AU2019355498B2

AU2019355498B2 - Method for culturing fresh water microalga

Info

Publication number: AU2019355498B2
Application number: AU2019355498A
Authority: AU
Inventors: Shunsuke Hirooka; Shin-Ya Miyagishima
Original assignee: Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency
Priority date: 2018-10-02
Filing date: 2019-10-02
Publication date: 2023-02-02
Anticipated expiration: 2039-10-02
Also published as: AU2019355498A1; JPWO2020071444A1; WO2020071444A1; JP7455386B2; CN113015791A

Abstract

A method for culturing a fresh water microalga, said method comprising a culture step for culturing the fresh water microalga in a medium, in which hydrogen ion concentration is adjusted to pH 1.0-6.0 and sodium ion concentration is adjusted to 0.1-0.4 M, at a culture temperature of 15-60

Description

[DESCRIPTION] [TITLE OF INVENTION] METHOD FOR CULTURING FRESH WATER MICROALGA

[Technical Field]

[0001]

The present invention relates to a method for culturing a freshwatermicroalga.

In addition, the present invention also relates to a method for producing a freshwater

microalga to which salt tolerance is imparted and a salt-tolerant freshwater microalga

obtained by the production method. More specifically, the present invention relates to a

freshwater microalga suitable for outdoor mass culture, particularly outdoor mass culture

in seawater, and a method for producing the same.

Priority is claimed on Japanese Patent Application No. 2018-187763, filed

October 2, 2018, the content of which is incorporated herein by reference.

[Background Art]

[0002]

Microalgae have a carbon dioxide fixation capacity higher than that of terrestrial

plants and do not compete with agricultural products for growing places. Therefore,

some species of microalgae are cultured in large quantities and used industrially as feed,

functional foods, cosmetic materials, and the like.

The industrial use of microalgae is limited to a use form of expensive functional

foods and the like from the viewpoint of cost. In order to suppress the production cost

of microalga and promote industrial use, outdoor mass culture is preferable. Although

outdoor mass culture has characteristics such as easy management, there are problems in

that there is a risk of contamination, theniicroalga is directly affected by the external

environment, and there is also the possibility of invasion of algae predators and the like.

In order to evade such risks, the microalgae that can be cultured outdoors in large

quantities need to meet requirements such as being tolerant to environmental changes

(light, temperature, and the like), being cultivable under conditions where other

organisms cannot survive, and being able to be grown to high density.

Accordingly, hitherto, only a few species such as Chlorella, Euglena, Dunaliella,

and Spirulina have been practically used in industry. These algae species have been

successfully cultured outdoors in large quantities and are used as raw materials for

functional foods or supplements.

[0003]

An environment in which other organisms cannot survive includes an

environment with a high salt concentration such as seawater. Regarding culturing a

useful microalga in a medium of high salt concentration, for example, there are reports of

Patent Documents I to 3.

Patent Document I discloses a method for producing a fat and oil component,

characterized in that salt-tolerant algae are cultured in a medium in which the salt

concentration is gradually increased. In the method disclosed in Patent Document 1, in

a case where the nitrate content in the medium is measured at a wavelength of 220 nm,

the salt concentration in the second step is increased when the nitrate content is 10mg/L

or less.

Patent Document 2 discloses that in the culture of algae Pseudochoricystis

ellipsoidea, which inhabit freshwater and have a hydrocarbon-producing capacity, salt is

added to the medium during a time from the start of the culturing to a time when an

optical density of the medium reaches half of the optical density indicating a saturated

state, in order to enhance productivity of the hydrocarbon-producing capacity.

Patent Document 3 discloses a method of culturing the genus Crypthecodinium

that produces docosahexaenoic acid by setting a salt concentration of a culture solution to

a value 0.1% to 10% by weight higher than the salt concentration suitable for growth of

the algae in order to accumulate docosahexaenoic acid in the algae.

The methods disclosed in Patent Documents 1 to 3 are intended to enhance

hydrocarbon-producing capacity or docosahexaenoic acid-producing capacity by

applying salt stress to microalga, and do not aim to suppress a risk of contamination in

outdoor culture.

[0004]

Meanwhile, the microalga belonging to Cyanidiophyceae, which is a unicellular

primitive red alga, preferentially grow in acidic hot springs containing sulfuric acid.

Cyanidiophyceae includes the genus Cyanidioschyzon, the genus Cyanidium, and the

genus Galdieria. Cyanidioschyzon merolae that belongs to the genus Cyanidioschyzon

has no rigid cell wall. Cyanidioschyzon merolae is constituted with a very simple set of

cell organelles, and the genome sequence thereof has been completely decoded.

Therefore, Cyanidioschyzon merolae is used as a model organism for basic research on

photosynthetic organisms, and the development of techniques for modifying the gene

thereof is also making progress (Non-Patent Documents 1 and 2).

[Citation List]

[Patent Documents]

[0005]

[Patent Document 1]

PCT International Publication No. WO 2015/25552

[Patent Document 2]

Japanese Unexamined Patent Application, First Publication No. 2013-102748

[Patent Document 3]

Japanese Unexamined Patent Application, First Publication No. H07-075557

[Non-Patent Documents]

[0006]

[Non-Patent Document 1]

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.

[Non-Patent Document 2]

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.

[Summary of Invention]

[Technical Problem]

[0007]

A freshwater acidophilic mieroalga belonging to Cyanidiophyceae can grow in

an acidic environment where other organisms cannot grow, and are suitable for outdoor

culture. In a case where the microalga can be imparted with high salt concentration

tolerance, the microalga can be cultured in an acidic and high salt concentration

environment, and the risk of contamination during outdoor culture is further reduced. In

addition, in a case where seawater can be used for culture, the culture cost can be

suppressed.

[0008]

Therefore, an object of the present invention is to provide a method for culturing

a freshwater microalga that can favorably grow a freshwater microalga in a low pH and high sodium ion concentration environment, a freshwater microalga that can be favorably grown in a low pH and high sodium ion concentration environment, and a method for producing the freshwater microalga.

[Solution to Problem]

[0009]

The present invention includes the following aspects.

[1] A method for culturing a freshwater microalga, including a culture step of

culturing a freshwater microalga at a culture temperature of 15°C to 60°C in a medium

prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and the sodium ion

concentration is 0.1 to 0.4 M.

[2] The method for culturing a freshwater microalga according to [1], including

a pre-culture step of culturing a freshwater microalga in a medium prepared such that the

hydrogen ion concentration is pH 1.0 to 6.0 and the sodium ion concentration is 0.1 to

0.4 M, and a main culture step of culturing a freshwater microalga after the pre-culture

step 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.

[3] The method for culturing a freshwater nicroalga according to [1] or [2], in

which the medium in the main culture step is a medium prepared such that the hydrogen

ion concentration is pH 1.0 to 6.0 and the sodium ion concentration is 0.5 M or more.

[4] The method for culturing a freshwater microalga according to [1] or [2], in

ion concentration is pH 1.0 to 6.0 and the sodium ion concentration is 0.4 M or more.

[5] The method for culturing a freshwater microalga according to any one of [1]

to [4], in which 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 such that the hydrogen ion concentration is pH 1.0 to 6.0.

[6] The method for culturing a freshwater microalga according to any one of [1]

to [5], in which the freshwater microalga is a microalga belonging to Cyanidiophyceae.

[7] A method for producing a freshwater microalga that can grow in a medium

concentration is 0.5 M or more, the method including a step of culturing a freshwater

microalga that cannot grow in a medium having a sodium ion concentration of 0.5 M or

more in a medium prepared such that the hydrogen ion concentration is pH 1.0 to 6.0 and

the sodium ion concentration is 0.1 to 0.4 M.

[8] The method for producing a freshwater microalga according to [7], in which

the freshwater microalga is a haploid of microalga belonging to the genus Cyanidium.

[9] The method for producing a freshwater microalga according to [7], in which

the freshwater microalga is a haploid of microalga belonging to the genus Galdieria.

[10] A haploid of microalga belonging to Cyanidiophyceae, in which a value

calculated by the following Formula (1) in a case of culturing in a static state for 7 days

at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and a continuous

light of an illuminance of 60 mol/m 2s in an M-Allen medium prepared such that a

hydrogen ion concentration is pH 2.0 and a sodium ion concentration is 0.5 M is 2 or

more, and a value calculated by the following Formula (1) in a case of culturing in a

static state for 7 days at a culture temperature of 42°C, a carbon dioxide concentration of

2%, and a continuous light of an illuminance of 60 pmol/m 2 s in an M-Allen medium

prepared such that a hydrogen ion concentration is pH 2.0 is less than 2.

(OD 7 5 ovalue after 7 days from time of culture start - OD 7 5 0 value at time of

culture start)/(7 x OD 7 5 ovalue at time of culture start) (1)

[11] The haploid of microalga belonging to Cyanidiophyceae according to [10],

in which cells are ruptured in an isotonic solution having a hydrogen ion concentration of

pH 7 or in a distilled water.

[12] The haploid of microalga belonging to Cyanidiophyceae according to [10]

or [11],

in which in a case where algal cells are dried and the cells after the drying

treatment are suspended in an isotonic solution having a pH 7, the cells are ruptured.

[13] A method for culturing a haploid of microalga belonging to

Cyanidiophyceae, including culturing the haploid of microalga belonging to

Cyanidiophyceae according to any one of [10] to [12] in a medium prepared by adding at

least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt

to seawater such that a hydrogen ion concentration is pH 1.0 to 6.0.

[14] A method for culturing a haploid of microalga belonging to

Cyanidiophyceae, including culturing the microalga belonging to Cyanidiophyceae

according to any one of [10] to [12] outdoors.

[15] A method for culturing a freshwater microalga, including culturing a

haploid of microalga belonging to Cyanidiophyceae as a freshwater microalga, in which

a value calculated by the following Formula (1) in a case of culturing in a static state for

7 days at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and a

continuous light of an illuminance of 60 mol/m 2 s in an M-Allen medium prepared such

that a hydrogen ion concentration is pH 2.0 and a sodium ion concentration is 0.5 M is 2

or more, and a value calculated by the following Formula (1) in a case of culturing in a

2%, and a continuous light of an illuminance of 60 mol/m2 s in an M-Allen medium

prepared such that a hydrogen ion concentration is pH 2.0 is less than 2, in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.4 M or more.

(OD 7 5 ovalue after 7 days from time of culture start - OD 7 5 ovalue at time of

culture start)/(7 x OD7 5 0 value at time of culture start) (1)

[16] A method for producing a freshwater microalga belonging to

Cyanidiophyceae as a freshwater microalga, including culturing a haploid ofmicroalga

belonging to Cyanidiophyceae, in which a value calculated by the following Formula (1)

in a case of culturing in a static state for 7 days at a culture temperature of 42°C, a carbon

dioxide concentration of 2%, and a continuous light of an illuminance of 60 mol/m 2s in

an M-Allen medium prepared such that a hydrogen ion concentration is pH 2.0 and a

sodium ion concentration is 0.5 M is 2 or more, and a value calculated by the following

Formula (1) in a case of culturing in a static state for 7 days at a culture temperature of

42°C, a carbon dioxide concentration of 2%, and a continuous light of an illuminance of

60 pmol/m 2 s in an M-Allen medium prepared such that a hydrogen ion concentration is

pH 2.0 is less than 2, in a medium prepared such that a hydrogen ion concentration is pH

1.0 to 6.0 and a sodium ion concentration is 0.4 M or more.

(OD 7 5 0 value after 7 days from time of culture start - OD 7 5 0 value at time of

culture start)/(7 x OD 750 value at time of culture start) (1)

[Advantageous Effects ofInvention]

[0010]

According to the present invention, a method for culturing a freshwater

microalga that can favorably grow a freshwater microalga in a low pH and high sodium

ion concentration environment, a freshwater microalga that can favorably grow in a low

pH and high sodium ion concentration environment, and a method for producing the

freshwater microalga are provided.

8a

[0010a]

A method for culturing a haploid of a freshwater microalga belonging to

Cyanidiophyceae, comprising:

a pre-culture step of culturing a haploid of a freshwater microalga belonging to

Cyanidiophyceae at a culture temperature of 15°C to 60°C in a medium prepared such

that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.25

to 0.35 M, and

a main culture step of culturing the haploid of the freshwater microalga

belonging to Cyanidiophyceae after the pre-culture step in a medium prepared such that a

sodium ion concentration is 0.5 to 1 M and a hydrogen ion concentration is pH 1.0 to 6.0.

[001Ob]

A method for producing a haploid of a freshwater microalga belonging to

Cyanidiophyceae that grows in a medium prepared such that a hydrogen ion

concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.5 M or more, the

method comprising in the following order:

a step of culturing a haploid of a freshwater microalga belonging to

Cyanidiophyceae that does not grow in a medium having a sodium ion concentration of

0.5 M or more in a medium prepared such that a hydrogen ion concentration is pH 1.0 to

6.0 and a sodium ion concentration is 0.25 to 0.35 M, and

a step of culturing the haploid of the freshwater microalga belonging to

Cyanidiophyceae in a medium prepared such that a hydrogen ion concentration is pH 1.0

to 6.0 and a sodium ion concentration is 0.5 M or more.

[001Oc]

Throughout this specification the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated

8b element, integer or step, or group of elements, integers or steps, but not the exclusion of

any other element, integer or step, or group of elements, integers or steps.

