JP7455386B2 - Cultivation method for freshwater microalgae - Google Patents

Description

IPOD FERM BP-22333 IPOD FERM BP-22334

Application of Article 30, Paragraph 2 of the Patent Act On February 13, 2018, on the website (address: http://www.jst.go.jp/mirai/jp/program/lowcarbon/index.html#theme01) Published.

Application of Article 30, Paragraph 2 of the Patent Act Published on the website (address: http://www.jst.go.jp/mirai/jp/uploads/saitaku2017/JPMJMI17EF_miyagishima.pdf) on February 13, 2018 .

Application of Article 30, Paragraph 2 of the Patent Act Published on the website (address: https://www.jst.go.jp/mirai/jp/news/2017/index.html) on February 13, 2018 .

The present invention relates to a method for culturing freshwater microalgae. The present invention also relates to a method for producing salt-tolerant freshwater microalgae and salt-tolerant freshwater microalgae obtained by the production method. More specifically, the present invention relates to freshwater microalgae suitable for outdoor mass cultivation, especially outdoor mass cultivation in seawater, and a method for producing the same.
This application claims priority based on Japanese Patent Application No. 2018-187763 filed in Japan on October 2, 2018, the contents of which are incorporated herein.

Microalgae have a higher carbon fixation ability than land plants, and because they do not compete with agricultural products for growing space, some species are cultivated in large quantities and used as feed, functional foods, and cosmetic materials. It is used industrially as such.
Due to cost considerations, the industrial use of microalgae is limited to expensive functional foods and the like. In order to suppress the production cost of microalgae and promote industrial use, outdoor mass cultivation is preferable. Although outdoor mass cultivation is characterized by easy management, there is a risk of contamination, it is directly affected by the external environment, and there is also the problem of invasion by algae predators. To avoid such risks, microalgae that can be cultivated outdoors in large quantities must be resistant to environmental fluctuations (light, temperature, etc.), can be cultured under conditions where other organisms cannot survive, and must be able to grow to high densities. Conditions such as being possible are required.
Therefore, to date, only a few species, such as Chlorella, Euglena, Dunaliella, and Spirulina, have been put into practical use industrially. These algae species have been successfully cultivated outdoors in large quantities and are used as raw materials for functional foods and supplements.

Examples of environments in which other organisms cannot survive include environments with high salt concentrations such as seawater. Regarding culturing useful microalgae in a high salt concentration medium, for example, there are reports in Patent Documents 1 to 3.
Patent Document 1 describes a method for producing oil and fat components, which is characterized by culturing salt-tolerant algae in a medium with a stepwise increase in salt concentration. In the method described in Patent Document 1, when the nitrate content in the medium is measured at a wavelength of 220 nm and the content is 10 mg/L or less, increasing the salt concentration in the second step is performed. It is a feature.
Patent Document 2 discloses that in culturing the alga Pseudochoricystis ellipsoidea, which lives in fresh water and has the ability to produce hydrocarbons, in order to increase the productivity of its hydrocarbon-producing ability, the culture is started. It has been disclosed that salt is added to the medium between the time when the optical density of the medium reaches one half of the optical density indicating the saturation state.
Patent Document 3 discloses that in the culture of the genus Crypthecodinium that produces docosahexaenoic acid, the salt concentration of the culture solution is lowered from the salt concentration suitable for the growth of the algae in order to accumulate docosahexaenoic acid in the algae. A method of culturing by setting a value 0.1 to 10% higher by weight is disclosed.
The methods described in Patent Documents 1 to 3 aim to increase hydrocarbon production ability and docosahexaenoic acid production ability by applying salt stress to microalgae, and are aimed at suppressing contamination risk in outdoor culture. It's not a thing.

On the other hand, microalgae belonging to the class Cyanidiophyceae, which are unicellular primitive red algae, preferentially proliferate in sulfuric acid hot springs. The class Ideyukogome includes the genera Cyanidioschyzon, Cyanidium, and Galdieria. Cyanidioschyzon merolae, which belongs to the genus Cyanidioschyzon, does not have a strong cell wall. Cyanidioscizon melolae is composed of a very simple set of organelles, and its genome sequence has been completed. Therefore, it is used as a model organism for basic research on photosynthetic organisms, and genetic modification technology is also being developed (Non-patent Documents 1 and 2).

International Publication No. 2015/25552 JP2013-102748A Japanese Patent Application Publication No. 07-075557

Fujiwara T et al. (2013) Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers. PLOS ONE. Sep 5;8(9):e73608. Fujiwara T et al. (2015) A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae. Front Plant Sci. Aug 26;6:657.

Freshwater acidophilic microalgae belonging to the class Idyukogome can grow in an acidic environment where other organisms cannot grow, and are suitable for outdoor cultivation. If high salt concentration tolerance can be imparted to these microalgae, they can be cultured in an acidic and high salt concentration environment, further reducing the risk of contamination during outdoor cultivation. Furthermore, if seawater can be used for culture, culture costs can be reduced.

Therefore, the present invention provides a method for cultivating freshwater microalgae that allows freshwater microalgae to grow well in an environment of low pH and high sodium ion concentration, and a method for culturing freshwater microalgae that allows the freshwater microalgae to grow well in an environment of low pH and high sodium ion concentration. An object of the present invention is to provide freshwater microalgae and a method for producing the freshwater microalgae.

The present invention includes the following aspects.
[1] A method for culturing microalgae, in which freshwater microalgae are cultured in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M, at a culture temperature of A method for cultivating freshwater microalgae, including a culturing step of culturing at 15 to 60°C.
[2] A method for culturing freshwater microalgae, in which freshwater microalgae are cultured in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M. The freshwater microalgae after the pre-culturing step have a sodium ion concentration of 1.2 to 5 times the sodium ion concentration in the pre-culturing step and a hydrogen ion concentration of pH 1.0 to 6.0. The method for culturing freshwater microalgae according to [1], comprising a main culturing step of culturing in a medium prepared so as to be.
[3] The medium according to [1] or [2], wherein the medium in the main culture step is a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.5 M or more. A method for culturing freshwater microalgae.
[4] The medium according to [1] or [2], wherein the medium in the main culture step is a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.4 M or more. A method for culturing freshwater microalgae.
[5] The medium in the main culture step is a medium prepared by adding at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt to seawater and having a hydrogen ion concentration of pH 1.0 to 6.0. The method for culturing freshwater microalgae according to any one of [1] to [4].
[6] The method for cultivating freshwater microalgae according to any one of [1] to [5], wherein the freshwater microalgae is a microalgae belonging to the class Idyukogome.
[7] Freshwater microalgae that cannot grow in a medium with a sodium ion concentration of 0.5M or higher were prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M. A method for producing freshwater microalgae that can be grown in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.5M or more, including the step of culturing in a medium.
[8] The method for producing freshwater microalgae according to [7], wherein the freshwater microalgae is a haploid microalgae belonging to the genus Cyanidium.
[9] The method for producing freshwater microalgae according to [7], wherein the freshwater microalgae is a haploid microalgae belonging to the genus Gardelia.
[10] A haploid microalgae belonging to the class Idyukogome, cultured at a culture temperature of 42°C and a carbon dioxide concentration in M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M. M prepared so that the value calculated by the following formula (1) is 2 or more and the hydrogen ion concentration pH is 2.0 when cultured statically for 7 days under continuous light with an illuminance of 60 μmol/m ² % and an illuminance of 60 μmol/m 2 s. - The value calculated by the following formula (1) is less than 2 when statically cultured for 7 days in Allen medium at a culture temperature of 42 ° C., a carbon dioxide concentration of 2%, and an illuminance of 60 μmol/m ² s of continuous light. A haploid microalgae belonging to the class Idyukogome.
(OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture) / (7 x OD ₇₅₀ value at the start of culture) (1)
[11] The haploid microalgae belonging to the class Idyucogatonia according to [10], whose cells rupture in an isotonic solution with a hydrogen ion concentration of pH 7 or in distilled water.
[12] The microalgae belonging to the class Idyukogome according to [10] or [11], wherein when the algal cells are subjected to a drying treatment and the cells after the drying treatment are suspended in an isotonic solution of pH 7, the cells rupture. haploid.
[13] Adding at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt to seawater of the haploid microalgae belonging to the Idyukogome class according to any one of [10] to [12] above, A method for culturing a haploid microalgae belonging to the class Iducogos, which further comprises culturing in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0.
[14] A method for culturing haploid microalgae belonging to the class Idyukogoma, which comprises culturing outdoors the microalgae belonging to the class Idyukogoma according to any one of [10] to [12] above.
[15] As a freshwater microalgae, it is a haploid microalgae belonging to the class Idyukogome, and is cultured in M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M. The value calculated by the following formula (1) when statically cultured for 7 days at 42°C, carbon dioxide concentration 2%, and continuous light with an illuminance of 60 μmol/m ² s is 2 or more, and the hydrogen ion concentration pH is 2.0. The value calculated by the following formula (1) when statically cultured for 7 days in M-Allen medium prepared as follows at a culture temperature of 42 ° C., a carbon dioxide concentration of 2%, and a continuous light intensity of 60 μmol/m ² s. is less than 2, and culturing haploid microalgae belonging to the class Idyukogome in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.4 M or more. A method for culturing freshwater microalgae.
(OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture) / (7 x OD ₇₅₀ value at the start of culture) (1)
[16] As a freshwater microalgae, it is a haploid microalgae belonging to the class Idyukogome, and is cultured at a culture temperature in M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M. The value calculated by the following formula (1) when statically cultured for 7 days at 42°C, carbon dioxide concentration 2%, and continuous light with an illuminance of 60 μmol/m ² s is 2 or more, and the hydrogen ion concentration pH is 2.0. The value calculated by the following formula (1) when statically cultured for 7 days in M-Allen medium prepared as follows at a culture temperature of 42 ° C., a carbon dioxide concentration of 2%, and a continuous light intensity of 60 μmol/m ² s. is less than 2, and culturing haploid microalgae belonging to the class Idyukogome in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.4 M or more. Production method of freshwater microalgae.
(OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture) / (7 x OD ₇₅₀ value at the start of culture) (1)

According to the present invention, there is provided a method for cultivating freshwater microalgae that allows freshwater microalgae to grow well in an environment of low pH and high sodium ion concentration, and a method for cultivating freshwater microalgae that allows good growth of freshwater microalgae in an environment of low pH and high sodium ion concentration. Freshwater microalgae and a method for producing the freshwater microalgae are provided.

