JP7698882B2 - Method for manufacturing artificial pond for cultivating halophilic microalgae and artificial pond - Google Patents

Description

The present invention relates to a method for manufacturing an artificial pond for cultivating halophilic microalgae, and more specifically, to a method for manufacturing an artificial pond for cultivating halophilic microalgae in which a water-impermeable structure is provided on the bottom and sides of the artificial pond using a mixed particle group of a specific composition, an artificial pond manufactured by this method, and a method for producing halophilic microalgae cultivated in the artificial pond.

There is a demand to meet much of the energy demand through renewable energy sources such as solar and wind power, but renewable energy has a low energy density, and a rapid shift away from fossil energy is a high hurdle.
In order to lower this hurdle by producing fuel oil using microalgae, the idea of "producing fuel oil using microalgae" has been proposed (for example, Patent Documents 1 and 2).
However, considering the current situation, many issues remain to be resolved before it can be put into practical use, including the culture method, economies of scale, and costs.

It has also been proposed to obtain useful organic substances from cultured and grown microalgae, and specific studies have been conducted on various useful substances such as sugars, polypeptides, and vitamins (for example, Patent Documents 3 and 4).
Patent Document 3 describes a method in which a halophilic cyanobacterium is cultured at a high salt concentration until it reaches a stationary phase, and then cultured at a low salt concentration for several hours to several days to produce polysaccharides, and the polysaccharides are separated and purified.
Furthermore, Patent Document 4 describes a method for solvent extraction of β-carotene from an aqueous algal biomass suspension.
However, many problems remain to be solved, including the cultivation method, the enjoyment of economies of scale, and cost issues.

On the other hand, waterproof sheets using bentonite are known.
Patent Document 5 describes a waterproof sheet in which granular pieces of clay such as bentonite are adhered to a sheet of high-density polyethylene or the like with an adhesive such as polyisobutylene or polybutylacrylate.
Patent Document 6 describes a waterproof sheet obtained by applying a bentonite elastomer, which is made by kneading bentonite with water, to the back surface of a "sheet material such as a polyvinyl chloride sheet" to which a fiber aggregate is fixed.

However, these are sheets used for waterproofing in the fields of civil engineering and construction, and the material used in large quantities is a petroleum-derived polymer, which goes against the purpose of meeting energy demand with renewable energy.

As a technique that uses bentonite but not in sheet form, Patent Document 7 describes a "method of forming a water-stopping film in the concave surface of an excavated pond," in which a mixture made by adding an inorganic substance such as bentonite to an emulsion of synthetic rubber or synthetic resin is allowed to penetrate to a specified depth into the concave surface of the excavated pond.

Patent Document 8 describes a waterproofing method for ground, in which a solidification material such as cement and bentonite are almost simultaneously spread onto the ground, and then the target soil of the ground is mixed with the bentonite and the solidification material to form a waterproofing layer.
Patent Document 9 describes an artificial greening method in which a mixed soil layer of bentonite and locally generated soil is laid on the inner surface of a recess in an artificial waterway, a crushed stone layer is laid on top of that, and waterproof mortar is laid on top of the crushed stone layer.

However, these technologies are used for "waterproofing in the fields of civil engineering and construction" and were not invented with the realization that they were particularly suitable for cultivating halophilic microalgae. In addition, they involve the addition of synthetic rubber or synthetic resin emulsions, solidifying agents such as cement, and then a layer of crushed stone or waterproof mortar is laid on top of that.

Whether it is "fuel oil production from microalgae" or "production of useful organic matter using microalgae," reducing costs is essential for practical application.
In particular, in order to realize economies of scale and reduce costs, it is extremely important to use extremely large artificial ponds as culture tanks, but there were no artificial ponds suitable for culturing halophilic microalgae.

Regarding "obtaining fuel oil contained in microalgae" as an alternative to petroleum or natural gas, for example, large-scale cultivation of microalgae has not been suitably achieved; the amount of energy required is large; etc., and further improvements are required in terms of cost and efficiency, and thus.
In addition, with regard to "obtaining useful organic matter from microalgae," further improvements are required from the standpoint of cost (efficiency), such as eliminating contamination from artificial compounds, heavy metals, and trace harmful elements, and large-scale cultivation of microalgae.

JP 2015-231363 A JP 2015-146785 A Japanese Patent Application Publication No. 5-049491 Japanese Patent Application Publication No. 7-002761 Japanese Patent Application Publication No. 3-132508 Japanese Patent Application Publication No. 9-248877 Japanese Patent Application Publication No. 55-085717 Japanese Patent Application Publication No. 10-331152 JP 2001-224243 A

The present invention has been made in consideration of the above-mentioned background technology, and its object is to solve the above-mentioned problems and provide a method for manufacturing an artificial pond for cultivating halophilic microalgae that does not use petroleum-derived products such as synthetic polymers, artificial substances such as solidifying agents and flocculants, or binding agents such as mortar, cement and silica, and is therefore free of various types of contamination from these.

Another object of the present invention is to provide a method for manufacturing an artificial pond for cultivating halophilic microalgae that satisfies various performance requirements, such as cost reduction, ease of manufacturing, large-scale cultivation (taking advantage of economies of scale), and no hindrance to the cultivation of halophilic microalgae.

As a result of extensive research into solving the above problems, the inventors discovered that a structure (layer) formed by pressing a group of particles containing bentonite powder is suitable for use as the side and/or bottom of an artificial pond for cultivating halophilic microalgae due to the subsequent swelling caused by water absorption and salt precipitation, and that this can be produced cheaply and easily, which led to the completion of the present invention.

That is, the present invention is for culturing halophilic microalgae, and is a method for producing an artificial pond for storing at least the halophilic microalgae and seawater or artificial seawater,
The present invention provides a method for producing an artificial pond, characterized in that a water-impermeable structure is provided on the side and/or bottom of the artificial pond by compressing a particle group containing bentonite powder.

The present invention also relates to a method for culturing halophilic microalgae, comprising at least the halophilic microalgae and a method for producing an artificial pond for storing seawater or artificial seawater,
The present invention also provides a method for producing an artificial pond, comprising the steps of: providing a water-impermeable structure on the side and/or bottom of the artificial pond by pressing a mixture of particles containing bentonite powder and sand.

The present invention also provides a method for producing an artificial pond in an area that is originally sandy, adding bentonite from outside to the sand that constitutes the sandy area and mixing it to obtain the mixed particle group, and then pressurizing the mixed particle group to provide a water-impermeable structure.

The present invention also provides a method for manufacturing the artificial pond, in which the seawater is deep ocean water.

The present invention also relates to an artificial pond for culturing halophilic microalgae, which is produced using the above-mentioned method for producing an artificial pond, and which contains at least the halophilic microalgae and seawater or artificial seawater,
The present invention provides an artificial pond, characterized in that the water-impermeable structure is formed on the side and/or bottom surface in a state in which the structure absorbs water.

The present invention also provides a method for producing halophilic microalgae, which comprises placing halophilic microalgae and seawater or artificial seawater in the artificial pond and culturing the halophilic microalgae.

The present invention also provides a method for producing useful organic matter, which is characterized by obtaining useful organic matter from "halophilic microalgae produced using the above-mentioned method for producing halophilic microalgae."

