Algae and oil producing method

Abstract

To provide a technique for improving production efficiency of a carbon component by algae in the presence of seawater salt.SOLUTION: Algae have oil production capability, are one or more strains selected from the group of Nannochloropsis gaditana MCNa1 strain (accession number: FERM BP-22470), Nannochloropsis gaditana MCNb1 strain (accession number: FERM BP-22471), Nannochloropsis gaditana MCNc1 strain (accession number: FERM BP-22472), Nannochloropsis gaditana MCNd1 strain (accession number: FERM BP-22473) and Nannochloropsis gaditana MCNe1 strain (accession number: FERM BP-22474), or have a 18SrDNA sequence which consists of a base sequence having 90% or more of sequence identity among the strains.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a method for producing algae and oil. More specifically, it relates to microalgae having an oil-producing ability and a method for producing oil using the same.

Background Art

[0002] Examinations have been actively conducted on the production of various carbon components by culturing algae. In the process of culturing algae, various useful carbon components are produced by the immobilization of CO 2 For example, Patent Document 1 discloses microalgae Chlamydomonas sp. MT-JE-SH-1 belonging to the genus Chlamydomonas, which grows at the salinity concentration of seawater, accumulates starch in cells, and produces ethanol from the intracellular starch by keeping it in a dark and anaerobic atmosphere.

[0003] On the other hand, Non-Patent Document 1 has examined the relationship between oil production using marine algae and the salt concentration during culturing, and it has been reported that the growth of algae is suppressed when the salt concentration increases.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The main object of the present disclosure is to provide a technique for improving the production efficiency of carbon components by algae in the presence of seawater salt.

Means for Solving the Problems

[0007] To solve the above problems, the present disclosure provides the following [1] - [8]. [1] Algae having the ability to produce oil and being any one or more strains selected from the following group, or having an 18S rDNA sequence consisting of a nucleotide sequence having 90% or more sequence identity with those strains. Algae. Nannochloropsis gaditana MCNa1 strain (Accession No.: FERM BP - 22470) Nannochloropsis gaditana MCNb1 strain (Accession No.: FERM BP - 22471) Nannochloropsis gaditana MCNc1 strain (Accession No.: FERM BP - 22472) Nannochloropsis gaditana MCNd1 strain (Accession No.: FERM BP - 22473) Nannochloropsis gaditana MCNe1 strain (Accession No.: FERM BP - 22474) [2] Algae being any one or more strains selected from the following group. Nannochloropsis gaditana MCNa1 strain (Accession No.: FERM BP - 22470) Nannochloropsis gaditana MCNb1 strain (Accession No.: FERM BP - 22471) Nannochloropsis gaditana MCNc1 strain (Accession No.: FERM BP - 22472) Nannochloropsis gaditana MCNd1 strain (Accession No.: FERM BP - 22473) Nannochloropsis gaditana MCNe1 strain (Accession No.: FERM BP - 22474) [3] Algae having an 18S rDNA sequence consisting of a nucleotide sequence having 90% or more sequence identity with any of the strains in [2] and having the ability to produce oil.

[0008] [4] A method for producing oil, comprising the step of culturing algae in a medium containing seawater salt, wherein the algae are any one or more strains selected from the following group, having an 18S rDNA sequence consisting of a nucleotide sequence having 90% or more sequence identity with those strains and having an oil-producing ability, production method. Nannochloropsis gaditana MCNa1 strain (Accession No.: FERM BP-22470) Nannochloropsis gaditana MCNb1 strain (Accession No.: FERM BP-22471) Nannochloropsis gaditana MCNc1 strain (Accession No.: FERM BP-22472) Nannochloropsis gaditana MCNd1 strain (Accession No.: FERM BP-22473) Nannochloropsis gaditana MCNe1 strain (Accession No.: FERM BP-22474) [5] The production method according to [4], further comprising the step of extracting oil from the algae. [6] The production method according to [4] or [5], wherein the culturing is performed under outdoor open conditions while maintaining preferential growth of the algae. [7] The production method according to any one of [4] to [6], wherein the pH of the medium is 7 - 10. [8] The production method according to any one of [4] to [7], wherein the culturing is performed in an atmosphere with a CO 2 concentration of 0.04 - 10%.

