Method for producing seaweed cells

Abstract

The present application provides a method for producing seaweed cells, which comprises: (A) culturing at least one selected from spores of seaweed, single cells derived from the spores, and cell aggregates of the spores and / or the single cells in a medium 1 substantially free of seaweed morphogenesis inducers under stirring.

Description

Technical Field

[0001] This invention relates to methods for manufacturing seaweed cells, etc. Background Technology

[0002] Seaweed cultivation is generally carried out by propagating the mother algae and then making seedlings from the spores obtained from the mother algae.

[0003] Seedlings are typically produced by attaching spores to a solid phase such as a thread or plate. However, in this method, since the seaweed is cultivated in a state attached to a solid phase, the operation of removing the seaweed from the solid phase during harvest is rather cumbersome. On the other hand, Patent Document 1 discloses a seaweed cultivation method in which seaweed spores are inoculated at high density onto a plate, and aggregates are obtained by allowing the spores to attach to each other, and the resulting aggregates are then cultivated. Using this method, seaweed can be cultivated in a suspended state in the culture medium, simplifying harvesting.

[0004] However, in either method, obtaining spores as raw materials for seaweed production requires a complicated process of cultivating the mother thallus and then obtaining spores from it.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent No. 3828359 Summary of the Invention

[0008] The technical problem that the invention aims to solve

[0009] The technical problem of this invention is to provide a technique for producing seaweed in a simple and efficient manner.

[0010] Technical solutions for solving technical problems

[0011] The inventors of this invention, through in-depth research into the aforementioned technical problems, discovered that by culturing at least one of the following—spores of seaweed, single cells derived from the spores, and cell clumps of the spores and / or single cells—under stirring conditions in a culture medium 1 that substantially does not contain seaweed morphogenesis inducing factors, seaweed cells that can be used as raw materials for seaweed production can be easily and effectively manufactured. Based on this discovery, further research was conducted, resulting in this invention.

[0012] That is, the present invention includes the following embodiments.

[0013] Item 1. A method for manufacturing seaweed cells, comprising:

[0014] (A) A process of culturing at least one of the following in a culture medium 1 that is substantially free of algal morphogenesis inducing factors, under stirring conditions: spores of algae, single cells derived from the spores, and cell clumps of the spores and / or the single cells.

[0015] Item 2. The manufacturing method as described in Item 1, wherein,

[0016] The seaweed mentioned above belongs to the order Ulotrichales.

[0017] Item 3. The manufacturing method as described in Item 1 or 2, wherein,

[0018] The above-mentioned seaweed belongs to the genus Monostroma.

[0019] Item 4. The manufacturing method as described in any one of items 1 to 3, wherein,

[0020] The aforementioned algal cells contain single cells.

[0021] Item 5. The manufacturing method as described in any one of items 1 to 4, comprising:

[0022] (A1) The process of culturing seaweed spores in the above-mentioned culture medium 1; and

[0023] (A2) The process of culturing the culture of step A1 in the above culture medium 1 while stirring.

[0024] Item 6. The manufacturing method as described in Item 5, wherein,

[0025] The culture in step A1 above is a static culture.

[0026] Item 7. The manufacturing method as described in any one of items 1 to 6, wherein,

[0027] The culture medium 1 mentioned above is an artificial seawater culture medium.

[0028] Item 8. The manufacturing method as described in any one of items 1 to 7, wherein,

[0029] The seaweed mentioned above is edible seaweed.

[0030] Item 9. The manufacturing method as described in any one of items 1 to 8, wherein,

[0031] The culture under the above stirring conditions is an aerated culture.

[0032] Item 10. A method for manufacturing seaweed, comprising:

[0033] The step of culturing seaweed cells obtained by the manufacturing method according to any one of items 1 to 9 in a culture medium 2 containing seaweed morphogenesis inducing factors.

[0034] Item 11. A type of seaweed thallus or seaweed without attached roots.

[0035] Invention Effects

[0036] According to the present invention, seaweed cells that can be used as raw materials for seaweed production can be manufactured easily and efficiently, thereby enabling the easy and efficient production of seaweed. Attached Figure Description

[0037] Figure 1 This is a photograph of Monostroma oxyspermum cultured cells after 32 days of culture in the Monostroma oxyspermum culture experiment of Example 1.

[0038] Figure 2 Photographs of *Triplophysa latifolia* cultured cells after 32 days of culture with the addition of morphogenetic material, as part of a culture conducted concurrently with the *Triplophysa latifolia* culture experiment in Example 1.

