WO2022234788A1

WO2022234788A1 - Method for culturing algae

Info

Publication number: WO2022234788A1
Application number: PCT/JP2022/018908
Authority: WO
Inventors: 進也宮城島; 俊亮廣岡; 崇之藤原; 諒杉本
Original assignee: 大学共同利用機関法人情報・システム研究機構
Priority date: 2021-05-07
Filing date: 2022-04-26
Publication date: 2022-11-10

Abstract

The present invention relates to a method for culturing algae, the method comprising culturing algae in a culture medium containing an organic carbon source under conditions that reduce the NADH/NAD+ ratio in the cells of the algae, wherein the algae are at least one kind selected from the group consisting of unicellular red algae, green algae, diatoms, and blue-green algae.

Description

Algae culture method

The present invention relates to a method for culturing algae.
This application claims priority based on Japanese Patent Application No. 2021-079124 filed in Japan on May 7, 2021, the content of which is incorporated herein.

Because algae have a high carbon dioxide fixing capacity compared to land plants, and because they do not compete with agricultural products for habitat, some algae species are cultivated in large quantities and used in feeds, functional foods, and cosmetics. It is used industrially as a material.

　When using algae for industrial purposes, it is preferable to adopt culture conditions that allow easy and efficient cultivation due to factors such as cost and ease of culture management.

For example, Non-Patent Document 1 and Non-Patent Document 2 show that Cyanidioschyzon merolae can be heterotrophically cultured.

In heterotrophic culture, it is thought that algae can be cultivated with high efficiency by supplying an organic carbon source. However, in some cases, such as Cyanidioschizon mellolae reported in Non-Patent Document 1 and Non-Patent Document 2, the growth is suppressed under heterotrophic conditions more than under autotrophic conditions.

The present invention has been made to solve the above problems, and adds an organic carbon source of at least one type of algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria. It is an object of the present invention to provide a culture method that improves the growth of algae in culture in a medium that has undergone a process of culturing.

The present invention includes the following aspects.
(1) culturing algae in a medium supplemented with an organic carbon source under conditions that reduce the intracellular NADH/NAD ⁺ ratio of said algae;
A method for culturing algae, wherein the algae is at least one type of algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria.
(2) The method for culturing algae according to (1) above, wherein the culture under the conditions is aerobic culture.
(3) The method for culturing algae according to (2) above, wherein in the aerobic culture, the oxygen concentration of the gas blown into the medium in which the algae are cultured exceeds the oxygen concentration of normal air.
(4) The above (2) or (3), wherein the medium is a liquid medium, and the amount of gas blown into the liquid medium per 1 mL of the liquid medium is 1 to 400 mL/min·mL. A method for cultivating algae.
(5) The culture medium under the conditions contains, as a nitrogen source, at least one selected from the group consisting of nitrates, nitrites, nitrates (NO ₃ ⁻ ), and nitrite ions (NO ₂ ⁻ ). The method for culturing algae according to any one of (1) to (4) above, which is carried out using
(6) The method for culturing algae according to any one of (1) to (5) above, wherein the culturing is performed in the dark.
(7) The method for culturing algae according to any one of (1) to (6) above, wherein the algae belong to the genus Cyanidioschyzon.

According to the present invention, at least one type of algae selected from the group consisting of unicellular red algae, green algae, diatoms, and cyanobacteria is cultivated in a medium supplemented with an organic carbon source, and the cultivation improves the growth of the algae. can provide a method.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram explaining an example of the process of NADH production|generation and oxidation in a cell. It is the result of measuring the intracellular NADH/NAD ⁺ ratio of Cyanidioschizon mellorae cultured under each culture condition. Fig. 2 is a graph showing the results of comparing the growth conditions of Cyanidioschizon mellolae between the swirling culture and the aerobic culture (1). 4 is a graph showing the growth of Cyanidioschizon mellolae in aerobic culture (2). 1 is a graph showing the state of growth of Cyanidioschizon mellolae after passage in rotary culture. 1 is a graph showing the state of growth of Cyanidioschizon mellolae after passage in aerobic culture. Fig. 3 is a graph showing the results of comparing the growth conditions of Cyanidioschizon mellorae between aerobic culture in a normal atmosphere and aerobic culture in an oxygenated atmosphere. Fig. 3 is a graph showing the results of comparing the growth conditions of Cyanidioschizon mellorae between aerobic culture in a normal atmosphere and aerobic culture in an oxygenated atmosphere. Fig. ₃ is a graph showing the results of comparing the growth conditions of Cyanidioschizone mellorae in orbital cultures in which the nitrogen source contained in the medium was changed from NH4 ⁺ ^to _NO3- . Fig. ₃ is a graph showing the results of comparing the growth conditions of Cyanidioschizone mellorae in aerobic cultures in which the nitrogen source contained in the medium was changed from NH4 ⁺ ^to _NO3- .

An embodiment of the method for culturing algae of the present invention will be described below.