After pre-cultivating Cyanidium sp. HKN1 (haploid) in M-Allen medium (MA medium), MA medium, MA medium with 0.3M NaCl added (MA+0.3M NaCl medium), or MA medium Growth curve when main culture was performed in a medium (MA + 0.5M NaCl medium) to which 0.5M NaCl was added, and growth when main culture was performed in MA + 0.5M NaCl medium after preculture in MA + 0.3M NaCl medium. It is a graph showing a curve (change in OD ₇₅₀ over time). It is a graph showing a growth curve when Cyanidium sp. HKN1 (haploid) was precultured in MA+0.3M NaCl medium and then main cultured in seawater medium or MA+0.5M NaCl medium. This is a graph showing the OD ₇₅₀ of Cyanidium sp. HKN1 (haploid) after being precultured in MA+0.3M NaCl medium and then main cultured in a seawater medium at pH 2 to 7 for 7 days. Growth curves when Cyanidium sp. HKN1 (diploid) was precultured in MA medium and then main cultured in MA medium, MA+0.3M NaCl medium, or MA+0.5M NaCl medium, and precultured in MA+0.3M NaCl medium. It is a graph showing a growth curve when main culture is performed in MA+0.5M NaCl medium after culturing. It is a graph showing a growth curve when Cyanidium sp. HKN1 (diploid) was precultured in MA+0.3M NaCl medium and then main cultured in seawater medium or MA+0.5M NaCl medium. Growth curves when Cyanidioscizon melolae 10D was precultured in MA medium and then main cultured in MA medium, MA+0.3M NaCl medium, or MA+0.5M NaCl medium, and precultured in MA+0.3M NaCl medium. This is a graph showing a growth curve when the cells were then main cultured in MA+0.5M NaCl medium. It is a graph showing a growth curve when Cyanidioscizon melolae 10D was precultured in MA+0.3M NaCl medium and then main cultured in seawater medium or MA+0.5M NaCl medium. This is a fluorescence micrograph showing polyphosphoric acid and vacuoles of a culture of Cyanidium sp. HKN1 (haploid) cultured in MA medium, MA medium + 0.5M NaCl medium, and seawater medium. This is a fluorescence micrograph showing polyphosphoric acid and vacuoles of a culture of Cyanidium sp. HKN1 (diploid) cultured in MA medium, MA medium + 0.5 M NaCl medium, and seawater medium. It is a graph showing a growth curve when Cyanidium sp. HKN1 (haploid) is cultured in 10 L of seawater medium. FIG. 2 shows a molecular phylogenetic tree of microalgae belonging to the class Ideyucogonaceae based on the chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene. Local bootstrap values determined by the maximum likelihood method (only 50 or higher are listed, left) and posterior probabilities determined by the Bayesian method (only 0.95 or higher are listed, right) are shown near each branch.

As used herein, "MA medium" means M-Allen medium. Specifically, it refers to a medium having the composition shown in Table 1, which has been adjusted using sulfuric acid so that the hydrogen ion concentration is pH 1.0 to 6.0. When simply described as "MA medium" or "M-Allen medium", it means a medium to which NaCl is not added. "M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0" means an MA medium to which NaCl is not added and which is adjusted to have a pH of 2.0.

As used herein, "MA base + 0.3M NaCl medium" means MA medium adjusted to have a NaCl concentration of 0.3M. Specifically, it refers to a medium having the composition shown in Table 4, which has been adjusted using sulfuric acid so that the hydrogen ion concentration is pH 1.0 to 6.0. "M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.3M" means an MA+0.3M NaCl medium adjusted to have a pH of 2.0.

As used herein, "MA+0.5M NaCl medium" means an MA medium adjusted to have a NaCl concentration of 0.5M. Specifically, it refers to a medium having the composition shown in Table 5, which has been adjusted using sulfuric acid so that the hydrogen ion concentration is pH 1.0 to 6.0. "M-Allen medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M" means an MA+0.5M NaCl medium adjusted to have a pH of 2.0.

In MA medium, MA+0.3M NaCl medium, and MA+0.5M NaCl medium, the hydrogen ion concentration (pH) can be any value in the range of pH 1.0 to 6.0 unless otherwise stated.

In this specification, "M" used with respect to component concentration in a medium represents "mol/L".

<Culture method of freshwater microalgae>
In one embodiment, the present invention provides a method for culturing freshwater microalgae. In the culture method of this embodiment, freshwater microalgae are cultured at a culture temperature of 15 to 60°C in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M. It includes a culture step of culturing at ℃. The culture method includes a preculture step of culturing freshwater microalgae in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M; Cultivate the freshwater microalgae after the process in a medium prepared such that the sodium ion concentration is 1.2 to 5 times the sodium ion concentration in the pre-culture step and the hydrogen ion concentration is pH 1.0 to 6.0. It is preferable to include a main culture step.

In one embodiment, the culture method of the present embodiment is characterized by performing preculture in an acidic medium with a sodium ion concentration of 0.1 to 0.4M before main culture in an acidic medium with a high salt concentration. do. By performing such pre-cultivation, even freshwater microalgae that do not have salt tolerance can be successfully grown in a medium with a high salt concentration comparable to that of seawater.

In this specification, the sodium ion concentration and hydrogen ion concentration (pH) of the medium in the preculture step mean the sodium ion concentration and pH at the start of culture, respectively, in the preculture step. As long as the medium at the start of the culture in the preculture step has a "sodium ion concentration of 0.1 to 0.4M" and a "pH of 1.0 to 6.0", the sodium ion concentration or pH will fluctuate during the preculture period. Even if the amount is outside the above range, it is included in the pre-culture step of the culture method of the present embodiment.
The sodium ion concentration and pH of the medium in the main culture step refer to the sodium ion concentration and pH at the start of the main culture step, respectively. As long as the medium at the start of the main culture process has a sodium ion concentration of 1.2 to 5 times the sodium ion concentration in the preculture process and a pH of 1.0 to 6.0, Even if the sodium ion concentration or pH fluctuates and deviates from the above range, it is included in the main culture step of the culture method of the present embodiment.

(Freshwater microalgae)
The culture method of this embodiment is applicable to freshwater microalgae that can grow under acidic conditions of pH 1.0 to 6.0. "Freshwater microalgae" means microalgae that live in freshwater areas. The sodium ion concentration of fresh water is usually less than 0.05% by mass. The freshwater area is not particularly limited, and examples include rivers, lakes, hot springs, underground water, etc., but freshwater areas under acidic conditions with a pH of 1.0 to 6.0 are preferable. A preferred example of a freshwater area under such acidic conditions is an acidic hot spring (such as a sulfuric acid hot spring). As used herein, "microalgae" means unicellular algae.

Examples of freshwater microalgae that can grow under acidic conditions of pH 1.0 to 6.0 include microalgae belonging to the class Cyanidiophyceae. The class Rhodophyta is taxonomically classified into the phylum Rhodophyta and the class Cyanidiophyceae. Currently, three genera, Cyanidioschyzon genus, Cyanidium genus, and Galderia genus, are classified in the class Idyukogomena. Many types of microalgae have been known to date and will not be listed here, but Figure 11 shows the bases of the chloroplast rbcL gene of microalgae belonging to the Idyukogome class. A phylogenetic tree using sequences is shown.

The culture method of the present embodiment is preferably applied to freshwater microalgae that do not have salt tolerance among freshwater microalgae. Here, "not salt tolerant" means that when cultured in seawater or a medium with a sodium ion concentration equivalent to seawater (approximately 0.5M), ) means that growth is suppressed (including inability to grow) compared to when cultured in The suppression of proliferation is preferably such that the growth rate is reduced by 60% or more, more preferably 80% or more, compared to the growth rate in the freshwater microalgae culture medium. Examples of freshwater microalgae that can proliferate under acidic conditions of pH 1.0 to 6.0 and have no salt tolerance include microalgae belonging to the class Ideyukogoma, shown in FIG. 11. Examples include haploid microalgae belonging to the genus Cyanidium, Cyanidioscizon melolae, and haploid microalgae belonging to the genus Gardelia.

Some microalgae belonging to the class Idyukogometa can take on a haploid cell form or a diploid cell form. The present inventors have developed a method for obtaining a cell group with a haploid cell morphology from a cell group with a diploid cell morphology, and conversely, a method for obtaining a cell group with a haploid cell morphology from a cell group with a haploid cell morphology. The present invention provides a method for obtaining a cell group with a specific morphology (International Publication No. WO2019/107385).

As shown in Examples described below, diploid microalgae belonging to the genus Cyanidium have salt tolerance, but haploid microalgae do not have salt tolerance. Specific examples of microalgae belonging to the genus Cyanidium having haploid and diploid cell morphology include Cyanidium sp. YFU3 strain (FERM P-22334) (hereinafter referred to as "YFU3 strain"). ), and Cyanidium sp. HKN1 strain (FERM P-22333) (hereinafter referred to as "HKN1 strain"), as well as their related species, mutants, and descendants.
Hereinafter, when describing the YFU3 strain by distinguishing between diploid cell morphology and haploid cell morphology, the diploid cell morphology will be referred to as "YFU3 strain (diploid)" and the haploid cell morphology will be described as "YFU3 strain (diploid)". The cell morphology is described as "YFU3 strain (haploid)". Similarly, when describing the HKN1 strain by distinguishing between diploid cell morphology and haploid cell morphology, diploid cell morphology should be described as "HKN1 strain (diploid)" and haploid cell morphology. The cell morphology is described as "HKN1 strain (haploid)". When simply described as "YFU3 strain" or "HKN1 strain", both diploid cell morphology and haploid cell morphology are included.

Whether an algae is diploid or haploid can be determined by checking the copy number of the same gene locus. That is, if the number of copies of the same gene locus is 1, it is determined that the organism is haploid. Furthermore, it is also possible to determine whether algae are haploid using a next-generation sequencer or the like. For example, sequence reads for the entire genome are obtained using a next-generation sequencer, etc., those sequence reads are assembled, and then the sequence reads are mapped to the assembled sequence. In diploids, base differences between alleles are found in various regions on the genome, but in haploids, there is only one allele, so no such regions are found.
Alternatively, cells are stained with a nuclear staining reagent such as DAPI, and compared with cells known to be haploid, cells that show the same fluorescence intensity are determined to be haploid, and the fluorescence is approximately twice as high. Cells exhibiting brightness may be determined to be diploid. Alternatively, cells are stained with a nuclear staining reagent such as DAPI, and compared to cells known to be diploid, cells that show the same fluorescence intensity are determined to be diploid, and approximately 1/2 Cells exhibiting a fluorescence intensity of .

YFU3 strain (haploid) is a unicellular red algae isolated from the high temperature acidic water of a hot spring in Yufu City, Oita Prefecture, Japan. The YFU3 strain was deposited on May 30, 2017 with the accession number FERM P-22334 at the National Institute of Technology and Evaluation, Patent Microorganism Depositary (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). It has been transferred to the International Deposit as of April 20, 2018 with accession number FERM BP-22334.
Strain HKN1 is a unicellular red algae isolated from the high temperature acidic water of a hot spring in Hakone Town, Ashigarashimo District, Kanagawa Prefecture, Japan. The HKN1 strain (haploid) was deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary under the accession number FERM P-22333 on May 30, 2017, and as of 2018 under the accession number FERM BP-22333. It was transferred to an international deposit on April 20, 2015.