Advantages of the Invention

[0009] According to the present disclosure, a technique for producing oil using algae is provided.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0011] Hereinafter, preferred embodiments for carrying out the present disclosure will be described. Note that the embodiments described below show an example of typical embodiments of the present disclosure, and the scope of the present disclosure is not construed narrowly thereby.

[0012] 1. Oil-producing algae The oil-producing algae according to the present disclosure grow well in the presence of seawater salt and exhibit high oil-producing ability. In the present disclosure, "seawater salt" is obtained by evaporating seawater to dryness and means a substance containing various inorganic salts mainly composed of sodium chloride. Also, the "medium containing seawater salt" may be seawater, a concentrated solution or a diluted solution of seawater, or dissolved water of seawater salt. Further, the "medium containing seawater salt" may be artificial seawater. Artificial seawater is obtained by dissolving sodium chloride and other inorganic salts in distilled water or tap water imitating the composition of seawater. The artificial seawater may contain a pH adjuster to suppress changes in pH, and may be added with vitamins (thiamine, biotin, vitamin B12) or additional specific inorganic salts to promote growth and oil productivity.

[0013] The oil-producing algae according to the present disclosure can be, in one embodiment, any of the following strains. Nannochloropsis gaditana MCNa1 strain (Accession No.: FERM ABP-22470) Nannochloropsis gaditana MCNb1 strain (Accession No.: FERM ABP-22471) Nannochloropsis gaditana MCNc1 strain (Accession No.: FERM ABP-22472) Nannochloropsis gaditana MCNd1 strain (Accession No.: FERM ABP-22473) Nannochloropsis gaditana MCNe1 strain (Accession No.: FERM ABP-22474)

[0014] These strains have been internationally deposited with the International Patent Organism Depositary, National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (IPOD) (Room 120, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture), which is a depositary institution based on Articles 27-2 and 27-3 of the Implementing Regulations of the Patent Act and an international depositary authority based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, under the above accession numbers. The receipt date of the 5 strains is March 7, 2023.

[0015] The oil-producing algae according to the present disclosure can grow well in a medium with a seawater salt concentration of 3-4% by mass. In addition, the oil-producing algae according to the present disclosure can preferentially grow over other microorganisms even under outdoor open conditions where environmental fluctuations such as light and temperature are large and there is an influence of contamination by other microorganisms, etc. Here, the "outdoor open conditions" or "outdoor open culture" means culture conditions in the wild, where the culture environment of the algae (specifically, the culture solution of the algae) is not isolated from the natural environment in the wild (air, temperature, light, rainfall, bacterial flora in the air, etc.).

[0016] As the excellent growth property of the oil-producing algae according to the present disclosure, for example, when the inoculum amount is 0.1 to 0.4 g / L and the culture is started, the cell weight on the 10th day of culture can be 0.5 to 2 g / L, preferably 2 to 5 g / L, more preferably 5 to 10 g / L in dry weight.

[0017] In addition, as the excellent oil-producing ability of the oil-producing algae according to the present disclosure, for example, when starting with an inoculum amount of 0.1 to 0.4 g / L, the oil production rate from the start of culture to the 15th day can be 10 to 50 mg / L / day, preferably 50 to 200 mg / L / day, more preferably 200 to 1000 mg / L / day.

[0018] The measurement of the amount of oil can be carried out by methods known to those skilled in the art, and the methods are not limited. For example, it can be carried out as follows. Weigh the dried algal thalli into a microtube dedicated for crushing and use it for measurement. Add glass beads with a diameter of 0.5 mm to the microtube and crush the cells with a multi-bead shocker device. The oil in the cells is methylated using a fatty acid methylation kit (such as those manufactured by Nacalai), and the fatty acid methyl esters thus produced are quantified by gas chromatography-mass spectrometry (GC-MS), and the oil content rate (% by weight) per dry algal thalli weight and the oil production amount (mg / L) per culture solution volume can be calculated. In addition, the oil production amount (mg / L) per culture solution volume is calculated by multiplying the oil content rate and the biomass amount.

