Method for producing thallus of seaweed

Abstract

To provide a technique for suppressing maturation of seaweed.SOLUTION: The foregoing problem is solved by a seaweed culturing method comprising a step of culturing seaweed under supply of microbubbles and a step of collecting the seaweed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing algal bodies of seaweed. [Background technology]

[0002] Many seaweed species grow vegetatively at low water temperatures and mature at high water temperatures, releasing spores and causing the algae to decrease or disappear. Therefore, in conventional seaweed cultivation, seaweed is cultivated only during short periods when the seawater temperature is suitable for vegetative growth without maturing.

[0003] In recent years, land-based seaweed cultivation methods, which are less susceptible to seasonal increases in seawater temperature, have been attracting attention. Non-Patent Document 1 describes a land-based cultivation method used in which low-temperature deep-sea water is supplied several times a day to cool the seawater in the culture tank, making it possible to harvest Green Laver year-round. However, this method requires energy and equipment to pump the deep-sea water, resulting in high production costs. Additionally, it is necessary to increase the amount of seawater taken during the hot summer months. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hiraoka and Oka, “Tank cultivation of Ulva prolifera in deep seawater using a new “germling cluster” method,” J Appl Phycol 20:97-102, 2008 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a technique for inhibiting the maturation of seaweeds and obtaining algal bodies in higher yields. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted extensive research and found that the maturation of seaweed can be suppressed by culturing the seaweed under a supply of fine bubbles. Based on this finding, further research has led to the completion of the present invention.

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

[0008] Section 1. A method for producing algal cells of seaweed, comprising the steps of culturing seaweed under a supply of fine bubbles and recovering the algal cells of the seaweed. Section 2. 2. The method according to claim 1, wherein the seaweed is an alga whose somatic cells differentiate into spores. Section 3. 3. The manufacturing method according to claim 1 or 2, wherein the seaweed is selected from the group consisting of: Enteromorpha spp., Usbaaonori, Hiraaonori, Bowaonori, Ulva perforatum, Hitotsugusa, Hirohanohitsugusa, Asakusa-nori, Susabinori, Habanori, and Kayamonori. Section 4. 4. The method according to claim 1, wherein the seaweed is Porphyra sieboldii, Hinodemus hilohanoegusa, or Porphyra yezoensis. Section 5. 5. The method according to claim 1, wherein the seaweed is cultured under stirring conditions. Section 6. 6. The method according to claim 1, wherein the fine bubbles include fine bubbles having a particle diameter of 500 nm or less. Section 7. 7. The method according to claim 1, wherein the microbubbles are supplied at a supply rate of 0.0001 L / min to 1 L / min per 1 L of culture solution. Section 8. 8. The method according to claim 1, wherein the microbubbles are generated by a micropore method. Section 9. 9. An algal body of the seaweed obtained by the production method according to claim 1. [Effects of the Invention]

[0009] According to the present invention, by suppressing the maturation of seaweed, it is possible to suppress the reduction of seaweed bodies at temperatures suitable for growth and increase the yield. [Brief explanation of the drawings]

[0010] [Figure 1A-1B] 1A and 1B are distribution diagrams of particle diameters and particle numbers of bubbles when fine bubbles are generated under the conditions of Reference Example 1. FIG. [Figure 2] Figure 2(a) is a schematic diagram of the air supply method of the production method of the present invention in the culture experiments of Examples 1 and 2. Figure 2(b) is a schematic diagram of the air supply method of a conventional production method. [Figure 3] FIG. 3 is a photograph of Enteromorpha laver 7 days after cultivation in the Enteromorpha cultivation experiment of Example 1. [Figure 4] FIG. 4 shows the wet weight of algal bodies over 7 days in the Enteromorpha culture experiment in Example 1. [Figure 5] FIG. 5 is a photograph of the tip of Enteromorpha spp. after 7 days of cultivation in the Enteromorpha cultivation experiment of Example 1. [Figure 6] FIG. 6 shows the average daily growth rate for each strain over seven days in the Enteromorpha culture experiment in Example 2. [Figure 7] FIG. 7 is a schematic diagram of the gas supply method used in the culture experiments of Examples 3 to 5. [Figure 8] FIG. 8 shows the wet weight of algal bodies over 7 days in the Enteromorpha culture experiment of Example 3. [Figure 9] FIG. 9 shows the wet weight of algal bodies over 8 days in the culture experiment of Hibiscus tularensis in Example 4. [Figure 10] FIG. 10 shows the wet weight of algae bodies over 8 days in the Porphyra yezoensis culture experiment in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0011] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