[001Od]

Any discussion of documents, acts, materials, devices, articles or the like which

has been included in the present specification is not to be taken as an admission that any

or all of these matters form part of the prior art base or were common general knowledge

in the field relevant to the present disclosure as it existed before the priority date of each

of the appended claims.

[The rest of this page is left intentionally blank]

[Brief Description of Drawings]

[0011]

Fig. 1 is a graph showing a growth curve when performing pre-culture on

Cyanidium sp. HKNI (haploid) in an M-Allen medium (MA medium), and then

performing main culture thereon in an MA medium, a medium obtained by adding 0.3 M

of NaCL to an MA medium (MA + 0.3 M of NaCl medium), or a medium obtained by

adding 0.5 M of NaCl to an MA medium (MA + 0.5 M of NaCl medium), and a growth

curve (time-dependent change of OD 7 5 o) when performing pre-culture in an MA + 0.3 M

of NaCl medium, and then performing main culture in an MA + 0.5 M of NaCl medium.

Fig. 2 is a graph showing a growth curve when performing pre-culture on

Cyanidium sp. HKN1 (haploid) in an MA + 0.3 M of NaCl medium, and then performing

main culture thereon in a seawater medium or an MA + 0.5 M of NaC medium.

Fig. 3 is a graph showing OD7 5 o after performing pre-culture on Cyanidium sp.

HKNI (haploid) in an MA + 0.3 M of NaCl medium, and then performing main culture

thereon in a seawater medium having a pH 2 to 7 for 7 days.

Fig. 4 is a graph showing a growth curve when performing pre-culture on

Cyanidium sp. HKNI (diploid) in an MA medium, and then performing main culture

thereon in an MA medium, an MA + 0.3 M of NaCl medium, or an MA + 0.5 M of NaCI

medium, and a growth curve when performing pre-culture in an MA + 0.3 M of NaCl

medium, and then performing main culture in an MA + 0.5 M of NaCl medium.

Fig. 5 is a graph showing a growth curve when performing pre-culture on

Cyanidium sp. HKN I(diploid) in an MA + 0.3 M of NaCl medium, and then performing

main culture in a seawater medium or an MA + 0.5 M of NaCl medium.

Fig. 6 is a graph showing a growth curve when performing pre-culture on

Cyanidioschyzon merolae IOD in an MA medium, and then performing main culture thereon in an MA medium, an MA + 0.3 M of NaCl medium, or an MA + 0.5 M of NaCl medium, and a growth curve when performing pre-culture in an MA + 0.3 M of NaCl medium, and then performing main culture in an MA + 0.5 M of NaCl medium.

Fig. 7 is a graph showing a growth curve when performing pre-culture on

Cyanidioschyzon merolae 1OD in an MA + 0.3 M of NaCl medium and then performing

main culture thereon in seawater medium or an MA + 0.5 M of NaCl medium.

Fig. 8 is a fluorescence micrograph of observing polyphosphoric acid and

vacuoles of a culture product obtained by culturing Cyanidium sp. HKN1 (haploid) in an

MA medium, an MA medium + 0.5 M of NaCl medium, and a seawater medium.

Fig. 9 is a fluorescence micrograph of observing polyphosphoric acid and

vacuoles of a culture product obtained by culturing Cyanidium sp. HKN1 (diploid) in an

MA medium, an MA medium + 0.5 M of NaCl medium, and a seawater medium.

Fig. 10 is a graph showing a growth curve when culturing Cyanidium sp. HKNI

(haploid) in 10 L of a seawater medium.

Fig. 11 shows a molecular phylogenetic tree ofmicroalga belonging to

Cyanidiophyceae based on a chloroplast ribulose 1,5-bisphosphate

carboxylase/oxygenase large subunit (rbcL) gene. A local bootstrap value (only 50 or

more is described, left) by the maximum likelihood method and a posterior probability

(only 0.95 or more is described, right) by the Bayes method are shown near each branch.

[Description of Embodiments]

[0012]

In the present specification, "MA medium" means an M-Allen medium.

Specifically, it means a medium having the composition shown in Table I and adjusted

using sulfuric acid such that the hydrogen ion concentration is pH 1.0 to 6.0. In a case

of simply being referred to as "MA medium" or "M-Allen medium", it means a medium to which NaCl is not added. "M-Allen medium prepared such that a hydrogen ion concentration is pH 2.0" means an MA medium which is adjusted such that a hydrogen ion concentration is pH 2.0 and to which NaCl is not added.

[0013]

In the present specification, "MA medium + 0.3 M of NaCI medium" means an

MA medium adjusted such that the NaCl concentration is 0.3 M. Specifically, it means

a medium having the composition shown in Table 4 and adjusted using sulfuric acid such

that the hydrogen ion concentration is pH 1.0 to 6.0. "M-Allen medium prepared such

that the hydrogen ion concentration pH is 2.0 and the sodium ion concentration is 0.3 M"

means an MA + 0.3 M of NaCl medium adjusted such that the hydrogen ion

concentration is pH 2.0.

[0014]

In the present specification, "MA + 0.5 M of NaCl medium" means an MA

medium adjusted such that the NaCl concentration is 0.5 M. Specifically, it means a

medium having the composition shown in Table 5 and adjusted using sulfuric acid such

that a hydrogen ion concentration is pH 2.0 and a sodium ion concentration is 0.5 M"

means a MA + 0.5 M of NaCl medium adjusted such that the hydrogen ion concentration

is pH 2.0.

[0015]

In the MA medium, the MA + 0.3 M of NaCl medium, and the MA + 0.5 M of

NaCI medium, the hydrogen ion concentration (pH) can be an optional value in a range

of pH 1.0 to 6.0 unless otherwise specified.

[0016]

In the present specification, "M" used with respect to the concentration of

components in the medium represents "mol/L".

[0017]

In an embodiment, the present invention provides a method for culturing

freshwater microalgae. The culture method of the present embodiment includes a

culture step of culturing a freshwater microalga at a culture temperature of 15°C to 60°C

in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a

sodium ion concentration is 0.1 to 0.4 M. The culture method preferably includes a pre

culture step of culturing freshwater microalgae in a medium prepared such that a

hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.1 to 0.4

M, and a main culture step of culturing a freshwater microalga after the pre-culture step

in a medium prepared such that a sodium ion concentration is 1.2 to 5 times the sodium

ion concentration in the pre-culture step and a hydrogen ion concentration is pH 1.0 to

6.0.

[0018]

In an embodiment, the culture method of the present embodiment is

characterized in that a pre-culture is performed in an acidic medium having a sodium ion

concentration of 0.1 to 0.4 M before performing a main culture in an acidic medium

having a high salt concentration. By performing the pre-culture, even a freshwater

microalga having no salt tolerance can favorably grow in a medium having the same high

salt concentration to that of seawater.

[0019]

In the present specification, the sodium ion concentration and the hydrogen ion

concentration (pH) of the medium in the pre-culture step mean both of the sodium ion concentration and the pH at a time of culture start in the pre-culture step. As long as the medium at the time of culture start in the pre-culture step has a "sodium ion concentration 0.1 to 0.4 M" and has a "pH 1.0 to 6.0", the medium is included in the pre culture step of the culture method of the present embodiment even in a case where the sodium ion concentration or the pH fluctuates out of the range during the pre-culture period.

The sodium ion concentration and the pH of the medium in the main culture step

mean both of the sodium ion concentration and the pH at the time of culture start in the

main culture step. As long as in the medium at the time of culture start in the main

culture step, "the sodium ion concentration" is "pH 1.0 to 6.0" which is "1.2 to 5 times

the sodium ion concentration in the pre-culture step", the medium is included in the main

culture step of the culture method of the present embodiment even in a case where the

sodium ion concentration or the pH fluctuates out of the range during the main culture

period.

[0020]

(Freshwatermicroalga)

The culture method of the present embodiment is applicable to a freshwater

nicroalga that can be grown under an acidic condition of pH 1.0 to 6.0. "Freshwater

microalga" means microalga that inhabits in a freshwater area. The sodium ion

concentration of freshwater is typically less than 0.05% by mass. The freshwater area is

not particularly limited, and examples thereof include rivers, lakes, hot springs,

groundwater, and the like, and a freshwater area having an acidic condition of pH 1.0 to

6.0 is preferable. Acidic hot springs (such as sulfuric acid acidic hot springs) are

preferable examples of freshwater areas under such an acidic condition. In the present

specification, "microalga" means unicellular algae.

[0021]

Examples of a freshwater microalga that can be grown under an acidic condition

of pH 1.0 to 6.0 include a microalga belonging to Cyanidiophyceae. Taxonomically,

Cyanidiophyceae is classified into Rhodophyta and Cyanidiophyceae. Currently, three

genera of Cyanidioschyzon, Cyanidium, and Galdieria are classified into

Cyanidiophyceae. As a microalga belonging to Cyanidiophyceae, many types of

microalgae are known so far and are not listed here. However, Fig. 11 shows the

phylogenetic tree using a base sequence of the chloroplast rbcL gene of the microalgae

belonging to Cyanidiophyceae.

[0022]

The culture method of the present embodiment is preferably applied to a

freshwater microalga having no salt tolerance among freshwater microalgae. Here,

"having no salt tolerance" means that in a case of culturing in seawater or a medium

having the same sodium ion concentration (about 0.5 M) to that of seawater, growth is

suppressed (including the case that growth is not possible) compared to a case of

culturing in a medium for freshwater microalgae (sodium ion concentration of 0.05 M or

less). The suppression of growth is preferably such that the growth rate is reduced by

60% or more, and more preferably reduced by 80% or more, as compared with the

growth rate in the medium for freshwater microalgae. Examples of the freshwater

microalga that can be grown under an acidic condition of pH 1.0 to 6.0 and have no salt

tolerance include a microalga belonging to Cyanidiophyceae shown in Fig. 11.

Examples thereof include a haploid of microalga belonging to the genus Cyanidium,

Cyanidioschyzon merolae, and a haploid of microalga belonging to the genus Galdieria,

and the like.

[0023]

Some microalgae belonging to Cyanidiophyceae can have a haploid cell form or

a diploid cell form. The present inventors provide a method of obtaining a cell

population having a haploid cell form from a cell population having a diploid cell form,

or conversely, a method of obtaining a cell population having a diploid cell form from a

cell population having a haploid cell form (PCT International Publication No. WO

2019/107385).

[0024]/

As shown in examples which will be described later, a diploid of microalga

belonging to the genus Cyanidium have salt tolerance, but haploids have no salt

tolerance. Specific examples of the microalga belonging to the genus Cyanidium

having both a haploid cell form and a diploid cell form include a Cyanidium sp. YFU3

strain (FERM P-22334) (hereinafter, referred to as "YFU3 strain"), a Cyanidium sp.

HKN1 strain (FERM P-22333) (hereinafter, referred to as "HKNI strain"), and allied

species, mutants, and progenies of these.

Hereinafter, in a case where the YFU3 strain is described by distinguishing

between a diploid cell form and a haploid cell form, the diploid cell form will be

described as "YFU3 strain (diploid)", and the haploid cell form will be described as

"YFU3 strain (haploid)". Likewise, in a case where the HKN1 strain is described by

distinguishing between the diploid cell form and the haploid cell form, the diploid cell

form will be described as "HKN1 strain (diploid)", and the haploid cell form will be

described as "HKN1 strain (haploid)". In a case where the strains are simply referred to

as "YFU3 strain" or "HKNI strain", each of the strains includes both the diploid cell

form and the haploid cell form.

[00251

Whether algae are diploid or haploid can be determined by checking the number

of copies of the same loci. That is, in a case where the number of copies of the same

loci is 1, the algae are determined to be haploid. In addition, whether a certain alga is

haploid or not can also be determined using a next-generation sequencer and the like.

For example, whole genome sequence reads are acquired by a next-generation sequencer

and the like, the sequence reads are assembled, and then the sequence reads are mapped

for the sequence obtained by the assembling. In diploids, the difference in base

between alleles are found in various regions on the genome. However, in haploids,

since only one allele is present, no such regions are found.

Alternatively, cells are stained with a nuclear staining reagent such as DAPI, and

cells showing the same fluorescence brightness as those cells known to be haploid may

be determined to be haploid, and cells showing about two times of the fluorescence

brightness may be determined to be diploid. Alternatively, the cells are stained with a

nuclear staining reagent such as DAPI, and the cells showing the same fluorescence

brightness as those cells known to be diploid may be determined to be diploid, and cells

showing about 1/2 times of the fluorescence brightness may be determined to be haploid.

[0026]

The YFU3 strain (haploid) is a unicellular red alga isolated from hot acidic

water of a hot spring in Yufu-shi, Oita prefecture, Japan. The YFU3 strain was

deposited in the Patent Microorganism Depositary Center of National Institute of

Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba prefecture,

Japan) on May 30, 2017 under the accession No. FERM P-22334, and transferred to the

International Depositary on April 20, 2018 under the accession No. FERM BP-22334.

The HKNl strain is a unicellular red alga isolated from hot acidic water of a hot

spring in Hakone-machi, Ashigarashimo-gun, Kanagawa prefecture, Japan. TheHKN1 strain (haploid) was deposited in the Patent Microorganism Depositary Center of

National Institute of Technology and Evaluation on May 30, 2017 under the accession

No. FERM P-22333, and transferred to the International Depositary on April 20, 2018

under the accession No. FERM BP-22333.