Furthermore, freshwater microalgae to which the culture method of the present embodiment is applied may be isolated from freshwater areas such as acidic hot springs, or may be obtained from culture collections and the like. For example, Cyanidioscizon melolae is stored at the American Type Culture Collection (ATCC; 10801 University Boulevard Manassas, VA 20110 USA), National Institute for Environmental Studies, Microbial System Collection Facility (16-2 Onogawa, Tsukuba City, Ibaraki Prefecture, Japan). ) etc.

Further, the freshwater microalgae to which the culture method of the present embodiment is applied are not limited to those isolated from nature, and may be natural freshwater microalgae with mutations. Mutations may be naturally occurring or may be artificially generated. For example, Cyanidioscizon melolae has a small genome size (approximately 16 Mbp) and the genome sequence has been completed (Matsuzaki M et al., Nature. 2004 Apr 8;428(6983):653-7 .), easy to genetically modify. Therefore, for example, the culture method of the present embodiment may be applied to a Cyanidioscizon melolae transformant (for example, a transformant enriched with nutritional components) produced by genetic modification. Furthermore, the culture method of this embodiment may be applied to transformants of other freshwater microalgae as long as genetic modification is possible.

Furthermore, some microalgae belonging to the genus Gardelia can assume a haploid cell morphology or a diploid cell morphology. For example, a haploid cell morphology can be obtained by culturing diploid microalgae belonging to the genus Gardelia for a certain period of time (for example, about 1 to 3 weeks). As a medium for culturing diploid cells to obtain haploid cells, for example, acidic hot spring drainage medium, Tsukahara Kosen medium (Hirooka et al. 2016 Front in Microbiology), etc. can be suitably used. The culture method of the present embodiment can also be applied to haploid microalgae belonging to the genus Gardelia or transformants thereof. Examples of microalgae belonging to the genus Gardelia include G. partita (NBRC 102759), and G. sulphuraria (SAG108.79, etc.). Microalgae belonging to the genus Gardelia may be isolated from freshwater bodies such as acidic hot springs, or may be obtained from culture collections and the like. In addition to the culture collection mentioned above for Cyanidioscizon melorae, the culture collection includes NITE Biological Resource Center (NRBC; 2-49-10 Nishihara, Shibuya-ku, Tokyo, Japan), GEORG-AUGUST-UNIVERSITY GOTTINGEN culture collection of Algae (SAG) and the like.

Among microalgae belonging to the class Idyukogome, haploid algal cells often do not have a strong cell wall. Algal cells with a haploid cell morphology that do not have such a strong cell wall can be destroyed by relatively mild treatments such as neutralization treatment, hypotonic treatment, and freeze-thaw treatment. In this specification, "not having a strong cell wall" means that cell rupture occurs in any of the following cell rupture treatments (A) to (C).

(A) Algal cells are suspended in an isotonic solution of pH 7 and left for one week or more.
(B) Suspend algal cells in distilled water and leave for 1 minute or more.
(C) Algal cells are dried and suspended in an isotonic solution of pH 7.
In (A) to (C) above, when the algal cells are cultured cells, the medium may be removed by centrifugation or the like and the algal cells may be washed with an isotonic solution or the like before each treatment.
In (A) and (C) above, the isotonic solution includes a pH 7 buffer containing 10% sucrose and 20mM HEPES.
In the above (C), examples of the drying treatment include drying in a refrigerator (4° C.), freeze drying, and the like. For the drying process, algal cell precipitates collected by centrifugation are used. When drying in a refrigerator, the drying time depends on the amount of algae cells, but is exemplified as 3 days or more.

In addition, to determine whether cell rupture has occurred, centrifuge the algal cell suspension (1,500 x g, 3 minutes) after the cell rupture treatments in (A) to (C) above. The determination can be made by determining the ratio of the amount of protein in the centrifuged supernatant to the total amount of protein in the supernatant. Specifically, when the rupture rate determined by the following formula is 20% or more, it can be determined that cell rupture has occurred.

Alternatively, observe the algal cells in the algal cell suspension with an optical microscope (e.g., 600x magnification) and find that the proportion of cells in which cell rupture has occurred is approximately 10% or more, preferably approximately 20% of the total algal cells. If this is the case, it may be determined that cell rupture has occurred.

In the cell rupture treatments (A) to (C) above, an isotonic solution with a pH of 7 can be used. It can be said that these cells undergo cell rupture under these conditions.
Microalgae whose cells burst under pH 7 conditions will have difficulty growing in the external environment if they flow out of the culture tank, and contamination to the environment can be suppressed.
When algal cells do not have a strong cell wall, the cell wall is usually not observed when observed using an optical microscope (for example, at a magnification of 600 times). Note that whether or not cell rupture occurs due to mild hypotonic treatment under conditions of pH 6 or lower does not affect the determination of whether or not the microalgae does not have a strong cell wall.

[Pre-culture step]
The pre-culture step is a step of culturing freshwater microalgae in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M.

The medium used in the pre-culture step is not particularly limited as long as it has a sodium ion concentration of 0.1 to 0.4M and a pH of 1.0 to 6.0. For example, a common freshwater microalgae culture medium to which 0.1 to 0.4 M sodium ions are added and the pH is adjusted to 1.0 to 6.0 can be preferably used.
The culture medium for freshwater microalgae is not particularly limited, and an appropriate one may be selected depending on the type of freshwater microalgae to be cultured. Examples of the culture medium for freshwater microalgae include an inorganic salt culture medium containing a nitrogen source, a phosphorus source, an iron source, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, etc.), and the like. For example, nitrogen sources include ammonium salts, nitrates, nitrites, urea, amines, etc., phosphorus sources include phosphates, phosphites, etc., and iron sources include iron chloride, Examples include iron sulfate and iron citrate. Specific examples of the culture medium for freshwater microalgae include 2×Allen medium (Allen MB. Arch. Microbiol. 1959 32:270-277.), M-Allen medium (Minoda A et al. Plant Cell Physiol. 2004). 45: 667-71.), MA2 medium (Ohnuma M et al. Plant Cell Physiol. 2008 Jan;49(1):117-20.), and the like. Note that in this specification, M-Allen medium may be referred to as "MA medium."

The sodium ion concentration may be appropriately selected within the range of 0.1 to 0.4M depending on the sodium ion concentration in the main culture step described below. More specifically, a sodium ion concentration that is 0.2 to 0.8 times the sodium ion concentration expected in the main culture step can be selected. The sodium ion concentration is preferably 0.5 times or more, more preferably 0.5 to 0.7 times, and 0.5 to 0.6 times the sodium ion concentration in the main culture step. is even more preferable.
For example, when the sodium ion concentration in the main culture step is the same as seawater (approximately 0.5M), the sodium ion concentration in the preculture step is preferably 0.25M or more, and 0.25 to 0.35M. It is more preferable that the amount is 0.25 to 0.3M.
The sodium ion concentration of the medium may be adjusted using a commercially available sodium ion reagent or by using common salt. In addition, natural seawater, concentrated seawater, artificial seawater, etc. are diluted to a sodium ion concentration of 0.1 to 0.4M, and nitrogen sources, phosphorus sources, iron sources, trace elements, etc. are added as appropriate. It's okay. As natural seawater, filtered surface water or deep ocean water can be used, and commercially available products can also be used. Commercially available natural seawater products include, for example, Naseem 10 (Izu 10m surface seawater) and Naseem 800 (Izu Akasawa 800m deep seawater) (both produced by Japan QCE bluelab division). Commercial products of artificial seawater include, for example, Daigo IMK medium and Daigo artificial seawater SP (both manufactured by Nippon Pharmaceutical Co., Ltd.).

For example, the following composition is known as the concentration of major ions contained in natural seawater, particularly in the surface layer of seawater. This composition is thought to be a common composition in the surface layer of the ocean, and it is well known that salinity concentration varies from region to region. Therefore, it is difficult to define the salinity of seawater, but there is no doubt that sodium is the main metal ion. Therefore, the inventors generally determined that when the sodium ion concentration exceeds 0.4M, it is a seawater condition. The sodium ion concentration closer to seawater conditions is 0.45M or more, more preferably 0.5M or more.
Sodium ion 1.0556% by mass
Magnesium ion 0.1272% by mass
Calcium ion 0.0400% by mass
Potassium ion 0.0380% by mass
Strontium ion 0.0008% by mass
Chloride ion 1.8980% by mass
Sulfate ion 0.2649% by mass
Bromide ion 0.0065% by mass
Hydrogen carbonate ion 0.0140% by mass
Fluoride ion 0.0001% by mass
Boric acid 0.0026% by mass

The hydrogen ion concentration may be appropriately selected within the pH range of 1.0 to 6.0 depending on the type of freshwater microalgae. For example, when the freshwater microalgae are microalgae belonging to the class Idyukogoma, the pH is preferably 1.0 to 5.0, more preferably 1.0 to 3.0.
The pH of the culture medium can be adjusted using, for example, an inorganic acid such as sulfuric acid or hydrochloric acid, or an inorganic base such as potassium hydroxide. Furthermore, a pH buffer may be optionally added to the medium in order to suppress pH fluctuations during culture.

The preculture step can be started by inoculating freshwater microalgae cells into a medium. The algae concentration at the start of culture is not particularly limited, but can be set to, for example, a cell turbidity of OD ₇₅₀ =0.05 to 0.5. " _OD750 " means absorbance at 750 nm. The cell turbidity at the start of culture is preferably OD ₇₅₀ =0.05 to 0.3.

The temperature conditions in the preculture step may be appropriately selected depending on the type of freshwater microalgae. Generally, the culture temperature can be exemplified by 15 to 60°C, preferably 15 to 50°C, and more preferably 30 to 50°C. When the freshwater microalgae are microalgae belonging to the class Idyukogome, the culture temperature is preferably 30 to 50°C.
The light conditions in the preculture step may be appropriately selected depending on the type of freshwater microalgae. Generally, 5 to 2000 μmol/m ² s can be exemplified. When the freshwater microalgae are microalgae belonging to the class Ideyucogomatidae, the amount is preferably 5 to 1500 μmol/m ² s. The light condition may be continuous light or may have a light/dark cycle (10L:14D, etc.). The preculture step may be performed under natural light.
The CO ₂ conditions in the preculture step may be appropriately selected depending on the type of freshwater microalgae. Generally, 0.04 to 5% CO ₂ conditions can be exemplified. When the freshwater microalgae are microalgae belonging to the class Idyukogome, 0.04 to 3% CO ₂ conditions are preferable. Among the microalgae belonging to the genus Gardelia, the genus Gardelia has high tolerance to high _CO2 concentrations and can grow even in 100% _CO2 . The CO ₂ condition may also be used. Moreover, the CO ₂ condition may be the atmospheric CO ₂ concentration.