≪Method of cultivating algae≫
A culture method of an embodiment comprises culturing algae in a medium supplemented with an organic carbon source under conditions that reduce the intracellular NADH/NAD ⁺ ratio of said algae, wherein said algae are unicellular red This is the method using at least one type of algae selected from the group consisting of algae, green algae, diatoms and cyanobacteria.

“Culturing in a medium containing an organic carbon source” means culturing so that algae grow by assimilating the organic carbon source. Growing by assimilating the above organic carbon sources means that at least a part of the organic carbon sources utilized by the algae to be cultured is supplied from the outside to the medium, other than the organic carbon sources biosynthesized by the algae themselves through photosynthesis. It means that it is supplied to and assimilated. "Culturing in a medium supplemented with an organic carbon source" is a concept that includes culturing under "heterotrophic conditions" and "mixed trophic conditions.""Heterotrophicconditions" refer to conditions in which there is no light irradiation and an organic carbon source is present. "Mixotrophic conditions" refer to conditions in which there is light irradiation and an organic carbon source is present. In addition, "autotrophic conditions" refer to conditions in which there is light irradiation and no organic carbon source is present. The organic carbon source referred to here is an organic carbon source newly added to the culture system, which does not correspond to the organic carbon source biosynthesized by photosynthesis by the cultured algae themselves or the organic carbon source derived therefrom.
In the culture method of the embodiment, the algae may be cultured under heterotrophic conditions or mixed trophic conditions.

In conventional culture methods, for example, Cyanidioschizon mellolae (hereinafter also simply referred to as "schizon") stops growing after 6 to 7 cell divisions in the dark (under heterotrophic conditions). There is an upper limit to the algae density that can be reached. Since the growth also stopped even after passage, it is considered that the growth stop is not caused by an increase in algae density or depletion of nutrient sources. In other words, in the conventional culture method, when cultured under heterotrophic conditions, there is a problem that long-term culture cannot be maintained by subculturing.

According to the studies of the present inventors, as shown in the examples below, the value of the NADH/NAD ⁺ ratio in the cells of schizon cultured under "heterotrophic conditions" and "mixed nutrition conditions" was "independent It was found that the ratio of NADH/NAD ⁺ in the cells of schizomon cultured under "nutrient conditions" was higher than that in the cells.
Based on this finding, it is possible to improve the growth of algae by culturing algae in a medium supplemented with an organic carbon source under conditions that reduce the NADH/NAD ⁺ ratio in algae cells. I found a headline.

The improved growth of the algae is under "heterotrophic conditions" or "mixotrophic conditions" compared to conventional culture methods that do not apply conditions that reduce the NADH/NAD ⁺ ratio in algae cells. When cultured by a culture method applying conditions that reduce the NADH/NAD ⁺ ratio in algae cells, the growth rate of the algae is improved, the maximum attainable algae density is improved, and passage is included. It can be confirmed by achieving any one or more of the following:

FIG. 1 is a schematic diagram illustrating an example of the process of intracellular NADH production and oxidation. Regarding the production of NADH, for example, in the process of utilizing organic carbon sources such as glucose and glycerol in glycolysis and TCA cycle, NAD ⁺ is reduced to produce NADH.
Regarding the production of NAD ⁺ , for example, NADH is consumed by respiration or anaerobic fermentation (not shown) to produce NAD ⁺ .
Therefore, in culture under "heterotrophic conditions" or "mixotrophic conditions" with the addition of organic carbon sources, organic carbon source consumption (NADH production) and/or respiration restriction (NADH consumption suppression) is likely to occur. It is considered that the amount of intracellular NADH increases and an imbalance of NADH/NAD ⁺ occurs.

The method for culturing algae of the embodiment is a culture in a medium supplemented with an organic carbon source, which helps consumption of intracellular NADH and reduces the value of the intracellular NADH/NAD ⁺ ratio, thereby inhibiting the growth of algae. It is an improvement. The mechanism by which algae growth is improved by reducing the amount of intracellular NADH is not necessarily clear. However, for example, it is known that when the amount of NADH increases, glycolysis and the TCA cycle, which are involved in the production of NADH, are suppressed. It is inferred to be improved.

"Conditions for reducing the intracellular NADH/NAD ⁺ ratio" are not particularly limited, but from the viewpoint of being easy to implement and having excellent effects of improving growth, the following two conditions are used. can be exemplified.
1) increasing the oxygen supply to the medium; 2) using a medium containing electron acceptors;

As the above 2), the following is preferable.
2-1) Using a medium containing a specific nitrogen source

As shown in the examples below, 1) only by increasing oxygen supply to the medium, an excellent effect of improving growth can be obtained. Also, 2-1) an excellent effect of improving growth can be obtained only by using a medium containing a specific nitrogen source. Furthermore, by performing both of 1) increasing the supply of oxygen to the medium and 2-1) using a medium containing a specific nitrogen source, a more excellent growth improvement effect can be obtained.
The details of 1) and 2) above will be described below.

1) Oxygen supply It is believed that increasing the oxygen supply promotes, for example, the following reactions, thereby promoting the consumption of NADH within algae cells.
2H ⁺ + 1/2O ₂ + 2NADH → H ₂ O + 2NAD ⁺

The method for culturing algae of the embodiment is an example of the method of increasing oxygen supply to the medium in 1) above, which is exemplified as "conditions for reducing the intracellular NADH/NAD ⁺ ratio", wherein the culture is aerobic culture. Illustrate a method.