According to the present invention, the above problems and issues can be solved, and an artificial pond for cultivating halophilic microalgae can be produced that is free of petroleum, low cost, and free of the risk of pollution.
Since the present invention does not rely on "synthetic polymers or synthetic polymer laminates" such as vinyl sheets or bentonite sheets, does not rely on synthetic chemicals such as solidifying agents or flocculants, and does not rely on artificial binders such as mortar, cement, concrete, silica, etc., there is no need to worry about various types of contamination from these. According to the present invention, an artificial pond can be produced using safe natural materials that are all widely available on Earth, so there is no risk of organic contamination or heavy metal contamination.

Furthermore, if petroleum-derived products such as (synthetic) polymers (sheets) and (organic) flocculants were used, the purpose of obtaining fuel oil and useful organic matter without relying on petroleum would be defeated. However, according to the present invention, artificial ponds can be produced entirely without relying on petroleum, and fuel oil and useful organic matter can be obtained from halophilic microalgae without relying on petroleum, thereby fulfilling the above-mentioned purpose.

When cultivating and growing halophilic microalgae to obtain fuel oil and other useful substances, reducing costs becomes an extremely big challenge.
According to the present invention, it has the following advantages: artificial ponds are easy to manufacture; the material costs for artificial ponds are inexpensive; halophilic microalgae can be easily cultivated on a large scale; economies of scale can be enjoyed through the large scale cultivation; and costs can be reduced.
In particular, by simply compressing a particle group containing bentonite powder, preferably a mixed particle group obtained by mixing bentonite powder and sand, and further by compressing the mixed particle group with the aid and presence of (sea) water, a perfect water-impermeable structure can be produced extremely easily.

According to the present invention, since bentonite is abundant on earth, if an artificial pond is constructed in a place where bentonite is mined, further cost reduction is possible.
Conversely, if an artificial pond is constructed on a sandy area and bentonite is added to the sand in the area, further cost reduction is possible.
In addition, in the present invention, it is preferable to culture halophilic microalgae in "seawater, deep ocean water, or artificial seawater obtained by adjusting the components thereof," and in that case, it is preferable to install the artificial pond near the sea, and since the sea is often sandy, a synergistic effect can be achieved, which can further reduce costs.

The growth rate of halophilic microalgae is unexpectedly higher when the water-impermeable structure is made of "mixed particles containing sand and bentonite" than when it is made of "sand only." The growth rate also depends on the type of bentonite.
Therefore, by using a type of bentonite suitable for the "halophilic microalgae to be cultured," an artificial pond with even better growth properties can be produced.

In addition, when constructing an artificial pond in the desert, it is possible to use sand from the desert as the sand for the water-impermeable structure, but if the sand contains a lot of silica sand ( _SiO2 ), the introduced seawater will seep underground and it will be difficult to store it. In that case, if bentonite is mixed with the desert sand, an artificial pond with low water permeability can be constructed.

Various types of contamination can hinder the cultivation of halophilic microalgae, but according to the present invention, the basic performance of the pond described above is achieved and all natural materials are used, avoiding the use of non-natural materials, so an artificial pond suitable for cultivating halophilic microalgae can be provided.
Furthermore, when halophilic microalgae are produced in an artificial pond filled with seawater or artificial seawater, there is no contamination (contamination by other organisms) from "organisms that cannot survive (cannot grow) in the presence of dissolved salts at or above the concentration of seawater." From this point of view, it is preferable to cultivate halophilic microalgae in an artificial pond in the first place, but among them, the artificial pond of the present invention, which does not use any non-natural products, is even more suitable for culturing halophilic microalgae because it is less contaminated by both other organisms and non-natural products.

According to the present invention, as specifically described in the Examples, cost reduction and environmental conservation are achieved. Therefore, large-scale cultivation of microalgae becomes feasible, and it becomes possible to obtain non-petroleum energy and low-cost useful organic matter.
That is, the method is sufficient in terms of obtaining fuel oil contained in microalgae in terms of cost, efficiency, etc. Also, the method is sufficient in terms of obtaining useful organic matter from microalgae in terms of the required energy consumption, the recovery rate of microalgae, cost, efficiency, etc.

In order to profitably obtain useful organic matter and oils and fats (fuel) from halophilic microalgae, a certain level of production scale is necessary, but the present invention is suitable for large-scale production (matched to large-scale production) due to its reduced costs, increased efficiency in obtaining the desired products, and almost no environmental impact.

1 is a schematic cross-sectional view showing an example of a process for producing an artificial pond by providing a water-impermeable structure on the sides and bottom of a fallow field and pouring seawater into it, and the produced artificial pond: (a) A fallow field used for production; (b) The water-impermeable structure before pouring seawater; (c) An artificial pond produced by pouring seawater containing halophilic microalgae, in which the thickness of the water-impermeable structure does not change before and after the introduction of seawater, etc.; (d) An artificial pond produced by pouring seawater containing halophilic microalgae, in which the thickness of the water-impermeable structure increases before and after the introduction of seawater, etc. FIG. 1 is a schematic cross-sectional view of an artificial pond manufactured and used in the enlargement test of Example 9. 1 is a graph showing the growth rate of halophilic microalgae in the expansion test of Example 9. 1 is a graph showing the growth rate of halophilic microalgae in the laboratory test of Example 10.

The present invention is described below, but is not limited to the specific forms below and can be modified as desired within the scope of the technical concept.

The method for producing an artificial pond of the present invention is for culturing halophilic microalgae, and is a method for producing an artificial pond for storing at least the halophilic microalgae and seawater or artificial seawater,
The artificial pond is characterized in that a water-impermeable structure is provided on the side and/or bottom of the pond by compressing a particle group containing bentonite powder.

<Particle group>
In the present invention, a water-impermeable structure is provided on the side and/or bottom of the artificial pond, the structure being made of a particle group containing at least bentonite powder.
<<Bentonite>>
Here, "bentonite" is a general term for clays whose main component is montmorillonite, and is mainly composed of layered silicate minerals (mainly containing aluminum phyllosilicate). In addition to aluminum (Al), bentonite also contains sodium (Na), calcium (Ca), potassium (K), magnesium (Mg), etc., and there are many types depending on the type of cation. Many bentonites are formed by the metamorphism of volcanic ash with water. They may be acidic white clay, which is acidic, or neutral to alkaline.
The interlayer cations of montmorillonite include Na ⁺ , Ca2 ⁺ , K ⁺ , Mg2 ^+, etc., and some bentonites contain a large amount of one of these ions. The bentonite used in the present invention may be sodium bentonite, which contains a large amount of Na ⁺ and has a high swelling power, or calcium bentonite, which contains a large amount of Ca2 ⁺ and has a low swelling power.

The bentonite used in the present invention is excellent in terms of ease of manufacturing artificial ponds when mixed with sand, and in terms of the performance of the resulting artificial ponds. Since the proliferation of halophilic microalgae also depends on the type of bentonite, a bentonite that is suitable for the halophilic microalgae to be cultured (one that has excellent proliferation properties) is selected.
The bentonite used in the present invention is determined based on suitable chemical composition (composition formula), swelling power, pH, particle size, structural viscosity (thixotropy), bulk density (apparent specific gravity), dispersibility in water (dispersion stability), etc., taking into consideration mainly the two points mentioned above (the properties and manufacturability of the artificial pond, and the growth properties of algae).