The culture method in the pre-culture step is not particularly limited, and any method commonly used for culturing microalgae may be used. Specific examples include static culture, aeration culture (200 to 400 mL air/min, etc.), shaking culture (100 to 200 rpm, etc.), and the like.

The culture period in the pre-culture step is not particularly limited, but it requires a period from the end of the lag period to the start of the logarithmic phase, usually 3 days or more, preferably 5 days or more, and more Preferably it is 7 days or more. By performing the preculture step for a period equal to or longer than the minimum number of days, the growth of freshwater microalgae in the main culture step can be maintained better. The upper limit of the preculture period is not particularly limited, but if it is carried out until the end of the logarithmic phase and into the stationary phase, it is inappropriate to maintain good growth of microalgae in the main culture. The preculture period is preferably 3 to 20 days, more preferably 5 to 15 days, and even more preferably 7 to 10 days.
The scale of preculture can be selected depending on the scale of main culture. When main culture is carried out on a small scale, such as breeding of microalgae or selection of mutant strains, preculture can be carried out on a small scale, and is usually carried out in a volume of 0.1 to 1000 mL. Further, when industrial mass production is carried out by main culture, pre-culture may be carried out in about 1 to 10 L.

[Main culture process]
In the main culture step, the freshwater microalgae after the pre-culture step are grown so that the sodium ion concentration is 1.2 to 5 times the sodium ion concentration in the pre-culture step and the hydrogen ion concentration is pH 1.0 to 6.0. This is a step of culturing in a medium prepared in the following manner.

The medium used in the main culture step has a sodium ion concentration 1.2 to 5 times that of the medium used in the pre-culture step, and is 0.4M or more, preferably 0.45M or more, in terms of sodium ion concentration. Preferably, 0.5M or more can be used. The hydrogen ion concentration is not particularly limited as long as the medium has a pH of 1.0 to 6.0. For example, it is preferable to use a common freshwater microalgae culture medium to which sodium ions at the above concentration are added and the pH is adjusted to 1.0 to 6.0. As the culture medium for freshwater microalgae, the same ones as those mentioned in the above "[Pre-culture step]" can be mentioned.

The medium used in the main culture step may be seawater to which a nitrogen source, phosphorus source, iron source, trace elements, etc. are added as appropriate, and the pH is adjusted to 1.0 to 6.0. The seawater may be natural seawater, artificial seawater, or diluted concentrated seawater. Commercially available natural seawater and artificial seawater include those listed in the "[Pre-culture step]" section above. In the main culture step, when performing mass culture, costs can be reduced by using natural seawater. Natural seawater may be surface water or deep ocean water. Natural seawater is preferably filtered to remove impurities. The nitrogen source, phosphorus source, iron source, trace elements, etc. to be added to seawater may be appropriately selected depending on the type of freshwater microalgae.

When the freshwater microalgae are microalgae belonging to the class Idyukogoma, it is preferable to add at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt to the seawater.
Examples of nitrogen-containing salts include nitrogen-containing inorganic salts such as ammonium salts, nitrates, and nitrites. Among these, ammonium salts (such as ammonium sulfate) are preferred as the nitrogen-containing salt. The amount of ammonium salt added to seawater is exemplified by an ammonium ion concentration of 20 to 100 mM.
Examples of phosphorus-containing salts include phosphorus-containing inorganic salts such as phosphates and phosphites. Among these, phosphates (potassium dihydrogen phosphate, etc.) are preferred as the phosphorus-containing salt. An example of the amount of phosphate added to seawater is 2 to 10 mM in terms of phosphate ion concentration.
Examples of iron-containing salts include iron (III) chloride, iron (II) sulfate, iron (II) citrate, and hydrates thereof. Among these, iron (III) chloride is preferred as the iron-containing salt. An example of the amount of iron-containing salt added to seawater is 0.1 to 2 mM in terms of iron ion concentration.
In addition, trace elements such as boric acid, manganese, zinc, molybdenum, cobalt, and copper are preferably added to the seawater.
When the freshwater microalgae are microalgae belonging to the class Idyukogoma, a specific example of the culture medium used in the main culture step is preferably a "seawater culture medium" listed in Table 9 below.

The sodium ion concentration of the medium used in the main culture step is not particularly limited, but is 1.1 to 5 times, preferably 1.2 to 5 times, 1.4 to 2 times the sodium ion concentration in the preculture step. It is more preferably twice as large, and even more preferably 1.5 to 2 times. When seawater is used in the main culture step, the sodium ion concentration is about 0.4M or more, and a medium with a sodium ion concentration of 1M or less is used depending on the type of microalgae used.
Furthermore, when the main culture step is performed outdoors, a sodium ion concentration of 0.5 M or more can be used to suppress contamination with other organisms. For example, the sodium ion concentration may be 0.5 to 1M.
The sodium ion concentration of the medium can be adjusted in the same manner as the medium in the pre-culture step.

The hydrogen ion concentration may be appropriately selected within the pH range of 1.0 to 6.0 depending on the type of freshwater microalgae. For example, when the freshwater microalgae are microalgae belonging to the class Idyukogoma, the pH is preferably 1.0 to 5.0, more preferably 1.0 to 3.0.
Furthermore, when the main culture step is performed outdoors, a lower pH (such as pH 1.0 to 2.0) can be used in order to suppress contamination with other organisms.
The pH of the medium can be adjusted in the same manner as the medium in the pre-culture step.

The main culture step can be started by inoculating the culture solution from the pre-culture step into the medium for the main culture step. The algae concentration at the start of culture is not particularly limited, but can be set to, for example, a cell turbidity of OD ₇₅₀ =0.05 to 0.5. " _OD750 " means absorbance at 750 nm. The cell turbidity at the start of culture is preferably OD ₇₅₀ =0.05 to 0.3.

The culture scale in the main culture step can be appropriately selected depending on the purpose. For example, when breeding microalgae or selecting mutant strains, main culture can be carried out on a small scale, and is usually carried out in about 10 mL to 10 L. In addition, when industrial mass production is performed by main culture, the culture may be carried out in about 20 to 5000 L. When performing mass culture of 500 L, outdoor culture may be performed.

The temperature conditions, light conditions, CO ₂ conditions, and culture method in the main culture step can be the same as the preculture conditions described above.
Moreover, the main culture step may be performed outdoors. In this case, the temperature conditions, light conditions, and CO ₂ conditions can be the conditions of the external environment in which the culture tank is installed. Even when the main culture step is carried out outdoors, since the culture is carried out under acidic conditions of pH 1.0 to 6.0, contamination with other organisms can be suppressed. In addition, when haploid microalgae belonging to the class Idyukogometa are used as freshwater microalgae, it is difficult to survive in an environment with a pH of 7, so even if they are released from an outdoor culture tank into the external environment. Environmental pollution is suppressed.

The culture period in the main culture step is not particularly limited, and culture can be continued until a desired amount of biomass is obtained. Alternatively, the growth status of microalgae may be checked and cultured until the stationary phase is reached.

According to the culture method of this embodiment, freshwater microalgae can be grown favorably in a medium with low pH and high sodium ion concentration in a short induction period from the start of culture. Therefore, even in the case of outdoor cultivation, invasion of other organisms can be effectively suppressed. In addition, since microalgae belonging to the Idyukogome class (especially haploid) die under neutral conditions, there is a possibility that microalgae belonging to the Idyukogome class (especially haploid) may be released into the environment from a mass culture system. Even if there is, biological containment is possible. Therefore, it can be suitably applied to outdoor mass cultivation of freshwater microalgae that produce useful substances.

<Production method of freshwater microalgae>
In one embodiment, the present invention provides a method for producing freshwater microalgae that can be grown in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.5M or higher. The method for producing freshwater microalgae of this embodiment is to produce freshwater microalgae that cannot grow in a medium with a sodium ion concentration of 0.5M or more, with a hydrogen ion concentration of 1.0 to 6.0 and a sodium ion concentration of 0.1. It includes a step of culturing in a medium prepared to have a concentration of ~0.4M.

The freshwater microalgae used in the method of this embodiment are freshwater microalgae that cannot grow in a medium with a sodium ion concentration of 0.5M or more.
To determine whether freshwater microalgae cannot grow in a medium with a sodium ion concentration of 0.5M or higher, the target freshwater microalgae can be cultured in a medium with a sodium ion concentration of 0.5M or higher, and over time. This can be confirmed by measuring cell turbidity (OD ₇₅₀ ). If the OD ₇₅₀ does not increase from the start of culture, it can be determined that the freshwater microalgae cannot grow in a medium with a sodium ion concentration of 0.5M or more.
Further, it is preferable that the freshwater microalgae can grow at a pH of 1.0 to 6.0.
Examples of such freshwater microalgae include haploid microalgae belonging to the genus Cyanidium. Specific examples of haploid microalgae belonging to the genus Cyanidium include HKN1 strain (haploid) and YFU3 strain (haploid).

The method of this embodiment includes the step of culturing freshwater microalgae as described above in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M. This step can be performed in the same manner as "[Pre-culture step]" in "<Method for culturing freshwater microalgae>" above.

According to the method of this embodiment, salt tolerance can be imparted to freshwater microalgae that do not have salt tolerance, and they can grow in a medium with a pH of 1.0 to 6.0 and a sodium chloride concentration of 0.5 M or more. freshwater microalgae can be obtained. In an environment with high salt concentration and low pH in which the freshwater microalgae can grow, invasion of other organisms is suppressed. In addition, since microalgae belonging to the Idyukogome class (especially haploid) die under neutral conditions, there is a possibility that microalgae belonging to the Idyukogome class (especially haploid) may be released into the environment from a mass culture system. Even if there is, biological containment is possible. Therefore, the freshwater microalgae can be suitably used for outdoor mass culture.