[0027]

In addition, a freshwater microalga to which the culture method of the present

embodiment is applied may be isolated from a freshwater area such as an acidic hot

spring, or may be obtained from a culture collection or the like. For example,

Cyanidioschyzon merolae can be obtained from the National Institute for Environmental

Studies MICROBIAL CULTURE COLLECTION (16-2 Onogawa, Tsukuba-shi, Ibaraki

prefecture, Japan), American Type Culture Collection (ATCC; 10801 University

Boulevard Manassas, VA 20110 USA), and the like.

[0028]

In addition, the freshwater microalga to which the culture method of the present

embodiment is applied is not limited to those isolated from the natural world, and a

natural freshwater microalga may be mutated. The mutation may be spontaneous

mutation or induced mutation. As the genome size of Cyanidioschyzon merolae is

small (about 16 Mbp) and the genome sequence thereof has been completely decoded

(Matsuzaki M et al., Nature. 2004 Apr 8; 428 (6983): 653-7.), Cyanidioschyzon merolae

can be easily genetically modified. Therefore, for example, the culture method of the

present embodiment may be applied to a transformant of Cyanidioschyzon merolae

prepared by genetic modification (for example, a transformant having a fortified

nutrient). In addition, the culture method of the present embodiment may be applied to

transformants of other freshwater microalgae as long as the gene can be modified.

[0029]

In addition, some microalgae belonging to the genus Galdieria can have both a

haploid cell form and a diploid cell form. For example, it is possible to obtain a haploid

cell form by culturing a diploid ofmicroalga belonging to the genus Galdieria for a

certain period of time (for example, about 1 to 3 weeks). As a medium for culturing

diploid cells in order to obtain haploid cells, for example, an acidic hot spring drainage

medium, a Tsukahara mineral spring medium (Hirooka et al. 2016 Front in

Microbiology), and the like can be preferably used. The culture method of the present

embodiment can also be applied to a haploid of microalga belonging to the genus

Galdieria or transformants thereof. Examples of microalgae belonging to the genus

Galdieria include G. partita (NBRC 102759), G. sulphuraria (SAG108.79 and the like),

and the like. Microalgae belonging to the genus Galdieria may be isolated from

freshwater areas such as acidic hot springs, or may be obtained from a culture collection

and the like. As for the culture collection, in addition to the culture collection

mentioned in Cyanidioschyzon merolae, 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 can be mentioned.

[0030]

Among the microalgae belonging to Cyanidiophyceae, haploid algal cells often

do not have a rigid cell wall. The haploid cell form of algal cells that have no such rigid

cell wall can be disrupted by a relatively mild treatment such as a neutralization

treatment, a hypotonic treatment, or a freeze-thaw treatment. In the present

specification, "having no rigid cell wall" means that cell rupture occurs by any of the

following cell rupturing treatments (A) to (C).

[00311

(A) Algal cells are suspended in an isotonic solution at pH 7 and left for one week or longer.

(B) Algal cells are suspended in distilled water and left for 1 minute or longer.

(C) Algal cells are subjected to a drying treatment and suspended in an isotonic

solution at pH 7.

In the (A) to (C), in a case where the algal cells are cultured cells, the medium

may be removed by centrifugation or the like before each treatment is performed, and the

algal cells may be washed with an isotonic solution or the like.

In the (A) and (C), examples of the isotonic solution include a pH 7 buffer

containing 10% sucrose and 20 mM of HEPES.

In the (C), examples of the drying treatment include drying in a refrigerator

(4°C), freeze-drying, and the like. For the drying treatment, the precipitate of algal cells

collected by centrifugation is used. In a case where the cells are dried in a refrigerator,

the drying treatment time depends on the amount of algal cells, and is, for example, 3

days or longer.

[0032]

In addition, whether or not the cell rupture has occurred can be determined by

performing centrifugation (1,500x g, 3 minutes) on the algal cell suspension after the cell

rupturing treatments (A) to (C) described above, and calculating the ratio of the mass of

proteins in the centrifugal supernatant to the total mass of proteins in the algal cell

suspension. Specifically, in a case where a rupture rate obtained by the following

equation is equal to or higher than 20%, it can be determined that a cell rupture has

occurred.

[0033]

[Equation 1]

Rupture rate= Protein mass of centrifuged supernatant (mass g) x 100 Total protein mass in algae suspension (mass g)

[0034]

Alternatively, in a case where the algal cells in the algal cell suspension are

observed using an optical microscope (for example, at a magnification of 600X), and the

ratio of ruptured cells to the total number of the algal cells is found to be about equal to

or higher than 10% and preferably about equal to or higher than 20%, it may be

determined that cell rupture has occurred.

[0035]

In the cell rupturing treatments (A) to (C) described above, an isotonic solution

at pH 7 can be used.

Accordingly, it can be said that the cells ruptured by any of the cell rupturing

treatments (A) to (C) are cells that are ruptured under the condition of pH 7.

A microalgae whose cells are ruptured under the condition of pH 7 is difficult to

grow in an external environment in a case where the microalga flows out of a culture

tank, and contamination to the environment can be suppressed.

In a case where the algal cell has no a rigid cell wall, by the observation using an

optical microscope (for example, at a magnification of 600X), generally, a cell wall is not

observed. Therefore, whether or not cell rupture occurs due to the mild hypotonic

treatment under the condition of pH equal to or lower than 6 does not exert an influence

on determining whether or not the microalga have a rigid cell wall.

[0036]

[Pre-culture step]

The pre-culture step is a step of culturing a freshwater microalga in a medium

prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion

concentration is 0.1 to 0.4 M.

[0037]

The medium used in the pre-culture step is not particularly limited as long as the

sodium ion concentration is 0.1 to 0.4 M and the pH is 1.0 to 6.0. For example, a

medium prepared by adding 0.1 to 0.4 M of sodium ion to a general medium for

freshwater microalgae to adjust the pH to 1.0 to 6.0 can be preferably used.

The medium for freshwater microalgae is not particularly limited, and may be

appropriately selected according to the type of freshwater microalgae to be cultured.

Examples of the medium for freshwater microalgae include an inorganic salt medium

containing a nitrogen source, a phosphorus source, an iron source, trace elements (such

as zinc, boron, cobalt, copper, manganese, and molybdenum), and the like. For

example, examples of the nitrogen source include ammonium salt, nitrate, nitrite, urea,

amine, and the like, examples of the phosphorus source include phosphate and phosphite,

and examples of the iron source include iron chloride, iron sulfate, iron citrate, and the

like. Specific examples of the medium for freshwater microalgae include a 2 x Allen

medium (Allen MB. Arch. Microbiol. 1959 32: 270-277.), an M-Allen medium (Minoda

A et al. Plant Cell Physiol. 2004 45: 667-71) ), an MA2 medium (Ohnuma M et al. Plant

Cell Physiol. 2008 Jan; 49 (1): 117-20.), and the like. In addition, in the present

specification, the M-Allen medium is described as "MA medium" in some cases.

[0038]

The sodium ion concentration may be appropriately selected within a range of

0.1 to 0.4 M according to the sodium ion concentration in the main culture step to be

described later. More specifically, a sodium ion concentration which is 0.2 to 0.8 times the sodium ion concentration planned 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 further more preferably 0.5 to 0.6 times the sodium ion concentration in the main culture step.

For example, in a case where the sodium ion concentration in the main culture

step is about the same as that of seawater (about 0.5 M), the sodium ion concentration in

the pre-culture step is preferably 0.25 M or more, more preferably 0.25 to 0.35 M, and

further more preferably 0.25 to 0.3 M.

The sodium ion concentration of the medium may be adjusted by using a

commercially available sodium ion reagent or by using salt. In addition, the sodium ion

concentration of natural seawater, concentrated seawater, artificial seawater, and the like

are diluted such that the sodium ion concentration is 0.1 to 0.4 M, and a nitrogen source,

a phosphorus source, an iron source, trace elements, and the like may be appropriately

used by being appropriately added. As the natural seawater, filtered surface layer water

or deep sea water can be used, and commercially available products can also be used.

Examples of commercially available product of natural seawater include Naseem 10 (10

m surface seawater of Izu) and Naseem 800 (800 m deep sea water of Izu Akazawa)

(both from the Japan QCE bluelab business division). Examples of commercially

available product of artificial seawater include Daigo IMK medium and Daigo artificial

seawater SP (both from Nihon Pharmaceutical Co., Ltd.).

[0039]

As the concentration of major ions contained in natural seawater, particularly in

a surface layer of seawater, the following composition is known, for example. This

composition is considered a general composition on the surface layer of the sea, and it is

well known that the salt concentration varies from region to region. Therefore, it is difficult to define the salt concentration of seawater, but there is no doubt that sodium is a major metal in metal ions. Therefore, the inventors have assumed that a case where the sodium ion concentration generally exceeds 0.4 M is a seawater condition. The sodium ion concentration closer to the seawater condition is 0.45 M or more, and more preferably 0.5 M 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

Bicarbonate ion 0.0140% by mass

Fluoride ion 0.0001% by mass

Boric acid 0.0026% by mass

[0040]

The hydrogen ion concentration may be appropriately selected in the range of

pH 1.0 to 6.0 according to the type of freshwater microalgae. For example, in a case

where the freshwater microalgae are microalgae belonging to Cyanidiophyceae, the

hydrogen ion concentration is preferably pH 1.0 to 5.0, and more preferably pH 1.0 to

3.0.

The pH of the medium can be adjusted by using, for example, an inorganic acid

such as sulfuric acid or hydrochloric acid, an inorganic base such as potassium hydroxide, and the like. In addition, a pH buffer may be optionally added to the medium in order to suppress pH fluctuation during culturing.

[0041]

The pre-culture step can be started by inoculating cells of a freshwater microalga

into the medium. The algae concentration at the time of culture start is not particularly

limited, but can be, for example, a cell turbidity of OD7 5 o = 0.05 to 0.5. "OD7 5o" means

the absorbance at 750 mu. The cell turbidity at the time of culture start is preferably

ODy5o = 0.05 to 0.3.

[0042]

The temperature conditions in the pre-culture step may be appropriately selected

according to the type of freshwater microalgae. In general, the example of the culture

temperature is 15°C to 60°C, preferably 15°C to 50°C, and more preferably 30°C to

50°C. In a case where the freshwater microalga is a microalga belonging to

Cyanidiophyceae, the culture temperature is preferably 30°C to 50°C.

The light conditions in the pre-culture step may be appropriately selected

according to the type of freshwater microalgae. In general, 5 to 2000 mol/m 2 s can be

an exemplary example. In a case where the freshwater microalga is a microalga

belonging to Cyanidiophyceae, 5 to 1500 tmol/m 2s is preferable. The light condition

may be continuous light, and a light and darkness cycle (1OL: 14D and the like) may be

provided. The pre-culture step may be performed in natural light.

The C02 condition in the pre-culture step may be appropriately selected

according to the type of freshwater microalgae. In general, 0.04% to 5% C02

conditions can be an exemplary example. In a case where the freshwater microalga is a

microalga belonging to Cyanidiophyceae, 0.04% to 3% CO 2 conditions are preferable.

Among the microalgae belonging to Cyanidiophyceae, the genus Galdieria is highly tolerant to high CO 2 concentration and can grow even with 100% CO 2. Therefore, in a case where the freshwater microalga is a microalga belonging to the genus Galdieria, the

100% C02 condition maybe set. In addition, the C02 condition maybe an atmospheric

CO 2 concentration.

[0043]

The culturing method in the pre-culture step is not particularly limited, and a

method generally used as a culture method for a microalga may be used. Specific

examples include static culture, aeration culture (200 to 400 mL air/min and the like),

shaking culture (100 to 200 rpm and the like), and the like.

[0044]

The culture period in the pre-culture step is not particularly limited, but a period

from the end of the induction period to the beginning of the logarithmic phase is

required, usually 3 days or more is required, preferably 5 days or more is required, and

more preferably 7 days or more is required. By performing the pre-culture step for a

period of the minimum number of days or more, the growth of a freshwater microalga in

the main culture step can be favorably maintained. An upper limit of the pre-culture

period is not particularly limited, but if it is performed from the logarithmic phase to the

stationary phase, it is inappropriate for maintaining good growth of a microalga in the

main culture. The pre-culture period is preferably 3 to 20 days, more preferably 5 to 15

days, and further more preferably 7 to 10 days.

The scale of the pre-culture can be selected according to the scale of the main

culture. In a case of culturing on a small scale such as breeding a microalga or selecting

a mutant strain in the main culture, pre-culture can be performed on a small scale, and is

usually performed at 0.1 to 1,000 mL. In addition, in a case where mass production is carried out industrially in the main culture, the pre-culture can be performed at about I to

10 L in some cases.

[0045]

[Main culture step]

The main culture step is a step of culturing a freshwater microalga after the pre

culture step 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 a hydrogen ion

concentration is pH 1.0 to 6.0.

[0046]

The medium used in the main culture step has a sodium ion concentration of 1.2

to 5 times that of the medium used in the pre-culture step, is 0.4 M or more, preferably

0.45 M or more, more preferably 0.5 M or more, in terms of sodium ion concentration.

The hydrogen ion concentration is not particularly limited as long as the medium is a

medium having a pH 1.0 to 6.0. For example, a medium prepared by adding sodium

ions at the concentration to a general medium for freshwater microalgae to adjust the pH

to 1.0 to 6.0 can be preferably used. Examples of the medium for freshwater

microalgae include those similar to that mentioned in the "[pre-culture step]".