[0019] Also, the algal thalli can be collected over time, and the increase and decrease over time in the oil content rate (% by weight) per dry algal thalli and the oil production amount (mg / L) per culture solution volume can be confirmed. Furthermore, by measuring the amount of oil obtained per day from a unit culture solution (L), the oil production rate (mg / L / day) can be calculated. Note that the oil production rate can be calculated by dividing the oil production amount by the number of days of the culture period.

[0020] In one embodiment, the oil-producing algae according to the present disclosure can be closely related strains or derived strains of the above-mentioned strains.

[0021] Examples of closely related strains include microalgae having an 18S rDNA sequence consisting of a nucleotide sequence having 90% or more sequence identity with any of the above strains and showing oil production ability under nitrogen-sufficient conditions. The closely related strains can belong to the genus Nannochloropsis, preferably Nannochloropsis gaditana. It is expected that such closely related species will have the same growth ability and oil production ability as the above strains in the presence of seawater salt.

[0022] From the perspective of maintaining fertility and oil production ability in the presence of seawater salt, the related strains have an 18S rDNA sequence consisting of a nucleotide sequence showing a sequence identity of 95% or more, 96% or more, more preferably 97%, 98%, still more preferably 99% or more, 99.5% or more with respect to the 18S rDNA sequence of any of the above strains. The related strains can be obtained by selecting candidate algae from a database based on the sequence identity of the nucleotide sequence of the 18S rDNA sequence and evaluating the oil production ability of the candidate algae.

[0023] Examples of the induced strain include a genetically modified variant of any of the above strains. For the genetic modification, those skilled in the art can appropriately select a conventionally known method such as culturing using a mutagenic substance or a gene introduction method using a vector. Examples of the genetic modification include modifications for improving sugar assimilation ability, light collection ability, CO2 fixation ability, nutrient uptake ability, accelerating cell division, promoting oil synthesis, suppressing oil decomposition, etc. Further, examples of the genetic modification also include improvements in high growth ability, high lipid content, wide optimal culture temperature range, high temperature resistance, high photosynthesis efficiency, light damage resistance, high salt concentration resistance, high shear stress resistance, floating property, polar environment resistance, low oxygen adaptability, low respiration rate, colony formation ability, weak cell wall, low self-inhibition ability, lipid composition, etc.

[0024] 2. Method for producing oil The method for producing oil according to the present disclosure includes a step of culturing the above-described oil-producing algae and a step of extracting oil from the algae.

[0025] [Oil] Examples of the oil and fat include aliphatic ester compounds composed of an aliphatic carboxylic acid and a monohydric or trihydric alcohol. The aliphatic carboxylic acid is not particularly limited as long as it is produced by algae. For example, it may be an aliphatic carboxylic acid having 14 to 22 carbon atoms, specifically including palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, and the like. In addition, the oil and fat may also include phospholipids, free fatty acids, steroid compounds, photosynthetic pigments such as carotenoids, and the like.

[0026] [Cultivation step] The cultivation conditions can be set according to conventional methods. In particular, the pH condition is 7.0 or higher, preferably 7.0 - 10.0, more preferably 7.5 - 9.5, and particularly preferably about 8.0 (see Example 5). Also, the CO 2 condition is 0.04% - 10%, preferably 2% - 10% (see Example 6).

[0027] The light condition is, for example, a day-night cycle condition such as a 12 - 20 hour light period and a 4 - 12 hour dark period by a cool white fluorescent lamp of about 50 - 1000 μmol photons / m 2 ·second. For the cultivation period, for example, a pre-cultivation is carried out for about 2 - 12 days, preferably 5 - 9 days, more preferably about 7 days. When the growth is normal and the number of cells becomes sufficient, an expansion culture is carried out. For example, a main culture is carried out for 10 - 20 days, preferably 12 - 16 days, more preferably about 14 days. As the cultivation method, a static cultivation method, a shaking cultivation method, or a deep aeration stirring cultivation method can be applied. The shaking cultivation may be a reciprocating shaking or a rotary shaking.