The method for culturing algae of the embodiment is preferably a method including aerobic culture of the algae in a medium supplemented with an organic carbon source.

That is, the method for culturing algae of the embodiment includes aerobic culture of the algae in a medium supplemented with an organic carbon source, and the algae is selected from the group consisting of unicellular red algae, green algae, diatoms, and cyanobacteria. is preferably at least one type of algae.

Here, aeration culture is a method of blowing gas (also called aeration or bubbling) into the medium for culturing algae to increase the amount of oxygen supplied to the medium. From the viewpoint of efficient oxygen supply, an air bubble generator may be used to refine the air bubbles of the gas to be aerated. Preferably, the medium to be aerated is a liquid medium.

The gas to be ventilated may be a gas containing oxygen, and from the viewpoint of ease of implementation, it may be normal air. The normal atmospheric oxygen concentration is about 21% by volume. The gas to be ventilated includes, for example, a gas having an oxygen concentration of 21% by volume or more.

The flow rate of aerated gas may be, for example, 50 to 20000 mL/min, 50 to 10000 mL/min, 100 to 1000 mL/min, 200 to 900 mL with respect to 50 mL of liquid medium. /min, 300-800 mL/min, 400-700 mL/min. The aeration amount per 1 mL of the liquid medium may be, for example, 1 to 400 mL/min-mL, 1 to 200 mL/min-mL, 2 to 20 mL/min-mL, 4 to It may be 18 mL/min·mL, 6 to 16 mL/min·mL, or 8 to 14 mL/min·mL.

The oxygen concentration of the gas to be ventilated is not particularly limited, but it is preferable to ventilate a gas having an oxygen concentration higher than that of the normal atmosphere because the oxygen supply efficiency can be easily improved.
In the method for cultivating algae of the embodiment, it is preferable that the oxygen concentration of the gas that is passed through the medium in which the algae are cultured is higher than the oxygen concentration of the normal atmosphere.

As an example of the oxygen concentration of the gas to be vented, it may be a gas containing 25 to 100% by volume of oxygen, or a gas containing 30 to 90% by volume of oxygen, relative to the total volume of the gas. , a gas containing 40 to 80% by volume of oxygen.

As the gas, a mixed gas of air and oxygen obtained by adding oxygen to the air can be exemplified.
An example of the gas to be vented may be atmospheric oxygenated gas containing 25 to 100 vol. It may be a gas in which oxygen is added to the atmosphere, or a gas in which oxygen is added to the atmosphere so as to contain 40 to 80% by volume of oxygen.

A method for culturing algae of an embodiment includes aeration culturing the algae in a medium supplemented with an organic carbon source, and the aeration culture is carried out, for example, by adding 25 to 100% by volume of oxygen to the total volume of gas to be aerated. A gas containing is aerated at a flow rate of 50 to 20000 mL / min for 50 mL of liquid medium (1 to 400 mL / min · mL as an aeration rate per 1 mL of liquid medium).
A gas containing 30 to 90% by volume of oxygen with respect to the total volume of gas to be aerated is 100 to 1000 mL / min for 50 mL of liquid medium (2 to 20 mL / min · mL as aeration rate per 1 mL of liquid medium) The culture may be aerated at a flow rate of
A gas containing 40 to 80% by volume of oxygen with respect to the total volume of gas to be aerated is 300 to 800 mL / min for 50 mL of liquid medium (6 to 16 mL / min · mL as aeration rate per 1 mL of liquid medium) The culture may be aerated at a flow rate of

An example of the amount of dissolved oxygen (DO, Dissolved Oxygen) of the medium at 40° C. may be 0.5 mg/L or more, or 6.3 mg/L or more. Although the upper limit of the dissolved oxygen content of the medium at 40° C. is not particularly limited, it may preferably be 6.3 to 30 mg/L, more preferably 13 to 27 mg/L, More preferably, it may be 19-24 mg/L.
A medium containing a dissolved oxygen content within the above numerical range can effectively improve the growth of the algae.

Here, aerobic culture was mainly exemplified as an example of efficient oxygen supply to the medium. Cultivation or the like may be performed to increase oxygen supply to the medium.

<Culture medium>
The medium used in the algae culture method of the embodiment is preferably a liquid medium as described above, but may be a solid medium and preferably a liquid medium.

The composition of the medium is not particularly limited, and an appropriate one may be selected according to the type of algae to be cultured. Examples of the medium include inorganic salt media containing nitrogen sources, phosphorus sources, iron sources, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, etc.) and the like. For example, nitrogen sources include ammonium salts, nitrates, nitrites, urea, amines, and the like; phosphorus sources include phosphates, phosphites, and the like; iron sources include iron chloride, iron sulfate, iron citrate, and the like. Specific examples of the medium include, for example, 2 × Allen medium (Allen MB. Arch. Microbiol. 1959 32:270-277.), M-Allen medium (Minoda A et al. Plant Cell Physiol. 2004 45: 667-71 ), MA2 medium (Ohnuma M et al. Plant Cell Physiol. 2008 Jan;49(1):117-20.). In this specification, the M-Allen medium is sometimes referred to as "MA medium".