[0012] Method for producing seaweed bodies In one aspect, the present invention relates to a method for producing algal cells of seaweed (sometimes referred to herein as the "production method of the present invention"), which comprises the steps of culturing seaweed under a supply of microbubbles and recovering the algal cells of the seaweed. This method will be described below.

[0013] The seaweed is not particularly limited as long as it is a multicellular marine algae that is large enough to be visible to the naked eye, and examples include seaweed such as green algae, red algae, and brown algae. Seaweed that is desired to be cultivated in industrial quantities and is used as food or as a raw material for biofuel is particularly preferred, and the effects of the production method of the present invention can be more effectively achieved with seaweed that grows vegetatively and matures at temperatures of 15 to 25°C, the temperature used in industrial aquaculture. Furthermore, from the viewpoint of further increasing the yield during cultivation using the production method of the present invention, seaweed whose somatic cells differentiate into spores is more preferred, and examples of such seaweed include seaweed belonging to the Ulva class of the Chlorophyta division, the Bangiophyceae class of the Rhodophyta division, and the Phaeophyceae class of the Xanthophyta division. Examples of seaweeds belonging to the Ulva class include: Green laver, Usbaaonori, Flat laver, Bow laver, Southern laver, Taretsuaonori, Kinuitoaonori, Ulva perforatum, Ulva longana, Ribbon laver, Ulva giantana, Ulva buttonana, Ulva giantana, Ulva serrata, Ulva dulcis, Hitotsugusa, Hitotsugu broadana, etc. Examples of seaweeds belonging to the Bangiophyceae class include Asakusa nori and Susabinori. Examples of seaweeds belonging to the Phaeophyceae class include: Ecklonia cava, Kurome, Undaria pinnatifida, Laminaria japonica, Habanori, and Kayamonori. Preferred examples include edible seaweed such as Green Laver, Usba Aonori, Hira Aonori, Bow Aonori, Ulva perforatum, Hitoegusa, Hinodegusa, Habanori, Kayamonori, etc. The seaweed may be used alone or in combination of two or more types.

[0014] According to the production method of the present invention, it is possible to obtain seaweed algae bodies that have an increased wet weight or volume compared to the spores, germinated bodies, etc. used as starting materials. The wet weight of the obtained seaweed algae bodies is, for example, 10 times or more, 50 times or more, or 100 times or more, and 150 times or less, 200 times or less, 250 times or less, 300 times or less, 400 times or less, 500 times or less, 1000 times or less, 2000 times or less, 5000 times or less, 10000 times or less, 100,000 times or less, or 1,000,000 times or less, of the wet weight at the start of cultivation.

[0015] Maturity is the stage at which seaweed bodies begin to form and release spores. Generally, seaweed bodies undergo vegetative growth at low water temperatures, mature at high water temperatures, and then decrease or disappear after releasing spores. Therefore, in seaweed farming, it is desirable to harvest before maturation occurs and the number of algae decreases. When seaweeds such as Ulva, Bangiophyceae, and Phaeophyceae mature, somatic cells differentiate directly into germ cell sacs, and the differentiated somatic cells fall off from the algae, resulting in a decrease in leaf length and algae weight. In addition, the areas where the cells have fallen off turn white, reducing their commercial value as a food product.