[0039] Figure 3 The proliferation curves of single-cell populations of *Triplophysa harziana* obtained by culturing in a medium without morphogenesis-inducing factors are shown.

[0040] Figure 4 To be Figure 3 The proliferation curves were obtained by logarithmically transforming the absorbance data. From day 2 to day 7 of culture, a linear increase was observed, which can be considered the logarithmic phase. On the other hand, deviations from the linear phase occurred on days 0-2 and 8-9, which can be considered the induction phase and the transition to the stationary phase, respectively.

[0041] Figure 5 The above is the logarithmic growth curve of a single cell population of Monostroma latissimum obtained in Example 2 in a culture medium without morphogenesis inducing factors.

[0042] Figure 6 This is a photograph of the thallus obtained after cultivation in Example 4.

[0043] Figure 7 This is a photograph taken after the thallus obtained from the culture in Example 4 was laid on a glass slide.

[0044] Figure 8 This is a photograph of the thallus of a typical reef membrane that grows from spores. The arrows indicate attached roots. Detailed Implementation

[0045] In this specification, the terms “containing” and “comprising” include the concepts of “containing”, “comprising”, “substantially constituted by” and “consisting solely of”.

[0046] 1. Methods for manufacturing seaweed cells

[0047] In one embodiment of the present invention, a method for manufacturing seaweed cells is disclosed, comprising: (A) a step of culturing at least one of the following: seaweed spores, single cells derived from the spores, and cell clumps of the spores and / or the single cells, in a culture medium 1 substantially free of seaweed morphogenesis inducing factors, under stirred conditions (in this specification, it is sometimes referred to as "the method for manufacturing seaweed cells of the present invention"). This will be described below.

[0048] There are no particular restrictions on the type of seaweed, which is usually green algae. From the viewpoint of further improving the efficiency of seaweed cell production, seaweed belonging to the order Ulotrichales is preferred. Examples of seaweed belonging to the order Ulotrichales include those belonging to families such as Gomontiaceae, Gayraliaceae, Capsosiphonaceae, Collinsiellaceae, and Ulotrichaceae. Seaweed belonging to families such as Gomontiaceae and Gayraliaceae are preferred, and seaweed belonging to the family Gomontiaceae is even more preferred. Examples of seaweed belonging to the family Gomontiaceae include those belonging to the genera Monostroma and Gomontia. Seaweed belonging to the genus Monostroma is preferred. Examples of seaweed belonging to the genus *Monostroma* include *Monostroma latissimum*, *Monostroma nitidum*, *Monostroma grevillei*, *Monostroma angicava*, *Monostroma alittoralis*, *Monostroma crassissimum*, and *Monostroma crassidermum*, with edible seaweeds such as *Monostroma latissimum* being preferred. Seaweed can be a single species or a combination of two or more species.

[0049] Spores are spores obtained from seaweed, without particular restrictions. Spores refer to reproductive cells. The state of a spore is not particularly limited; examples of spores include zoospores, gametes, zygotes, tetraspores, carpospores, monoascozoospores, and neutral zoospores. Spores can be a single species or a combination of two or more species.

[0050] There are no particular restrictions on whether a single cell originating from a spore is an undifferentiated single cell (cells constituting differentiated thallus are not included in the term "single cell" here). Single cells are typically roughly spherical. The diameter of a single cell is, for example, 3–20 μm or 5–15 μm. A single cell can be a single species or a combination of two or more species.

[0051] Cell clusters are formed by the aggregation of at least one type selected from the above-mentioned spores and single cells, and there are no particular restrictions as long as they do not contain cell clusters constituting differentiated thallus. The number of cells constituting the cell cluster is, for example, about 2 to 300, 5 to 200, or 10 to 100. The cells constituting the cell cluster are usually roughly spherical. The diameter of the cell cluster is, for example, 10 to 500 μm, 20 to 300 μm, or 30 to 200 μm. Cell clusters can be a single type or a combination of two or more types.

[0052] The seaweed cell manufacturing method of the present invention uses at least one of the above-mentioned spores, single cells and cell masses as starting materials.

[0053] There are no particular limitations on the types of substances reported to date that can induce algal morphogenesis. It is known that when algae such as green algae are cultured in synthetic media without seawater, they fail to form or maintain their morphology, resulting in algal cell destruction. The cause is known to be substances produced by microorganisms present in seawater (algal morphogenesis inducing factors) (International Patent Publication No. 2004 / 007510, Japanese Patent Application Publication No. 2003-189845, etc.). Examples of such microorganisms include strains belonging to the Cytophaga-Flavobacterium-Bacteriodes complex, such as *Flavobacterium*, *Zobellia*, and *Tenacibaculum*, as well as variants derived from these strains. Examples of algal morphogenesis inducing factors include *Thallusin*.