[0028] The culture medium is not particularly limited as long as it is a medium in which algae can grow. For example, as the basal medium, Modified Bold 12N (MB12N) medium, TAP medium, HSM medium, BG-11 medium, BBM medium, and the like can be mentioned. In addition, the Daigo IMK medium, f / 2 medium, ESM medium, MNK medium, and the like used for marine and brackish water algae can also be used. The medium may contain sea salt, seawater, concentrated seawater or artificial seawater. For example, the added concentration of sea salt is 0.5 - 5% by weight, preferably 2 - 5% by weight, more preferably 3 - 4% by weight, based on the basal medium. When assuming large-scale cultivation of algae, convenient seawater can also be used as the base of the medium.

[0029] In addition to the nitrogen source, the medium may contain a carbon source, inorganic substances, and other nutrients. As the carbon source, saccharides, sugar alcohols, acidic sugars or biomass containing these can be used without particular limitation. As the inorganic substances, potassium dihydrogen phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, ferric chloride, manganese sulfate, calcium chloride, calcium carbonate, zinc sulfate, copper sulfate, ammonium borate·molybdate, potassium iodide, etc. can be used. Antibiotics or the like that do not affect the growth of algae may be added to the medium.

[0030] The oil-producing algae according to the present disclosure can preferentially grow against other microorganisms even under conditions where environmental fluctuations such as light and temperature are large and there is an influence of contamination by other microorganisms or the like. Therefore, outdoor open conditions can be applied to the culture process, and large-scale outdoor culture using seawater can be applied.

[0031] [Extraction step] The cultured algae are separated from the medium, dried if necessary, and then the oil is extracted. Separation is preferably carried out by using solid-liquid separation means such as filtration or centrifugation to separate the algae from the medium.

[0032] Drying can be carried out under general conditions. Specific drying methods include freeze-drying and drying by heating. By drying, a solid powder of algae can be obtained. Note that the algae separated from the medium may be subjected to oil extraction without drying. Also, without separation, following the culture process, the culture solution containing algae may be directly subjected to oil extraction.

[0033] For oil extraction, conventional oil extraction methods can be used. In particular, general extraction methods using organic solvents such as chloroform / methanol systems typified by the Folch method and the Bligh-Dyer method can be used, but it is not limited to these.

Examples

[0034] [Example 1: Evaluation of novel microalgae] Novel microalgae were searched for in seawater collected on Amami Oshima Island, Kagoshima Prefecture, and six strains (microalgae MCNa1, MCNb1, MCNc1, MCNd1, MCNe1, MCNf1) were isolated by the micromanipulation method. The growth ability and oil production ability of the six obtained strains were evaluated in a medium containing seawater salts. As a comparison target, the Chlamydomonas sp. JSC4 strain, which has the ability to produce oil in a medium containing seawater salts, was used.

[0035] (1) Culture conditions [Pre-culture conditions] Medium: 90% artificial seawater + 2-fold concentrated Daigo IMK medium (filter sterilized) (Table 1) Culture solution volume, culture vessel: 50 mL culture solution, 100 mL Erlenmeyer flask Culture temperature: 25 - 28 °C, under air-conditioning control Stirring conditions: Static culture (occasionally stirred manually) Light source: White LED, irradiated from above Light intensity: Photosynthetic photon flux density (PPFD) 50 - 80 μmol / m 2 s Light-dark cycle: 14 hr light / 10 hr dark Culture period: 2 weeks

[0036] [Production conditions] Medium: 2-fold concentrated Daigo IMK + NaNO 3 Fortified + 90% artificial seawater (filter sterilized) (Table 2) Culture solution volume, culture vessel: 800 mL culture solution, 1 L square plastic bottle Initial inoculation concentration: 0.1 - 0.4 g / L Culture temperature: 28 °C or higher (ambient temperature 28 °C + fluorescent lamp heat) Stirring conditions: 1 - 2% CO 2 Aerate and stir by introducing mixed compressed air at about 0.2 - 0.5 vvm Light source: Irradiate from one side of the fluorescent lamp Light intensity: For the first 5 days after the start of cultivation, PFD is 100 μmol / m 2 s, and after the 6th day, PFD is 200 - 250 μmol / m 2 s Light - dark cycle: Light for 24 hours (continuous irradiation) Cultivation period: 20 days