[0016] The evaluation of whether or not algae maturation is inhibited can be performed by visually or microscopically checking the presence and number of spores in the culture solution. In the case of seaweeds such as Ulva, Bangiophyceae, and Phaeophyceae, in addition to the above methods, it can be confirmed by checking whether the wet weight of the algae after cultivation or the average daily growth rate during the cultivation period is increased compared to the control group. The average daily growth rate is an index that represents the rate of increase in wet weight of the algae per day, where the initial weight is W0 and the weight after t days is W t It can be calculated by the following formula: Average daily growth rate (%)=[(W t / W0) 1 / t -1] × 100

[0017] In addition, for seaweeds such as Ulva, Bangiophyceae, and Phaeophyceae, whether or not algae maturation is inhibited can also be evaluated by visually checking for the presence or absence of bleached areas at the tips of the algae after cultivation. The fewer bleached areas there are, the more inhibited maturation is.

[0018] Spores are reproductive cells, and examples of spores include zoospores, gametes, zygotes, tetraspores, carpospores, uniospores, neutozoospores, and monospores.

[0019] In the production method of the present invention, spores or germinated bodies can be used as starting materials. The spores are not particularly limited as long as they are obtained from seaweed. The spores may be clumped spores in which spores adhere to each other. The clumped spores can be obtained, for example, by the method described in Japanese Patent No. 3828359. The germinated bodies can be obtained by transferring clumped spores, which have been finely crushed to a diameter of approximately 5 mm or less as needed, to a culture vessel, culturing them with aeration, and allowing them to germinate. The spores may be of one type alone or a combination of two or more types.

[0020] As used herein, "fine bubbles" refers to bubbles with a diameter of less than 100 μm. Fine bubbles with a diameter of less than 100 μm are defined as "fine bubbles" (registered trademark) by the International Organization for Standardization (ISO). Fine bubbles with a diameter of 1 to 100 μm are defined as "microbubbles," and those with a diameter of less than 1 μm are defined as "ultrafine bubbles" (registered trademark) (ISO). Bubbles with a diameter of less than 1 μm are also called "nanobubbles." Fine bubbles have a negatively or positively charged surface, and are characterized by attracting objects with an opposite charge and repelling objects with the same charge. The fine bubbles used in the manufacturing method of the present invention preferably have a diameter of less than 100 μm, 50 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, 1 μm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 100 nm or less. Using microbubbles with small diameters can reduce damage to algae when they are generated. Furthermore, microbubbles with diameters less than 100 μm are characterized by their slow rising tendency. Supplying gas in the form of microbubbles allows the effects of gas molecules to be exerted in water for a longer period of time. Microbubbles with diameters less than 1 μm can remain in water for particularly long periods of time, allowing the effects of gas molecules to be exerted even longer. Microbubbles with diameters less than 1 μm transmit visible light and therefore appear transparent to the naked eye. The particle size and number of microbubbles can be measured using nanoparticle tracking analysis, particle trajectory analysis, laser diffraction / scattering, dynamic light scattering, resonant current mass spectrometry, electrical detection zone analysis, dynamic image analysis, and shading methods.

[0021] Microbubbles generated using a commonly available microbubble generator can be used. The microbubble generation method is not particularly limited. Microbubbles can be generated using methods such as a swirling liquid flow system, a static mixer system, a micropore system, an ejector system, a Venturi system, a pressurized dissolution system, a cooling dissolution system, or a mixed steam condensation system. Microbubbles with a diameter of less than 1 μm can be generated using methods such as a micropore system, a static mixer system, a swirling liquid flow system, an ultrasonic system, or a pressurized dissolution system. Microbubbles may be generated within the culture vessel, or, for example, in a separate vessel connected to the culture vessel. In the production method of the present invention, microbubbles are preferably generated within the culture vessel using a micropore system, from the viewpoints of simplicity and minimal impact on the seaweed. The micropores used for aeration are not particularly limited, but nonwoven fabrics and ceramics can be used, for example. When installing a microbubble generator within the culture vessel, the installation location is not particularly limited, but it can be installed on the bottom or side, for example.

[0022] The gas used as the source of generating fine bubbles is not particularly limited, but examples thereof include the atmosphere, and gases including gases contained in the atmosphere such as H2, O2, CO2, Ar, and water vapor.