[0054] The culture medium (culture medium 1) used in the seaweed cell manufacturing method of the present invention is substantially free of the seaweed morphogenesis inducing factor. Here, "substantially free" takes into account the unavoidable contamination of the seaweed morphogenesis inducing factor, in which case it can be said to be "substantially free" of the seaweed morphogenesis inducing factor. In other words, a culture medium substantially free of the seaweed morphogenesis inducing factor means a culture medium in which no seaweed morphogenesis inducing factor and / or microorganisms that produce the factor are added (e.g., no natural seawater or natural seawater components are added). Since seawater contains the seaweed morphogenesis inducing factor and microorganisms that produce the factor, an artificial seawater culture medium, for example, free of the seaweed morphogenesis inducing factor and microorganisms that produce the factor, can be used as culture medium 1. There are no particular limitations on the artificial seawater culture medium, and examples include artificial seawater itself, and culture media in which nutrients (excluding the seaweed morphogenesis inducing factor and microorganisms that produce the factor) are appropriately added to artificial seawater as needed.

[0055] Culture is carried out under stirred conditions. There are no particular restrictions on the method of stirring the culture medium, as long as a portion or the entire medium is stirred. For example, this can be done by aerating the medium using a ventilator (aerated culture), introducing liquid (e.g., culture medium) into the medium using a pump, moving a stir bar (e.g., rotating), or shaking the culture vessel. During culture, the cells are cultured in a suspension state.

[0056] There are no particular restrictions on the cultivation temperature as long as it is suitable for cultivating seaweed, such as 5 to 35°C, preferably 10 to 30°C, and more preferably 15 to 25°C.

[0057] As for the light conditions during cultivation, there are no particular restrictions as long as the light conditions are suitable for cultivating seaweed. Examples include natural light light-dark cycle conditions, artificial light conditions, and artificial light light-dark cycle conditions.

[0058] There are no special restrictions on the cultivation time, for example, it can be 1 to several tens of days, or 2 to 7 days.

[0059] The seaweed cell manufacturing method of the present invention, as one embodiment, includes the following two steps.

[0060] (A1) The process of culturing seaweed spores in the above-mentioned culture medium 1; and

[0061] (A2) The process of culturing the culture of step A1 in the above culture medium 1 while stirring.

[0062] The culture in step A1 is preferably a static culture. Furthermore, the culture time in step A1 is, for example, 10 days to several months, or 20 days to 1.5 months. Through step A1, a culture containing single cells or cell clusters derived from spores can be obtained. Definitions of other terms, etc., are the same as described above.

[0063] The seaweed cell manufacturing method of the present invention enables the simple and efficient production of large quantities of seaweed cells (mainly containing single cells derived from spores) that can be used as raw materials for seaweed production.

[0064] In existing technologies, obtaining seaweed production raw materials (spores) requires a cumbersome process of cultivating a mother thallus and then extracting spores from it. However, according to the present invention, seaweed cells suitable for seaweed production can be prepared in large quantities from a small number of spores. Furthermore, it is possible to prepare a small quantity of the obtained seaweed cells and then use them to produce large quantities of seaweed production raw materials. In other words, according to the present invention, when obtaining seaweed production raw materials (spores), large quantities of seaweed production raw materials can be prepared without the cumbersome process of cultivating a mother thallus and then extracting spores from it, or by limiting this process to the minimum necessary steps.

[0065] The resulting seaweed cells can be used in the seaweed production process described later (as cells for seaweed production). They can also be used as raw materials for purposes such as food and biofuels.

[0066] 2. Seaweed manufacturing methods

[0067] One embodiment of the present invention relates to a method for manufacturing seaweed (in this specification, it is sometimes referred to as "the seaweed manufacturing method of the present invention"), which includes a step of culturing seaweed cells obtained by the manufacturing method of the present invention in a culture medium 2 containing seaweed morphogenesis inducing factors. This will be described below.

[0068] There are no particular limitations on the culture medium 2 containing algal morphogenesis inducing factors. Examples include natural seawater itself or a culture medium in which natural seawater is appropriately supplemented with nutrients as needed. Alternatively, culture medium 2 can also be a culture medium in which algal morphogenesis inducing factors and microorganisms that produce these factors are added to culture medium 1.