Examples of organic carbon sources in media containing organic carbon sources include sugar alcohols, sugars, and amino acids. Sugar alcohols include, for example, glycerol. Sugars include, for example, glucose, mannose, fructose, sucrose, maltose, lactose sugars. When the algae belong to the class Idycogome, examples of the organic carbon source include glucose and glycerol, with glycerol being preferred.

2) Electron acceptor In the method for culturing algae of the embodiment, a medium containing an electron acceptor can be used as 2) the above-mentioned “condition for reducing the intracellular NADH/NAD ⁺ ratio”.
A method for culturing algae of an embodiment includes culturing the algae in a medium to which an organic carbon source is added, and the medium preferably contains an electron acceptor.
As another aspect, the method for culturing algae of the embodiment is preferably a method including culturing the algae in a medium supplemented with an organic carbon source and an electron acceptor.
Examples of the electron acceptor include pyruvic acid and malic acid.

2-1) Nitrogen source Further, as the electron acceptor in 2) above, a specific nitrogen source can be exemplified, and the specific nitrogen source includes nitrate, nitrite, nitrate ion (NO ₃ ⁻ ), and nitrite ion ( NO ₂ ⁻ ) is preferably used for the culture.
Nitrates contained in the medium are usually ionized to produce nitrate ions. Nitrous acid formulated in the medium usually ionizes to produce nitrite ions.

A method for culturing algae of an embodiment includes culturing the algae in a medium to which an organic carbon source is added, and the medium contains nitrates, nitrites, nitrates (NO ₃ ⁻ ), and nitrite ions as nitrogen sources. (NO ₂ ⁻ ), and the algae is preferably at least one algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria.

A method for culturing algae according to an embodiment includes aerobic culture of the algae in a medium supplemented with an organic carbon source, wherein the medium contains nitrate, nitrite, nitrate ion (NO ₃ ⁻ ), and nitrite as nitrogen sources. It is preferable that the algae include at least one selected from the group consisting of ions (NO ₂ ^- ), and the algae be at least one algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria.

The nitrogen source contained in the medium substantially contains only at least one selected from the group consisting of nitrates, nitrites, nitrate ions (NO ₃ ⁻ ), and nitrite ions (NO ₂ ⁻ ). is preferred.

Using a medium containing the above specific nitrogen sources promotes the consumption of NADH in cultured algae. This is because, in order to utilize the above nitrate ions, it is necessary to convert nitrate ions (NO ₃ ⁻ )→nitrite ions (NO ₂ ⁻ )→ammonium ions (NH ₄ ⁺ ), and in this process NADH is This is thought to be due to consumption.

It is more preferable to perform the culture using a medium containing at least one selected from the group consisting of nitrate and nitrate ion (NO ₃ ⁻ ) as a nitrogen source, since more NADH consumption can be expected. That is, it is preferable that the nitrogen source contained in the medium substantially contains only nitrate and/or nitrate ion. For example, the medium is preferably free of ammonium salts and ammonium ions ₍ NH4 ⁺ ).

Conventionally, when cultivating Cyanidioschizon mellorae in a bright place, it was known that the growth rate was lower when nitrate ions were used as the nitrogen source than when ammonium ions were used as the nitrogen source (S. Imamura et al. al., Plant Cell Physiol. 51(5): 707-717 (2010).
Based on such findings, it is an unexpected result that the growth of algae is improved by using the medium containing the above-described specific nitrogen source under heterotrophic conditions.

_Nitrate ^ions ₍ _NO ₃ ^- ⁾ ^and The total amount of nitrite ions (NO ₂ ⁻ ) is preferably 50% or more, more preferably 80% or more, on a molar basis, and is substantially nitrate ions (NO ₃ ⁻ ) or nitrite ions ( NO ₂ ⁻ ) only is more preferred.

As described above, 1) aerobic culture was exemplified as an example of increasing oxygen supply to the medium, but as shown in the examples below, for example, 2) using a medium containing a specific nitrogen source only Even if it is, the effect of the outstanding growth improvement is acquired. Therefore, the culture method when adopting "conditions that reduce the intracellular NADH/NAD ⁺ ratio" other than aerobic culture is not limited to aerobic culture, and a method generally used as a culture method for algae can be used as appropriate. can be used. Specific examples include static culture, rotary culture (100 to 200 rpm, etc.), and the like.

In addition, a combination with aerobic culture may be performed, and examples of aerobic culture include the methods exemplified above.

The method for culturing algae of the embodiment includes culturing algae in a medium to which an organic carbon source is added, and the air flow rate is 50 to 10,000 mL/min for 50 mL of liquid medium (1 to 200 mL/min as an aeration rate per 1 mL of liquid medium).・It is preferable that the method is a method of performing an aeration culture in which aeration is performed at a flow rate of 1 mL), and the culture is performed using nitrate, nitrite, nitrate ion (NO ₃ ⁻ ), and nitrite ion (NO ₂ ⁻ ) as a nitrogen source. A method using a medium containing at least one selected from the group consisting of is preferred.