[0037]

Table 1

[0038]

Table 2

[0039] (2) Measurement of dry cell weight As an index of cell mass, the dry cell weight was measured Cells were collected into a weighed microtube by centrifugation, washed once with distilled water, and then freeze - dried. The weight of the microtube containing the dried cells was measured, and the weight of the empty microtube was subtracted to obtain the dry weight of the cells (mg). The dry cell weight was divided by the volume of the culture medium to obtain the dry cell weight (mg / L) contained in the culture medium. The dried algal bodies were also used for the measurement of oil described below

[0040] (3) Measurement of oil accumulation Approximately 3 mg of dry cells were weighed into a microtube dedicated for disruption. Glass beads with a diameter of 0.5 mm were added to the microtube, and the cells were disrupted using a multi-bead shocker device. The oil and fat in the cells were methylated using a fatty acid methylation kit (manufactured by Nacalai), and the generated fatty acid methyl esters were quantified by gas chromatography-mass spectrometry (GC-MS). A capillary column DB-23 (Length: 60 m, diam: 0.25 mm, Flim: 0.15 μm) was used. Margaric acid was added as an internal standard substance during fatty acid methylation. Quantitative analysis was performed by the internal standard method based on the value of methyl margarate.

[0041] (4) Results The results are shown in Figure 1. The dry cell weights of microalgae MCNa1, MCNb1, MCNc1, MCNd1, MCNe1, and MCNf1 on the 10th day were 4.1, 4.4, 1.7, 5.2, 4.2, and 1.1 g / L, respectively. Superior growth was confirmed in MCNa1, MCNb1, MCNd1, and MCNe1 compared to the control strain JSC4 (1.9 g / L). The oil production rates of microalgae MCNa1, MCNb1, MCNc1, MCNd1, MCNe1, and MCNf1 on the 12th day were 103, 192, 63, 124, 92, and 50 mg / L / day, respectively. Superior growth was confirmed in MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1 compared to the target strain JSC4 (50 mg / L / day).

[0042] [Example 2: Outdoor Evaluation of Novel Microalgae] Regarding the six strains of microalgae (microalgae MCNa1, MCNb1, MCNc1, MCNd1, MCNe1, and MCNf1) evaluated in Example 1, the growth and oil accumulation amount in a medium containing seawater salt outdoors were evaluated. The Chlamydomonas sp. JSC4 strain was used as a comparison target. The culture was carried out under the following conditions. The measurement of the dry cell weight and the measurement of the oil accumulation amount were performed according to the method described in Example 1.

[0043] (1) Culture conditions [Pre-culture conditions] Medium: Artificial seawater for outdoor culture + Daigo IMK + NaNO 3 Enriched medium (Table 3) Medium A: MCNa1, MCNb1, MCNc1, MCNd1, MCNe1 Medium B: MCNf1 Medium C: JSC4 Volume of culture solution, culture vessel: 800 mL of culture solution, 1 L square plastic bottle Culture temperature: 25 - 28 °C, under air-conditioning control Stirring conditions: Aeration stirring (compressed air + 2% carbon dioxide gas at 0.25 - 0.5 vvm) Light source: Two-sided fluorescent lamp (inside the growth chamber) Light intensity: Photosynthetic photon flux density (PPFD) of 150 - 200 μmol / m 2 s Light-dark cycle: 16 hours light / 8 hours dark Culture period: 5 days

[0044] [Production conditions] Medium: Artificial seawater for outdoor culture + Daigo IMK + NaNO 3 Enriched medium (Table 3) Medium A: MCNa1, MCNb1, MCNc1, MCNd1, MCNe1 Medium B: MCNf1 Medium C: JSC4 Volume of culture solution, culture vessel: 5 L of culture solution, 10 L bucket (material: polycarbonate, outer dimensions: 273mm × 255mm, inner dimensions: 253mm × 245mm) Initial inoculation concentration: 0.1 - 0.2 g / L Culture temperature: Outdoor environment (as it goes) Stirring conditions: 1 - 2% CO 2 Bubble aeration stirring by introducing about 0.5 vvm of compressed air mixed with it Light source: Natural light (as it goes) Light intensity: Natural light (as it goes) Light-dark cycle: Natural light (as it goes) Culture period: 20 days Seeding amount: Sowing 500 ml of pre-culture