[0069] There are no particular limitations on the cultivation method used in the seaweed manufacturing method of the present invention. For example, as described in Patent Document 1, seaweed cells can be seeded at a high density onto a plate to allow the seaweed cells to adhere to each other, forming aggregates, and seaweed can be manufactured by culturing the aggregates obtained through suspension culture. Alternatively, as in existing methods, seaweed cells can be seeded at a relatively low density to allow them to adhere to a solid phase such as a thread or plate, and seaweed can be manufactured by culturing in this state. In addition, seaweed cells can be cultured in a suspension state (preferably under stirring conditions). The culture temperature, light conditions, etc., are the same as in step (A) described above.

[0070] In one embodiment of the present invention, the seaweed manufacturing method of the present invention can also produce thallus or seaweed without attached roots.

[0071] The resulting seaweed can be used for purposes such as food and as a raw material for biofuels.

[0072] Example

[0073] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0074] Example 1

[0075] From the mature thallus of the multicellular green algae *Pterocarya spp.*, motile spores (zoospores) capable of motility using flagella are released into a glass culture dish containing sterile seawater. When light is shone from one direction, the negatively phototactic motile spores aggregate in the direction opposite to the light-shielded side. Utilizing this phototaxis, the motile spores are aseptically separated by causing them to move in the sterile seawater.

[0076] 200-300 isolated zoospores were inoculated into sterile plastic petri dishes (6 cm in diameter) containing nutrient-fortified sterilized seawater and incubated statically in an incubator. The nutrient-fortified sterilized seawater was prepared by adding 20 mL of ES medium nutrient solution to 1 L of artificial seawater. The ES medium nutrient solution was prepared according to the formula described on pages 501-502 of *Algal culturing techniques* (edited by RA Andersen, published in 2005, Elsevier Academic Press). The artificial seawater was prepared by dissolving artificial seawater preparation powder (MARINE ART SF-1·25L, Tomita Pharmaceutical) in 25 L of distilled water and steam sterilizing at 121°C and 1 atmosphere for 5 minutes. The culture conditions were set as follows: white fluorescent lamp as the light source, light intensity 100 μmol / s. -1 m 2 The light / dark cycle is 12 hours / 12 hours, and the temperature is 20℃.

[0077] Under these culture conditions, the spores of the pointed reef membrane are as follows Figure 1 As shown, after about one month of culture, a cell population consisting of dozens of cells was formed. Additionally, in a culture experiment conducted concurrently with this experiment, morphogenetic material was added, as... Figure 2 As shown, the spores of the pointed reef membrane develop into a leaf-like morphology.

[0078] Figure 1 When the cell population is stirred in a glass culture dish using a glass dropper, it can be easily separated into individual cells or loosely bound clumps of a few cells, forming a cell suspension. 10 mL of this cell suspension was transferred to a 1 L flask containing nutrient-fortified sterilized seawater and cultured under the same water temperature and light conditions as described above, with stirring and aeration. To determine the change in cell number over the number of culture days, the culture seawater was drawn from the flask daily, and the absorbance at a wavelength of 730 nm was measured using an absorbance meter.

[0079] As a result, such Figure 3 As shown, the reef cells of the *Syntheticus* species proliferate in an exponential function manner. Further as... Figure 4 After performing a logarithmic transformation to analyze the data as shown, the same induction phase, logarithmic phase, and transition to the stationary phase were observed, similar to the proliferation patterns seen in unicellular microalgae or bacteria. That is, slow proliferation occurs until day 2 of flask culture (induction phase), followed by stable logarithmic proliferation until day 7 (logarithmic phase), and the growth rate begins to decrease from day 8 (transition to the stationary phase).

[0080] The average daily specific growth rate during the logarithmic phase, from day 2 to day 7 of culture, was 0.55 (R0). 2 =0.999), and the cells multiplied 1.7 times per day. Furthermore, the relationship between absorbance and cell density was determined using a hemocytometer, yielding the formula: cell density (cells / mL) = 1830 × absorbance. This formula allows estimation that over 5 days, from day 2 to day 7 of culture, the cell count in a 1L flask increased from 15,000 to 230,000.

[0081] As described above, by stirring the *Pteris vittata* in a sterile culture medium devoid of morphogenesis-inducing factors, a large population of single-celled cells that do not develop into multicellular organisms can be proliferated.