A method for cultivating algae of an embodiment comprises culturing algae in a medium supplemented with an organic carbon source, wherein the culture aerates a gas containing 25 to 100% by volume of oxygen with respect to the total volume of gas, It is preferable to perform aerobic culture in which 50 mL of liquid medium is aerated at a flow rate of 50 to 10000 mL / min (1 to 200 mL / min · mL as an aeration rate per 1 mL of liquid medium), and the culture is performed by It is preferable to use a medium containing at least one selected from the group consisting of nitrates, nitrites, nitrates (NO ₃ ⁻ ) and nitrite ions (NO ₂ ⁻ ) as a nitrogen source.

The pH of the medium can be determined appropriately according to the algae to be cultured. For example, in the case of algae belonging to the class Idycogome, pH 1 to 6 is preferable, pH 1 to 5 is more preferable, and pH 1 to 3 is even more preferable, since they can grow better under acidic conditions.

<Other culture conditions>
The temperature conditions for the culture may be appropriately selected according to the type of algae. Generally, the culture temperature can be 15 to 60°C, preferably 15 to 50°C, more preferably 30 to 50°C. When the algae belong to the class Idycogome, the culture temperature is preferably 30 to 50°C.

The _CO2 conditions in culture may be appropriately selected according to the type of algae. Generally, 0.04-5 vol% CO ₂ conditions can be exemplified. 0.04-3 vol% CO ₂ conditions are preferred when the algae belong to the class Idycogome.

Light conditions in the pre-culture may be appropriately selected according to the type of algae. Generally, 5 to 2000 μmol/m ² s can be exemplified. When the algae belong to the class Idycogome, it is preferably 5 to 1500 μmol/m ² s, more preferably 5 to 100 μmol/m ² s. The light condition may be continuous light, or a light-dark cycle (10L:14D, etc.) may be provided. Culturing may be performed under natural light.

From the viewpoint of facilitating the provision of heterotrophic conditions or mixed nutrition conditions, the light conditions in the culture ^are preferably below the light compensation point. It may be ˜10 μmol/m ² s, and may be 0-5 μmol/m ² s.

In the algae culture method of the embodiment, the culture is preferably performed in a dark place. Here, "dark place" means less than 5 μmol/m ² s, preferably 0 μmol/m ² s.

In a dark place, algae are cultivated under heterotrophic conditions, consume organic carbon sources (NADH production), and photosynthesis is also restricted, so respiration restriction (suppression of NADH consumption) is likely to occur. Therefore, the effect of improving the growth of algae by reducing the intracellular NADH/NAD ⁺ ratio is exhibited more effectively.

During the culture period, the algae may be subcultured as appropriate. When grown in liquid medium, passage intervals include, for example, 10 to 50 days, or 15 to 30 days.

The culture period is not particularly limited, but the culture period including passage is preferably 30 days or longer, more preferably 40 days or longer, and even more preferably 80 days or longer. The culture period in the dark, including passage, is preferably 30 days or more, more preferably 40 days or more, and even more preferably 80 days or more in a continuous dark period.

<Algae>
Algae to be cultured in the culture method of the embodiment is at least one type of algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria, and unicellular red algae are preferred.

Unicellular green algae include algae belonging to the genus Chlamydomonas. Unicellular diatoms include algae belonging to the genus Phaeodactylum. Unicellular blue-green algae include algae belonging to the phylum Cyanobacteria, preferably algae belonging to the genus Synechocystis, Synechocystis sp. PCC 6803 is more preferred.

Unicellular red algae include, for example, algae belonging to the class Cyanidiophyceae. The class Cyanidiphyta is taxonomically classified into the phylum Rhodophyta and the class Cyanidiophyceae. Three genera, Cyanidioschyzon, Cyanidium, and Galdieria, are currently classified in the class Idycogome.

The algae are preferably algae belonging to the genus Cyanidioschyzon, more preferably Cyanidioschyzon mellolae.

Among the algae, the culture method of the present embodiment is preferably applied to algae whose growth is more suppressed when cultured under heterotrophic conditions or mixed nutrition conditions than when cultured under autotrophic conditions. Here, the above-mentioned "growth is suppressed when cultured under heterotrophic conditions or mixed nutrition conditions" means that growth is suppressed (including inability to grow) compared to the case of culture under autotrophic conditions. , the growth rate is reduced, the maximum algae density is improved, and the growth period including passage is shortened.