[0045] [Table 3]

[0046] (2) Results As a result of the culture evaluation in the outdoor open system for 20 days, the dry cell weights of the five strains of microalgae MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1 on the 12th day were 2.6, 3.1, 2.2, 2.1, 2.1, and 0.5 g / L, respectively. Superior growth was confirmed in MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1 compared to the control strain JSC4 (1.5 g / L). The lipid production rates of microalgae MCNa1, MCNb1, MCNc1, MCNd1, MCNe1, and MCNf1 on the 12th day were 42, 51, 30, 29, 30, and 16 mg / L / day, respectively. Superior growth was confirmed in MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1 compared to the control strain JSC4 (26.3 mg / L / day). (Figure 2). It was confirmed that these five strains are microalgae that can grow well even under outdoor open conditions where environmental fluctuations such as light and temperature are large and there is an influence of contamination by other microorganisms, etc.

[0047] [Example 3: Identification of novel microalgae] The sequences of the 18S rDNA genes of five strains of microalgae (microalgae MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1) were determined. Chromosomal DNA was extracted from the algae. Using the obtained chromosomal DNA as a template, PCR reaction was performed using the primers shown in Table 4 to amplify the 18S rDNA gene. After the reaction, confirmation by agarose electrophoresis showed that an 18S rDNA gene fragment was confirmed at around 1.1 kbp.

[0048] [Table 4]

[0049] The nucleotide sequences of the 18S rDNAs of the 5 strains are shown in SEQ ID NOs: 1-5, respectively. As a result of sequence analysis, it was suggested that the 5 strains, MCNa1, MCNb1, MCNc1, MCNd1, and MCNe1, are novel microalgae belonging to Nannochloropsis gaditana. These 5 strains have been internationally deposited with the Independent Administrative Institution, National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (IPOD) (Room 120, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture), an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, based on the provisions of Articles 27-2 and 27-3 of the Implementing Regulations of the Patent Act, under the following accession numbers. The receipt date of the 5 strains was March 7, 2023. Nannochloropsis gaditana MCNa1: Accession number FERM BP-22470 Nannochloropsis gaditana MCNb1: Accession number FERM BP-22471 Nannochloropsis gaditana MCNc1: Accession number FERM BP-22472 Nannochloropsis gaditana MCNd1: Accession number FERM BP-22473 Nannochloropsis gaditana MCNe1: Accession number FERM BP-22474 Five strains of microalgae (Nannochloropsis gaditana MCNa1, MCNb1, MCNc1, MCNd1) were single-colonized on agar medium.

[0050] [Example 4: Laboratory Evaluation of Novel Microalgae] Using the two single-colonized microalgae strains (N. gaditana MCNa1, MCNb1) obtained in Example 3, growth comparison was performed with other microalgae belonging to the genus Nannochloropsis, Nannochloropsis salina NBRC102719 and Nannochloropsis oceanica NBRC102738.

[0051] The cultivation was carried out under the following conditions. For the evaluation condition 1, a two-stage flask was used for the container (Figure 3). By placing an aqueous solution of potassium carbonate in the lower part of the two-stage flask, the CO concentration in the gas phase of the algal culture environment in the upper part can be adjusted. At the sampling timing, the aqueous potassium carbonate solution was replaced. 2 The growth degree, dry cell weight, and lipid accumulation amount at each culture time were quantified. The growth degree was measured by a spectrophotometer (750 nm) for the absorbance of a solution obtained by appropriately diluting the culture sample with deionized water. The dry cell weight and lipid accumulation amount were carried out according to the method described in Example 1.