[0023] In the production method of the present invention, the microbubbles are preferably supplied at a supply rate of 0.0001 to 1 L / min per 1 L of culture solution, more preferably at a supply rate of 0.0005 to 0.5 L / min per 1 L, and even more preferably at a supply rate of 0.001 to 0.2 L / min per 1 L.

[0024] Culturing is preferably carried out under agitation conditions. Culturing under agitation conditions is not particularly limited as long as the culture medium is partially or entirely agitated. For example, culturing can be carried out by aerating the culture medium using an aeration pump or the like (aeration culture), passing a liquid (e.g., culture medium) through the culture medium using a pump or the like, moving a stirrer (e.g., rotating), or shaking the culture vessel. During culture, the seaweed may grow, for example, in a floating state, or partially fixed to a wall or suitable carrier. Culturing under agitation conditions removes oxygen bubbles that have adhered to the surface of the algae during photosynthesis, allowing for efficient photosynthesis. Furthermore, fluidizing the algae mass allows each algae mass to be uniformly irradiated with light, resulting in uniform growth.

[0025] The culture temperature is not particularly limited as long as it is a temperature suitable for the vegetative growth and maturation of seaweed, and is, for example, 5 to 35°C, preferably 10 to 30°C, and more preferably 15 to 25°C.

[0026] The light conditions during cultivation are not particularly limited as long as they are light conditions that allow seaweed to grow, and examples include natural light light-dark cycle conditions, artificial light conditions, and artificial light light-dark cycle conditions.

[0027] The culture period is not particularly limited, but in one embodiment, according to the production method of the present invention, the yield can be increased more quickly by suppressing maturation while culturing at a temperature suitable for seaweed growth. From this perspective, in one embodiment, the culture period is, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, and is preferably 60 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 10 days or less, or 7 days or less.

[0028] The culture of the present invention is usually carried out in a container. The culture of the present invention is usually static culture. There are no particular limitations on the container as long as it can be used for culturing algae, and a suitable capacity can be selected depending on the scale of the culture. For example, glass or plastic flasks, beakers, aquaria, large circular tanks, and connected tanks can be used. The culture container may be installed indoors or outdoors.

[0029] The cultivation of the present invention is carried out in water. The liquid used for cultivation is not particularly limited as long as it is suitable for growing seaweed, but is usually seawater. The seawater is not particularly limited as long as it is suitable for growing 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 that has been artificially adjusted by adding salts such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and magnesium sulfate, pH buffers, and other trace components to mimic the composition of natural seawater. It also includes seawater from which salt and other components have been removed and / or from which active ingredients have been added. Seawater may be sterilized before use.

[0030] Nutrients that promote seaweed growth may be added to the seawater as needed. When artificial seawater is used, a seaweed morphogenesis inducer may be added. The seaweed morphogenesis inducer refers to a substance produced by a microorganism present in seawater, and various reported substances may be used without particular limitation (WO 2004 / 007510, JP 2003-189845, etc.). Examples of seaweed morphogenesis inducers include salusin.

[0031] The production method of the present invention suppresses the maturation of seaweed and the associated reduction in algae mass, thereby enabling efficient vegetative growth of seaweed and increasing harvest yields. Furthermore, at aquaculture sites where the water temperature must be maintained below a certain level, the energy required to lower the water temperature can be reduced, thereby reducing production costs.

[0032] In one embodiment of the present invention, the algae bodies of seaweed can be obtained by the production method of the present invention. The obtained algae bodies of seaweed can be used, for example, as food, biofuel, raw materials for effective substances such as pharmaceuticals, etc. [Example]