[0047]

The medium used in the main culture step may be one in which a nitrogen

source, a phosphorus source, an iron source, trace elements, and the like are appropriately

added to seawater and the pH is adjusted to 1.0 to 6.0. The seawater may be natural

seawater, artificial seawater, or diluted concentrated seawater. Examples of

commercially available product of natural seawater and artificial seawater include those

similar to those mentioned in the "[pre-culture step]". In the main culture step, in a case

where mass culturing is performed, the cost can be suppressed by using natural seawater.

The natural seawater may be surface layer water or deep sea water. Natural seawater is

preferably one from which impurities have been removed by filtration or the like. The

nitrogen source, the phosphorus source, the iron source, the trace elements, and the like

to be added to seawater may be appropriately selected according to the type of freshwater

microalgae.

[0048]

In a case where the freshwater microalga is a microalga belonging to

Cyanidiophyceae, it is preferable to add at least nitrogen-containing salt, phosphorus

containing salt, and iron-containing salt to seawater.

Examples of the nitrogen-containing salt include nitrogen-containing inorganic

salt such as ammonium salt, nitrate, nitrite, and the like. Among these, as the nitrogen

containing salt, an ammonium salt (ammonium sulfate and the like) is preferable.

Examples of the amount of the ammonium salt added to seawater include 20 to 100 mM

as an ammonium ion concentration.

Examples of the phosphorus-containing salt include phosphorus-containing

inorganic salts such as phosphates and phosphite. Among these, as the phosphorus

containing salt, a phosphate (potassium dihydrogen phosphate and the like) is preferable.

Examples of the amount of phosphate added to seawater include 2 to 10 mM as the

phosphate ion concentration.

Examples of the iron-containing salt include iron chloride (III), iron sulfate (II),

iron citrate (II), and hydrates thereof. Among these, as the iron-containing salt, iron

chloride (IlI) is preferable. Examples of the amount of the iron-containing salt added to

seawater include 0.1 to 2 mM as the iron ion concentration, for example.

In addition, it is preferable to add trace elements such as boric acid, manganese,

zinc, molybdenum, cobalt, and copper to seawater.

In a case where the freshwater microalga is a microalga belonging to

Cyanidiophyceae, specific examples of the medium used in the main culture step

preferably include "seawater medium" described in Table 9 to be described later.

[0049]

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, more preferably 1.4 to

2 times, and further more preferably 1.5 to 2 times the sodium ion concentration in the

pre-culture step. In a case where seawater is used in the main culture step, a sodium ion

concentration is about 0.4 M or more, and a medium of 1 M or less is used depending on

the type of microalga used.

In addition, in a case where the main culture step is performed outdoors, a

sodium ion concentration of 0.5 M or more can be used in order to suppress

contamination of other organisms. For example, the sodium ion concentration may be

0.5 to I M.

The sodium ion concentration of the medium can be adjusted in the same

manner as in the medium in the pre-culture step.

[0050]

The hydrogen ion concentration may be appropriately selected in the range of

where the freshwater microalga is a microalga belonging to Cyanidiophyceae, the

concentration is preferably pH 1.0 to 5.0, and more preferably pH 1.0 to 3.0.

In addition, in a case where the main culture step is performed outdoors, a lower

pH (pH 1.0 to 2.0 and the like) can be used in order to suppress contamination of other

organisms.

The pH of the medium can be adjusted in the same manner as in the medium in

the pre-culture step.

[0051]

The main culture step can be started by inoculating the medium in the main

culture step with the culture solution of the pre-culture step. The algae concentration at

the time of culture start is not particularly limited, but can be, for example, a cell

turbidity of OD 75 0 = 0.05 to 0.5. "OD7 5 0 " means the absorbance at 750 nm. The cell

turbidity at the time of culture start is preferably OD7 5 o = 0.05 to 0.3.

[0052]

The culture scale in the main culture step can be appropriately selected

depending on the purpose. For example, in a case of breeding a microalga or selecting a

mutant strain, the main culture can be performed on a small scale, and usually performed

in about 10 mLto 10L. In addition, in a case where industrial mass production is

performed in the main culture, the culture may be performed at about 20 to 5000 L. In a

case where a mass culture of 500 L is performed, outdoor culture may be performed.

[0053]

The temperature condition, the light condition, the C02 condition, and the

culture method in the main culture step can be the same as the pre-culture condition

described above.

In addition, in the main culture step, the culture may be performed outdoors. In

this case, the temperature condition, the light condition, and the CO 2 condition can be

conditions of the external environment in which a culture tank is installed. Even in a

case where the main culture step is outdoor culture, since the culture is performed under

acidic conditions of pH 1.0 to 6.0, contamination with other organisms can be

suppressed. In addition, in a case where a haploid of microalga belonging to

Cyanidiophyceae are used as a freshwater microalga, it is difficult for the haploids to

survive in an environment of pH 7, contamination of the environment is suppressed even

in a case of being released to the external environment from an outdoor culture tank.

[0054]

The culture period in the main culture step is not particularly limited, and the

culture can be continued until a desired amount of biomass is obtained. Alternatively,

the growth status of a microalga may be checked and cultured until the stationary phase

is reached.

[0055]

According to the culture method of the present embodiment, a freshwater

microalga can be favorably grown in a medium having a low pH and a high sodium ion

concentration in a short induction period from the time of culture start. Therefore, even

in the case of outdoor culture, invasion of other organisms can be effectively suppressed.

In addition, since the microalga belonging to Cyanidiophyceae (in particular, haploid)

dies under a neutral condition, biological containment is possible even in a case where a

microalga belonging to Cyanidiophyceae (in particular, haploids) is released into the

environment from the mass culture system. Therefore, the microalga can be suitably

applied to outdoor mass culture of a freshwater microalga that produces useful

substances.

[0056]

In an embodiment, the present invention provides a method for producing a

freshwater microalga that can be grown in a medium prepared such that a hydrogen ion

concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.5 M or more. The

method for producing a freshwater microalga of the present embodiment includes a step of culturing a freshwater microalga that cannot be grown in a medium having a sodium ion concentration of 0.5 M or more in a medium prepared such that the hydrogen ion concentration is pH 1.0 to 6.0 and the sodium ion concentration is 0.1 to 0.4 M.

[0057]

The freshwater microalga used in the method of the present embodiment is a

freshwater microalga that cannot be grown in a medium having a sodium ion

concentration of 0.5 M or more.

Whether or not the freshwater microalga can be grown in a medium having a

sodium ion concentration of 0.5 M or more can be checked by culturing a target

freshwater microalga in a medium having a sodium ion concentration of 0.5 M or more

and measuring the cell turbidity (OD 75 o) over time. In a case where OD75 o is not

increased from the time of culture start, it can be determined that the freshwater

microalga is a freshwater microalga that cannot be grown in a medium having a sodium

ion concentration of 0.5 M or more.

In addition, it is preferable that a freshwater microalga can be grown at pH 1.0 to

6.0.

Examples of such a freshwater microalga include haploids of microalgae

belonging to the genus Cyanidium, for example. Specific examples of the haploid of

microalga belonging to the genus Cyanidium include HKN1 strain (haploid) and YFU3

strain (haploid).

[0058]

The method of the present embodiment includes a step of culturing the

freshwater microalga in a medium of pH 1.0 to 6.0 having a sodium ion concentration of

0.1to0.4M. The step can be performed in the same manner as the "[Pre-culture step]"

of the "<Culture method of freshwater microalga>".

[0059]

According to the method of the present embodiment, it is possible to obtain a

freshwater microalga that can be grown in a medium of pH 1.0 to 6.0 having a sodium

chloride concentration of 0.5 M or more by imparting salt tolerance to a freshwater

microalga having no salt tolerance. In the environment of high salt concentration and

low pH where the freshwater microalga can grow, invasion of other organisms is

suppressed. In addition, since a microalga belonging to Cyanidiophyceae (in particular,

haploids) dies under a neutral condition, biological containment is possible even in a case

where a microalga belonging to Cyanidiophyceae (in particular, haploids) is released into

the environment from the mass culture system. Therefore, the freshwater microalga can

be suitably used for outdoor mass culture.

[0060]

The haploid of the microalga belonging to Cyanidiophyceae according to one

embodiment of the present invention grows in a medium having a high salt concentration

such as a sodium ion concentration of 0.5 M, but the haploid of themicroalga belonging

to natural Cyanidiophyceae cannot grow. However, in a case where salt tolerance is

acquired during the pre-culture period by the culture method according to one

embodiment of the present invention, it becomes possible for the haploid of microalga to

grow even in a medium having a sodium ion concentration of 0.5 M. At this time, there

is a characteristic that logarithmic growth is started after a shorter induction period than

during the pre-culture period, and this characteristic is the same even in a case where the

culture is further subcultured. Moreover, the haploid algae of the present invention that

have acquired salt tolerance are characterized in that the haploid algae do not grow or grow poorly in a case where the haploid algae are subcultured in an MA medium to which NaCl is not added.

Whether or not the growth is favorable can be examined by comparing the

culture condition with the culture condition of the control, but in one embodiment of the

present invention, it is examined by the size of the ratio of the number of cells in the

initial state of self-culture and the number of cells increased over a predetermined period

of time. Specifically, it is calculated by Formula (1). The number of cells is obtained

by measuring the absorbance OD in the culture solution at 750 nm, the predetermined

period is set to 7 days, and in a case where a value of Formula (1) is 2 or more, the

growth is favorable and in a case where a value of Formula (1) is less than 2, the growth

is not favorable. The cause of poor growth is considered that it takes time to adapt to

environmental conditions, or that the haploid ofmicroalga belonging to Cyanidiophyceae

do not have a rigid cell wall, and thus the cells cannot withstand the osmotic pressure of

hypertonic solution and are destroyed and die. It is considered to perform observation

with a microscope for checking that the cells are destroyed, but it is difficult to ensure the

quantitativity. Therefore, in one embodiment of the present invention, a quantitative

consideration is made by measuring the amount of phycocyanin, which is the content of

cells.

[0061]

In one embodiment, the present invention provides a haploid ofmicroalga

dioxide concentration of 2%, and continuous light with an illuminance of 60 mol/m 2s in

an MA medium prepared such that the hydrogen ion concentration is pH 2.0 and the

60 pmol/m 2 s in the MA medium is less than 2.

(OD 7 5 0value after 7 days from time of culture start - OD 75 0 value at time of

culture start)/(7 x OD 7 5 0 value at time of culture start) (1)

[0062]

The "MA medium prepared such that the hydrogen ion concentration is pH 2.0

and the sodium ion concentration is 0.5 M" is the medium shown in Table 5 in the

examples (hereinafter, also referred to as "MA + 0.5 M of NaCl medium"). The haploid

of microalga belonging to Cyanidiophyceae of the present embodiment can grow in an

MA + 0.5 M of NaC medium. In addition, the haploid of microalga have

characteristics that the growth rate calculated by the following Formula (1) in a case of

culturing in a static state under conditions of a culture temperature of 42°C, a carbon

an MA + 0.5 M of NaCI medium is 2 or more.

culture start)/(7 x OD7 5 0 value at time of culture start) (1)

[0063]

In the above Formula (1), the "OD7 5 o value at the time of culture start" means

the absorbance of the culture solution measured at a wavelength of 750 nm at the time of

culture start (0 hour). Usually, the haploid of microalga belonging to Cyanidiophyceae

are inoculated into an MA + 0.5 M of NaCl medium such that the OD7 5 o value at the time

of culture start is 0.1, and culture is started. In the Formula (1), the "OD750 value after 7

days from time of culture start" means the absorbance of the culture solution measured at

a wavelength of 750 nm after 7 days (168 hours) from the time of culture start. The absorbance of the culture solution can be measured with an absorbance meter.

The culture is performed by culturing in a static state for 7 days under conditions

of a culture temperature of 42°C, a carbon dioxide concentration of 2%, and continuous

light with an illuminance of 60 tmol/m 2s. The culture scale is not particularly limited,

but for example, a 24-well plate can be used for culturing in I mL of the culture solution.

[0064]

The haploid of microalga belonging to natural Cyanidiophyceae cannot grow in

an MA + 0.5 M of NaC medium. Therefore, in a case where the haploid of microalga

belonging to natural Cyanidiophyceae are cultured in an MA + 0.5 M of NaC medium,

the value calculated by the Formula (1) is less than 2. However, since the haploid of the

microalga belonging to Cyanidiophyceae of the present embodiment have tolerance to

high sodium ion concentration, the haploid of the microalga can favorably grow even in

an MA + 0.5 M of NaCl medium. Therefore, in a case where the haploid of microalga

belonging to Cyanidiophyceae of the present embodiment are cultured in an MA + 0.5 M

of NaCI medium, the value calculated by the Formula (1) is 2 or more. In the haploid of

the microalga belonging to Cyanidiophyceae of the present embodiment, a value

calculated by the Formula (1) in a case of culturing in an MA + 0.5 M of NaCl medium is

preferably 3 or more, and more preferably 4 or more.

[0065]

In addition to the characteristics, the haploid of the microalga belonging to

Cyanidiophyceae of the present embodiment have characteristics that the value

calculated by the Formula (1) is less than 2, in a case of culturing in a static state in an

MA medium at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and

a continuous light of an illuminance of 60 pmol/m2 s. The MA medium is the medium

shown in Table I in the examples. Usually, the haploid of microalga belonging to

Cyanidiophyceae are inoculated into an MA medium such that the OD7 5 0 value at the

time of culture start is 0.1, and culture is started. The culture is performed by culturing

in a static state 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 mol/m 2s.