<Haploid microalgae belonging to the class Idyukogometa that has salt tolerance>
The haploid microalgae belonging to the Idyukogoma class according to one embodiment of the present invention grows in a medium with a high salt concentration such as a sodium ion concentration of 0.5M, but the haploid microalgae belonging to the Idyukogoma class according to one embodiment of the present invention grows in a medium with a high salt concentration such as a sodium ion concentration of 0.5M. Ploids cannot grow. However, if salt tolerance is acquired during the preculture period using the culture method according to one embodiment of the present invention, it becomes possible to grow even in a medium with a sodium ion concentration of 0.5M. At this time, there is a characteristic that logarithmic growth begins after a lag period that is shorter than during the preculture period, and this characteristic remains the same even when the medium is subcultured. Furthermore, the haploid algae of the present invention that has acquired salt tolerance has the characteristic that if it is subcultured to an MA medium to which NaCl is not added, it will not grow or will grow poorly.
Good growth can also be determined by comparing with control culture conditions, but in one embodiment of the present invention, the number of cells in the initial culture state and the number of cells that have increased over a predetermined period of time can be determined. Consider the size of the ratio. Specifically, it is calculated using equation (1). The number of cells is determined by measuring the absorbance OD of the culture solution at 750 nm, and the predetermined period is 7 days. If the value of formula (1) is 2 or more, growth is good, and if it is less than 2, growth is poor. Suppose that The causes of poor growth include the need for time to acclimatize to the environmental conditions, and haploid microalgae belonging to the Idyukogome class, which do not have strong cell walls, cannot withstand the osmotic pressure of hypertonic solutions, resulting in cell failure. It is possible that they were destroyed and died. A possible way to confirm that cells are destroyed is to observe them using a microscope, but it is difficult to ensure quantitative results. Therefore, in one embodiment of the present invention, quantitative considerations are made by measuring the amount of phycocyanin, which is a cell content.

In one embodiment, the present invention is directed to haploid microalgae belonging to the class Ideyukogoma, cultured at 42° C. in an MA medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M. , the value calculated by the following formula (1) when statically cultured for 7 days under continuous light with a carbon dioxide concentration of 2% and an illuminance of 60 μmol/m ² s is 2 or more, in MA medium, at a culture temperature of 42 ° C. Haploid microalgae belonging to the class Ideyukogoma, which has a value calculated by the following formula (1) of less than 2 when cultured stationary for 7 days under continuous light with a carbon dioxide concentration of 2% and an illuminance of 60 μmol/m ² s. I will provide a.
Culture rate expressed as (OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture)/(7 x OD ₇₅₀ value at the start of culture) (1)

"MA medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5M" is the medium described in Table 5 in Examples (hereinafter also referred to as "MA+0.5M NaCl medium"). . The haploid microalgae belonging to the genus Idyukogoma of this embodiment can grow in an MA+0.5M NaCl medium. Furthermore, when statically cultured in MA+0.5M NaCl medium under the conditions of culture temperature 42°C, carbon dioxide concentration 2%, and continuous light with illuminance 60 μmol/m ² s, the growth rate calculated by the following formula (1) is 2 or more.
(OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture) / (7 x OD ₇₅₀ value at the start of culture) (1)

In the above formula (1), "OD ₇₅₀ value at the start of culture" means the absorbance of the culture solution measured at a wavelength of 750 nm at the start of culture (0 hour). Normally, a haploid microalgae belonging to the class Ideyucogonaceae is inoculated into an MA+0.5M NaCl medium so that the OD ₇₅₀ value at the start of culture is 0.1, and culture is started. In the above formula (1), "OD ₇₅₀ value 7 days after the start of culture" means the absorbance of the culture solution measured at a wavelength of 750 nm 7 days (168 hours) after the start of culture. The absorbance of the culture solution can be measured with an absorbance meter.
The culture is performed by static culture for 7 days under the conditions of a culture temperature of 42° C., a carbon dioxide concentration of 2%, and continuous light with an illuminance of 60 μmol/m ² s. Although the culture scale is not particularly limited, for example, a 24-well plate can be used for culturing with 1 mL of culture solution.

Natural haploid microalgae belonging to the class Idyukogoma cannot grow in MA+0.5M NaCl medium. Therefore, when a natural haploid microalgae belonging to the class Idyukogome is cultured in an MA+0.5M NaCl medium, the value calculated by the above formula (1) is less than 2. However, the haploid microalgae of the present embodiment belonging to the genus Idyukogoma has tolerance to high sodium ion concentrations, and therefore can grow well even in MA+0.5M NaCl medium. Therefore, when the haploid microalgae belonging to the class Idyukogome of this embodiment is cultured in MA+0.5M NaCl medium, the value calculated by the above formula (1) will be 2 or more. In the haploid microalgae belonging to the class Idyukogome of this embodiment, the value calculated by the above formula (1) when cultured in MA+0.5M NaCl medium is preferably 3 or more, and preferably 4 or more. It is more preferable.

In addition to the above-mentioned characteristics, the haploid microalgae belonging to the class Idyukogome of this embodiment can be statically cultured in an MA medium at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and a continuous light illuminance of 60 μmol/m ² s. In this case, the value calculated by the equation (1) is less than 2. The MA medium is the medium described in Table 1 in the Examples. Usually, a haploid microalgae belonging to the class Idyukogoma is inoculated into an MA medium so that the OD ₇₅₀ value at the start of culture is 0.1, and culture is started. The culture is performed by static culture for 7 days under the conditions of a culture temperature of 42° C., a carbon dioxide concentration of 2%, and continuous light with an illuminance of 60 μmol/m ² s. Although the culture scale is not particularly limited, for example, a 24-well plate can be used for culturing with 1 mL of culture solution.

Natural haploid microalgae belonging to the class Idyukogoma can grow well in MA medium. Therefore, when a natural haploid microalgae belonging to the class Idyukogome is cultured in an MA medium, the value calculated by the above formula (1) is usually 2 or more. However, the haploid microalgae belonging to the class Ideyukogomera of this embodiment has resistance to high sodium ion concentrations, but has a reduced ability to proliferate at low sodium ion concentrations. Therefore, when the haploid microalgae belonging to the class Idyukogome of this embodiment is cultured in an MA medium, the value calculated by the above formula (1) will be 2 or more. It is preferable that the haploid microalgae belonging to the class Idyukogome of this embodiment has a value calculated by the above formula (1) when cultured in an MA medium of less than 1.8, and less than 1.5 or more. It is more preferable that

The haploid microalgae belonging to the class Idyukogome of this embodiment preferably has a mortality rate of 30% or more, more preferably 40% or more, and 50% or more when suspended in an MA medium. It is more preferable that If the mortality rate is 30% or more, even if the algal cells are released into the environment, it is difficult for the algal cells to survive in an environment with a low sodium ion concentration. Therefore, even when cultured in an outdoor open culture system, contamination to the environment is unlikely to occur.

The mortality rate is calculated as follows.
After culturing in MA+0.5M NaCl medium, algal cells in an amount that gives an OD ₇₅₀ =1 when suspended in 2 mL of the medium are collected and subjected to the following treatments 1 to 3.
Treatment 1: Suspend in 2 mL of MA medium and shake with vortex for 10 minutes.
Treatment 2: Suspend in 2 mL of MA+0.5M NaCl medium and shake by vortexing for 10 minutes.
Treatment 3: Suspend in 0.1 mL of MA+0.5M NaCl medium, freeze at -196°C, make up to 2 mL with the above medium, and shake by vortexing for 10 minutes.

The mortality rate is then calculated using the following formula:
Mortality rate (%)={(PC concentration after treatment 2−PC concentration after treatment 1)/(PC concentration after treatment 2−PC concentration after treatment 3)}×100
In the above formula, "PC concentration" represents the phycocyanin concentration. The PC concentration can be determined by measuring the absorbance at 620 nm and 678 nm using a spectrophotometer equipped with an integrating sphere for the suspension after any one of treatments 1 to 3. The PC concentration can be calculated from the absorbance at 620 nm and 678 nm using the following formula.
PC concentration (μg/ml) = 138.5 × A ₆₂₀ - 35.49 × A ₆₇₈

The haploid microalgae belonging to the class Idyukogome of this embodiment preferably has any one or more of the following characteristics (a) to (c), and any two of the following (a) to (c). It is more preferable to have one feature, and even more preferably to have all the features (a) to (c) below.
(a) Compared to the natural haploid microalgae, the growth rate is higher for 7 days after the start of culture in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.5M.
(b) Compared to the natural haploid microalgae, the growth rate is low for 7 days after the start of culture in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.05M or less.
(c) Compared to the growth rate for 7 days after the start of culture in a medium with a pH of 1.0 to 6.0 where the sodium ion concentration is 0.05M or less, the growth rate at pH 1.0 to 6.0 where the sodium ion concentration is 0.5M or less The growth rate was high for 7 days after the start of culture in the 6.0 medium.

"Natural microalgae belonging to the class Idyukogome" refers to microalgae belonging to the class Idyukogomena that inhabits in the natural world, or microalgae of the type having similar properties to the above-mentioned microalgae. Natural microalgae may be isolated from nature and maintained by culturing in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.05M or less.
"Natural haploid microalgae belonging to the Idyukogome class" refers to haploid microalgae belonging to the Idyukogome class living in the natural world or diploid microalgae belonging to the Idyukogome class living in the natural world. Refers to haploid. Natural diploid microalgae belonging to the Idyukogome class are grown for a certain period of time (for example, about 1 to 3 weeks) in a medium prepared to have a sodium ion concentration of 0.05 M or less and a pH of 1.0 to 6.0. ), microalgae that have undergone meiosis after being cultured under certain conditions are physically selected under a microscope to obtain haploid microalgae belonging to the class Ideyucogomatidae.

Whether or not a haploid microalgae belonging to the class Idyukogometa has the feature (a) above is determined by the fact that the haploid microalgae and the natural microalgae, which is the same species as the microalgae, have sodium ions. The determination can be made by culturing in a medium with a pH of 1.0 to 6.0 and a concentration of 0.5M, and comparing the growth rate for 7 days after the start of culture. When the growth rate is higher than that of a haploid natural microalgae, it is determined that the microalgae has the characteristic (a) above.

Whether or not a haploid microalgae belonging to the class Idyukogometa has the feature (b) above is determined by the fact that the haploid microalgae and the natural microalgae, which is the same species as the microalgae, are separated by sodium ions. The determination can be made by culturing in a medium with a pH of 1.0 to 6.0 and a concentration of 0.05 M or less, and comparing the growth rate for 7 days after the start of culture. If the growth rate is lower than that of a haploid natural microalgae, it is determined that the microalgae has the characteristic (b) above.

Whether or not a haploid microalgae belonging to the class Idyukogometa has the characteristic (c) above is determined by testing the haploid microalgae at a pH of 1.0 to 6.0 with a sodium ion concentration of 0.05M or less. The determination can be made by culturing in a pH 1.0 to 6.0 medium with a sodium ion concentration of 0.0 and a pH 1.0 to 6.0 medium with a sodium ion concentration of 0.5 M, and comparing the growth rates for 7 days after the start of culture in both mediums. The growth rate in a pH 1.0-6.0 medium with a sodium ion concentration of 0.5M is higher than the growth rate in a pH 1.0-6.0 medium with a sodium ion concentration of 0.05M or less. If the size is large, it is determined that the microalgae has the characteristic (c) above.