[0033] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0034] Reference example 1 Using a micropore-type microbubble generator (NAC Corporation, model FP20-70), 500 mL of artificial seawater was aerated at a pressure of 0.15 MPa and a flow rate of 0.033 L / min for 1 hour, and the particle size and number of bubbles with diameters of 20 to 1000 nm were measured. As a control, the particle size and number were compared with those obtained during normal aeration at the same flow rate using a standard aeration tube. Measurements were performed using a ZetaView (Particle Metrix). The results are shown in Figure 1A. When aeration was performed using the microbubble generator, many bubbles with particle sizes less than 100 nm were observed. Using the same generator, 2 L of artificial seawater (with 1 ml of Porphyran Conco added) was treated at a pressure of 0.15 MPa and a total aeration rate of 0.08 L / min (aeration rate from the aeration tube:aeration rate from the fine bubble generator = 1:1) for 30, 60, 120, and 240 minutes. The particle size and number of bubbles with diameters between 1 and 500 μm were measured (see Figure 2(a)). As a control, normal aeration at 0.08 L / min using the aeration tube alone was performed under the same conditions for 240 minutes (see Figure 2(b)). The particle size and number of bubbles were compared. Measurements were performed using a PartAn SI (Microtrac Bell). The results are shown in Figure 1B. Aeration using the fine bubble generator generated many bubbles with diameters of 100 μm or less. In the following examples, fine bubbles were generated using a micro-hole type fine bubble generator (Nac Corporation, model FP20-70).

[0035] Example 1 The wet weight of Enteromorpha spp. was compared when the culture was performed while supplying fine bubbles in addition to aeration using a standard aeration tube (Fig. 2(a)), and when the culture was performed using only aeration using an aeration tube (Fig. 2(b)). Five clumps of Enteromorpha spp. (starting wet weight: 0.01 g / 5 clumps) were used as test algae. They were cultured in a medium containing 2 L of sterilized artificial seawater and 1 ml of a nutrient solution (Porphyran Conco, Daiichi Steel Corporation) at 20°C, with a 12-h light / 12-h dark cycle and a total aeration rate of 0.06 L / min (aeration rate from the aeration tube:aeration rate from the microbubble generator = 1:1). The medium was changed on days 3 and 5. The algae were cultured for 7 days under standard aeration alone or with standard aeration plus microbubble supply. The wet weight (g) of the algae was measured (Figure 4). Daily growth rates were calculated from the results. As shown in Figures 3 and 4, the Enteromorpha laver grown more when microbubbles were supplied. The average daily growth rate (%) over seven days was 113.2% with microbubbles, 1.2 times higher than the 92.6% achieved with normal cultivation. Furthermore, when the algae were cultivated with microbubbles supplied, there were fewer areas (arrows) releasing spores from the tip, and the degree of maturity was lower (Figure 5).

[0036] Example 2 The Kamosho, Yoshino, and Matsuzaki strains of Enteromorpha laver were used, and the wet weights of the algae were measured and the daily growth rates calculated under the same conditions as in Example 1 after 7 days of culture, except that the total aeration rate was 0.04 L / min (aeration rate from the aeration tube:aeration rate from the microbubble generator = 1:1). The initial wet weights of the algae were 0.015 g / 5 clumps for the Kamosho strain, 0.02 g / 5 clumps for the Yoshino strain, and 0.027 g / 5 clumps for the Matsuzaki strain. As a result, as shown in Figure 6, the daily growth rate of the green laver strains to which fine bubbles were supplied also tended to be higher, even for strains other than the duck bed strains.

[0037] Example 3 To prevent the water from becoming cloudy due to the supply of bubbles from the bottom and to eliminate the difference in light intensity across the vessel, a micropore generator was installed on the side of the culture vessel as shown in Figure 7, and a growth saturation light intensity of 500 μmol / m was set. 2 The wet weight (g) of Enteromorpha prolifera after 8 days of cultivation at 1 / s was compared with the wet weight (g) of the algae when only aeration was performed using a conventional aeration tube (Figure 2(b)). Specifically, five clumps of aggregated algae of the Kamosho strain of Enteromorpha spp. (initial wet weight of algae: 0.01 g / 5 clumps) were used as test algae and placed in a medium containing 2 L of sterilized artificial seawater and 1 mL of a nutrient supplement (Porphyran Conco, Daiichi Steel Corporation). The culture conditions were: water temperature 20°C, light / dark cycle 12 h / 12 ​​h, total aeration rate 0.08 L / min (aeration rate from the aeration tube:aeration rate from the microbubble generator = 1:1), and medium change on days 2, 4, and 6. The wet weight (g) of the algae was measured, and the daily growth rate was calculated. As shown in Figure 8, even when a microbubble generator was installed on the side of the container, the green laver that received microbubble supply grew more. The average daily growth rate (%) over eight days was 119.2% with microbubbles, 1.1 times higher than the 107.2% with normal cultivation. Furthermore, when observing the algae bodies, the green laver that received microbubble supply had fewer areas releasing spores from the tip and was less mature.

