproduction, to obtain thallus parts, and thus completed the present invention. The present invention will now be described.

[0009] [1] A method for cultivating seaweed, comprising: Step A: Shredding seaweed leaves; Step B: culturing the shredded leaves; and Step C: Culturing zoospores produced from the cultured thallus, A method characterized by repeating the steps A and B five or more times, and then performing the step C. [2] The method according to [1] above, wherein the zoosporangia produced from the cultured thallus are physically disrupted to remove the zoospores, which are then cultured. [3] The method according to [1] or [2], wherein in step A, the leaf-like portion is chopped into pieces of more than 40 μm. [4] The method according to any one of [1] to [3], wherein in step A, the leaf-like parts are cut into pieces of 1 cm or less. [5] The method according to any one of [1] to [4], wherein the seaweed is a seaweed belonging to the order Hibiscus. [6] The method according to [5], wherein the seaweed is seaweed belonging to the genus Streptomyces. [7] The method according to any one of [1] to [6], wherein steps A to C are carried out on land. [Effects of the Invention]

[0010] The method of the present invention allows for extremely efficient seaweed cultivation. While the seedlings produced by the method of the present invention can be attached to nets and cultivated in the sea or brackish waters, the method of the present invention can also be applied to land-based aquaculture. Therefore, the method of the present invention is industrially extremely advantageous as a technology for efficiently producing seaweed, for which poor harvests and shortages have been reported in recent years across Japan. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a schematic diagram of the life cycle of Hinodermata. [Figure 2] Figure 2 is a photograph of gametophytes obtained by culturing the thinly sliced ​​leaf parts of Monostroma punctata. [Figure 3] Figure 3 shows photographs of gametophytes obtained by repeatedly cutting and culturing the leaf parts of Monostroma punctata. [Figure 4] Figure 4 is a photograph of zoosporangia produced by culturing zygotes of Monostroma . [Figure 5] Figure 5(1) is a low-magnification photograph of a gametophyte obtained by zoospore culture, and Figure 5(2) is a microscopic photograph of the same gametophyte. DETAILED DESCRIPTION OF THE INVENTION

[0012] The seaweed cultivation method according to the present invention will be described below step by step, but the present invention is not limited to the following specific examples. Note that "cultivation" generally refers to the artificial rearing and propagation of aquatic organisms, and in industrial terms is synonymous with the production of aquatic organisms.

[0013] Process A: Shredding seaweed leaves In this process, the fronds of seaweed are shredded. Seaweed is a general term for algae that grow in the sea and generally refers to algae that are visible to the naked eye and belong to three divisions: green algae, brown algae, and red algae. The fronds of seaweed refer to the parts other than the appressorium that secures the seaweed to rocks and the stalk connecting the center of the frond to the appressorium. Many of these are morphologically similar to the leaves of terrestrial organisms, but some grow very large or are very elongated.

[0014] Seaweed is not particularly limited as long as it is useful for cultivation and produces zoosporangia, but can be classified into, for example, green algae such as seaweeds of the family Azoaceae of the order Hibulophytales and seaweeds of the family Ulva of the order Ulvales; brown algae such as seaweeds of the family Ulva of the order Laminariales, seaweeds of the family Undaria of the order Laminariales, seaweeds of the family Laminariales, and seaweeds of the family Sargassum of the order Fucales; and red algae. Seaweeds of the family Azoaceae of the order Hibulophytales include Azoaceae; seaweeds of the family Ulva of the order Ulvales include Ulva; seaweeds of the family Undaria of the order Laminariales include Undaria; seaweeds of the family Undaria of the order Laminariales include Laminaria; and seaweeds of the family Sargassum of the order Fucales include Hijiki.

[0015] The means for shredding the leaves is not particularly limited, but may be mechanically shredded using a mixer or blender, or manually shredded using scissors, for example.

[0016] The size of the starting leaf parts before chopping is not particularly limited, and they may be grown appropriately until they are suitable for chopping, but for example, they can be 3 cm or more.