As a medium with a pH of 1.0 to 6.0 with a sodium ion concentration of 0.5M and a medium with a pH of 1.0 to 6.0 with a sodium ion concentration of 0.05M or less, The same medium as the culture medium mentioned in ``Culture method'' may be used. Examples of the culture conditions in (a) to (c) above include static culture under conditions of a culture temperature of 42° C., a carbon dioxide concentration of 2%, and continuous light at an illuminance of 60 μmol/m ² s. Although the culture scale is not particularly limited, for example, a 24-well plate can be used for culturing with 1 mL of culture solution.

The haploid microalgae belonging to the class Idyukogome of this embodiment can be obtained by the method for producing freshwater microalgae of the embodiment described above. Specific examples of haploid microalgae belonging to the class Idyukogome include haploid microalgae belonging to Cyanidioscizon melolae, Cyanidium, and haploid microalgae belonging to the genus Gardelia. Specific examples of haploid microalgae belonging to the genus Cyanidium include HKN1 strain (haploid) and YFU3 strain (haploid). A specific example of haploid microalgae belonging to the genus Gardelia is G. partita, and G. sulphuraria and the like. Among these, haploid microalgae belonging to the genus Cyanidium or microalgae belonging to the genus Gardelia are preferred, and haploid microalgae belonging to the genus Cyanidium are more preferred, including strain HKN1 (haploid) and strain YFU3 (haploid). ) is more preferred, and HKN1 strain (haploid) is particularly preferred.

It is preferable that the microalgae belonging to the class Ideyukogomera of this embodiment can grow in a medium with a pH of 1.0 to 6.0 in which the sodium ion concentration is 0.6M or higher, and a pH 1.0 medium with a sodium ion concentration of 0.7M or higher. It is more preferable to be able to grow in a medium with a pH of 1.0 to 6.0, and even more preferable to be able to grow in a medium with a pH of 1.0 to 6.0, where the sodium ion concentration is 0.8M or more, and it is more preferable to be able to grow in a medium with a pH of 1.0 to 6.0, where the sodium ion concentration is 0.9M or more. It is particularly preferred that the cells can be grown in a medium with a concentration of .0 to 6.0. The microalgae belonging to the genus Idyukogome of this embodiment may be one that can grow in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 1M or more.

In this embodiment, the microalgae belonging to the Idyukogome class are cultured in a medium with a sodium ion concentration of 0.5M and a pH of 1.0 to 6.0, and then cultured in a medium with a sodium ion concentration of 0.5M at a pH of 1.0 to 6.0. It is preferable that the growth rate is maintained even when the cells are subcultured into a new medium containing 0. Thereby, the microalgae of this embodiment can be subcultured and maintained in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.5M. Furthermore, even when the microalgae of this embodiment maintained as described above are cultured in large quantities at any time in a medium with a pH of 1.0 to 6.0 and a sodium ion concentration of 0.5M, the microalgae of this embodiment can be grown favorably. be able to.

The haploid microalgae belonging to the class Idyukogome of this embodiment can proliferate well at a sodium ion concentration comparable to that of seawater. Propagation can be achieved using a medium containing the above-mentioned P. acetate and adjusted to a pH of 1.0 to 6.0. Specific examples of such a culture medium include those similar to those listed in "[Main culture step]" of the above "<Culture method of freshwater microalgae>".

The haploid microalgae of the present embodiment belonging to the class Idyukogoma can proliferate favorably in an environment with high sodium ion concentration and low pH, and therefore can be suitably used for outdoor mass culture.

EXAMPLES Hereinafter, the present invention will be explained with reference to examples, but the present invention is not limited to the following examples.

(Preparation of medium)
M-Allen medium (MA medium) having the composition shown in Table 1 was prepared. Specifically, medium components other than A2 Fe stock were mixed, the pH was adjusted to 2.0 with sulfuric acid, and the mixture was sterilized using an autoclave. After autoclave sterilization, 4 mL of filter-sterilized A2 Fe stock was added to prepare an MA medium.
Tables 2 and 3 show the compositions of A2 trace element and A2 Fe stock, respectively.

0.3M NaCl was added to MA medium to prepare MA+0.3M NaCl medium. Furthermore, 0.5M NaCl was added to the MA medium to prepare an MA+0.5M NaCl medium. The compositions of MA+0.3M NaCl medium and MA+0.5M NaCl medium are shown in Tables 4 and 5, respectively. A2 trace element and A2 Fe stock in Tables 4 and 5 are shown in Tables 2 and 3, respectively.

In the following examples, when the medium is used for pre-culture or main culture, each medium may be expressed as follows.
When used for preculture MA medium: Medium (A)
MA+0.3M NaCl medium: Medium (B)
MA+0.5M NaCl medium: Medium (C)
When used for main culture MA medium: Medium (a)
MA+0.3M NaCl medium: medium (b)
MA+0.5M NaCl medium: medium (c)

(Measurement of growth status)
The growth status of microalgae was confirmed by cell turbidity (OD ₇₅₀ ). Specifically, the absorbance of the algal culture solution at 750 nm was measured using an absorbance meter (BIO-RAD's SmartSpec Plus) to determine cell turbidity (OD ₇₅₀ ).

(Culture method)
Cyanidium sp. HKN1 was cultured by static culture in a CO ₂ incubator for both preculture and main culture unless otherwise specified. The culture temperature was 42° C., continuous light with an illuminance of 60 μmol/m ² s was used, and the CO ₂ concentration was 2%. Unless otherwise specified, culturing was performed in 24-well plates in 1 mL of culture medium.
Cyanidioscizon melolae 10D was cultured by aeration culture (300 mL ambient air/min) for both preculture and main culture unless otherwise specified. The culture temperature was 42° C., and continuous light with an illuminance of 60 μmol/m ² s was used. Unless otherwise specified, culturing was performed in 24-well plates in 1 mL of culture medium.
For both Cyanidium sp. HKN1 and Cyanidioscizon melolae 10D, the cell turbidity at the start of preculture and at the start of main culture was set to OD ₇₅₀ =0.1. Unless otherwise specified, culturing was performed in 24-well plates in 1 mL of culture medium.

(Qualitative analysis of polyphosphoric acid)
The presence of polyphosphoric acid was observed by DAPI staining as follows.
After adding glutaraldehyde to the culture solution to a final concentration of 1% (w/v), DAPI was added to a final concentration of 3 μg/mL, and observation was made using a fluorescence microscope.

(Qualitative analysis of vacuoles)
The presence of vacuoles was observed by quinacrine staining as follows.
After adding 1M Tris-HCl (pH 8.0) buffer to a final concentration of 100 mM to the culture solution, quinacrine was added to a final concentration of 40 μg/mL, and the mixture was allowed to stand at room temperature for 15 minutes. . After centrifugation (1500 g, 5 minutes), the supernatant was discarded, MA medium was added to the precipitate, the mixture was allowed to stand at 37°C for 30 minutes, and observed under a fluorescence microscope.

[Example 1]
Cyanidium sp. HKN1 (haploid) (hereinafter sometimes abbreviated as "HKN1 (haploid)") was precultured for one week using MA medium (medium (A)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (haploid).
In addition, HKN1 (haploid) was precultured for one week using MA+0.3M NaCl (medium (B)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (haploid).
The results are shown in Table 6 and Figure 1. FIG. 1 is a graph of the changes in OD ₇₅₀ shown in Table 1.

As shown in Table 6 and Figure 1, when the preculture was on medium (A), similar growth was observed when the main culture was on medium (a) and medium (b), but when the main culture was on medium (c ) did not proliferate.
On the other hand, when the preculture was in medium (B), even when the main culture was in medium (c), the same growth as in the cases of medium (a) and medium (b) was shown.

[Example 2]
Naseem 10 (surface seawater: Japan QCE bluelab division) was used as natural seawater. HKN1 (haploid) was cultured by adding each component of the MA medium to seawater (Naseem 10), and it was confirmed which components in the MA medium contributed to the growth of HKN1 (haploid).
The media used for main culture are shown in Tables 7 and 8. MA medium components shown in Tables 7 to 8 were added to Naseem 10, and the pH was adjusted to 2.0 with sulfuric acid to prepare media 1 to 17. Note that since magnesium and calcium are abundant in seawater, MgSO ₄ and CaCl ₂ were not added to mediums 1 to 16. For medium 17, as a positive control, MgSO ₄ and CaCl ₂ equivalent to MA medium were added.

After HKN1 (haploid) was precultured in medium (B) for one week, main culture was performed in any of mediums 1 to 17 for 10 days. After 10 days of main culture, the OD ₇₅₀ of the medium was measured to confirm the growth status of HKN1 (haploid). The results are shown in Tables 7-8.

As shown in Tables 7-8, medium 16 and medium 17 showed comparable growth. From these results, by adding (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ , A2 Fe stock, and A2 trace metal element to seawater, it was possible to create a seawater medium suitable for the growth of HKN1 (haploid). It was confirmed that it can be used.
The composition of the medium 16 is shown in Table 9. In the following Examples, what is referred to as "seawater culture medium" is a culture medium having the composition shown in Table 9. A2 trace element and A2 Fe stock in Table 9 are shown in Table 2 and Table 3, respectively.

[Example 3]
HKN1 (haploid) was precultured in MA+0.3M NaCl medium (medium (B)) for 1 week, and then main cultured for 7 days in MA+0.5M NaCl medium (positive control) (medium (c)) or seawater medium. did. During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (haploid).
The results are shown in Table 10 and FIG. 2. FIG. 2 is a graph of the changes in OD ₇₅₀ shown in Table 10.

As shown in Table 10 and FIG. 2, HKN1 (haploid) showed equivalent growth when main culture was performed in either medium (c) or seawater medium.

[Example 4]
In order to confirm the effect of pH on the growth of HKN1 (haploid), seawater media with pH varied between 2 and 7 were prepared. Adjustment of pH was performed using sulfuric acid or potassium hydroxide.
HKN1 (haploid) was precultured for one week in MA+0.3M NaCl (medium (B)), and main culture was performed for 7 days in each seawater culture medium whose pH was adjusted as described above. After the main culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of HKN1 (haploid).
The results are shown in Table 11 and FIG. 3. FIG. 3 is a graph of the OD ₇₅₀ shown in Table 11.

As shown in Table 11 and FIG. 3, HKN1 (haploid) could proliferate at pH 6 or lower, but could not proliferate at pH 7. Generally, when algae are grown in a medium that uses ammonia as a nitrogen source, the pH decreases as the algae grow, so it is necessary to maintain the pH near neutrality by adding a buffer or alkaline substance. . However, there was no need for pH adjustment with the acidophilic HKN1 (haploid).