[0038] Example 4 A germ-free strain of Hibiscus globulus (initial wet weight of algae: 0.017 g / 5 individuals) was used as the test algae and placed in a medium containing 2 L of sterilized artificial seawater and 1 ml of a nutrient solution (Porphyran Conco, Daiichi Seimo Co., Ltd.). The saturation light intensity for Hibiscus globulus growth was 200 μmol / m 2 The culture conditions were the same: 1 / s, water temperature 20°C, light / dark cycle = 12 hours / 12 hours, total aeration rate 0.08 L / min (aeration rate from the aeration tube:aeration rate from the microbubble generator = 1:1), and medium change on days 2, 4, and 6. The culture was then continued for 8 days under either normal aeration only (Figure 2(b)), or normal aeration plus the supply of microbubbles from a microbubble generator installed on the side of the container (Figure 7), and the wet weight of the algae (g) was measured. As shown in Figure 9, the Hibiscus hirsutus that received microbubbles grew larger, and no decrease in wet weight due to maturation was observed. Furthermore, when observing the algae, those with microbubbles had fewer holes and chipped edges, and were less mature.

[0039] Example 5 The Saijo strain of Porphyra yezoensis (initial wet weight of algae: 0.19 g / 5 individuals) was used as the test algae and placed in a medium containing 2 L of sterilized artificial seawater and 1 ml of a nutrient solution (Porphyran Conco, manufactured by Daiichi Seimo Co., Ltd.). The light intensity for the growth saturation of Porphyra yezoensis was 300 μmol / m 2 The culture conditions were: 1 / s, water temperature 20°C, light / dark cycle 12 h / 12 ​​h, total aeration rate 0.08 L / min (aeration rate from the aeration tube:aeration rate from the microbubble generator = 1:1). The medium was changed on days 2 and 4, and on day 6, the medium was changed to 2 L of sterile artificial seawater supplemented with 2 ml of Porphyran-Conco. The culture was then performed for 8 days under either normal aeration only (Figure 2(b)), or normal aeration plus microbubble supply from a microbubble generator attached to the side of the container (Figure 7). The wet weight of the algae (g) was measured. The daily growth rate was calculated from the results. As shown in Figure 10, the Porphyra yezoensis grown to a greater extent when microbubbles were supplied. The average daily growth rate (%) over eight days was 50.5% with microbubbles, which was about 10% higher than the 46.4% achieved with normal cultivation.

Claims

1. A method for producing algal cells of seaweed, comprising the steps of culturing seaweed under a supply of fine bubbles and recovering the algal cells of the seaweed.

2. The method according to claim 1 , wherein the seaweed is an alga whose somatic cells differentiate into spores.

3. 2. The method according to claim 1, wherein the seaweed is selected from the group consisting of Enteromorpha spp., Enteromorpha umbellata, Enteromorpha spp., Enteromorpha hiraensis, Enteromorpha arvensis, Enteromorpha perforatum, Enteromorpha hilohanoi, Porphyra asakusa, Porphyra susabiensis, Porphyra habanori, and Porphyra kayamoneori.

4. The method according to claim 1, wherein the seaweed is Porphyra sieboldii, Hinodemus hilohanoegusa, or Porphyra sieboldii.

5. The method according to claim 1 , wherein the seaweed is cultured under stirring conditions.

6. The method according to claim 1 , wherein the microbubbles include microbubbles having a particle diameter of 500 nm or less.

7. 2. The method according to claim 1, wherein the fine bubbles are supplied at a supply rate of 0.0001 L / min to 1 L / min per 1 L of culture solution.

8. The manufacturing method according to claim 1 , wherein the microbubbles are generated by a micropore method.

9. 9. The algae body of the seaweed obtained by the manufacturing method according to any one of claims 1 to 8.

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