The type of bentonite powder used in the present invention is not particularly limited. In other words, as long as it is bentonite, there are no particular limitations on its origin, chemical composition (cation species, etc.), proportion of main components, crystal structure (degree of crystallinity), average particle size, particle size distribution, acidity/alkalinity, and other aspects.
Bentonite with low swelling power having a swelling power of 13 mL/2 g or less, bentonite with medium swelling power having a swelling power of 13 to 18 mL/2 g, or bentonite with high swelling power having a swelling power of 18 mL/2 g or more can be suitably used.
"Swelling power" is defined as the volume of water (in mL) that 2 g of bentonite will absorb.

Either artificially modified or naturally mined and unmodified may be used, but from the viewpoint of reducing costs, naturally mined or crushed materials are preferable rather than artificially modified materials.
Furthermore, the place of origin is not particularly limited and may be North America, Central America, South America, Europe, China, Japan, India, Africa, etc., and any place of origin may be used.

The upper limit of the volume average particle size is preferably the particle size generally referred to as "clay", specifically, 5 μm or less is preferable, 4 μm or less is more preferable, 3 μm or less is even more preferable, and 2 μm or less is particularly preferable.

If the upper limit of the volume average particle size is equal to or less than the above, there is an effect that the volume can be easily expanded by swelling with water. Furthermore, when a "mixed particle group containing bentonite powder and sand" described below is used as the "particle group containing bentonite powder", the bentonite powder is sufficiently fine that it can easily penetrate into the gaps in the sand, improving water impermeability.

The bentonite used is desirably one that expands upon contact with seawater to a swelling ratio of preferably 5 to 16 times, and particularly preferably 7 to 13 times. Such bentonite has good swelling efficiency within the water-impermeable structure upon introduction of seawater, etc., and is preferable from the standpoints of water impermeability, workability, etc.
For example, if a water-impermeable structure is created by adjusting the bentonite content to 10 mass% relative to the total mass of the bentonite powder and sand, the volume or thickness of the water-impermeable structure will theoretically double when seawater or the like is introduced.
Even if the thickness of the water-impermeable structure does not increase when bentonite and (artificial) seawater are used in combination with sand, the bentonite (particles) swell and penetrate between the coarse sand particles, improving water impermeability.

Commercially available products can also be suitably used. Examples of such commercially available products include Hotaka, TB series such as TB-250, TB-S, and TB-200, TS series such as TS-01, Akagi, Asama, Haruna, clay series such as Neo Clay and Super Clay, all manufactured by Tachibana Material Co., Ltd. In addition, examples of the Kunigel (registered trademark) series, such as Kunigel V1, Kunigel V2, and Kunigel GS, all manufactured by Kunimine Kogyo Co., Ltd., can also be mentioned.

<Mixed particle group>
In the present invention, it is preferable to use a "mixed particle group containing bentonite powder and sand" as the "particle group containing bentonite powder" from the viewpoint of reducing costs, etc.
In other words, the present invention is a method for culturing halophilic microalgae, and also a method for manufacturing an artificial pond for storing at least the halophilic microalgae and seawater or artificial seawater, in which a water-impermeable structure is provided on the side and/or bottom of the artificial pond by pressing a mixed particle group containing bentonite powder and sand.

<<Sand>>
In the present invention, it is preferable to provide a water-impermeable structure on the side and/or bottom of the artificial pond by using a mixed particle group containing at least bentonite powder and sand. Here, "sand" refers to debris having an average particle size of 2 mm (2000 μm) to 1/16 mm (62.5 μm).
Any of the following may be used: very coarse sand (2 mm to 1 mm), coarse sand (1 mm to 1/2 mm (1000 μm to 500 μm), medium sand (1/2 mm to 1/4 mm (500 μm to 250 μm), fine sand (1/4 mm to 1/8 mm (250 μm to 125 μm), and very fine sand (1/8 mm to 1/16 mm (125 μm to 62.5 μm).
Among them, fine sand (1/4 mm to 1/8 mm (250 μm to 125 μm) and/or extra-fine sand (1/8 mm to 1/16 mm (125 μm to 62.5 μm)) is particularly preferred. Here, "and" also means a mixture of fine sand and extra-fine sand.

Rocks and the like may be artificially crushed to meet the above definition, but in order to reduce material costs, it is preferable to use natural sand as it is.
In addition, it is preferable to use sand that is already present at the site where the artificial pond is being manufactured in order to further reduce costs.
In other words, the present invention also relates to a method for manufacturing an artificial pond in an area that is originally sandy, adding bentonite from the outside to the sand that constitutes the sandy area and mixing it to obtain the above-mentioned mixed particle group, and then pressurizing the mixed particle group to provide a water-impermeable structure.

In the present invention, it is preferable to use seawater, deep ocean water, or water obtained by artificially adjusting the salinity of these as the medium (culture solution), and it is preferable to obtain these from a location close to the sea or a salt lake. Therefore, since the ground in such locations is often sandy, using sand from the sandy area can further reduce costs through a synergistic effect.

According to the method for manufacturing an artificial pond of the present invention, by using halophilic microalgae to produce fuel oil and useful organic matter, a significant synergistic effect is obtained in terms of the ease of obtaining seawater/deep ocean water used as the culture medium (culture solution) and sand used as the material for the water-impermeable structure.

<<Bentonite and sand content>>
When a mixed particle group of bentonite powder and sand is used, the mass of the bentonite powder in the mixed particle group is not particularly limited, but the lower limit of the content of the bentonite powder relative to the total mass of the bentonite powder and sand in the mixed particle group is preferably 2 mass% or more, more preferably 3 mass% or more, even more preferably 4 mass% or more, particularly preferably 6 mass% or more, and most preferably 8 mass% or more.
Furthermore, with respect to the total mass described above, the upper limit of the bentonite powder content is preferably 80 mass% or less, more preferably 60 mass% or less, even more preferably 40 mass% or less, particularly preferably 18 mass% or less, and most preferably 15 mass% or less.

If the mass of the bentonite powder in the mixed particle group (the content ratio of the bentonite powder) is too small, the water-impermeable structure obtained may have poor water impermeability or may not absorb water and not be able to expand in volume sufficiently.
On the other hand, if the value is too high, bentonite is more expensive than sand, so the mixed particle group (manufacturing material) may be expensive, or the viscosity of the mixed particle group may be too high, requiring too much energy for stirring.

<<Other particles (powders)>>
In addition to the bentonite powder and sand described above, the particle group in the present invention may contain "other particles (powders)" having a particle size smaller than sand, such as soil, mud, clay, etc. The "other particles (powders)" may be used in an amount of preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less, based on the total particle group. However, it is most preferable that the "other particles (powders)" are not substantially used.

<Manufacturing method of artificial ponds and manufactured artificial ponds>
The manufacturing method of the present invention is characterized in that a water-impermeable structure is provided on the side and/or bottom of an artificial pond by pressurizing a mixed particle group.
Here, the location from which an "artificial pond" is made may be any of the following: a simple flat area in nature, a location in nature that is almost unchanged, or a location that has been specially processed, altered, reworked, divided, reinforced, etc., or a man-made object or an artificially created object that has been specially processed, altered, reworked, divided, reinforced, etc., etc.