[0052] (1) Culture conditions [Pre-culture conditions] Medium: 100% artificial seawater + 2-fold concentrated Daigo IMK + NaNO 3 Enriched medium (filter sterilized) (Table 5) Culture solution volume, culture vessel: 50 mL culture solution, 100 mL volumetric Erlenmeyer flask Culture temperature: 28 °C, under air-conditioning control Stirring condition: 150 rpm Light source: Irradiated from the side by white LED Light intensity: Photosynthetic photon flux density (PPFD) 150 μmol / m 2 s Light-dark cycle: 16 hr light / 8 hr dark Culture period: 2 weeks

[0053] [Evaluation condition 1] Medium: 100% artificial seawater + 2-fold concentrated Daigo IMK + NaNO 3 Enriched medium (filter sterilized) (Table 5) Culture vessel: Two-stage Erlenmeyer flask Culture solution volume: 50 mL culture solution (upper part), 50 mL 2M potassium carbonate / 2M potassium hydrogen carbonate mixed solution (lower part) Assumed CO concentration in the gas phase: 2% (lower part: 10 mL of 2M potassium carbonate and 40 mL of 2M potassium hydrogen carbonate mixed solution) 2 Initial inoculation concentration: 0.1 - 0.4 g / L Culture temperature: 28 °C, under air-conditioning control Stirring condition: 110 rpm Light source: Irradiated from the side by white LED Light quantity: Photosynthetic photon flux density (PPFD) 150 μmol / m 2 s Light-dark cycle: 16 hr light / 8 hr dark Cultivation period: 15 days

[0054] [Evaluation condition 2] Medium: 2-fold concentration of Daigo IMK + NaNO 3 Enriched + 90% artificial seawater (filtered and sterilized by filter) (Table 2) Volume of culture solution, incubator: 800 mL of culture solution, 1 L square plastic bottle Cultivation temperature: 28°C or higher (ambient temperature 28°C + heat from fluorescent lamp) Stirring condition: 1-2% CO 2 Aerate and stir by passing compressed air mixed to about 0.2-0.5 vvm Light source: Irradiated from the side by fluorescent lamp 1 Light quantity: Photosynthetic photon flux density (PPFD) 250 μmol / m 2 s Light-dark cycle: 13.5 hr light / 11.5 hr dark Cultivation period: 24 days

[0055]

Table 5

[0056] (2) Results As a result of the 15-day in-house flask culture evaluation (evaluation condition 1), the dry cell weights of the MCNa1 strain and the MCNb1 strain on the 10th day were 2.1 and 3.0 g / L respectively, showing higher growth productivity compared to the respective values (1.6 and 1.3 g / L) of Nannochloropsis salina NBRC102719 and Nannochloropsis oceanica NBRC102738 (Figure 4). Furthermore, the lipid accumulation rate of the MCNb1 strain on the 15th day was 78 mg / L / day, which was higher than that of the NBRC102719 strain and the NBRC102738 strain.

[0057] Growth evaluation (evaluation condition 2) was carried out while supplying 2% CO gas for 21 days with an indoor scale of 800 ml. As a result, the dry cell weights of the MCNa1 strain and the MCNb1 strain on the 11th day were 1.0 and 1.6 g / L, respectively, showing a higher growth rate compared to the NBRC102719 strain and the NBRC102738 strain (Figure 5). 2

[0058] [Example 5: Growth pH of novel microalgae] Using the MCNa1 strain, growth comparison was carried out in a medium containing seawater salts under different pH environments. The culture was carried out under the following conditions. For the seawater salt medium, the artificial seawater medium in Table 5 was used. The pH of the medium was changed by adding a buffer solution adjusted to each pH to the seawater salt medium. Under the pH 5 condition, 2-morpholinoethanesulfonic acid (MES) adjusted to pH 5.0 was added at a final concentration of 25 mM to prepare an artificial seawater medium at pH 5. Similarly, under the pH 6 condition, an MES buffer solution adjusted to pH 6.0, under the pH 7 condition, a 1,4-piperazinediethanesulfonic acid (PIPES) buffer solution adjusted to pH 7.0, under the pH 7 and 8 conditions, a tricine buffer solution adjusted to pH 8.0 and 9.0, and under the pH 10 condition, a 3-cyclohexylaminopropanesulfonic acid (CAPS) buffer solution adjusted to pH 10.0 were used to prepare pH-adjusted artificial seawater media. The growth degree at each culture time was evaluated according to the aforementioned method.