In another aspect, the culturing method of the present embodiment has a higher intracellular NADH/NAD ⁺ value when cultured under heterotrophic conditions or mixed trophic conditions than when cultured under autotrophic conditions among the algae. It is preferable to apply to algae that increases. The intracellular NADH/NAD ^{+ value when cultured under heterotrophic conditions or mixed nutrition conditions is greater than the intracellular NADH/NAD +} ^value when cultured under autotrophic conditions, and is heterotrophic The intracellular NADH/NAD ^{+ value when cultured under conditions or mixed nutrition conditions is 1.3 times or more the intracellular NADH/NAD +} ^value when cultured under autotrophic conditions Preferably, it is 1.5 times or more. The intracellular NADH/NAD ^{+ value when cultured under heterotrophic or mixotrophic conditions is greater than the intracellular NADH/NAD +} ^value when cultured under autotrophic conditions, and NADH/NAD Algae with a ⁺ value of 0.05 or more are preferred, algae with a + value of 0.05 to 0.5 are more preferred, and algae with a + value of 0.1 to 0.4 are even more preferred. The intracellular NADH/NAD ⁺ value can be obtained by the method described in Examples below.

In addition, algae to which the culture method of the present embodiment is applied may be isolated from the environment or obtained from culture collections and the like. For example, Cyanidioschizone mellorae has been collected from the National Institute for Environmental Studies Microbiology Collection Facility (16-2 Onogawa, Tsukuba City, Ibaraki Prefecture, Japan), American Type Culture Collection (ATCC; 10801 University Boulevard Manassas, VA 20110 USA). ), etc.

In addition, algae to which the culture method of the present embodiment is applied are not limited to those isolated from nature, and may be natural algae mutated. Mutations may be naturally occurring or artificially occurring. For example, Cyanidioschizon mellorae has a small genome size (about 16 Mbp), and the genome sequence has been completed (Matsuzaki M et al., Nature. 2004 Apr 8;428(6983):653-7 ), and are susceptible to genetic modification. Therefore, for example, the culture method of the present embodiment may be applied to a transformant of Cyanidioschizon mellolae produced by genetic modification (for example, a transformant with enhanced nutritional components). In addition, the culture method of the present embodiment may be applied to transformants of other algae as long as they can be genetically modified.

According to the method for culturing algae of the embodiment, the growth of the algae can be improved in the culture in the medium to which the organic carbon source is added.
According to the method for culturing algae of the embodiment, it is possible to improve the growth rate of algae whose growth rate is lower under heterotrophic conditions than under autotrophic conditions.
According to the method for culturing algae of the embodiment, it is possible to improve the maximum algal density of algae whose maximum algae density is lower under heterotrophic conditions than under autotrophic conditions.

The method for culturing algae of the embodiment enables subculture of algae, which has been difficult to maintain for a long period of time under heterotrophic conditions, and is an epoch-making method that makes a great contribution to the cultivation of a wide range of algae. culture method.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Experiment 1 Confirmation of NADH/NAD ⁺ ratio The inventors focused on the intracellular redox balance (NADH/NAD ⁺ ratio) as a factor in the low growth ability of Cyanidioschizone mellorae in the dark. . Therefore, the intracellular NADH/NAD ⁺ ratio was determined under each culture condition.

Cyanidioschizon mellolae strain 10D (NIES-3377, maintained at the National Institute of Genetics, hereinafter referred to as "schizon") was grown using MA2 medium supplemented with 350 mM glycerol under the following autotrophic conditions. They were cultured under mixotrophic conditions or heterotrophic conditions, respectively.
Autotrophic condition: light intensity (25 μmol/m ² s), no addition of organic carbon source to MA2 medium Mixotrophic condition: light intensity (25 μmol/m ² s), addition of organic carbon source (glycerol) to MA2 medium Yes Heterotrophic conditions: Dark place (light intensity 0 μmol/m ² s), addition of organic carbon source (glycerol) to MA2 medium Each culture here was carried out in an incubator (IS 600, Yamato Scientific) maintained at 40°C. Using a bioshaker (NR-3, TAITEC), rotation culture (130 rpm rotary) was carried out under normal atmosphere.
The medium used is MA2 medium under autotrophic conditions, and MA2 + glycerol 350 mM medium under mixed and heterotrophic conditions (MA2 medium supplemented with glycerol so that the final concentration of glycerol is 350 mM. The following medium notation is the same. ) was used.
The initial inoculation amount was inoculated so that OD ₇₅₀ =0.2. Thereafter, culturing was performed for 5 days under autotrophic conditions and mixed nutrition conditions, and schisons were collected. Since the growth of schizons was poor under heterotrophic conditions, culturing was carried out under heterotrophic conditions for 2 days after culturing under mixed nutrition conditions for 5 days, and then schizons were collected.
The intracellular NADH/NAD ⁺ ratio of the harvested schizons was measured. NAD ⁺ /NADH Assay Kit, EnzyChrom (BioAssay Systems) was used for extraction and measurement, and absorbance at 565 nm was measured with a microplate reader (SYNERGY H1, BioTek).

Table 1 shows the composition of the MA2 medium.

The results are shown in FIG. Cyanidioschizon mellolae cells cultured under mixed and heterotrophic conditions showed an increased intracellular NADH/NAD ⁺ ratio value compared to cells under autotrophic conditions. became.

Based on the above findings, the following verification was performed to determine whether the growth ability of Schizon in the dark (heterotrophic conditions) can be improved by culturing under conditions that increase NADH consumption. rice field.