The culture scale is not particularly limited, but for example, a 24-well plate can be used

for culturing in I mL of the culture solution.

[0066]

The haploid of microalga belonging to natural Cyanidiophyceae can favorably

grow in an MA medium. Therefore, in a case where the haploid of microalga belonging

to natural Cyanidiophyceae are cultured in an MA medium, the value calculated by the

Formula (1) is usually 2 or more. However, while the haploid of the microalga

belonging to Cyanidiophyceae of the present embodiment have tolerance to high sodium

ion concentrations, the ability of growth at a low sodium ion concentration is reduced.

Therefore, in a case where the haploid of microalga belonging to Cyanidiophyceae of the

present embodiment are cultured in an MA medium, the value calculated by the Formula

(1) is 2 or more. In the haploid of microalga belonging to Cyanidiophyceae of the

present embodiment, the value calculated by the Formula (1) in a case of culturing in an

MA medium is preferably less than 1.8, and more preferably less than 1.5.

[0067]

In the haploid of microalga belonging to Cyanidiophyceae of the present

embodiment, a death rate when suspended in an MA medium is 30% or more, more

preferably 40% or more, and further more preferably 50% or more. In a case where the

death 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 the environment where the sodium ion concentration is low. Therefore, even in a case of being cultured in an outdoor open culture system, contamination of the environment is unlikely to occur.

[0068]

The death rate is calculated as follows.

After performing culture in an MA + 0.5 M of NaCI medium, algal cells in an

amount of OD 7 5 o= I when suspended in 2 mL of the medium are collected, and the

following treatments 1 to 3 are performed.

Treatment 1: Suspension is performed in 2 mL of an MA medium and shaking is

performed with vortex for 10 minutes.

Treatment 2: Suspension is performed in 2 mL of an MA + 0.5 M of NaCl

medium and shaking is performed with vortex for 10 minutes.

Treatment 3: After suspension in 0.1 mL of an MA + 0.5 M of NaC medium,

freezing is performed at -196°C, dilution is performed to be 2 mL in the medium, and

shaking is performed with vortex for 10 minutes.

[0069]

Subsequently, the death rate is calculated by the following formula.

Death rate (%)= {(PC concentration after Treatment 2 - PC concentration after

Treatment 1)/(PC concentration after Treatment 2 - PC concentration after Treatment 3)1

x 100

In the formula, "PC concentration" represents the phycocyanin concentration.

The PC concentration can be obtained by measuring the absorbance at 620 nm and 678

nm of the suspension after performing any of Treatments I to 3 using a

spectrophotometer equipped with an integrating sphere. The PC concentration can be

calculated from the absorbance at 620 nm and 678 nm by the following formula.

PC concentration (pg/ml) = 138.5 x A620 -35.49 x A678

[0070]

The haploid of the microalga belonging to Cyanidiophyceae of the present

embodiment preferably has one or more characteristics of the following (a) to (c),

preferably has two characteristics of the following (a) to (c), and further more preferably

has all characteristics of the following (a) to (c).

(a) Compared with the haploids of the natural microalga, the growth rate for 7

days after the time of culture start in a medium of pH 1.0 to 6.0 having a sodium ion

concentration of 0.5 M is high.

(b) Compared with the haploid of the natural microalga, the growth rate for 7

concentration of 0.05 M or less is low.

(c) Compared with the growth rate for 7 days after the time of culture start in a

medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.05 M or less, the growth

rate for 7 days after the time of culture start in a medium of pH 1.0 to 6.0 having a

sodium ion concentration of 0.5 M is high.

[0071]

"Microalga belonging to natural Cyanidiophyceae" refers to microalga

belonging to Cyanidiophyceae that inhabits in nature or microalgae of the type having the

same properties as the microalga. The natural microalga may be those isolated from

nature and maintained by culturing in a medium of pH 1.0 to 6.0 having a sodium ion

concentration of 0.05 M or less.

"The haploid of microalga belonging to natural Cyanidiophyceae" refers to

haploid of microalga belonging to Cyanidiophyceae that inhabits in nature or haploid

obtained from diploid of microalga belonging to Cyanidiophyceae that inhabits in nature.

A diploid of microalga belonging to natural Cyanidiophyceae is cultured in a medium prepared such that a sodium ion concentration is 0.05 M or less and pH is 1.0 to 6.0 for a certain period of time (for example, about 1 to 3 weeks), cultured under certain conditions, and a meiotic microalga is physically selected under a microscope to obtain a haploid of microalga belonging to natural Cyanidiophyceae.

[0072]

Whether or not the haploid of microalga belonging to Cyanidiophyceae has the

characteristics of (a) can be determined by culturing a natural microalga that is the same

species as the microalga in a medium of pH 1.0 to 6.0 having a sodium ion concentration

of 0.5 M, and comparing the growth rate for 7 days after the time of culture start. In a

case where the growth rate is higher than that of the haploid of natural microalga, it is

determined that the microalgae have the characteristics of (a).

[0073]

Whether or not the haploid of the microalga belonging to Cyanidiophyceae has

the characteristics of (b) can be determined by culturing a natural microalga that is the

same species as the microalga in a medium of pH 1.0 to 6.0 having a sodium ion

concentration of 0.05 M or less, and comparing the growth rate for 7 days after the time

of culture start. In a case where the growth rate is lower than that of the haploid of

natural microalga, it is determined that the microalga has the characteristics of (b).

[0074]

Whether or not the haploid of the microalga belonging to Cyanidiophyceae has

the characteristics of (c) can be determined by culturing the haploid of microalga in a

medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.05 M or less and a

medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.5 M, and comparing the

growth rates for 7 days after the time of culture start in both of the media. In a case

where the growth rate in the medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.5 M is higher than the growth rate in the medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.05 M or less, it is determined that the microalga has the characteristics of (c).

[0075]

Examples of the medium of pH 1.0 to 6.0 having a sodium ion concentration of

0.5 M and the medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.05 M or

less include the same medium as the medium mentioned in the "<Culture method of

freshwater microalga>". Examples of the culture conditions in (a) to (c) include

for culturing in 1 mL of the culture solution.

[0076]

The haploid of microalga belonging to Cyanidiophyceae of the present

embodiment can be obtained by the method for producing a freshwater microalga of the

embodiment. Specific examples of the haploid of the microalga belonging to

Cyanidiophyceae include Cyanidioschyzon merolae, a haploid of microalga belonging to

the genus Cyanidium, and a haploid of microalga belonging to the genus Galdieria.

Specific examples of the haploid of microalga belonging to the genus Cyanidiui include

HKNI strain (haploid) and YFU3 strain (haploid). Specific examples of the haploid of

microalga belonging to the genus Galdieria include G. partita and G. sulphuraria.

Among these, the haploid of microalga belonging to the genus Cyanidium or the genus

Galdieria are preferable, the haploid of microalga belonging to the genus Cyanidium is

more preferable, HKN1 strain (haploid) or YFU3 strain (haploid) is further more

preferable, and the HKN Istrain (haploid) is particularly preferable.

[0077]

The microalga belonging to Cyanidiophyceae of the present embodiment can

preferably grow in a medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.6

M or more, more preferably grow in a medium of pH 1.0 to 6.0 having a sodium ion

concentration of 0.7 M or more, further more preferably grow in a medium of pH 1.0 to

6.0 and having a sodium ion concentration of 0.8 M or more, and particularly preferably

grow in a medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.9 M or more.

The microalga belonging to Cyanidiophyceae of the present embodiment may grow in a

medium of pH 1.0 to 6.0 having a sodium ion concentration of I M or more.

[0078]

The microalga belonging to Cyanidiophyceae of the present embodiment

preferably maintains the growth rate even in a case of being cultured in a medium of pH

1.0 to 6.0 having a sodium ion concentration of 0.5 M and subcultured in a new medium

of pH 1.0 to 6.0 having a sodium ion concentration of 0.5 M. In this way, the microalga

of the present embodiment can be maintained by performing subculture in a medium of

pH 1.0 to 6.0 having a sodium ion concentration of 0.5 M. In addition, even in a case

where the microalga of the present embodiment maintained as described above is mass

cultured in a medium of pH 1.0 to 6.0 having a sodium ion concentration of 0.5 M at an

optional time, the microalga of the present embodiment can favorably grow.

[0079]

Since the haploid of the microalga belonging to Cyanidiophyceae of the present

embodiment can favorably grow at the same sodium ion concentration as that of

seawater, the haploid of the microalga can favorably grow by using a medium obtained

by adding at least nitrogen-containing salt, phosphorus-containing salt, and iron

containing salt to the seawater and adjusting the pH to 1.0 to 6.0. Specific examples of such a medium include those similar to those mentioned in "[Main culture step]" of the

"<Culture method of freshwater microalga>".

[0080]

Since the haploid of the microalga belonging to Cyanidiophyceae of the present

embodiment can favorably grow in an environment of high sodium ion concentration and

low pH, the haploid of the microalga can be suitably used for outdoor mass culture.

[Examples]

[0081]

Hereinafter, the present invention will be described based on examples, but the

present invention is not limited to the following examples.

[0082]

(Preparation of medium)

An M-Allen medium (MA medium) having the composition shown in Table I

was prepared. Specifically, medium components other than A2 Fe stock were mixed,

adjusted to pH 2.0 with sulfuric acid, and then sterilized by 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 both of the A2 trace element and the

A2 Fe stock.

[0083]

[Table 1]

M-Allen medium (MA medium) (NH 4 ) 2 SO 4 2.64 g KH2PO4 0.54 g MgSO4-7H20 0.5 g CaC12'2H20 0.14 g A2 trace element 2 mL Distilled water 994 mL Prepared with sulfuric acid at pH 2.0 4 mL of A2 Fe stock added after autoclave

[0084]

[Table 2]

A2 trace element H3B03 2.85 g MnC12-4H20 1.8 g ZnCl 2 0.105 g Na2M0 4 -2H20 0.39 g CoC12 6H20 40 mg CuC12-2H20 43 mg Distilled water 1000 mL

[0085]

[Table 3]

A2 Fe stock EDTA-2Na 7g FeCI3-6H 20 4g Distilled water 1000 mL Filter sterilization

[0086]

0.3 M of NaCl was added to an MA medium to prepare an MA + 0.3 M of NaCl

medium. In addition, 0.5 M of NaCl was added to an MA medium to prepare an MA +

0.5 M of NaCl medium. The compositions of the MA + 0.3 M of NaCl medium and the

MA + 0.5 M of NaCI medium are shown in Tables 4 and 5. The A2 trace element and

A2 Fe stock in Tables 4 and 5 are shown in both of Tables 2 and 3.

[0087]

[Table 4]

MA + 0.3 M of NaCI medium (NH 4 ) 2 SO 4 2.64 g KH 2 PO4 0.54 g MgSO 4 -7H20 0.5 g CaCl2-2H 20 0.14 g NaCl 17.5 g A2 trace element 2 mL Distilled water 994 mL Prepared with sulfuric acid at pH 2.0 4 mL of A2 Fe stock added after autoclave

[0088]

[Table 5]

MA + 0.5 M of NaCI medium (NH4) 2 SO 4 2.64 g KH2PO 4 0.54 g MgSO4-7H20 0.5 g CaCl2'2H20 0.14 g NaCl 29.2 g A2 trace element 2 mL Distilled water 994 mL Prepared with sulfuric acid at pH 2.0 4 mL of A2 Fe stock added after autoclave

[0089]

In the following examples, in a case where the medium is used for pre-culture or

main culture, each medium may be denoted as follows.

In a case of being used for pre-culture

MA medium: medium (A)

MA + 0.3 M of NaCl medium: medium (B)

MA + 0.5 M of NaCl medium: medium (C)

In a case of being used for main culture

MA medium: medium (a)

MA + 0.3 M of NaCl medium: medium (b)

MA + 0.5 M of NaCl medium: medium (c)

[0090]

(Measurement of growth status)

The growth status ofmicroalga was checked by cell turbidity (OD7 5 o).

Specifically, the absorbance of the algae culture solution at 750 nm was measured using

an absorbance meter (SmartSpec Plus of BIO-RAD) to obtain cell turbidity (OD7 5 o).

[0091]

(Culture method)

Unless otherwise specified, the culture of Cyanidium sp. HKN1 was performed

by culturing in a static state in a CO 2 incubator in both the pre-culture and the main

culture. The culture temperature was set to 42°C, a continuous light of an illuminance

of 60 mol/n 2s was used, and the C02 concentration was set to 2%. Unless otherwise

specified, culture was performed on a 24-well plate with 1 mL of a culture solution.

The culture of Cyanidioschyzon merolae IOD was performed with aeration (300

mL ambient air/min) in both the pre-culture and the main culture unless otherwise

specified. The culture temperature was set to 42°C, and a continuous light of an

illuminance of 60 Imol/m 2 s was used. Unless otherwise specified, culture was

performed on a 24-well plate with 1 mL of a culture solution.

For both Cyanidium sp. HKIN1 and Cyanidioschyzon merolae 1OD, the cell

turbidity at the time of pre-culture start and at the time of main culture start was set to

OD 7 5 0 =0.1. Unless otherwise specified, culture was performed on a 24-well plate with

1 mL of a culture solution.

[0092]

(Qualitative analysis of polyphosphoric acid)

The presence of polyphosphoric acid was observed by DAPI staining as follows.

After adding glutaraldehyde to the culture solution such that a final

concentration is 1% (w / v), DAPI was added such that a final concentration is 3 g/mL,

and the mixture was observed with a fluorescence microscope.