[0059] The results are shown in Figure 6. It was confirmed that the MCNa1 strain can grow under conditions of pH 7.0 or higher. It could not grow at pH 5.0 and 6.0. It was able to grow at least at pH 7.0 - 10 and showed the best growth at pH 8.0 - 9.0. [Example 6: Growth CO 2 Concentration] Using the MCNa1 strain and the MCNb1 strain, growth comparison was carried out in a medium containing seawater salts under different CO 2 concentration environments. The culture was carried out under the evaluation condition 1 (two-stage flask) of Example 4. CO in the gas phase 2Under the assumed concentration of 0.04%, the potassium carbonate solution in the lower stage was prepared by mixing 38.9 mL of 2M potassium carbonate and 11.1 mL of 2M potassium bicarbonate. Under the assumption of 2% CO2, it was prepared by mixing 10 mL of 2M potassium carbonate and 40 mL of 2M potassium bicarbonate. Under the assumption of 5% CO 2 it was prepared by mixing 5 mL of 2M potassium carbonate and 45 mL of 2M potassium bicarbonate. Under the assumption of 10% CO 2 it was prepared by mixing 2.8 mL of 2M potassium carbonate and 47.2 mL of 2M potassium bicarbonate and used. The aqueous potassium carbonate solution was replaced at the sampling timing. For the upper-stage algal culture solution, a solution obtained by adding 1.25 mL of 1M tricine buffer (pH 9) to the artificial seawater medium in Table 5 was used. The pH was confirmed for each sampling, and when it fell below pH 8, 2M aqueous NaOH solution was added. The growth degree at each culture time was evaluated according to the above-described method.

[0060] The results are shown in Fig. 7. The growth under the condition of 0.04% CO assuming air is very slow, but the growth rate improved at a CO 2 concentration of 2% or more. It was confirmed that even when the CO 2 concentration was increased from 2% to 10%, growth was not inhibited and the growth rate could be maintained. 2

Sequence Listing Free-Text

[0061] SEQ ID NO: 1: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNa1 SEQ ID NO: 2: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNb1 SEQ ID NO: 3: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNc1 SEQ ID NO: 4: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNd1 SEQ ID NO: 5: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNe1 Accession number 6: Base sequence of primer Algae18S-F1 Accession number 7: Base sequence of primer Algae18S-R1 Accession number 8: Base sequence of primer NS3a Accession number 9: Base sequence of primer OPAlgae18S-R3

Claims

1. It has the ability to produce oil and fat, It is one or more strains selected from the following group, or has an 18S rDNA sequence consisting of a base sequence that has 90% or more sequence identity with these strains: algae. Nannochloropsis gaditana MCNa1 strain (Accession number: FERM BP-22470) Nannochloropsis gaditana MCNb1 strain (Accession number: FERM BP-22471) Nannochloropsis gaditana MCNc1 strain (Accession number: FERM BP-22472) Nannochloropsis gaditana MCNd1 strain (Accession number: FERM BP-22473) Nannochloropsis gaditana MCNe1 strain (Accession number: FERM BP-22474)

2. A method for producing fats and oils, comprising a step of culturing algae in a medium containing seawater salt, The algae is Is it one or more strains selected from the following group? The strain has an 18S rDNA sequence having a base sequence with 90% or more sequence identity with the strains, and has the ability to produce oils and fats. Manufacturing method. Nannochloropsis gaditana MCNa1 strain (Accession number: FERM BP-22470) Nannochloropsis gaditana MCNb1 strain (Accession number: FERM BP-22471) Nannochloropsis gaditana MCNc1 strain (Accession number: FERM BP-22472) Nannochloropsis gaditana MCNd1 strain (Accession number: FERM BP-22473) Nannochloropsis gaditana MCNe1 strain (Accession number: FERM BP-22474)

3. The method according to claim 2 , wherein the culturing is carried out under outdoor open conditions while maintaining preferential growth of the algae.

4. The method according to claim 3, wherein the pH of the medium is 7-10.

5. The culturing is performed by subjecting 2 The method for producing fats and oils according to claim 3 or 4, which is carried out in an atmosphere having a concentration of 0.04 to 10%.

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