Experiment 2-1 Aeration culture Schizon was cultured under the following culture conditions for rotary culture or aeration culture, and the growth conditions were compared using the algae density as an index.

・ Spinning culture (40°C, dark, MA2 + glycerol 350 mM, 20 mL culture, 125 rpm, normal atmosphere)
- Aeration culture (1) (40°C, dark, MA2 + glycerol 350 mM, 50 mL culture, 600 mL air/min, normal air aeration)
- Aeration culture (2) (40°C, dark, MA2 + glycerol 350 mM, 150 mL culture, 500 mL air/min, normal air aeration)

(Aerobic culture (1) conditions)
・Container: test tube diameter 30mm length 200mm
・ Air pump: Water center SSPP-2S manufactured by Mizusaku Co., Ltd.
Air was passed from an air pump, through a silicon tube, through a flow meter, and through a 0.22 μm filter in that order, and was blown into the culture solution in the test tube used for culturing through the test tube containing pure water. A silicone stopper (No. 9) was used to connect the silicone tube and the test tube.
(Aerobic culture (2) conditions)
・Container: iboy wide mouth bottle (capacity 250mL)
・ Air pump: Non-noise W-1000 manufactured by Nippon Animal Yakuhin Co., Ltd.
Air passed through a flow meter from an air pump through a silicon tube was blown through a bottle containing pure water and through a 0.22 μm filter into the culture solution in the vessel used for culture. A silicone stopper (No. 11) was used to connect the silicone tube and the test tube.

(Measurement of algae density)
The culture solution was sampled and turbidity (OD ₇₅₀ ) was measured with a spectrophotometer.

Fig. 3A shows the results of comparing the growth conditions of Cyanidioschizon mellolae between the rotary culture and the aerobic culture (1). In the case of culturing in the usual rotary culture, the growth stopped about 20 days after the start of the culture, and the algal density reached a plateau.
On the other hand, in aerobic culture (1), when the aeration rate was increased, the period during which schizons could grow was longer than in the vortex culture, and the algal density achieved was approximately 4.6 times that in the vortex culture.

FIG. 3B shows the results showing the growth of Cyanidioschizon mellolae in the aerobic culture (2).
In the aerobic culture, compared to the aerobic culture (1), even if a larger culture vessel is used and the amount of aeration is reduced, the period during which schisons can grow is longer than in the rotary culture, and the algae density reached is also higher in the rotary culture. Approximately 7.5 times higher than

Experiment 2-2 Growth after passage in aerobic culture Schizon was subcultured under the same culture conditions as those for the spin culture or aerobic culture in Experiment 2-1 above. The inoculum amount for subculturing was set so that OD ₇₅₀ was about 0.4. Air was constantly blown into the liquid medium (forced aeration) during the aeration culture period.

・Swirl culture (40°C, dark, MA2 + glycerol 350 mM, 50 mL culture, 125 rpm, normal atmosphere)
・Aeration culture (40°C, dark, MA2 + glycerol 400 mM, 50 mL culture, 600 mL air/min, normal air aeration)
Multiple passages and multiple time points were performed.

Fig. 4 shows the results of culturing in a normal swirling culture. A part of the culture solution cultured for 20 days was suspended (diluted) in a new culture solution and the culture was continued (arrows in FIG. 4 indicate passages). In the rotary culture, the growth after subculturing was not good, and growth stopped about 15 days after subculturing.

FIG. 5 shows the results of aeration culture. A portion of the culture solution cultured for 21 days, 30 days, or 44 days was suspended (diluted) in a new culture solution to continue culturing (the arrows in FIG. 5 indicate passages). As shown in FIG. 5, in the aeration culture with increased aeration volume, good growth was confirmed even after multiple passages, and it was confirmed that the culture can be cultured for a total of 95 days.
In the conventional methods for culturing Schizon, there was a problem that it was difficult to subculture in a dark place, but it was confirmed that good subculture can be carried out by performing aerobic culture.

Experiment 2-3 Aeration culture at high oxygen concentration (1)
Schizon was cultured under the following culture conditions.
During the culture, gas was constantly blown into the liquid medium (forced aeration). In the aeration culture (oxygenated atmosphere) culture system, at the start of the culture, aeration was performed in normal atmosphere (O ₂ concentration 21% by volume), and after growth stagnation was observed, oxygen was added to the normal atmosphere, Aeration culture was performed by aerating a gas with a stepwise increase in oxygen concentration (O ₂ concentration 21% by volume→42% by volume→63% by volume→80% by volume). The time to change the oxygen concentration is the number of days after culturing shown in FIG.
In addition, aerobic culture (normal atmosphere) was also performed in normal atmosphere (O ₂ concentration 21% by volume) without changing the oxygen concentration, and the growth conditions were compared using the algae density as an indicator.

・Aerobic culture (oxygenated atmosphere) (40°C, dark place, MA2 + glycerol 400mM, 50mL culture, 400mL/min, using an oxygen generator and a gas mixer, aeration of oxygenated gas against normal atmosphere)
・Aeration culture (normal atmosphere) (40°C, dark place, MA2 + glycerol 400 mM, 50 mL culture, 400 mL air/min, normal atmosphere)

The results are shown in Figure 6. It was confirmed that the maximum algae density was improved when the aeration culture was performed in an oxygenated atmosphere.