[0017] The size of the shredded thallus portions can be adjusted as needed, but is preferably greater than 40 μm. According to the inventors' experimental findings, shredding thallus portions to 40 μm or less sometimes resulted in spore clumps that did not grow into thallus portions during cultivation. While there is no particular upper limit on the size of the shredded thallus portions, a size of 1 cm or less is preferred, since the smaller the size, the more seaweed seedlings can be produced. In particular, when shredding thallus portions mechanically, the shape of the shredded thallus portions tends to become irregular, making it difficult to determine the size. Therefore, in the present disclosure, for example, when a suspension of shredded thallus portions is filtered through a filter with a mesh size of x μm without applying pressure or vacuum, the size of the thallus portions that cannot pass through the filter is defined as greater than x μm, and when a suspension of shredded thallus portions is filtered through a filter with a mesh size of y cm without applying pressure or vacuum, the size of the thallus portions that can pass through the filter is defined as y cm or less.

[0018] Process B: Cultivation process In this process, the seaweed fronds shredded in the previous process A are cultured. The culture conditions may be adjusted as appropriate depending on the type of seaweed to be cultivated, but for example, seawater is used. The type of seawater is not particularly limited, and examples that can be used include sterilized seawater, artificial seawater, underground seawater, surface seawater, and deep seawater. In the case of seaweed that can grow in brackish water, the seawater may be diluted with water or the salt concentration may be reduced. Nutrients such as nitrogen, phosphorus, amino acids, and plant growth factors, as well as diatom control agents, may also be added to the seawater. An example of a nutrient product containing nutritional components is "Porphyran Conco" manufactured by Daiichi Seimo Co., Ltd.

[0019] The amount of seawater used relative to the volume of chopped leaves can be adjusted as appropriate, but for example, the ratio of wet leaves to the volume of seawater can be set to 0.0001 w / v% or more and 5 w / v% or less, preferably 0.01 w / v% or more, more preferably 0.05 w / v% or more, and preferably 0.5 w / v% or less, more preferably 0.1 w / v% or less.

[0020] The culture temperature is preferably 10°C or higher and 35°C or lower, more preferably 15°C or higher and 30°C or lower, and even more preferably 20±2°C.

[0021] The photoperiod may be adjusted as appropriate, for example, to 10L:14D to 18L:6D, preferably 11L:13D to 13L:11D, and more preferably 12L:12D. 2 s or more 1000μmol / m 2 s or less, and 2 s or more 500μmol / m 2 s or less is preferable, and 100 μmol / m 2 s or more 200μmol / m 2 s or less is more preferable.

[0022] During the culture, the seawater may be stirred. Air may be supplied to the seawater for stirring or other purposes. The amount of air supplied may be adjusted appropriately depending on the amount of seawater used, and for example, the amount of air supplied per minute per volume of the culture tank may be 100 mL / min / L or more and 1 L / min / L or less, and preferably 400 mL / min / L or more and 800 mL / min / L or less.

[0023] The cultivation period can be adjusted as needed. For example, if the cultivated thallus is about 2 cm in size, it can be used as seedlings. Furthermore, although this varies depending on the type of seaweed, if the longest part is 5 cm or longer, it can be used as a product or raw material for a product.

[0024] In the method of the present invention, steps A and B are repeated five or more times. The more the shredding in step A and the culturing in step B are repeated, the more the number of seaweed seeds, products, and / or raw materials for the cultured seaweed increases in a relatively short period of time, which can be said to be a high production efficiency. However, this can result in partial poor growth, such as slow growth rate due to DNA replication errors. The number of repetitions is preferably eight or more, more preferably ten or more, and preferably 30 or less, more preferably 25 or less, and even more preferably 20 or less.

[0025] Step C: Zoospore cultivation step In this step, steps A and B are repeated multiple times, and then zoospores produced from the cultured thallus are cultured to obtain thallus. As mentioned above, the more times steps A and B are repeated, the more likely it is that poor growth will occur. Therefore, by obtaining and culturing zoospores that have undergone sexual reproduction in this step, it is possible to normalize growth.

[0026] Seaweeds generally have diphatic (2n) and haplomorphic (n) life cycles. As an example, the life cycle of Hibiscus nigricans (H. nigricans), a species of the genus Hibiscus in the family Aethioplastidae, is shown in Figure 1. As shown in Figure 1, the thallus of Hibiscus nigricans contains male gametophytes (n) and female gametophytes (n), and each gametophyte reproduces by parthenogenesis. However, each gametophyte also produces male gametes (n) and female gametes (n), which fertilize to form zygotes (2n), forming zoosporangia. Gametophytes reproduced by parthenogenesis from Hibiscus nigricans gametophytes have the same genome as the original gametophyte. Furthermore, gametophytes cultured by cutting the thallus of a gametophyte into pieces in steps A and B also have the same genome as the original gametophyte. As a result, the genome of gametophytes multiplied by parthenogenesis or repeated cutting and culturing of thallus parts is more likely to have replication errors as the number of repetitions increases, and the multiplication efficiency tends to decrease.