[Example 5]
Cyanidium sp. HKN1 (diploid) (hereinafter sometimes abbreviated as "HKN1 (diploid)") was precultured for one week using MA medium (medium (A)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (diploid).
In addition, HKN1 (diploid) was precultured for one week using MA+0.3M NaCl (medium (B)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (diploid).
The results are shown in Table 12 and FIG. 4. FIG. 4 is a graph of the changes in OD ₇₅₀ shown in Table 12.

As shown in Table 12 and Figure 4, HKN1 (diploid) was precultured in either medium (A) or medium (B); The main culture in medium (c) showed an equivalent growth rate.

[Example 6]
HKN1 (diploid) was precultured in MA + 0.3M NaCl medium (medium (B)) for 1 week, and then main cultured for 7 days in MA + 0.5M NaCl medium (positive control) (medium (c)) or seawater medium. did. During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of HKN1 (diploid).
The results are shown in Table 13 and FIG. 5. FIG. 5 is a graph of the changes in OD ₇₅₀ shown in Table 13.

As shown in Table 13 and FIG. 5, HKN1 (diploid) showed the same growth rate when main culture was performed in either medium (c) or seawater medium.

[Example 7]
Cyanidioscizon melolae 10D (hereinafter sometimes abbreviated as "10D") was pre-cultured for one week using MA medium (medium (A)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of 10D.
In addition, 10D was precultured for one week using MA+0.3M NaCl (medium (B)). After the pre-culture, the cells were statically cultured for 7 days using medium (a), (b), or (c) (main culture). During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of 10D.
The results are shown in Table 14 and FIG. 6. FIG. 6 is a graph of the changes in OD ₇₅₀ shown in Table 14.

As shown in Table 14 and FIG. 6, 10D showed similar growth when the preculture was on medium (A) and when the main culture was on medium (a) and medium (b). On the other hand, when the main culture was in medium (c), the _OD750 gradually decreased until the 3rd day of main culture, but recovered on the 5th day, and thereafter the growth rate was the same as in medium (a) and medium (b). showed that.
On the other hand, when the preculture was in medium (B), even when the main culture was in medium (c), the same growth as in the cases of medium (a) and medium (b) was shown from the start of culture.

[Example 8]
10D was precultured in MA+0.3M NaCl medium (medium (B)) for one week, and then main cultured for 7 days in MA+0.5M NaCl medium (positive control) (medium (c)) or seawater medium. During the main culture, the OD ₇₅₀ of the medium was measured over time to confirm the growth status of 10D.
The results are shown in Table 15 and FIG. FIG. 7 is a graph of the changes in OD ₇₅₀ shown in Table 15.

As shown in Table 15 and FIG. 7, 10D showed the same growth rate when main culture was performed in either medium (c) or seawater medium.

[Example 9]
HKN1 (haploid) cultured in MA medium for 1 week was transplanted to MA medium so that OD ₇₅₀ = 0.1, and statically cultured in a CO ₂ incubator (2% CO ₂ ) for 7 days. , HKN1 (haploid) cultured in MA medium. HKN1 (haploid) cultured in MA medium for 1 week was precultured in MA medium + 0.3 M NaCl for 1 week, then transferred to MA medium + 0.5 M NaCl medium, and kept in a CO ₂ incubator (2% The cells were statically cultured in CO ₂ ). This was used as a cultured product in MA medium + 0.5M NaCl medium.
HKN1 (haploid) cultured in MA medium for 1 week was pre-cultured in MA medium + 0.3M NaCl for 1 week, then transferred to seawater medium and left stationary in a CO ₂ incubator (2% CO ₂ ) for 7 days. It was cultured in situ. This was used as a seawater culture product.
For each cultured product, qualitative analysis of polyphosphoric acid was performed using DAPI, and qualitative analysis of vacuoles was performed using quinacrine.
The results are shown in FIG.
When MA medium culture products, MA medium + 0.5 M NaCl culture products, and seawater culture culture products were stained with DAPI, polyphosphoric acid was observed only in the MA medium culture products. When vacuoles, which are said to contain polyphosphoric acid, were stained with quinacrine, there was no difference in the way the vacuoles were stained.

[Example 10]
Culture was carried out in the same manner as in Example 9 except that HKN1 (diploid) was used as the algae, and three types of culture were obtained: MA medium culture, MA medium + 0.5 M NaCl medium culture, and seawater medium culture. manufactured a product. Similarly to Example 9, qualitative analysis of polyphosphoric acid was performed using DAPI, and qualitative analysis of vacuoles was performed using quinacrine.
The results are shown in FIG. Polyphosphoric acid was not confirmed in the MA medium cultured product, but polyphosphoric acid was observed mainly near the surface layer in the MA medium + 0.5 M NaCl cultured product and the seawater cultured product. Furthermore, vacuoles where polyphosphoric acid is said to exist were similarly observed in all culture products.

[Example 11]
10D was pre-cultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of 10D. The results are shown in Table 16.
In addition, after preculture in medium (B), algal cells obtained by main culturing 10D in MA+0.5M NaCl medium (medium (c)) for 7 days were cultured in MA medium (medium (a)) or MA+0.5M NaCl medium (medium (c)). The cells were subcultured into a 5M NaCl medium (medium (c)) and cultured for an additional 7 days. After the culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of 10D. The results are shown in Table 17.

The OD ₇₅₀ of the medium at the start of main culture and at the time of subplanting was set to 0.1. The same procedure was applied to Examples 12 to 17 below. In Table 17, "Equation (1)-(A)" indicates the value calculated using Equation (1) for microalgae that was precultured in medium (A) and then main cultured at the NaCl concentration shown in the table. "Equation (1)-(B)" indicates the value calculated using Equation (1) for microalgae that was precultured in medium (B) and then main cultured at the NaCl concentration shown in the table. The same applies to Examples 12 to 17 below. If the OD ₇₅₀ was <0.1, the value could not be calculated using formula (1), so it was marked as "-".

As shown in Table 16, the growth of cells precultured in medium (B) was suppressed when main cultured in a medium containing 0 mM NaCl compared to those precultured in medium (A). On the other hand, at high NaCl concentrations of 500mM or higher, no growth was observed in those precultured in medium (A), whereas growth was observed in those precultured in medium (B) even at high salt concentrations of 500mM. It was done.

As shown in Table 17, growth was suppressed in the main culture in medium (c) and then subcultured in medium (a), but good growth was observed in those subcultured in medium (c). It was done.
Furthermore, when the algal cells obtained by main culture in medium (c) were collected after centrifuging the culture solution and placed in distilled water at pH 7, it was confirmed that the cells burst.

[Example 12]
HKN1 (haploid) was precultured for 1 week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of HKN1 (haploid). The results are shown in Table 18.

In addition, after preculture in medium (B), algal cells obtained by main culturing HKN1 (haploid) for 7 days in MA + 0.5 M NaCl medium (medium (c)) were cultured in MA medium (medium (a )) or MA+0.5M NaCl medium (medium (c)), and cultured for an additional 7 days. After the culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of HKN1 (haploid). The results are shown in Table 19.

As shown in Table 18, in those precultured in medium (B), no growth was observed when main cultured in a medium containing 0 mM NaCl. On the other hand, at high NaCl concentrations of 400mM or higher, no growth was observed in those precultured in medium (A), whereas growth was observed in those precultured in medium (B) even at high salt concentrations of 400mM or higher. confirmed.

As shown in Table 19, growth was suppressed in the main culture in medium (c) and then subcultured in medium (a), but good growth was observed in those subcultured in medium (c). It was done.

[Example 13]
HKN1 (diploid) was precultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of HKN1 (diploid). The results are shown in Table 20.

In addition, after preculture in medium (B), algal cells obtained by main culturing HKN1 (diploid) for 7 days in MA + 0.5 M NaCl medium (medium (c)) were cultured in MA medium (medium (a )) or MA+0.5M NaCl medium (medium (c)), and cultured for an additional 7 days. After the culture was completed, the OD ₇₅₀ of the final medium was measured to confirm the growth status of HKN1 (diploid). The results are shown in Table 21.

As shown in Table 20, the growth of those precultured in medium (B) tends to be slightly suppressed when main culture is performed in a medium with a low NaCl concentration compared to that precultured in medium (A). It was there. On the other hand, at high NaCl concentrations of 800mM or higher, no growth was observed in those precultured in medium (A), whereas growth was observed in those precultured in medium (B) even at high salt concentrations of 800mM or higher. confirmed.

As shown in Table 21, after main culture in medium (c), good growth was observed in both those subcultured in medium (a) and those subcultured in medium (c).

[Example 14]
Galdieria partita (NBRC 102759) (haploid) was precultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured. The growth status of partita (haploid) was confirmed. The results are shown in Table 22.

In addition, after preculture in medium (B), G.I. Partita (haploid) was cultured for 7 days, and the resulting algal cells were subcultured into MA medium (medium (a)) or MA+0.5M NaCl medium (medium (c)), and further cultured for 7 days. After the completion of the culture, the OD ₇₅₀ of the final medium was measured and G. The growth status of partita (haploid) was confirmed. The results are shown in Table 23.

As shown in Table 22, the growth of the cells precultured in medium (B) was suppressed compared to the cells precultured in medium (A) when the main culture was performed in a medium containing 0 mM NaCl. On the other hand, at high NaCl concentrations of 700 mM or higher, the rate of growth inhibition was lower in those precultured with medium (B) than in those precultured with medium (A).

As shown in Table 23, growth was suppressed in the main culture in medium (c) and then subcultured in medium (a), but good growth was observed in those subcultured in medium (c). It was done.

[Example 15]
G. partita (diploid) was precultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured. The growth status of partita (diploid) was confirmed. The results are shown in Table 24.

In addition, after pre-culture in medium (B), G.I. Partita (diploid) was cultured for 7 days, and the algal cells obtained were subcultured into MA medium (medium (a)) or MA+0.5M NaCl medium (medium (c)), and further cultured for 7 days. After the completion of the culture, the OD ₇₅₀ of the final medium was measured and G. The growth status of partita (diploid) was confirmed. The results are shown in Table 25.

As shown in Table 24, those precultured with medium (B) tended to have improved growth in the medium with a high NaCl concentration compared to those precultured with medium (A).

As shown in Table 25, after main culture in medium (c), good growth was observed in both those subcultured in medium (a) and those subplanted in medium (c).

[Example 16]
Galdieria sulfuraria (SAG108.79) (haploid) was precultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured. The growth status of sulfuraria (haploid) was confirmed. The results are shown in Table 26.

In addition, after preculture in medium (B), G.I. The algal cells obtained by main culturing Sulfuraria (haploid) for 7 days were subcultured into MA medium (medium (a)) or MA+0.5M NaCl medium (medium (c)), and cultured for an additional 7 days. After the completion of the culture, the OD ₇₅₀ of the final medium was measured and G. The growth status of sulfuraria (haploid) was confirmed. The results are shown in Table 27.