The above-mentioned "simple flat land in nature" includes deserts, sandy beaches, fields, natural land, etc.
Examples of the "substantially intact nature" include the sea, rivers, lakes, marshes, salt lakes, ponds, etc.
Specific examples of the "artificially created" include artificial reservoirs, pools, tanks, dedicated culture containers, rice fields, aquaculture ponds, tourist ponds, etc. Rice fields such as fallow rice fields are particularly preferred because they have ridges and are of suitable depth.

Figure 1 shows a "process of producing an artificial pond 10 for cultivating halophilic microalgae 13" (Figure 1), in which an unused rice field C is used to apply (e.g., attach) a "mixed particle group containing bentonite powder and sand" to the sides (ridges B) and bottom of the field C, which is then pressurized to create a water-impermeable structure 11a, into which seawater 12 (seawater, artificial seawater, deep ocean water) is poured to form a water-impermeable structure 11b during cultivation, and a schematic cross-sectional view of the "produced artificial pond 10" (Figure 1(c)).

The size is not limited to that shown in FIG. 1, but FIG. 1 shows an example of an artificial pond (see FIG. 1(c)) created by utilizing a fallow field (see FIG. 1(a)) that is 10 m long, 10 m wide, and has a ridge height (depth) of 30 cm.
As an example, Fig. 1 shows a schematic cross-sectional view of a water-impermeable structure 11a having a thickness of 5 cm (Fig. 1(b)) before seawater 12 is poured in after pressurization, and a water-impermeable structure 11b when the thickness does not increase after the seawater 12 is poured in (Fig. 1(c)), and when the thickness increases (Fig. 1(d)), and an artificial pond 10. Note that, although bentonite swells and enters between coarse particles, the volume and thickness of the water-impermeable structure 11a do not necessarily increase as shown in Fig. 1(d) above.

When an artificial pond is constructed artificially from the beginning, the area of the pond is preferably 1 ^m2 or more and 1,000 ^m2 or less, more preferably 3 ^m2 or more and 300 ^m2 or less, and particularly preferably 10 ^m2 or more and 100 ^m2 or less.
When using materials found in nature, the area is preferably 10 ^m2 or more and 10,000 ^m2 or less, more preferably 30 ^m2 or more and 3,000 ^m2 or less, and particularly preferably 100 ^m2 or more and 1,000 ^m2 or less.

If the area of one artificial pond is equal to or larger than the lower limit, there are effects such as economies of scale being obtained.
On the other hand, if the area of one "artificial pond" is equal to or less than the above upper limit, there are advantages such as not requiring too much capital investment, being easy to work with, and not being unnecessarily large.

The volume of one artificial pond is preferably 1 ^m3 or more, more preferably ^{3 m3} or more and 10,000 ^m3 or less, still more preferably 10 ^m3 or more and 3,000 ^m3 or less, and particularly preferably 30 ^m3 or more and 1,000 ^m3 or less.
It is desirable for the volume of one artificial pond to be equal to or larger than the above lower limit or equal to or smaller than the above upper limit, since this provides the same effect as in the case of the above-mentioned area.

The average depth of the artificial pond is preferably 0.07 m or more and 1.5 m or less, more preferably 0.10 m or more and 1.2 m or less, and particularly preferably 0.12 m or more and 0.8 m or less.
When the amount is within the above range, the halophilic microalgae can be easily exposed to sunlight, there is no risk of drying up, and the halophilic microalgae can be easily recovered.

<Construction method for water-impermeable structures>
The manufacturing method of the present invention is characterized in that a water-impermeable structure is provided on the side and/or bottom of an artificial pond by pressurizing a particle group such as a mixed particle group.

It is also preferable to compact the original ground before applying the particles containing bentonite powder, since then the particles can be compressed to provide a water-impermeable structure of uniform thickness, and the "compacted portion of the original ground" also contributes to water impermeability. The "compaction" can be performed in the same manner as the compression of the water-impermeable structure described below.

When applying the particles such as the mixed particles to the side and/or bottom of the artificial pond, it is preferable to first make the particles into a slurry using a dispersion medium before putting them in. It is also preferable to make a slurry of only the bentonite, and then add sand to make a slurry.
The dispersion medium is not limited, and examples thereof include pure water and seawater.
The amount of the dispersion medium is not particularly limited as long as a slurry of suitable viscosity is finally produced, but it is preferably from 1.5 to 15 times the mass of the bentonite, more preferably from 2 to 10 times the mass, and particularly preferably from 3 to 7 times the mass.

The method of pressurization is not particularly limited, and may be by manually compressing with weights or boards, by compressing with rollers or graders, or by hitting with a rammer. It is also preferable to determine the pressurization method depending on the size (scale) of the artificial pond.
The degree of pressure is not particularly limited, but is preferably 15 kPa or more and 150 kPa or less.

The thickness of the water-impermeable structure after pressurization is determined taking into consideration durability, manufacturing costs, etc., and is not particularly limited, but before seawater or artificial seawater is added, the average thickness depending on the location of the artificial pond is preferably 0.5 cm or more and 25 cm or less, more preferably 1.0 cm or more and 20 cm or less, even more preferably 2 cm or more and 15 cm or less, and particularly preferably 3 cm or more and 8 cm or less.

The thickness of the water-impermeable structure may increase when (artificial) seawater or deep sea water, which is a culture medium, is poured into the structure.
In order to obtain water impermeability, it is preferable to adjust the content of the bentonite powder so that the thickness expansion ratio is in the range of 1.0 to 3.0 times, more preferably in the range of 1.1 to 2.6 times, and particularly preferably in the range of 1.2 to 2.2 times.

Therefore, it is preferable to provide the water-impermeable structure so that the thickness of the water-impermeable structure is 1.0 cm or more and 50 cm or less, as an average value depending on the location of the artificial pond that has absorbed water and become thicker in a steady state where seawater or artificial seawater is filled and halophilic microalgae are cultivated, more preferably 2 cm or more and 30 cm or less, and particularly preferably 3 cm or more and 15 cm or less.

If the thickness of the water-impermeable structure after pressurization but before adding (artificial) seawater or deep-ocean water, or the thickness in the steady state when it is added and halophilic microalgae are cultivated, is too small, the water impermeability may be poor and water may leak, etc. Conversely, if it is too large, materials and construction work may be wasted.

In the present invention, it is not excluded to provide "another layer" thereon after compressing a particle group such as a mixed particle group to provide a water-impermeable structure.
In the present invention, after providing the water-impermeable structure, it is also preferable to pour soil on the water-impermeable structure and compact it to provide a soil layer before pouring in seawater or artificial seawater, as this can physically (mechanically) protect the water-impermeable structure (see Example 9 and Figure 2).
The soil of the soil layer is not particularly limited, but the soil that is originally present in the location where the artificial pond of the present invention is to be provided is preferred from the viewpoints of nature conservation, reduction in manufacturing costs, etc.
The "compaction" of the soil layer can be carried out in the same manner as in the compression of a water-impermeable structure described below.

The above soil layer is not essential, but if such a soil layer is provided, its thickness is preferably 1.0 cm or more and 60 cm or less, more preferably 2 cm or more and 50 cm or less, even more preferably 5 cm or more and 40 cm or less, and particularly preferably 10 cm or more and 30 cm or less.