Experiment 2-4 Aeration culture at high oxygen concentration (2)
In the aeration culture described above, at the start of culture, aeration was performed with normal air ( _O2 concentration of 21% by volume). Cultivation (oxygenated atmosphere) was performed ( _O2 concentration 42% by volume → 63% by volume → 80% by volume). The time to change the oxygen concentration is the number of days after culture shown in FIG.
In addition, aerobic culture (normal atmosphere) was also performed in normal atmosphere (O ₂ concentration 21% by volume) without changing the oxygen concentration, and the growth conditions were compared using the algae density as an index.

The results are shown in Figure 7. No difference was observed in the growth rate at the initial stage of the culture, but as the number of days of culture passed, it was confirmed that the maximum algae density was improved when the aerobic culture was carried out in an oxygenated atmosphere.

The above results indicated that it was possible to improve the maximum amount of schizon growth in the dark by increasing the amount of oxygen supplied.

Experiment 3-1 Change of nitrogen source (swirling culture)
The MA2+glycerol medium used in each of the above cultures contained ammonium ions (NH ₄ ⁺ ) as a nitrogen source, but glycerol was added to a medium (MA2 nitrate medium) in which the nitrogen source was changed to nitrate ions (NO ₃ ⁻ ). Schizon culture was carried out by swirl culture using the prepared medium, and the growth status was confirmed using the algae density as an index.

・NO ₃ ⁻ (40° C., dark place, MA2+glycerol nitrate 400 mM, 20 mL culture, 130 rpm, under normal atmosphere)
・NH ₄ ⁺ (40° C., dark, MA2+glycerol 400 mM, 20 mL culture, 130 rpm, under normal atmosphere)

Table 2 shows the composition of the MA2 nitrate medium.

The results are shown in FIG. Spinning culture in a medium containing nitrate ions (NO ₃ ⁻ ) as a nitrogen source showed a higher growth rate than spin culture in a medium containing ammonium ions (NH ₄ ⁺ ) as a nitrogen source.

Experiment 3-2 Change of nitrogen source (aerobic culture)
In Experiment 3-1 described above, schizon culture was performed under the same conditions as in Experiment 3-1, except that the rotation culture was used instead of the aeration culture.

・NO ₃ ^- (40°C, dark place, MA2 nitrate + glycerol 400 mM, 50 mL culture, 400 to 500 mL/min, normal air ventilation)
・NH ₄ ⁺ (40° C., dark place, MA2+glycerol 400 mM, 50 mL culture, 400-500 mL air/min, normal air ventilation)

The results are shown in FIG. The aerobic culture in a medium containing nitrate ions (NO ₃ ⁻ ) as a nitrogen source has a higher growth rate than the aerobic culture in a medium containing ammonium ions (NH ₄ ⁺ ) as a nitrogen source, and the maximum algae density has been reached. was improving.

From the above results, it was shown that the growth rate of schizon in a dark place can be improved by supplying nitrate ions (NO ₃ ^- ) as a nitrogen source.
In addition, the addition of nitrate ions (NO ₃ ⁻ ) as the nitrogen source in the aerobic culture improved the algae density more than the addition of nitrate ions (NO ₃ ⁻ ) as the nitrogen source in the rotary culture.
From this, it was suggested that the increase in oxygen supply and the use of nitrate ions (NO ₃ ⁻ ) each contributed to the improvement of the growth ability of schizon in the dark.

Each configuration and combination thereof in each embodiment is an example, and addition, omission, replacement, and other modifications of the configuration are possible without departing from the scope of the present invention. Moreover, the present invention is not limited by each embodiment, but is limited only by the scope of the claims.

Claims

culturing algae in a medium supplemented with an organic carbon source under conditions that reduce the intracellular NADH/NAD + ratio of said algae;
A method for culturing algae, wherein the algae is at least one type of algae selected from the group consisting of unicellular red algae, green algae, diatoms and cyanobacteria.
The method for culturing algae according to claim 1, wherein said culture under said conditions is aerobic culture.
The method for culturing algae according to claim 2, wherein in the aeration culture, the oxygen concentration of the gas blown into the medium in which the algae are cultured is an oxygen concentration that exceeds the oxygen concentration of normal air.
The method for culturing algae according to claim 2 or 3, wherein the medium is a liquid medium, and the amount of gas blown into the liquid medium per 1 mL of the liquid medium is 1 to 400 mL/min·mL.
The culture under the conditions uses the medium containing at least one selected from the group consisting of nitrate, nitrite, nitrate ion (NO 3 − ), and nitrite ion (NO 2 − ) as a nitrogen source. The method for culturing algae according to any one of claims 1 to 4, which is carried out.
The method for culturing algae according to any one of claims 1 to 5, wherein the culturing is performed in the dark.
The method for culturing algae according to any one of claims 1 to 6, wherein the algae belong to the genus Cyanidioschyzon.