[0027] On the other hand, in the present invention, zoospores are cultured in this step C. As shown in Figure 1, the zygote produced by fertilization of the male gamete (n) and female gamete (n) produced from each gametophyte is diploid (2n) and grows into a zoosporangium, but the zoospores contained in the zoosporangium are haploid (n), and the advantages of sexual reproduction can be enjoyed, such as the improvement of replication errors during meiosis and the recovery of multiplication efficiency.

[0028] In contrast to the species of Hitsugaya and the like, the leaf-like parts of kelp and the like are contained in sporophytes that grow from zygotes, and zoosporangia are formed, particularly in the lower part of such sporophytes, and the zoospores obtained from such zoosporangia can be cultured. Note that zoospores are also called spores, and among spores, zoospores have flagella and can swim around in the sea. Although it depends on the type of seaweed, it takes several months for the zygote to grow into a zoosporangium containing zoospores, but most of them die without releasing the zoospores. To obtain zoospores from the zoosporangium, for example, the zygote is cultured for 2 to 3 months, then cultured in the dark under relatively low temperature conditions for about 1 month, and then the temperature is raised and cultured for about 1 month. The culture temperature in the dark can be, for example, 20±2°C, and the subsequent culture temperature can be 26±2°C.

[0029] As mentioned above, it has been reported that obtaining zoospores from zygotes requires culturing the zygotes for 2 to 3 months, subjecting the resulting zoosporangia to dark treatment for about 1 month, and then treating them at elevated temperature for about 1 month to stimulate the zoosporangia. Physical destruction of the zoosporangia was not even considered. However, the present inventors have discovered that gametophytes containing thallus parts can be obtained more efficiently from zoospores by physically destroying the zoosporangia to extract the zoospores.

[0030] In the present invention, it is preferable to physically destroy the coating and remove the zoospores, for example, one month after zygote formation, preferably 1.5 months or 2 months after zygote formation, when the zoosporangia have grown to 30 μm or more, preferably 40 μm or more or 50 μm or more.

[0031] The means and conditions for physically disrupting the zoosporangia are not particularly limited, as long as they can disrupt the zoosporangial membrane and extract intact zoosporangia without damaging the internal zoosporangia. For example, the zoosporangia dispersion can be stirred using a mixer, blender, homogenizer, strong aeration, or the like. The concentration of the wet zoosporangia dispersion can be, for example, 20 mg / mL or more and 500 mg / mL or less. A concentration of 20 mg / mL or more can be considered to provide a sufficiently high zoosporangial extraction efficiency. On the other hand, a concentration of 20 mg / mL or more can sufficiently suppress zoosporangial damage due to zoosporangial collisions during stirring. The concentration is preferably 100 mg / mL or more and 200 mg / mL or less. Furthermore, the concentration of the dispersion, stirring speed, stirring time, etc. are preferably adjusted within a range that allows sufficient zoosporangial extraction and sufficient suppression of zoosporangial damage. Furthermore, when the scale of implementation is relatively small, the zoosporangia may be destroyed manually, for example by repeatedly drawing up and releasing the zoosporangia dispersion into a syringe until the zoosporangia coating is destroyed.

[0032] The zoospores obtained from the sporangia may be cultured under the same conditions as in the culture step B. The zoospores may be cultured, for example, until the gametophytes grown from the zoospores can be used as a product, or until the gametophytes have grown to a size that can be used in the chopping step A. That is, after obtaining leaf-like gametophytes or sporophytes in step C, the chopping step A and culture step B may be repeated again.

[0033] In addition to extracting and culturing zoospores that have undergone sexual reproduction, gametophytes may also be obtained by culturing zoospores that have developed from zoosporangia that have grown from parthenogenetic gametes from intact thallus gametophytes, or by culturing zoospores that have been extracted by physically disrupting zoosporangia in the same manner as zoosporangia. By using these processes in combination, seedlings can be obtained even more efficiently.