<Raw water for cultivation, culture medium, seawater, deep ocean water, artificial seawater, etc.>
The artificial pond produced by the production method of the present invention is for culturing halophilic microalgae, and is intended to hold at least the halophilic microalgae and seawater or deep ocean water.
In this specification, seawater and deep ocean water are sometimes collectively referred to as simply "seawater". Seawater or deep ocean water that is artificially prepared from the beginning, or seawater or deep ocean water that is used as a raw material and whose components have been artificially adjusted, are sometimes collectively referred to as simply "artificial seawater". Therefore, "artificial seawater" includes "artificial deep ocean water".

Cultivation is carried out in artificial ponds, seawater, deep ocean water, artificial seawater, etc.
That is, in the method for manufacturing an artificial pond, it is particularly preferable that the seawater is deep sea water or artificial deep sea water for the reasons described below.

Seawater has an average salinity of about 3.5% by mass and mainly contains cations such as sodium ions, magnesium ions, potassium ions, and calcium ions; and anions such as chloride ions and sulfate ions.

Deep ocean water has ideal conditions for cultivating halophilic microalgae, such as being free from artificial pollution, having few harmful bacteria due to its low temperature, and not being exposed to sunlight, meaning that there is no contamination by live phytoplankton.
In addition to these, it is rich in nitrogen (N) from nitrates, etc., phosphorus (P) from phosphates, etc., silicon (Si) from silicates, etc. In addition, because the salt concentration is high, bacteria that are harmful to the cultivation of halophilic microalgae are unlikely to survive (there are few of them).
Therefore, deep ocean water is particularly preferable as a culture medium for culturing halophilic microalgae in the present invention.

Seawater and/or deep ocean water can be concentrated by evaporating water using solar heat, etc., to increase the concentration of nitrogen (N) and phosphorus (P) sources required for culturing halophilic microalgae. As water evaporates (simultaneously), the salt (sodium chloride) concentration of seawater and deep ocean water also increases (for example, to about 15% by mass). However, many (highly) halophilic microalgae, such as microalgae belonging to the genus Dunaliella, can be cultured even at high salt (sodium chloride) concentrations (for example, about 15% by mass).
From this point of view, it is preferable to use solar heat to increase the concentrations of nitrogen (N) and phosphorus (P) sources.
Furthermore, a high salt (sodium chloride) concentration (for example, about 15% by mass) makes it difficult for bacteria to grow and reduces contamination, which is favorable for the growth of halophilic microalgae.

Seawater, deep ocean water, and raw water for cultivation made by evaporating and concentrating these using solar heat or other methods are inexpensive, and the present invention has economies of scale, so it is particularly suitable for taking advantage of these advantages.

The "artificial seawater" may be prepared by artificially adding all nutrients (including essential elements, etc.) and salts (including the salt in seawater) from the beginning, or by artificially adding such nutrients and salts to natural seawater or deep ocean water.

<Halophilic microalgae>
Specific examples of halophilic microalgae in the present invention include microalgae belonging to the genus Dunaliella. One or more types of halophilic microalgae can be cultured. As halophilic microalgae, those so-called "highly halophilic microalgae" are particularly preferred.

By culturing halophilic microalgae in (artificial) seawater, especially in seawater with a high salinity concentration, it is possible to reduce the risk of contamination (such as the introduction of foreign bacteria) at low cost.

Specific characteristics that make the subject of cultivation "halophilic microalgae" include, for example, the following.
From halophilic microalgae, useful organic matter such as carbohydrates or sugars, oligopeptides, polypeptides or proteins, vitamins or vitamin precursors, polyhydric alcohols, fats and oils, glycolipids, phospholipids or lipoproteins, or hydrocarbons can be obtained, carbon sources other than carbon dioxide can be used, there are many highly halophilic algae (marine microalgae), a wide range of adaptable growth temperatures can be used, and they are not subject to strong photoinhibition can be obtained. In addition, carbon dioxide in the atmosphere can be converted into a resource by halophilic microalgae.

Among them, microalgae belonging to the genus Dunaliella are particularly preferred as halophilic microalgae for the above-mentioned reasons.

The Dunaliella genus does not have a cell wall, making it easier to extract oil.
In addition, since Dunaliella stores glycerin in its cells for osmotic pressure adjustment, the glycerin can be recovered and effectively utilized. Since the glycerin is pure glycerin that does not contain solvents or catalysts, it is highly useful for use in cosmetics, food and beverages, and as a fermentation substrate.

In addition, the genus Dunaliella does not require bubbling (aeration with carbon dioxide), which reduces culture costs.
In addition, the genus Dunaliella is one of the few microalgae that can accumulate lipids while growing.

Furthermore, by accumulating β-carotene, it is possible to cultivate the plant without causing photoinhibition.
In addition, since the adaptable growth temperature range is wide, it is possible to culture it without artificial temperature control, which makes it possible to significantly reduce culture costs.

Examples of microalgae belonging to the genus Dunaliella include Dunaliella salina, Dunaliella viridis, Dunaliella bioculata, Dunaliella primolecta, Dunaliella tertiolecta, and Dunaliella bardawil.
It is particularly preferable to culture Dunaliella salina, since it can survive and grow at both low and high temperatures (4 to 60° C.), and can withstand high temperatures under high osmotic pressure conditions, etc. In the present invention, one or more types of halophilic microalgae belonging to the genus Dunaliella can also be simultaneously cultured.

<Action/Principle>
The action and principle by which the "water-impermeable structure in the presence of seawater" obtained by the manufacturing method of the present invention exhibits water impermeability, and the action and principle by which the artificial pond obtained by the present invention does not leak water and can be used for a long period of time, are not particularly limited, but are thought to be as follows. However, the present invention is not limited to the scope to which the following action and principle apply.

It is believed that each grain of bentonite powder swells with seawater and densely fills the gaps between adjacent grains of bentonite powder. Also, when bentonite powder is used in combination with sand, it is believed that the bentonite powder swelled by seawater densely fills the gaps between each grain of sand and its neighboring grains.
It is believed that the water-impermeable structure as a whole becomes dense, making it difficult for seawater to pass through.

In addition, the water impermeability of the culture water put into the artificial pond was higher when it was seawater than when it was pure water. This is thought to be because salt precipitated as extremely fine particles between the sand and/or bentonite particles, which further sealed the gaps between the particles, thereby further improving the water impermeability.

<Artificial pond>
The present invention also relates to an artificial pond for culturing halophilic microalgae, manufactured using the above-mentioned method for manufacturing an artificial pond, for storing at least the halophilic microalgae and seawater or artificial seawater, characterized in that the above-mentioned water-impermeable structure is formed on the sides and/or bottom in a state in which it has absorbed water.

As mentioned above, it is believed that the water impermeability has increased due to the swelling of the bentonite caused by the presence of seawater, but the chemical structure of such swollen bentonite, the shape of the bentonite powder, the way in which the bentonite fills the gaps between the sand particles, the form of the water impermeable structure (after coming into contact with seawater), etc. cannot be directly identified by their shape or parameters, and it is impossible or entirely impractical to carry out quantitative measurements to identify them.