As shown in Table 26, the growth of cells pre-cultured with medium (B) was suppressed compared to those pre-cultured with medium (A) when main culture was performed with a medium containing 0 mM NaCl. On the other hand, at high NaCl concentrations of 700mM or higher, no growth was observed in those precultured in medium (A), whereas growth was observed in those precultured in medium (B) even at high salt concentrations of 700mM or higher. confirmed.

As shown in Table 27, growth was suppressed in the main culture in medium (c) and then subcultured in medium (a), but good growth was observed in those subcultured in medium (c). It was done.

[Example 17]
G. sulfuraria (diploid) was precultured for one week using MA medium (medium (A)) or MA+0.3M NaCl (medium (B)). After the pre-culture, main culture was carried out for 7 days in MA medium in which the NaCl concentration was varied between 0 and 1000 mM. After the main culture was completed, the OD ₇₅₀ of the final medium was measured. The growth status of sulphuraria (diploid) was confirmed. The results are shown in Table 28.

In addition, after preculture in medium (B), G.I. The algal cells obtained by main culturing Sulfuraria (diploid) for 7 days were subcultured into MA medium (medium (a)) or MA+0.5M NaCl medium (medium (c)), and cultured for an additional 7 days. After the completion of the culture, the OD ₇₅₀ of the final medium was measured and G. The growth status of sulphuraria (diploid) was confirmed. The results are shown in Table 29.

As shown in Table 28, those precultured with medium (B) tended to have improved growth in the medium with a high NaCl concentration compared to those precultured with medium (A).

As shown in Table 29, after main culture in medium (c), good growth was observed in both those subcultured in medium (a) and those subcultured in medium (c).

Combining the above results, haploid freshwater microalgae can be pre-cultured at a NaCl concentration of 0.3M, compared to pre-culture at 0M NaCl. It was confirmed that proliferation was improved when main culture was carried out at this concentration. As a result, the value calculated by the above formula (1) became 2 or more. Furthermore, it was confirmed that after culturing at a NaCl concentration of 0.3 M or higher, proliferation at a 0 M NaCl concentration was suppressed compared to when pre-cultured at a 0 M NaCl concentration. Furthermore, by performing preculture at a NaCl concentration of 0.3M, the growth rate in a medium with a 0.5M NaCl concentration tended to be higher than that in a medium with a 0M NaCl concentration. It was also confirmed that after main culture at 0.5M NaCl concentration, subculture to a medium with 0.5M NaCl concentration was possible.
Furthermore, it was confirmed that by performing preculture at a NaCl concentration of 0.3M, even haploid freshwater microalgae can grow in a high NaCl concentration range of 500 to 1000 mM. When outdoor cultivation is assumed, the salt concentration may fluctuate during cultivation due to water evaporation or rainwater inflow. From the above results, it was shown that the haploid microalgae belonging to the class Ideyucogonaceae precultured at a NaCl concentration of 0.3 M exhibited tolerance to changes in salt concentration and could sufficiently withstand outdoor culture.

[Example 18]
HKN1 (1x) was pre-cultured in MA+0.3M NaCl medium (medium (B)) for 7 days, and then pre-cultured in seawater medium for 1 week. Next, the HKN1 (1x) was subcultured in 10 L of seawater medium for main culture. The main culture was performed in a vinyl house, and light, temperature, and _CO2 concentration were not controlled. The main culture was performed by aeration culture, and the culture period was from May 13, 2019 to July 1, 2019. The culture solution was sampled periodically and the absorbance at 750 nm was measured. The results are shown in FIG. 10.
As shown in FIG. 10, it was confirmed that the bacteria could be grown well in seawater medium even at a 10 L scale.

[Example 19]
HKN1 (haploid) was cultured in MA medium, MA+0.3M NaCl medium, or MA+0.5 NaCl medium for 7 days. A culture solution containing algal cells in an amount giving an OD ₇₅₀ =1 when suspended in 2 mL of medium was collected into three microtubes. The culture solution was centrifuged (1500×g, 5 minutes), the supernatant was removed, and the pellet was collected. Each algal cell collected as a pellet was treated with any of the following treatments 1 to 3.
Treatment 1: Suspended in 2 mL of MA medium and shaken with a vortex for 10 minutes.
Treatment 2: The cells were suspended in 2 mL of the same medium as that used for the culture, and shaken with a vortex for 10 minutes.
Treatment 3: Suspend in 0.1 mL of the same medium used for the above culture, freeze at -196°C, make up to 2 mL with the same medium used for the above culture, and vortex for 10 minutes. Shake.

When algal cells break, the cell contents are released into the medium and phycocyanin (PC) is exposed to the acidic medium and fades. Assuming that 100% of the algal cells were killed by the freezing treatment (Treatment 3), the death rate of the algal cells by Treatment 1 was calculated using the following formula.

Mortality rate (%) = {(PC concentration after treatment 2 - PC concentration after treatment 1) / (PC concentration after treatment 2 - PC concentration after treatment 3)} x 100

The PC concentration was measured by measuring absorbance at 620 nm and 678 nm using a spectrophotometer (UV-2600; Shimadzu Corporation) equipped with an integrating sphere (ISR-2600Plus; Shimadzu Corporation). The PC concentration was calculated using the following formula.
PC concentration (μg/ml) = 138.5 x A ₆₂₀ -35.49 x A ₆₇₈

The results are shown in Table 30.

As shown in Table 30, it was confirmed that algal cells cultured in MA+0.3M NaCl medium or MA+0.5M NaCl medium were more easily broken by resuspension in MA medium.

[Example 20]
The algal cell death rate in Treatment 1 was calculated in the same manner as in Example 19, except that 10D was used instead of HKN1 (haploid). The results are shown in Table 31.

As shown in Table 31, it was confirmed that the algal cells cultured in the MA+0.3M NaCl medium or the MA+0.5M NaCl medium became more easily broken by resuspension in the MA medium. Furthermore, the mortality rate of algal cells cultured in MA+0.5M NaCl medium was higher than that of algal cells cultured in MA+0.3M NaCl medium.

[Example 21]
G. in place of HKN1 (haploid). The mortality rate of algal cells in Treatment 1 was calculated in the same manner as in Example 19, except that Partita (haploid) was used. The results are shown in Table 32.

As shown in Table 32, it was confirmed that algal cells cultured in MA+0.3M NaCl medium or MA+0.5M NaCl medium were more easily broken by resuspension in MA medium. Furthermore, the mortality rate of algal cells cultured in MA+0.5M NaCl medium was higher than that of algal cells cultured in MA+0.3M NaCl medium.

[Example 22]
G. in place of HKN1 (haploid). The mortality rate of algal cells due to Treatment 1 was calculated in the same manner as in Example 19, except that Sulfuraria (haploid) was used. The results are shown in Table 33.

As shown in Table 33, it was confirmed that the algal cells cultured in MA+0.3M NaCl medium or MA+0.5M NaCl medium became easily broken by resuspension in MA medium. Furthermore, the mortality rate of algal cells cultured in MA+0.5M NaCl medium was higher than that of algal cells cultured in MA+0.3M NaCl medium.

According to the present invention, there is provided a method for cultivating freshwater microalgae that allows freshwater microalgae to grow well in an environment of low pH and high sodium ion concentration, and a method for cultivating freshwater microalgae that allows the freshwater microalgae to grow well in an environment of low pH and high sodium ion concentration. Provided are freshwater microalgae and a method for producing the freshwater microalgae.
According to the present invention, freshwater acidic microalgae can be cultured in large quantities using seawater that can be obtained at low cost, so that the present invention is useful for producing useful substances using algae. Furthermore, if freshwater acid microalgae are cultivated in large quantities, they are expected to absorb carbon dioxide from the atmosphere.

Claims (9)

A method for culturing freshwater microalgae, the method comprising:
A pre-culture step of culturing freshwater microalgae at a culture temperature of 15 to 60°C in a medium prepared to have a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M. ,
The freshwater microalgae after the pre-culturing step have a sodium ion concentration of 1.2 to 5 times the sodium ion concentration in the pre-culturing step, 0.4 M or more, and a hydrogen ion concentration of pH 1.0 to 6. A main culture step of culturing in a medium prepared so that the
including;
The freshwater microalgae is a microalgae belonging to the class Idyukogome.
A method for culturing freshwater microalgae. The medium in the main culture step is a medium prepared by adding at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt to seawater and having a hydrogen ion concentration of pH 1.0 to 6.0. Item 1. The method for culturing freshwater microalgae according to item 1. A medium prepared with a hydrogen ion concentration of pH 1.0 to 6.0 and a sodium ion concentration of 0.1 to 0.4M for microalgae belonging to the class Ideyukogo, which cannot grow in a medium with a sodium ion concentration of 0.5M or higher. A step of culturing with
A step of culturing the microalgae belonging to the Idyukogome class after the cultivation in a medium prepared such that the sodium ion concentration is 0.5 to 1M and the hydrogen ion concentration is pH 1.0 to 6.0;
A method for producing microalgae belonging to the class Idyukogoma that can grow in a medium containing hydrogen ion concentration pH 1.0 to 6.0 and sodium ion concentration 0.5 to 1M . 4. The method for producing microalgae belonging to the genus Cyanidium according to claim 3, wherein the microalgae belonging to the genus Cyanidinium are haploid. A haploid microalgae belonging to the class Idyukogome,
In MA medium prepared to have a hydrogen ion concentration of pH 2.0 and a sodium ion concentration of 0.5 M, the cells were statically cultured for 7 days at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and continuous light with an illuminance of 60 μmol/m ² s. When the value calculated by the following formula (1) is 2 or more,
^The following formula (1 ) is less than 2,
A haploid microalgae belonging to the class Idyukogome.
(OD ₇₅₀ value 7 days after the start of culture - OD ₇₅₀ value at the start of culture) / (7 x OD ₇₅₀ value at the start of culture) (1) 6. The haploid microalgae belonging to the class Ideyucogos according to claim 5, whose cells rupture in an isotonic solution with a hydrogen ion concentration of pH 7 or in distilled water. When the algal cells are dried and the cells after the drying treatment are suspended in an isotonic solution having a pH of 7, the cells burst.
7. The haploid microalgae belonging to the class Idyukogome according to claim 5 or 6. The haploid microalgae belonging to the Idyukogome class according to any one of claims 5 to 7 is added to seawater at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt, and the hydrogen ion concentration is adjusted to pH 1. Including culturing in a medium prepared to be 0 to 6.0,
A method for culturing haploid microalgae belonging to the class Idyukogome. A method for culturing microalgae belonging to the class Idyukogoma, which comprises culturing the haploid microalgae belonging to the class Idyukogoma according to any one of claims 5 to 7 outdoors.

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