[0034] The method of the present invention can be applied to conventional aquaculture, land-based aquaculture, or a combination thereof. For example, zoospores, gametophytes or their fragments, zygotes, etc. obtained by carrying out the method of the present invention on land may be attached to a net and cultured in the sea or in brackish waters, or all of the above steps A to C may be carried out in land-based aquaculture. [Example]

[0035] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0036] Example 1 (1) Culture of shredded phyllodes The leaves of fully grown Monostroma nitidum were shredded using scissors. The shredded leaves were dispersed in sterilized seawater and filtered without pressure through a 500 μm mesh filter ("Stainless Steel Sieve," manufactured by Sanpo Co., Ltd.). The resulting filtrate was filtered through a 40 μm mesh filter ("Stainless Steel Sieve," manufactured by Sanpo Co., Ltd.), and the shredded leaves that did not pass through the filter were collected. Separately, seawater sterilized in an autoclave (Tomy Seiko Co., Ltd.) was supplemented with "Porphyran Conco" (Daiichi Seimo Co., Ltd.) as a nutrient component at a ratio of 1 / 2000 (by mass). Chopped leaf parts (50 mg) were added to the resulting sterilized seawater (500 mL), and the mixture was cultured at 20°C under a 12-hour light / 12-hour dark cycle with aeration. The light intensity during the light period was 100-200 μmol / m 2 It was adjusted to s. After three weeks, the gametophytes had grown to a diameter of more than 2 cm (Figure 2). By continuing the culture, fully grown gametophytes of more than 5 cm were obtained.

[0037] (2) Repeated cultivation of shredded phyllodes The leaf-like gametophytes obtained in (1) above were cut into pieces and cultured in the same manner as in (1). The same procedure was repeated five times. Photographs of the gametophytes obtained in each culture, taken three weeks after the start of culture, are shown in Figure 3. As shown in the photograph in Figure 3, even after repeatedly chopping and culturing the Monostroma fronds four times, we were able to obtain fronds sufficient for use as seedlings. However, in the fifth culture, the gametophytes three weeks after the start of culture were smaller than those in the first to fourth cultures. However, by continuing culture, they were able to grow to a sufficient size.

[0038] (3) Culture from zoosporangia The zygotes (2n) of P. punctatus were attached to a petri dish and cultured under the same conditions as in (1) above, except that air was not blown into the sterilized seawater. After approximately two months of culture, the zoosporangia grew to approximately 50 μm in size (Figure 4). The zoosporangia were then removed from the petri dish using a scraper or spatula, and the seawater was injected into the dish using a syringe with a 26G needle, followed by release. This process was repeated until destruction of the zoosporangia membrane was confirmed. The culture was then statically cultured under the same conditions as in (1) above, except that aeration was not used and the temperature was adjusted to 18°C. Figure 5(1) shows a low-magnification photograph of the culture medium 20 days after the start of zoosporangial culture, and Figure 5(2) shows a microscopic photograph. As shown in Figure 5(2), it was confirmed that thallus parts had grown from the zoospores, and it was revealed that the green part in Figure 5(1) is the grown thallus part. If the culture is continued in this state, the thallus parts will be able to grow sufficiently, and since the nuclear phase of the zoospores is n after passing through the 2n zygote, it is thought that the genome replication error has been repaired and that the fragmentation and culture can be repeated.

Claims

1. 1. A method for cultivating seaweed, comprising: Step A: Shredding seaweed leaves; Step B: culturing the shredded leaves; and Step C: Culturing the zoospores produced from the cultured thallus, A method characterized by repeating the steps A and B five or more times and then carrying out the step C.

2. 2. The method according to claim 1, wherein the zoosporangia produced from the cultured thallus are physically disrupted to remove the zoospores and culture them.

3. 2. The method of claim 1, wherein in step A the leaves are chopped to a size of more than 40 μm.

4. The method according to claim 1, wherein in step A, the leaves are chopped into pieces of 1 cm or less.

5. The method according to any one of claims 1 to 4, wherein the seaweed is a seaweed belonging to the order Hypocreales.

6. The method according to claim 5, wherein the seaweed is a seaweed belonging to the genus Streptomyces.

7. The method according to claim 1, wherein steps A to C are carried out on land.

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