[0093]

(Qualitative analysis of vacuoles)

The presence of vacuoles was observed by quinacrine staining as follows.

After adding I M Tris-HCI (pH 8.0) buffer to the culture solution such that a

final concentration is 100 mM, quinacrine was added such that a final concentration is 40

ptg/mL, and the mixture was allowed to stand at room temperature for 15 minutes. After

centrifugation (1,500 g, 5 minutes), the supernatant was discarded, an MA medium was

added to the precipitate, and the mixture was allowed to stand at 37°C for 30 minutes and

observed with a fluorescence microscope.

[0094]

[Example 1]

Cyanidium sp. HKNI (haploid) (hereinafter, abbreviated as "HKN1 (haploid)"

in some cases) was pre-cultured for one week using an MA medium (medium (A)).

After the pre-culture, culture was performed in a static state for 7 days using the medium

(a), (b), or (c) (main culture). During the main culture, the OD 75 0 of the medium was

measured over time to check the growth status of HKN1 (haploid).

In addition, HKN I(haploid) was pre-cultured for one week using an MA + 0.3

M of NaCl (medium (B)). After the pre-culture, culture was performed in a static state

for 7 days using the medium (a), (b), or (c) (main culture). During the main culture, the

OD 7 5 o of the medium was measured over time to check the growth status of HKNI

(haploid).

The results are shown in Table 6 and Fig. 1. Fig. 1 is a graph showing changes

in OD 7 5 o shown in Table 1.

[0095]

[Table 6]

Pre-culture Main culture Number of culture days (OD7 5 o) 0day 1 day 3 days 5 days 7 days Medium (A) Medium (a) 0.1 0.405 1.066 1.875 2.655 Medium (A) Medium (b) 0.1 0.253 0.96 1.895 2.755 Medium (A) Medium (c) 0.1 0.077 0.063 0.052 0.047 Medium (B) Medium (c) 0.1 0.424 1.118 2.035 2.875

[0096]

As shown in Table 6 and Fig. 1, in a case where the pre-culture was performed

in the medium (A), the same growth was shown when the main culture was performed in

the medium (a) and the medium (b), but the growth was not shown when the main culture

was performed in the medium (c).

On the other hand, in a case where the pre-culture was performed in the medium

(B), even when the main culture was performed in the medium (c), the same growth was

shown as in the case of the medium (a) and the medium (b).

[0097]

[Example 2]

Naseem 10 (seawater of surface layer: Japan QCE bluelab division) was used as

natural seawater. The components of the MA medium were added to seawater (Naseem

10), HKNI (haploid) was cultured, and it was examined which component in the MA

medium contributed to the growth of HKN1 (haploid).

The medium used for the main culture is shown in Tables 7 and 8. The MA

medium components shown in Tables 7 and 8 were added to Naseem 10 and adjusted to

pH 2.0 with sulfuric acid to prepare the medium 1 to the medium 17. Since magnesium

and calcium are abundant in seawater, MgSO4 and CaC2 were not added to the medium I

to the medium 16. As a positive control, the medium 17 was added with MgSO4 and

CaCl2 corresponding to the MA medium.

[0098]

After pre-culturing HKNI(haploid) in the medium (B) for one week, main

culture was performed in any of the medium 1 to the medium 17 for 10 days. After 10

days of the main culture, the OD 75 0 of the medium was measured to check the growth

status of HKNI (haploid). The results are shown in Tables 7 and 8.

[0099]

[Table 7]

Medium 1 2 3 4 5 6 7 8 9 20 mM of (NH 4 ) 2 SO 4 + - - - + + + 4 mM of KH 2 PO4 + - - + - -

+ A2 Fe of stock + - - + -

+ A2 trace element 2 mM of MgSO 4 1 mM of CaC1 2 Culture result 0.122 0.218 0.191 0.167 0.181 0.016 0.583 0.18 0.194 (ODyso) +: Addition, -:Non-addition

[0100]

[Table 8]

Medium 10 11 12 13 14 15 16 17 20 mM of - + + + - + (NH 4 ) 2 SO4 + -

4mMof KH 2PO 4 A2Festock - + + - + + + +

A2 trace element + + - + + + + +

2 mM of +

MgSO 4 1mimof CaCl2 +

Culture result 0.185 0.18 2.885 0.011 0.555 0.173 5.76 5.73 (OD750 )

+: Addition, -:Non-addition

[0101]

As shown in Tables 7 and 8, the medium 16 and the medium 17 showed the same growth. From these results, it was confirmed that by adding (NH 4 ) 2 SO 4 , KH 2

P0 4 , A2 Fe stock, and A2 trace metal element to seawater, the medium can be used as

seawater medium suitable for the growth of HKN I(haploid).

Table 9 shows the composition of the medium 16. In the following examples,

what is denoted as "seawater medium" is a medium having the composition shown in

Table 9. The A2 trace element and A2 Fe stock in Table 9 are shown in both Tables 2

and 3.

[0102]

[Table 9]

Seawater medium (NH 4 )2SO 4 2.64 g KH2PO 4 0.54 g A2 trace element 2 mL Seawater (Naseem 10) 994 mL Prepared with sulfuric acid at pH 2.0 4 mL of A2 Fe stock added after autoclave

[0103]

[Example 3]

HKN1 (haploid) was pre-cultured in an MA + 0.3 M of NaC medium (medium

(B)) for one week, and main culture was performed in an MA + 0.5 M of NaC medium

(positive control) (medium (c)) or seawater medium for 7 days. During the main

culture, the OD 7 oof the medium was measured over time to check the growth status of

HKN1 (haploid).

The results are shown in Table 10 and Fig. 2. Fig. 2 is a graph showing

changes in the OD 7 50 shown in Table 10.

[0104]

[Table 10]

Pre-culture Main culture Number of culture days (OD75 o) 0 day 1 day 3 days 5 days 7 days Medium (B) Medium (c) 0.1 0.438 1.41 2.36 3.055 Medium (B) Seawater 0.1 0.395 1.345 2.32 3.28 medium

[0105]

As shown in Table 10 and Fig. 2, HKN1 (haploid) showed the same growth in a

case where the main culture was performed in any one medium of the medium (c) and

the seawater medium.

[0106]

[Example 4]

In order to check the influence of pH on the growth of HKN1 (haploid), a

seawater medium in which the pH was changed between pH 2 to 7 was prepared. The

pH was prepared using sulfuric acid or potassium hydroxide.

HKN1 (haploid) was pre-cultured in an MA + 0.3 M of NaCI (medium (B)) for

one week, and main-cultured in each seawater medium in which the pH was adjusted as

described above for 7 days. After the completion of the main culture, the OD75 oofthe

final medium was measured to check the growth status of HKN1 (haploid).

The results are shown in Table 11 and Fig. 3. Fig. 3 is a graph showing the

OD 7 5 o shown in Table 11.

[0107]

[Table 11]

pH (Time of main 2 3 4 5 6 7 culture start) pH (After 7 days from 1.89 2.42 2.61 2.65 4.83 6.43 main culture) Culture result 2.60 2.59 2.27 2.22 0.834 0.1 (OD 50 )

[0108]

As shown in Table 11 and Fig. 3, HKN1 (haploid) was able to grow at pH 6 or

lower, but was not able to grow at pH 7. In general, in a case where algae are grown in

a medium using ammonia as a nitrogen source, the pH decreases as the algae grow.

Therefore, it is necessary to add a buffer or an alkaline substance to maintain the pH to

be near neutrality. However, it was not necessary to adjust the pH with acidophilic

HKN1 (haploid).

[0109]

[Example 5]

Cyanidium sp. HKNI (diploid) (hereinafter, abbreviated as "HKN1 (diploid)" in

some cases) was pre-cultured for one week using an MA medium (medium (A)). After

the pre-culture, culture was performed in a static state for 7 days using the medium (a),

(b), or (c) (main culture). During the main culture, the OD7 5 0 of the medium was

measured over time to check the growth status of HKN1 (diploid).

In addition, HKN1 (diploid) was pre-cultured for one week using an MA + 0.3

OD 7 5 oof the medium was measured over time to check the growth status of HKN1

(diploid).

The results are shown in Table 12 and Fig. 4. Fig. 4 is a graph showing the

changes in OD7 5 o shown in Table 12.

[0110]

[Table 12]

Pre-culture Main culture Number of culture days (OD 75 0

) 0day 1 day 3 days 5 days 7 days Medium (A) Medium (a) 0.1 0.25 2.065 3.77 6.08 Medium (A) Medium (b) 0.1 0.333 1.725 3.59 5.77 Medium (A) Medium (c) 0.1 0.278 1.24 2.74 4.82 Medium (B) Medium (c) 0.1 0.276 1.08 2.64 4.46

[0111]

As shown in Table 12 and Fig. 4, in a case where the pre-culture was performed

in any one medium of the medium (A) and the medium (B), HKN1 (diploid) showed the

same growth rate in the main culture in the medium (a), the medium (b), and the medium

(c).

[0112]

[Example 6]

HKN1 (diploid) was pre-cultured in an MA + 0.3 M of NaC medium (medium

(B)) for one week, and the main culture was performed in an MA + 0.5 M of NaC

medium (positive control) (medium (c)) or a seawater medium for 7 days. During the

main culture, the OD 7 5 o of the medium was measured over time to check the growth

status of HKN1 (diploid).

The results are shown in Table 13 and Fig. 5. Fig. 5 is a graph showing

changes in OD7 5 o shown in Table 13.

[0113]

[Table 13]

Pre-culture Main culture Number of culture days (OD 7 50

) 0 day I day 3 days 5 days 7 days Medium (B) Medium (c) 0.1 0.281 1.11 2.89 4.68 Medium (B) Seawater 0.1 0.262 1.125 2.62 4.52 medium

[0114]

As shown in Table 13 and Fig. 5, HKN (diploid) showed the same growth rate

in a case where the main culture was performed in any one medium of the medium (c)

and the seawater medium.

[0115]

[Example 7]

Cyanidioschyzon merolae 1OD (hereinafter, abbreviated as "1OD" in some

cases) was pre-cultured for one week using an MA medium (medium (A)). After the

pre-culture, culture was performed in a static state for 7 days using the medium (a), (b),

or (c) (main culture). During the main culture, the OD7 5 o of the medium was measured

over time to check the growth status of1OD.

In addition, 10D was pre-cultured for one week using an MA + 0.3 M of NaCl

(medium (B)). After the pre-culture, culture was performed in a static state for 7 days

using the medium (a), (b), or (c) (main culture). During the main culture, the OD7 5 0 of

the medium was measured over time to check the growth status of 1OD.

The results are shown in Table 14 and Fig. 6. Fig. 6 is a graph showing

changes in OD 7 5 0 shown in Table 14.

[0116]

[Table 14]

Pre-culture Main culture Number of culture days (OD7 5 o) 0 day I day 3 days 5 days 7 days Medium (A) Medium (a) 0.1 0.291 1.815 4.74 7.17 Medium (A) Medium (b) 0.1 0.179 1.43 4.29 6.79 Medium (A) Medium (c) 0.1 0.036 0.019 0.061 0.547 Medium (B) Medium (c) 0.1 0.25 1.49 4.07 6.95

[0117]

As shown in Table 14 and Fig. 6, in a case where the pre-culture was performed

in the medium (A), 1OD showed the same growth when the main culture was performed

in the medium (a) and the medium (b). On the other hand, when the main culture was

performed in the medium (c), the OD7 5 o gradually decreased until the 3rd day of the main

culture, but was recovered on the 5th day, and after that, the same growth rate was shown

in the medium (a) and the medium (b).

On the other hand, in a case where the pre-culture was performed in the medium

(B), even when the main culture was performed in the medium (c), the same growth as in

the case of the medium (a) and the medium (b) was shown from the time of culture start.

[0118]

[Example 8]

1OD was pre-cultured in an MA + 0.3 M of NaCl medium (medium (B)) for 1

week and main-cultured in an MA + 0.5 M of NaCl medium (positive control) (medium

(c)) or a seawater medium for 7 days. During the main culture, the OD7 5 0 of the

medium was measured over time to check the growth status of 10D.

The results are shown in Table 15 and Fig. 7. Fig. 7 is a graph showing

changes in OD 7 50 shown in Table 15.

[0119]

[Table 15]

Pre-culture Main culture Number of culture days (OD7 5 0

) 0 day I day 3 days 5 days 7 days Medium (B) Medium (c) 0.1 0.262 1.935 4.39 7.25 Medium (B) Seawater 0.1 0.261 1.925 4.52 7.14 medium

[0120]

As shown in Table 15 and Fig. 7, 10D showed the same growth rate even in a

the seawater medium.

[0121]

[Example 9]

HKN1 (haploid) cultured in an MA medium for one week was transplanted into

an MA medium such that OD7 5 0 = 0.1, and cultured in a static state in a C02 incubator

(2% C0 2 ) for 7 days to obtain an MA medium culture product of HKN1 (haploid).

HKNI (haploid) cultured in an MA medium for one week was pre-cultured in an MA

medium + 0.3 M of NaCl for one week, then transplanted into an MA medium + 0.5 M

of NaCl medium, and cultured in a static state in a C02 incubator (2%) for 7 days. This

was used as an MA medium+ 0.5 M of NaCl medium culture product.