<Method for producing halophilic microalgae>
The present invention also relates to a method for producing halophilic microalgae, which comprises culturing the halophilic microalgae by placing halophilic microalgae and seawater or artificial seawater in the artificial pond produced by the above-mentioned method.
The production by culturing halophilic microalgae is not particularly limited, and known methods can be used.
From the standpoint of cost reduction and the like, it is preferable that the present invention be carried out outdoors, and therefore it is preferable to use sunlight as the light energy for propagation.

The seawater or the like (culture solution (medium solution)) for halophilic microalgae is adjusted to have a suitable composition in terms of contained elements and other factors depending on the type of halophilic microalgae to be produced.
It is preferable to appropriately adjust the culture temperature and culture time depending on the type of halophilic microalgae to be produced so that the productivity and performance of the product are suitable. In order to increase the production amount and reduce fixed costs, it is also preferable to carry out continuous culture, and it is also preferable to carry out continuous culture in a terraced rice field.

The produced halophilic microalgae are collected by a known method.
The recovery method is not particularly limited, and examples thereof include filtration, evaporation of water to dryness by heating, reducing pressure, air drying, etc., centrifugation, a method of sedimentation using a flocculant or electrodes, a method of sedimentation by blocking gases such as oxygen, nitrogen, carbon dioxide, etc., a method of sedimentation by changing the surrounding conditions, etc. These methods may be used alone or in combination of two or more.

<Production method of useful organic matter>
The present invention also relates to a method for producing useful organic matter, characterized in that useful organic matter is obtained from halophilic microalgae produced using the above-mentioned "method for producing halophilic microalgae."
The above useful organic matter may be obtained by any known method, including separation by squeezing, fractional distillation, various extraction methods, use of the residue after separation, separation after reaction, and the like.

The useful organic matter is not particularly limited as long as it is obtained from the produced halophilic microalgae, but is preferably carbohydrates or sugars; oligopeptides, polypeptides or proteins; vitamins or vitamin precursors; polyhydric alcohols; oils and fats; glycolipids, phospholipids or lipoproteins; or hydrocarbons.

Particularly preferred examples include, but are not limited to, oligopeptides such as glutathione; carotenoids such as β-carotene; polyhydric alcohols such as glycerin; and oils and fats such as glycerin fatty acid esters.

β-Carotene has a strong antioxidant effect and can be used, for example, in medicines, foods, food additives, supplements, and the like.
Glutathione has a strong antioxidant effect and can be used, for example, in medicines, foods, supplements, fertilizers, feed, etc.
Glycerin can be suitably used, for example, as a raw material for cosmetics, pharmaceuticals, etc., and also as a fuel.
The oils and fats can be used, for example, as edible oils, feed oils, fuel oils, etc.
The protein can be used, for example, as feed, fermentation raw material, etc.

The present invention will be explained in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples as long as it does not exceed the gist of the invention.

Example 1
<Comparison with other manufacturing methods>
The manufacturing method of the artificial pond of the present invention, the manufacturing method of the artificial pond using a polyvinyl chloride sheet, and the manufacturing method of the artificial pond using concrete were compared.

As shown in Table 1 below, methods for manufacturing artificial ponds using polymer sheets such as polyvinyl chloride sheets require land preparation and leveling in order to lay the sheets, and because polymer sheets are expensive, the manufacturing costs are too high.

Bentonite sheets are known in the civil engineering field for waterproofing, but these sheets are made by adding bentonite to synthetic polymers such as polyvinyl chloride in advance. However, these sheets use synthetic polymers derived from petroleum, which are expensive, and have a large environmental impact. Although the construction cost of these bentonite sheets is lower than that of concrete, which will be described later, the environmental impact and cost (approximately 1,000 yen/ ^m2 ) of the materials make them cost-effective for cultivating halophilic microalgae.

When synthetic polymers are used, the polymer sheets must be removed in their entirety when the product is abandoned or discarded, and failing to do so places a heavy burden on the environment. In particular, when polyvinyl chloride sheets are used, environmental destruction occurs, and disposal is extremely expensive when the product is discarded (Table 1).

In addition, the method of manufacturing an artificial pond using concrete requires preparation and leveling of the ground for pouring the concrete, which requires a long construction period. To build an artificial pond using concrete, it costs 10,000 to 30,000 yen per square ^meter .
In addition, trace elements contained in concrete (including mortar, cement, etc.) may impair the cultivation of halophilic microalgae. Moreover, when the pond is abandoned or discarded, the concrete must be removed, and if it is left without being removed, it will take a very long time to weather. In addition, although the environmental load from chemical substances is small, the large artificial pond will cause great structural environmental damage (Table 1).

On the other hand, in the present invention, if an artificial pond is built on a sandy site from the beginning, sand is extremely easy to obtain and there is no material cost involved.
Furthermore, when seawater is used as the water for the artificial pond (water for propagation), it is preferable that the installation (manufacturing) site be near the sea, and since there is a large amount of sea sand available and it is almost flat (with a flat surface), there is no need to develop land, further reducing costs.
In particular, when producing the plant on the site of a rice field or the like (which is extremely suitable in terms of depth), there is no need to prepare the land, and therefore preparation costs are almost zero.

Furthermore, according to the present invention, the bentonite powder can be swelled by simply applying pressure with a roller or the like after completion when (artificial) seawater is poured in, forming a water-impermeable structure, thereby making it possible to keep the manufacturing costs of the artificial pond extremely low.
The manufacturing cost per unit area of the manufacturing method of the present invention is lower than that of the above-mentioned case where a polymer sheet such as a polyvinyl chloride sheet is used, and also compared to that of the above-mentioned case where concrete is used.

In addition, the water-impermeable structure for an artificial pond obtained by the present invention does not cause any particular environmental damage even if it is left as is. Moreover, since it is made entirely of natural materials (natural sand and clay), it does not impose any burden on the environment.
In the case of synthetic organic polymers, of course they pose a large environmental burden if left unattended, but even inorganic substances such as cement involve artificial selection, concentration, and separation processes, so trace amounts of harmful components may be unnaturally concentrated.

Example 2
<Preparation of mixed particles containing bentonite powder and sand>
Bentonite powder and sand were mixed as follows to obtain a mixed particle group.
Pure water was added so that the bentonite powder was 5 times the total mass, and the mixture was thoroughly stirred and dispersed to prepare a "bentonite slurry."
Next, the "bentonite slurry" and sand were mixed in a container to obtain the composition shown in Table 2 below, and stirred with a stainless steel spoon until homogenous.
In this way, the following three types of "mixed particle groups with different ratios of bentonite powder and sand" were prepared.

The resulting mixed particle mass was compacted on-site using a roller or grader, but in the laboratory, the sample was placed in a special compaction tool (iron mould) and hit with a 2.5 kg rammer.
However, whether the pressure was applied using the rammer or whether the material was "pressed strongly by hand using a flat plate and then dried at 35 to 40°C," the same results were obtained in terms of water permeability (impermeability).
That is, after separately verifying that the method in the "" above gives results similar to those of compacting with dedicated equipment such as a rammer, roller, or grader, the following study was carried out using the above method.

Example 3
<Preparation of Water-Impermeable Structure>
The water impermeability of each of the mixed particle group samples No. 11 to No. 13 was determined using a large funnel as follows.