[0082] Example 2

[0083] The leaf-shaped multicellular green algae *Bambusa spp.*, widely cultivated as food throughout Japan, was cultured using the same method as in Example 1. *Bambusa spp.* is a reef alga with an alternating life cycle of large, leaf-shaped gametophyte generations and microsporidia with a diameter of approximately 0.05 mm. The microsporidia were first cultured until mature. Zoospores were then released from the mature microsporidia for further culture experiments.

[0084] As a result, in 1L flask cultures, the proliferation pattern of broad reefs, like that of pointed reefs, exhibited an induction phase, a logarithmic phase, and a transition to a stationary phase. The proliferation data for the logarithmic phase are presented below. Figure 5 The average daily specific growth rate was 0.67 (R0). 2 =0.989), and multiplied 2.0 times per day. 10 mL of a suspension of cells with broad reef membranes in the logarithmic growth phase was inoculated into another 1 L flask and cultured under the same conditions, resulting in proliferation at the same specific growth rate.

[0085] That is, when the broad reef membrane is cultured in a sterile medium without morphogenesis inducing factors and stirred, it can proliferate in large quantities as a single-celled cell population without developing into a multicellular organism. In addition, when this cell population is passaged, it can continue to proliferate at a rate of 2 times per day.

[0086] Example 3

[0087] 20 cm of 2 mm diameter Cremona thread was added to the cell suspension of the broad reef membrane prepared in Example 2 to ensure full adsorption of the cell population. The Cremona thread with adsorbed cells was then placed in a 500 mL beaker containing natural filtered seawater supplemented with nutrients for culture. The nutrient used was Porphyran-Conco (manufactured by Daiichi Seiki Co., Ltd.), with 0.5 mL added to 1 L of natural filtered seawater. The light and temperature were set to the same conditions as described in Example 1, and the natural seawater culture medium was changed daily.

[0088] After one month of cultivation, leaf-like broad reef membranes, approximately 1 cm in diameter, grew from the Cremona line. This is believed to be due to morphogenesis-inducing factors present in natural seawater, which induced leaf-like multicellular organisms from single-celled cells. This indicates that the single-celled populations of broad reef membranes proliferated in sterile culture medium using natural seawater possess the ability to develop into multicellular organisms.

[0089] Example 4

[0090] The single cells in the cell suspension of the broad reef membrane prepared in Example 2 were multicellularized using Thallusin. Specifically, this was done as follows: 5 mL of the cell suspension of the broad reef membrane prepared in Example 2 (1,000 to 5,000 cells) was transferred to a 500 mL flask, and 500 mL of culture medium containing Thallusin at a concentration of 1000 fmol / L was added to the nutrient-fortified sterilized seawater used in Example 1. The culture was carried out with aeration and stirring. The light and temperature were set to the same conditions as described in Example 1. After 10 days of culture, thallus composed of multiple cells developed.

[0091] Photographs of the thallus obtained after culture are shown. Figure 6 Additionally, photographs taken after the thallus was placed on a glass slide are shown below. Figure 7 On the other hand, a photograph of the thallus of a typical reef membrane, which grows from spores, is shown. Figure 8 .

[0092] It can be seen that the thallus obtained by conventional methods develops fibrous attachment roots for attaching to rocks, etc. Figure 8 (As indicated by the middle arrow), the foliation obtained through this embodiment did not form such attached roots.

Claims

1. A method for manufacturing single-celled seaweed cells, characterized in that, include: (A1) The procedure of statically culturing seaweed spores in a medium 1 that is substantially free of seaweed morphogenesis inducing factors; and (A2) The process of culturing the culture of step A1 in the culture medium 1 while stirring.

2. The manufacturing method as described in claim 1, characterized in that: The seaweed in question belongs to the order Hypophyta.

3. The manufacturing method as described in claim 1 or 2, characterized in that: The seaweed in question is a type of seaweed belonging to the genus *Reefme*.

4. The manufacturing method as described in claim 1 or 2, characterized in that: The culture medium 1 is an artificial seawater culture medium.

5. The manufacturing method as described in claim 1 or 2, characterized in that: The seaweed in question is edible seaweed.

6. The manufacturing method as described in claim 1 or 2, characterized in that: The culture under the stirring conditions is an aerated culture.

7. A method for manufacturing seaweed, characterized in that, include: The process of obtaining seaweed cells by the manufacturing method according to any one of claims 1 to 6; and The process of culturing algal cells in culture medium 2 containing algal morphogenesis inducing factors.

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