HKN1 (haploid) cultured in an MA medium for one week was pre-cultured in an

MA medium + 0.3 M of NaCl for one week, then transplanted into a seawater medium,

and cultured in a static state in a C02 incubator (2% C0 2 ) for 7 days. This was used as

a seawater medium culture product.

For each medium culture product, qualitative analysis of polyphosphoric acid

was performed with DAPI, and qualitative analysis of vacuoles was performed with

quinacrine.

The results are shown in Fig. 8.

As a result of staining the MA medium culture product, the MA medium + 0.5

M of NaCl medium culture product, and the seawater medium culture product by DAPI,

polyphosphate was observed only in the MA medium culture product. As a result of

staining the vacuoles which are said to contain polyphosphoric acid with quinacrine,

there was no difference in how the vacuoles were stained.

[0122]

[Example 10]

Culturing was performed in the same manner as in Example 9 except that HKNI

(diploid) was used as the algae to produce three types of culture products of an MA

medium culture product, an MA medium + 0.5 M of NaCl medium culture product, and a

seawater medium culture product. In the same manner as in Example 9, qualitative

analysis of polyphosphoric acid was performed with DAPI, and qualitative analysis of

vacuoles was performed with quinacrine.

The results are shown in Fig. 9. Although polyphosphoric acid could not be

observed in the MA medium culture product, polyphosphoric acid was observed around

the vicinity of the surface layer in the MA medium + 0.5 M of NaCl medium culture

product and the seawater medium culture product. In addition, vacuoles, which are said

to contain polyphosphoric acid, were similarly observed in all the culture products.

[0123]

[Example 11]

10D was pre-cultured in an MA medium (medium(A)) or an MA + 0.3 M of

NaCi (medium (B)) for one week. After the pre-culture, the main culture was

performed in an MA medium in which the NaCl concentration was changed between 0 to

1,000 mM for 7 days. After the completion of the main culture, the OD7 5 o of the final

medium was measured to check the growth status of IOD. The results are shown in

Table 16.

In addition, after pre-culture in the medium (B), the algal cells obtained by

main-culturing IOD in the MA + 0.5 M of NaCl medium (medium (c)) for 7 days were

subcultured in the MA medium (medium (a)) or the MA + 0.5 M of NaCl medium

(medium (c)), and further cultured for 7 days. After completion of the culture, the

OD 750 of the final medium was measured to check the growth status ofIOD. The results

are shown in Table 17.

[0124]

The OD7 50 of the medium at the time of the main culture start and the time of

subculture was set to 0.1. The same applies to the following Examples 12 to 17. In

Table 17, "Formula (1)-(A)" represents a value calculated by Formula (1) of microalga

which were pre-cultured in the medium (A) and then main-cultured at a NaC

concentration shown in the table. "Formula (1)-(B)" represents a value calculated by

Formula (1) of microalga which was pre-cultured in the medium (B) and then main

cultured at a NaCI concentration shown in the table. Hereinafter, the same applies to

Examples 12 to 17. When the OD 75 0 < 0.1, the value could not be calculated by

Formula (1), and thus was denoted as .

[0125]

[Table 16]

NaCl concentration of main culture (mM) Pre- 0 100 200 300 400 500 600 700 800 900 1000 culture _____

Medium 5.970 6.694 6.076 5.418 1.827 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 (A)__ _ _ _ __ _ _ _ __ _ _ _ __ _ _ _

Medium 4.429 5.194 5.117 5.109 4.879 4.128 3.523 2.400 2.010 1.742 2.015 (B)__ ___ _ __ _ __ ___ _

Formula 8.39 9.42 8.54 7.60 2.47 - - - - - (1)- (A) Formula 6.18 7.28 7.17 7.16 6.83 5.75 4.89 3.29 2.73 2.35 2.74 (I)- (B)

[0126]

[Table 17]

Subculture Medium (a) Medium (c) OD50 1.211 3.619 Increased 1.59 5.03 value of Formula (1)

[0127]

As shown in Table 16, in those pre-cultured in the medium (B), the growth in a

case of being main-cultured in the medium of NaCl 0mM was suppressed compared

with those pre-cultured in the medium (A). On the other hand, at a high NaCl

concentration of 500 mM or more, the growth was not observed in those pre-cultured in

the medium (A), whereas the growth was observed even at a high salt concentration of

500 mM in those pre-cultured in the medium (B).

[0128]

As shown in Table 17, in those main-cultured in the medium (c) and then

subcultured in the medium (a), the growth was suppressed, but in those subcultured in the

medium (c), favorable growth was observed.

In addition, it was confirmed that in the algal cells obtained by the main culture

in the medium (c), cells are ruptured as a result of recovering algal cells after

centrifuging the culture solution and putting thereof into distilled water having a pH 7.

[0129]

[Example 12]

HKN I(haploid) was pre-cultured using an MA medium (medium (A)) or an

MA + 0.3 M of NaCl (medium (B)) for one week. After the pre-culture, the main

culture was performed in an MA medium in which the NaCl concentration was changed

to between 0 to 1,000 mM for 7 days. After the completion of the main culture, the

OD 7 5 0 of the final medium was measured to check the growth status of HKNI (haploid).

The results are shown in Table 18.

[0130]

In addition, after pre-culture in the medium (B), the algal cells obtained by

main-culturing HKNI (haploid) in the MA + 0.5 M of NaCl medium (medium (c)) for 7

days were subcultured in the MA medium (medium (a)) or the MA + 0.5 M of NaCI

medium (medium (c)) and further cultured for 7 days. After completion of the culture,

the OD 7 50 of the final medium was measured to check the growth status of HKNI

(haploid). The results are shown in Table 19.

[0131]

[Table 18]

NaCl concentration of main culture (mM) Pre- 0 100 200 300 400 500 600 700 800 900 1000 culture Medium 4.813 6.167 5.838 4.381 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 (A) Medium <0.1 5.485 6.057 5.830 5.717 4.408 6.237 5.029 3.486 2.688 1.231 (B)__ ___ _

Formula 6.73 8.67 8.20 6.12 - - - - - - (1)-(A) Formula - 7.69 8.51 8.19 8.02 6.15 8.77 7.04 4.84 3.70 1.62 (l)-(B)

[0132]

[Table 19]

Subculture Medium Medium (a) (c) OD75 0 <0.1 5.258 Value of - 7.37 Formula (1)

[0133]

As shown in Table 18, in those pre-cultured in the medium (B), the growth in a

case of being main-cultured in the medium of NaCl 0 mM was not observed. On the

other hand, at a high NaCl concentration of 400 mM or more, the growth was not

observed in those pre-cultured in the medium (A), whereas the growth was observed

even at a high salt concentration of 400 mM or more in those pre-cultured in the medium

(B).

[0134]

As shown in Table 19, in those main-cultured in the medium (c) and subcultured

in the medium (a), the growth was suppressed, but in those subcultured in the medium

(c), favorable growth was observed.

[0135]

[Example 13]

HKN1 (diploid) was pre-cultured for one week using an MA medium (medium

(A)) or an MA + 0.3 M of NaCl (medium (B)). After the pre-culture, the main culture

was performed in an MA medium in which the NaCl concentration was changed between

0 to 1,000 mM for 7 days. After the completion of the main culture, the OD75 oof the

final medium was measured to check the growth status of HKN1 (diploid). The results

are shown in Table 20.

[0136]

In addition, after pre-culture in the medium (B), the algal cells obtained by

main-culturing HKN1(diploid) in the MA + 0.5 M of NaCl medium (medium (c)) for 7

days were subcultured in the MA medium (medium (a)) or the MA + 0.5 M of NaCl

the OD 750 of the final medium was measured to check the growth status of HKNI

(diploid). The results are shown in Table 21.

[0137]

[Table 20]

NaCl concentration of main culture (mM) Pre- 0 100 200 300 400 500 600 700 800 900 1000 culture Medium 11.43 10.99 11.643 11.810 8.678 8.187 3.149 0.622 <0.1 <0.1 <0.1 (A) 0 9 Medium 10.94 9.537 10.58 8.595 8.539 5.970 6.273 4.399 3.781 2.893 2.25 (B) 7 5111 1 Formula 16.19 15.57 16.49 16.73 12.25 11.55 4.36 0.75 - - (1)-(A) Formula 15.50 13.48 14.98 12.14 12.06 8.39 8.82 6.14 5.26 3.99 3.07 (1)-(B)______________

[0138]

[Table 21]

Subculture Medium Medium (a) (c) OD50 10.951 7.808 Value of 15.50 11.01 Formula (1)

[0139]

As shown in Table 20, in those pre-cultured in the medium (B), the growth tends

to be slightly suppressed in a case of being main-cultured in the medium having a low

NaCl concentration compared with those pre-cultured in the medium (A). On the other

hand, at a high NaCl concentration of 800 mM or more, the growth was not observed in

those pre-cultured in the medium (A), whereas the growth was observed at a high salt

concentration of 800 mM or more in those pre-cultured in the medium (B).

[0140]

As shown in Table 21, favorable growth was observed in both of those

subcultured in the medium (a) and in those subcultured in the medium (c) after being

main-cultured in the medium (c).

[0141]

[Example 14]

Galdieria partita (NBRC 102759) (haploid) was pre-cultured for one week using

an MA medium (medium (A)) or an MA + 0.3 M of NaCl (medium (B)). After the pre

culture, the main culture was performed in an MA medium in which the NaCl

concentration was changed between 0 to 1,000 mM for 7 days. After the completion of

the main culture, the OD7 5 0 of the final medium was measured, and the growth status of

G. partita (haploid) was checked. The results are shown in Table 22.

[0142]

In addition, algal cells obtained by pre-culturing G. partita (haploid) in the

medium (B), and main-culturing thereof for 7 days in a MA + 0.5 M of NaCl medium

(medium (c)) were subcultured in the MA medium (medium (a)) or the MA + 0.5 M of

NaCl medium (medium (c)), and further cultured for 7 days. After completion of the

culture, the OD 7 5 oof the final medium was measured, and the growth status of G. partita

(haploid) was checked. The results are shown in Table 23.

[0143]

[Table 22]

NaCl concentration of main culture (mM)

ture 0 100 200 300 400 500 600 700 800 900 1000 Medium 5.525 6.980 5.963 5.672 5.007 2.661 5.260 3.618 1.843 1.877 1.503 (A) Medium 0.719 7.686 7.420 7.011 5.565 2.997 5.887 5.308 4.377 3.512 3.415 (B) Formula 7.75 9.83 8.38 7.96 7.01 3.66 7.37 5.03 2.49 2.54 2.00 (I)-(A)I I Formula 0.88 10.84 10.46 9.87 7.81 4.14 8.27 7.44 6.11 4.87 4.74

[0144]

[Table 23]

Subculture Medium Medium (a) (c) OD7 5o 0.603 3.957 Value of 0.72 5.51 Formula (1)

[0145]

As shown in Table 22, in those pre-cultured in the medium (B), the growth in a

case of being main-cultured in the medium of NaCl 0 mM was suppressed compared

with those pre-cultured in the medium (A). On the other hand, at a high NaCl concentration of 700 mM or more, those pre-cultured in the medium (B) had a low growth suppression rate compared with those pre-cultured in the medium (A).

[0146]

As shown in Table 23, in those main-cultured in the medium (c) and then

medium (c), favorable growth was observed.

[0147]

[Example 15]

G. Partita (diploid) was pre-cultured for one week using an MA medium

(medium (A)) or an MA + 0.3 M of NaCl (medium (B)). After the pre-culture, the main

between 0 to 1,000 mM for 7 days. After the completion of the main culture, the OD7 5 o

of the final medium was measured, and the growth status of G. partita (diploid) was

checked. The results are shown in Table 24.

[0148]

In addition, the algal cells obtained by pre-culturing G. partita (diploid) in the

medium (B) and main-culturing thereof for 7 days in the MA + 0.5 M of NaCl medium

NaCI medium (medium (c)) and then further cultured for 7 days. After completion of

the culture, the OD7 5 0 of the final medium was measured, and the growth status of G.

partita (diploid) was checked. The results are shown in Table 25.

[0149]

[Table 24]

NaCl concentration of main culture (mM) Pre- 0 100 200 300 400 500 600 700 800 900 1000 culture Medium 7.140 9.457 9.329 8.022 4.739 3.256 3.437 2.049 1.822 1.437 0.856 (A) Medium 6.535 8.458 7.258 7.290 7.193 6.864 6.481 4.901 4.029 2.523 1.590 (B)__ ___ _

Formula 10.06 13.37 13.18 11.32 6.63 4.51 4.77 2.78 2.46 1.91 1.08 (1)-(A) Formula 9.19 11.94 10.23 10.27 10.13 9.66 9.12 6.86 5.61 3.46 2.13 (l)-(B)

[0150]

[Table 25]

Subculture Medium Medium (a) (c) OD75 0 5.417 5.180 Value of 7.60 7.26 Formula (1)

[0151]

As shown in Table 24, in those pre-cultured in the medium (B), the growth tends

to improve in the medium of high NaCl concentration compared with those pre-cultured

in the medium (A).

[0152]

As shown in Table 25, in any of those subcultured in the medium (a) and those

subcultured in the medium (c) after being main-cultured in the medium (c), favorable

growth was observed.

[0153]

[Example 16]

Galdieria sulphuraria (SAG108.79) (haploid) was pre-cultured for one week using an MA medium (medium (A)) or an MA + 0.3 M of NaCl (medium (B)). After the pre-culture, the main culture was performed in an MA medium in which the NaCI concentration was changed between 0 to 1,000 mM for 7 days. After the completion of the main culture, the OD 750 of the final medium was measured, and the growth status of sulphuraria (haploid) was checked. The results are shown in Table 26.