The outlet of the large funnel was blocked with a rubber plug, and 150 g of each of the mixed particle groups No. 11 to 13 was placed in the funnel, and the surface of the mixed particle groups was pressed uniformly by hand using a flat plate.
Thereafter, the product was dried in an oven (in the atmosphere) for 3 hours at 35° C. During the drying process, some cracks were observed, so the cracks were filled by further compressing the product with the board by hand.

As a result, three types of water-impermeable structures (No. 11, 12, and 13) with a thickness of 3.0 cm were obtained on top of the funnel. The water-impermeable structures had almost the same compaction properties as those compacted with a rammer.

Example 4
<Water impermeability study 1 (using a funnel)>
Onto the water-impermeable structure obtained in Example 3, 150 g of deep ocean water or artificial seawater (adjusted to 12 mass % salts including sodium chloride) containing Dunaliella salina (hereinafter, this will be simply abbreviated as "culture solution") was poured.

The water-impermeable structural body obtained by pressing the "mixed particle group consisting of bentonite powder and sand" was filled with culture medium in the same mass (150 g) as that of the water-impermeable structural body and allowed to stand.
When observed after one week, the water level of the culture solution that had permeated the water-impermeable structure had decreased by only 2 mm, and the hydraulic conductivity at this time was 3.3×10 ⁻⁹ [m/s].
The mixed particle groups Nos. 11, 12, and 13 of Example 2 each gave the same results as above.

Example 5
<Water impermeability study 2 (using a funnel)>
The scale was doubled for the study of Example 4. Specifically, 300 g of the "mixed particle group consisting of bentonite powder and sand" was used, and 300 g of the "culture solution" was injected.
After one week and two weeks had passed, the dripping of water from the bottom of the funnel was checked, but no water was dripping at all. No water permeation was observed.
The mixed particle groups Nos. 11, 12, and 13 of Example 2 each gave the same results as above.

Example 6
<Water impermeability study 3 (using a syringe)>
A large syringe was used instead of the large funnel in Example 4. That is, the mixed particle group Nos. 11, 12, and 13 of Example 2 were filled into the syringe and compressed by hand.
Specifically, 100 g of the mixed particle group (height 5.0 cm in the syringe) and 200 g of the same culture solution as in Example 4 (height 10.0 cm in the syringe) were placed vertically, and after 2 weeks, water leakage from under the syringe was observed.

As a result, the water-impermeable structure did not allow the culture medium to permeate (leak) at all.
The mixed particle groups Nos. 11, 12, and 13 of Example 2 each gave the same results as above.

Example 7
<Study on reducing salt concentration in culture medium>
In Example 4, a water-impermeable structure was prepared on a funnel in the same manner as in Example 4, and water permeability was observed, except that "seawater (salinity: 3.5% by mass)" and "freshwater not containing Dunaliella (salinity: 0.0% by mass)" were used instead of "deep ocean water or artificial seawater (adjusted to 12% by mass of salts including sodium chloride)," which is a culture medium containing Dunaliella salina.

When observed after one week, it was found that the order of water impermeability was deep ocean water or artificial seawater (salinity 12% by mass), followed by seawater (salinity 3.5% by mass), and finally freshwater (salinity 0.0% by mass). It was found that the higher the salinity, the higher the water impermeability, resulting in an excellent water-impermeable structure.
It was confirmed that high-concentration salts permeated the gaps between the bentonite and sand, acting as a water impermeable material. This was thought to be due to the precipitation of microscopic sodium chloride crystals.

Example 8
<Examination of differences in culturability depending on the presence or absence and type of bentonite>
20 g of the following three kinds of bentonite powders and 180 g of silica sand were mixed to obtain 200 g of mixed particle groups. In addition, 200 g of silica sand was prepared.
(1) Bentonite TB-S (manufactured by Tachibana Material Co., Ltd.; swelling power 23.0 mL/2 g (American-made, sodium bentonite)
(2) Bentonite TB-250 (manufactured by Tachibana Material Co., Ltd.; swelling power 9.0 mL/2 g (calcium bentonite from Gunma, Japan)
(3) Bentonite Kunigel (registered trademark) V1 (manufactured by Kunimine Kogyo Co., Ltd.; swelling power 16 mL/2 g (made in India)
(4) Bentonite: None (only silica sand)

A total of four types, 200 g each of the three types of mixed particle groups and sand (only silica sand), were placed in a 1,200 mL rectangular container, dried and molded to create a simulated artificial pond.
To this was added 900 mL of a 12% by mass salt medium (Red Sea Salt) containing Dunaliella salina. The mixture was left to stand for 18 days, and the cell density (cells/mL
) was measured.

The first three days were in the induction phase, and the cell density did not increase. Days 4 to 8 were in the logarithmic phase, and the cell density increased logarithmically. Days 9 to 17 were in the stationary phase, and the cell density did not increase or decrease much.
Up until the logarithmic phase (up to the 8th day), (2) TB-250, which had the lowest swelling power, showed active proliferation (growth) in the simulated artificial pond, followed by (3) Kunigel V, which had medium swelling power, and (1) TB-S, which had the highest swelling power, showed slow proliferation (growth).

The proliferation (growth) in the simulated artificial pond containing only silica sand without using bentonite (4) was lower during the logarithmic phase (up to the 8th day) than in any of the ponds containing bentonite (1), (2), or (3).
It was suggested that the artificial pond made with the mixed particle group using bentonite had a higher growth rate of halophilic microalgae than the artificial pond made with sand only.

Furthermore, the results of Examples 9 and 10 described below show that even more rapid proliferation (growth) was observed outdoors where the illuminance (photon density) and water temperature were high compared to laboratory tests.

Example 9
<Expansion test (using particles made of bentonite powder)>
To conduct the expansion test, two artificial ponds measuring 3 m x 5 m x 0.2 m (20 cm) deep were constructed outdoors in Chiba Prefecture, Japan, as shown in the schematic cross-section of Figure 2. They were named "Pool A" and "Pool B" (see Figures 2 and 3).

As shown in Figure 2, a hole was dug in the soil (ground) of the farm, the soil (ground) was compacted, particles of bentonite powder were added, and pressure was applied to form a water-impermeable structure 11a as shown in Figure 2.

The bentonite powder used was "TB-S" manufactured by Tachibana Material Co., Ltd.
The "TB-S" used is a commercial product made from raw sodium (Na) bentonite ore produced in Wyoming, USA, and has the following physical properties: moisture content is 10% by mass or less, more than 90% passes a 250 mesh particle size, swelling power is 23 [mL/2g] or more, apparent specific gravity is 0.70 to 0.85 g/cm ³ , pH of 1% by mass water is 9.0 to 10.5, and it absorbs 10 times its mass of water.

Furthermore, soil from the local area was placed on top of the above and compacted to create a soil layer 20 cm thick. A sandwich structure of "soil (ground) \ water-impermeable structure 11a \ soil layer" was created by applying pressure and compacting it from below (see Figure 2).