[0154]

In addition, algal cells obtained by pre-culturing G. sulphuraria (haploid) in the

medium (B) and main-culturing thereof in an MA + 0.5 M of NaC medium (medium (c))

for 7 days were subcultured in an MA medium (medium (a)) or an MA + 0.5 M of NaCl

the OD7 5 0of the final medium was measured, and the growth status of G. sulphuraria

(haploid) was checked. The results are shown in Table 27.

[0155]

[Table 26]

NaCl concentration of main culture (mM) Pre- 0 100 200 300 400 500 600 700 800 900 1000 culture Medium 3.932 3.911 4.549 3.543 1.871 0.366 0.233 <0.1 <0.1 <0.1 <0.1 (A) Medium 0.981 4.442 5.381 4.805 4.813 3.978 5.025 4.189 4.512 4.771 3.669 (B) Formula 5.47 5.44 6.36 4.92 2.53 0.38 0.19 - - -

Formula 1.6 6.20 7.54 6.72 6.73 5.54 7.04 5.84 6.30 6.67 51

[0156]

[Table 27]

Subculture Medium Medium (a) (c) OD50 1.006 4.125 Value of 1.29 5.75 Formula (1)

[0157]

As shown in Table 26, in those pre-cultured in the medium (B), the growth in a

case of being main-cultured in the medium of NaCl 0mM was suppressed compared

with those pre-cultured in the medium (A). On the other hand, at a high NaCl

concentration of 700 mM or more, the growth was not observed in those pre-cultured in

the medium (A), whereas the favorable growth was observed at a high salt concentration

of 700 mM or more in those pre-cultured in the medium (B).

[0158]

As shown in Table 27, in those main-cultured in the medium (c) and then

medium (c), favorable growth was observed.

[0159]

[Example 17]

G. sulphuraria (diploid) was pre-cultured for one week in an MA medium

of the final medium was measured, and the growth status of G. sulphuraria (diploid) was

checked. The results are shown in Table 28.

[0160]

In addition, the algal cells obtained by pre-culturing G. sulphuraria (diploid) in

the medium (B) and main-culturing thereof in the MA + 0.5 M of NaCl medium

(medium (c)) for 7 days were subcultured in the MA medium (medium (a)) or the MA

+ 0.5 M of NaCl medium (medium (c)) and further cultured for 7 days. After completion

of the culture, the OD7 5 o of the final medium was measured, and the growth status of G.

sulphuraria (diploid) was checked. The results are shown in Table 29.

[0161]

[Table 28]

NaCl concentration of main culture (mM)

ture 0 100 200 300 400 500 600 700 800 900 1000 Medium 7.492 6.495 6.502 6.228 6.514 3.966 5.180 2.789 2.315 1.851 1.293 (A) Medium 6.824 6.806 6.823 6.824 7.723 4.459 6.256 6.296 5.926 4.729 5.390 (B) Formula 10.56 9.14 9.15 8.75 9.16 5.52 7.26 3.84 3.16 2.50 1.70 (I)-(A )I IIIIIII Formula 9.61 9.58 9.60 9.61 10.89 6.23 8.79 8.85 8.32 6.61 7.56

[0162]

[Table 29]

Subculture Medium Medium (a) (c) OD7 5o 6.720 2.517 Value of 9.46 3.45 Formula (1)

[0163]

As shown in Table 28, in those pre-cultured in the medium (B), the growth in a

medium of a high NaCl concentration tends to improve compared with those pre-cultured

in the medium (A).

[0164]

As shown in Table 29, in both of those subcultured in the medium (a) and those

subcultured in the medium (c) after main culture in the medium (c), favorable growth

was observed.

[0165]

When combining the results, it was confirmed that in a haploid freshwater

microalga, the growth when main-cultured at a NaCl concentration of 0.5 M or more

improves by performing pre-culture at a NaCl concentration of 0.3 M compared with that

when pre-cultured at NaCl of 0 M. In this way, the value calculated by Formula (1)

became 2 or more. In addition, it was confirmed that after performing culture at a NaCl

concentration of 0.3 M or more, the growth at a NaCl concentration of 0 M was

suppressed compared with that when pre-cultured at a NaCl concentration of 0 M. In

addition, by performing pre-culture at a NaCl concentration of 0.3 M, the growth rate in

the medium of a NaCl concentration of 0.5 M tended to rise compared with the growth

rate in the medium of a NaCl concentration of 0 M. In addition, it was also confirmed

that after main culture at a NaCl concentration of 0.5 M, subculture to a medium having a

NaCl concentration of 0.5 M is possible.

In addition, it was confirmed that by performing pre-culture at a NaCl

concentration of 0.3 M, even in a haploid freshwater microalga, the growth is possible in

a range of high NaCl concentration of 500 to 1,000 mM. In a case of assuming outdoor

culture, it is considered that the salt concentration fluctuates during culture due to

evaporation of water or inflow of rainwater. From the above results, it was shown that

haploid of microalga belonging to Cyanidiophyceae pre-cultured at a NaCl concentration

of 0.3 M showed tolerance to fluctuations in salt concentration and sufficiently tolerated

outdoor culture.

[0166]

[Example 18]

HKN I(haploid) was pre-cultured in an MA + 0.3 M of NaCl medium (medium

(B)) for 7 days and then further pre-cultured in a seawater medium for one week.

Subsequently, the HKN1 (haploid) was subcultured in 10 L of a seawater medium for

main culture. The main culture was performed in a plastic greenhouse, and light,

temperature, and CO 2 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 periodically sampled and the absorbance at 750 nm was

measured. The results are shown in Fig. 10.

As shown in Fig. 10, it was confirmed that even at a scale of 10 L, favorable

growth is possible in a seawater medium.

[0167]

[Example 19]

HKN1 (haploid) was cultured in an MA medium, an MA + 0.3 M of NaC

medium, or an MA + 0.5 NaCl medium for 7 days. When suspended in 2 mL of a

medium, the culture solution containing algal cells in an amount of OD7 5 0 = I was

recovered in 3 microtubes. The culture solution was centrifuged (1,500 x g, 5 minutes),

the supernatant was removed, and the pellets were recovered. Each algal cell recovered

as pellets was treated by any of the following Treatments I to 3.

Treatment 1: Suspension was performed in 2 mL of an MA medium, and

shaking was performed with vortex for 10 minutes.

Treatment 2: Suspension was performed in 2 mL of the same medium used for

the culture, and shaking was performed with vortex for 10 minutes.

Treatment 3: After suspension in 0.1 mL of the same medium used for the

culture, freezing was performed at -196°C, dilution was performed to be 2 mL in the

same medium used for the culture, and shaking was performed with vortex for 10

minutes.

[0168]

When algal cells break, the cell contents are released into the medium and

phycocyanin (PC) is exposed to the acidic medium and fades. It was assumed that

100% of the algal cells were killed by the freezing treatment (Treatment 3), and the death

rate of the algal cells by Treatment I was calculated by the following formula.

[0169]

Death rate (%) {(PC concentration after Treatment 2 - PC concentration after

x 100

[0170]

The PC concentration was measured by measuring the 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 by the following formula.

PC concentration (pg/ml) = 138.5 x A 620 - 35.49 x A6 78

[0171]

The results are shown in Table 30.

CI r

c11

00oC' 00

cnCIA tf

C5 4

(z) 00C~ CL

[0173]

As shown in Table 30, it was confirmed that the algal cells cultured in an MA

+ 0.3 M of NaCl medium or an MA + 0.5 M of NaCl medium became fragile due to

resuspension in an MA medium.

[0174]

[Example 20]

The death rate of algal cells by Treatment I was calculated by the same method

as that in Example 19 except that 1OD was used instead of HKN1 (haploid). The results

are shown in Fig. 31.

cl,00 o' C'I 00

C)4 C)f)(f

~C 066z

CA 0

C) C CI N

Lf)Ch r00

\Crq

ft CL p0

[0176]

As shown in Table 31, it was confirmed that the algal cells cultured in the MA

+ 0.3 M of NaCl medium or the MA + 0.5 M of NaCl medium became fragile due to

resuspension in the MA medium. In addition, the death rate of algal cells cultured in

the MA + 0.5 M of NaCl medium was higher than that of algal cells cultured in the MA

+ 0.3 M of NaCl medium.

[0177]

[Example 21]

The death rate of algal cells by Treatment I was calculated by the same method

as that in Example 19 except that G. partita (haploid) was used instead of HKN1

(haploid). The results are shown in Fig. 32.

C) 0)

Z

I00 co

C5

4. c'nC

0 00

cf)

o 000

U 0) CIA -~C C,)00

I>C p \CC 0

[0179]

As shown in Table 32, it was confirmed that the algal cells cultured in the MA

+ 0.3 M of NaCl medium or the MA + 0.5 M of NaCl medium became fragile due to

+ 0.3 M of NaCl medium.

[0180]

[Example 22]

The death rate of algal cells by Treatment I was calculated by the same method

as that in Example 19 except that G. sulphuraria (haploid) was used instead of HKNI

(haploid). The result are shown in Fig. 33.

Z tr< ) r

(11 00 00

*~ ~ 00 +

C)

r- 0 0

C Or C)C

't C p0

0fc

[0182]

As shown in Table 33, it was confirmed that the algal cells cultured in the MA

+ 0.3 M of NaCl medium or the MA + 0.5 M of NaCl medium became fragile due to

+ 0.3 M of NaCl medium.

[Industrial Applicability]

[0183]

According to the present invention, a method for culturing a freshwater

microalga capable of favorably growing a freshwater microalga in a low pH and high

sodium ion concentration environment, a freshwater microalga that can favorably grow

in a low pH and high sodium ion concentration environment, and a method for producing

the freshwater microalga are provided.

According to the present invention, since a freshwater microalga can be cultured

in large quantities in inexpensively available seawater, it is useful for the production of

useful substances using algae. In addition, if freshwater microalgae are cultured in large

quantities, it is expected that the freshwater microalgae absorb carbon dioxide in the

atmosphere.

Claims

[CLAIMS]

[Claim 1] A method for culturing a haploid of a freshwater microalga belonging to Cyanidiophyceae, comprising: a pre-culture step of culturing a haploid of a freshwater microalga belonging to Cyanidiophyceae at a culture temperature of 15°C to 60°C in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.25 to 0.35 M, and a main culture step of culturing the haploid of the freshwater microalga belonging to Cyanidiophyceae after the pre-culture step in a medium prepared such that a sodium ion concentration is 0.5 to 1 M and a hydrogen ion concentration is pH 1.0 to 6.0.
[Claim 2] The method for culturing a haploid of a freshwater microalga according to Claim 1, wherein 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 such that a hydrogen ion concentration is pH 1.0 to 6.0.
[Claim 3] A method for producing a haploid of a freshwater microalga belonging to Cyanidiophyceae that grows in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.5 M or more, the method comprising in the following order: a step of culturing a haploid of a freshwater microalga belonging to Cyanidiophyceae that does not grow in a medium having a sodium ion concentration of 0.5 M or more in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.25 to 0.35 M, and a step of culturing the haploid of the freshwater microalga belonging to Cyanidiophyceae in a medium prepared such that a hydrogen ion concentration is pH 1.0 to 6.0 and a sodium ion concentration is 0.5 M or more.
[Claim 4] The method for producing a haploid of a freshwater microalga according to Claim 3, wherein the haploid of the freshwater microalga belonging to Cyanidiophyceae is a haploid of microalga belonging to the genus Cyanidium.
[Claim 5] A haploid of microalga belonging to Cyanidiophyceae obtained by the method according to Claim 3 or 4, wherein a value calculated by the following Formula (1) in a case of culturing in a static state for 7 days at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and a continuous light of an illuminance of 60 mol/m2s in an MA medium prepared such that a hydrogen ion concentration is pH 2.0 and a sodium ion concentration is 0.5 M is 2 or more, and a value calculated by the following Formula (1) in a case of culturing in a static state for 7 days at a culture temperature of 42°C, a carbon dioxide concentration of 2%, and a continuous light of an illuminance of 60 mol/m2 s in an MA medium prepared such that a hydrogen ion concentration is pH 2.0 is less than 2. (OD75 value after 7 days from time of culture start - OD75 value at time of culture start)/(7 x OD75 value at time of culture start) (1)
[Claim 6] The haploid of microalga belonging to Cyanidiophyceae according to Claim 5, wherein cells are ruptured in an isotonic solution having a hydrogen ion concentration of pH 7 or in a distilled water.
[Claim 7] The haploid of microalga belonging to Cyanidiophyceae according to Claim 5 or 6, wherein in a case where algal cells are dried and the cells after the drying treatment are suspended in an isotonic solution having a pH 7, the cells are ruptured.
[Claim 8] A method for culturing a haploid of microalga belonging to Cyanidiophyceae, comprising: culturing the haploid of microalga belonging to Cyanidiophyceae according to any one of Claims 5 to 7 in a medium prepared by adding at least a nitrogen-containing salt, a phosphorus-containing salt, and an iron-containing salt to seawater such that a hydrogen ion concentration is pH 1.0 to 6.0.
[Claim 9] A method for culturing a haploid of a microalga belonging to Cyanidiophyceae, comprising: culturing the haploid of microalga belonging to Cyanidiophyceae according to any one of Claims 5 to 7 outdoors.