Before seawater (deep ocean water in Example 9) was introduced into the artificial pond, the thickness of the water-impermeable structure made of bentonite powder ranged from 0.5 to 1 cm depending on the location, and the mass of the water-impermeable structure per bottom area of the pond ranged from 3.5 to 7.0 kg/ ^m2 depending on the location.
After the introduction of seawater (deep seawater in Example 9) into the artificial pond, the thickness of the water-impermeable structure was in the range of twice the above value (see FIG. 2).

<<Study of water impermeability>>
Since the apparent specific gravity (apparent density) of bentonite is 0.7 g/cm ³ , a sufficiently dense water-impermeable structure was formed by the introduction of seawater.
The hydraulic conductivity was in the range of 1.3×10 ⁻⁹ [m/s] to 1.7× ^{10 −10} [m/s], demonstrating excellent water impermeability.
In addition, because salt, mainly table salt (NaCl), crystallized, there was absolutely no water leakage. There was absolutely no water leakage over time during the study (excellent water impermeability was maintained).

<<Growth rate study>>
Dunaliella salina was introduced and cultured in pool A to the cell density shown on culture day 0 (left end) in Fig. 3(a) and in pool B to the cell density shown on culture day 0 (left end) in Fig. 3(b).

The photon density [μmol/m ² s] of the seawater surface and the seawater temperature [° C.] of the artificial ponds A and B installed outdoors are shown in Table 3 below, together with the measured values in the laboratory test of Example 10 described below.

Regarding the growth rate of Dunaliella salina, the results for pool A are shown in FIG. 3(a), and the results for pool B are shown in FIG. 3(b).
The scale of the vertical axis in Figures 3(a) and 3(b) and Figure 4, for example, "1.E+05" indicates ¹⁰⁵ [cells/mL]. The base of the logarithm of the vertical axis is "10". If the vertical axis is a numerical value of _log10 (cell density [cells/mL]), for example, "1.E+05" written on the vertical axis is "5".

As shown in Figure 3 (a) and (b), the cell density in both pools A and B increased tenfold in three days. A good growth rate was also observed in an outdoor artificial pond measuring 3 m x 5 m x 0.2 m (20 cm) deep. The bentonite used in the study was found to be superior.

Furthermore, the proliferation rate in the expansion test of Example 9 was faster than the proliferation rate in the laboratory test of Example 10 described below. That is, in the laboratory test of Example 10, it took about 7 to 10 days for the cell density to increase 10-fold, whereas in the expansion test of Example 9, it took about 3 to 4 days for the cell density to increase 10-fold.

Example 10
<Laboratory test conducted to compare with the expanded test of Example 9>
A water-impermeable structure was prepared in the same manner as in Example 3, using the particle group made of the bentonite powder used in Example 9 on a large funnel similar to that in Example 3. That is, a water-impermeable structure was provided in the same manner as in Example 3, except that the particle group made of the bentonite powder used in Example 9 was used instead of the "mixed particle group Nos. 11 to 13" in Example 3.

The growth rate was determined, and the results shown in the graph in Figure 4 were obtained. It took about 7 to 10 days for the cell density to increase 10-fold. On the other hand, as mentioned above, in the expansion test of Example 9, it took about 3 to 4 days for the cell density to increase 10-fold.

Example 11
<Expansion test (using a mixture of bentonite powder and sand)>
The expansion test was carried out in the same manner as in Example 9, except that the mixed particle group No. 12 in Examples 2 and 3 was used instead of the particle group made of bentonite powder used in Example 9, and a water-impermeable structure having a thickness of 3.0 cm was provided.

When evaluated in the same manner as in Example 9, good results were obtained in both water impermeability and growth rate, similar to those in Example 9.

According to the present invention, material costs and manufacturing costs are reduced, and even if the manufacturing period is short, an artificial pond for cultivating halophilic microalgae can be suitably manufactured. Furthermore, when the artificial pond is abandoned or discarded, there is no risk of environmental destruction structurally, and since no petroleum-derived materials are used at all, it does not burden the environment.
Therefore, large-scale cultivation of microalgae will become a reality, making it possible to obtain energy sources other than petroleum and useful organic matter at low cost.
Furthermore, it will enable the "greening of the desert," something that has not been possible until now, and will serve as a model for a sustainable society in which industrial activities can be carried out while reducing carbon dioxide emissions. As such, it will be widely used in fields such as energy supply, chemical and pharmaceutical manufacturing, and civil engineering.

10 Artificial pond 11a Water-impermeable structure (before introduction of seawater, etc.)
11b Water-impermeable structure (after introduction of seawater, etc., during cultivation)
12 Seawater or artificial seawater 13 Halophilic microalgae A Ground B Ridge C Rice field

Claims (11)

A method for producing an artificial pond for culturing halophilic microalgae, the artificial pond being for storing at least the halophilic microalgae and seawater or artificial seawater,
A method for manufacturing an artificial pond, comprising the steps of: providing a water-impermeable structure on the side and/or bottom of the artificial pond by compressing a particle group containing bentonite powder. A method for producing an artificial pond for culturing halophilic microalgae, the artificial pond being for storing at least the halophilic microalgae and seawater or artificial seawater,
2. The method for manufacturing an artificial pond according to claim 1, wherein a water-impermeable structure is provided on the side and/or bottom of the artificial pond by pressing a mixture of particles containing bentonite powder and sand. The method for manufacturing an artificial pond according to claim 2, wherein the mass of the bentonite powder in the mixed particle group is 2% by mass or more and 80% by mass or less with respect to the total mass of the bentonite powder and sand in the mixed particle group. The method for manufacturing an artificial pond according to any one of claims 1 to 3, wherein the thickness of the water-impermeable structure after pressurization is 0.5 cm or more and 25 cm or less as an average value depending on the location of the artificial pond before seawater or artificial seawater is poured in. A method for manufacturing an artificial pond according to any one of claims 1 to 4, in which the water-impermeable structure is provided so that the thickness of the water-impermeable structure is 1.0 cm to 50 cm as an average value depending on the location of the artificial pond when it absorbs water in a steady state in which seawater or artificial seawater is filled and halophilic microalgae are cultivated. A method for manufacturing an artificial pond according to any one of claims 1 to 5, wherein the area of the artificial pond is 10 ^m2 or more. In the case where a mixed particle group containing bentonite powder and sand is compressed to provide a water-impermeable structure,
A method for manufacturing an artificial pond according to any one of claims 1 to 6, comprising the steps of: constructing an artificial pond in a place that is originally sandy; adding and mixing bentonite from outside with the sand that constitutes the sandy area to obtain the mixed particle group; and pressurizing the mixed particle group to provide a water-impermeable structure. A method for manufacturing an artificial pond according to any one of claims 1 to 7, wherein the seawater is deep sea water. A method for producing halophilic microalgae, comprising: producing an artificial pond using the method for producing an artificial pond described in any one of claims 1 to 8; placing halophilic microalgae and seawater or artificial seawater in the artificial pond; and culturing the halophilic microalgae. A method for producing useful organic matter, comprising obtaining useful organic matter from halophilic microalgae produced by the method for producing halophilic microalgae according to claim 9 . The method for producing useful organic matter according to claim 10 , wherein the useful organic matter is a carbohydrate or sugar; an oligopeptide, a polypeptide or a protein; a vitamin or a vitamin precursor; a polyhydric alcohol; an oil or fat; a glycolipid, a phospholipid or a lipoprotein; or a hydrocarbon.

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