Method for producing oils and fats using algae and their symbiotic bacteria

Abstract

To provide a technique for producing oils and fats using algae.SOLUTION: Provided is a method for producing oils and fats, including a step of culturing algae and a step of extracting oils and fats from the algae, wherein the culturing is carried out in the presence of symbiotic bacteria.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a method for producing fats and oils using algae, and more particularly to a method for producing fats and oils using algae in combination with symbiotic bacteria. [Background technology]

[0002] Technologies using algae to produce industrial raw materials, fuels, feed, and raw materials for fine chemicals are being developed, and it is believed that promoting the growth of algae can contribute to improving productivity in these technologies.

[0003] Patent Document 1 describes that the growth of algae can be effectively promoted by adding a fulvic acid-containing composition having specific physical properties contained in brackish water (groundwater with a higher salt concentration than fresh water) to an algae culture medium. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-78155 A Summary of the Invention [Problem to be solved by the invention]

[0005] The main objective of the present disclosure is to provide techniques for producing fats and oils using algae. [Means for solving the problem]

[0006] In order to solve the above problems, the present disclosure provides the following [1]-[8]. [1] A method for producing fats and oils, comprising the steps of culturing algae and extracting fats and oils from the algae, The production method, wherein the culture is carried out in the presence of a symbiotic bacterium. [2] The production method according to [1], wherein the symbiotic bacterium is at least one type of microorganism selected from the group consisting of microorganisms belonging to the genus Croceibacter, microorganisms belonging to the genus Marinoscillum, microorganisms belonging to the genus Roseivirga, microorganisms belonging to the genus Parasphingorhabdus, and microorganisms belonging to the genus Marinobacter. [3] The production method according to [2], wherein the symbiotic bacteria is at least one type of microorganism selected from the group consisting of microorganisms belonging to Croceibacter atlanticus, microorganisms belonging to Marinoscillum luteum, microorganisms belonging to Roseivirga sp., microorganisms belonging to Parasphingorhabdus flavimaris, and microorganisms belonging to Marinobacter adhaerens. [4] The manufacturing method according to [3], wherein the symbiotic fungus is any one of the following strains or has a 16S rDNA sequence consisting of a base sequence that has 98% or more sequence identity with any of the following strains and promotes the growth of algae. Croceibacter atlanticus strain Na1 (Accession number: NITE BP-03911) Marinoscillum luteum Na5 strain (Accession number: NITE BP-03912) Roseivirga sp. Na8 strain (Accession number: NITE BP-03913) Parasphingorhabdus flavimaris Na11 strain (Accession number: NITE BP-03914) Marinobacter adhaerens Nb5 strain (Accession number: NITE BP-03915) [5] The method of any one of [1] to [4], wherein the algae belongs to the genus Nannochloropsis. [6] The method for producing the algae according to [5], wherein the algae belong to Nannochloropsis gaditana. [7] The method according to [6], wherein the algae is any one of the following strains, or has an 18S rDNA sequence consisting of a base sequence that has 98% or more sequence identity with those of these strains and has oil-producing ability. 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) [8] A method for producing [5], wherein the algae belongs to Nannochloropsis oceanica. Effect of the Invention

[0007] The present disclosure provides techniques for producing oils and fats using algae. [Brief description of the drawings]

[0008] [Figure 1] The growth-promoting effect of the symbiotic bacterium Croceibacter atlanticus Na1 strain on Nannochloropsis oceanica NIES-2146 is shown. [Diagram 2] The growth-promoting effect of the symbiotic bacterium Marinoscillum luteum Na5 strain on Nannochloropsis oceanica NIES-2146 is shown. [Diagram 3]The growth-promoting effect of the symbiotic bacterium Roseivirga sp. Na8 strain on Nannochloropsis oceanica NIES-2146 is shown. [Figure 4] The growth-promoting effect of the symbiotic bacterium Parasphingorhabdus flavimaris Na11 strain on Nannochloropsis oceanica NIES-2146 is shown. [Diagram 5] The growth-promoting effect of the symbiotic bacterium Marinobacter adhaerens Nb5 strain on Nannochloropsis oceanica NIES-2146 is shown. [Figure 6] The growth-promoting effect of the symbiotic bacterium Croceibacter atlanticus Na1 on Nannochloropsis gaditana MCNa1 is shown. [Figure 7] The growth-promoting effect of the symbiotic fungus Marinoscillum luteum Na5 strain on Nannochloropsis gaditana MCNa1 is shown. [Figure 8] The growth-promoting effect of the symbiotic bacterium Roseivirga sp. Na8 strain on Nannochloropsis gaditana MCNa1 is shown. [Figure 9] The growth-promoting effect of the symbiotic fungus Parasphingorhabdus flavimaris Na11 strain on Nannochloropsis gaditana MCNa1 is shown. [Figure 10] The growth-promoting effect of the symbiotic bacterium Marinobacter adhaerens Nb5 strain on Nannochloropsis gaditana MCNa1 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and the scope of the present disclosure should not be interpreted as being narrow.

[0010] The method for producing fats and oils according to the present disclosure includes a step of culturing algae and a step of extracting fats and oils from the algae, the culturing being carried out in the presence of symbiotic bacteria.

[0011] [Oils and fats] The fats and oils include aliphatic ester compounds consisting of aliphatic carboxylic acids and monohydric or trihydric alcohols. The aliphatic carboxylic acids are not particularly limited as long as they are produced by algae, and examples thereof include aliphatic carboxylic acids having 14 to 22 carbon atoms, specifically palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, and eicosapentaenoic acid. The fats and oils may also include phospholipids, free fatty acids, steroid compounds, and photosynthetic pigments such as carotenoids.

[0012] [Algae] The algae used in the present disclosure are not particularly limited as long as they have the ability to produce fats and oils, but are particularly preferred as microalgae. Microalgae are algae that are difficult to distinguish with the naked eye, and each individual has an actual size of several μm to several hundred μm. The algae used in the present disclosure include, for example, the genus Chlamydomonas, the genus Chlorella, the genus Dunaliella, the genus Nannochloropsis, the genus Botryococcus, the genus Chaetoceros, the genus Chlorococcum, the genus Euglena, the genus Haematococcus, Examples of algae that may be included include those belonging to the genera Haematococcus, Isochrysis, Navicula, Neochloris, Porphyridium, Prymnesium, Scenedesmus, Spirulina, Spirogyra, Synechococcus, and Tetraselmis. One or more types of algae may be used from among these.

[0013] Examples of algae belonging to the genus Chlamydomonas include Chlamydomonas reinhardtii. Examples of algae in the genus Chlorella include Chlorella vulgaris, Chlorella pyrenoidosa, and Chlorella sorokiniana. Examples of algae in the genus Dunaliella include Dunaliella bioculata, Dunaliella salina, and Dunaliella tertiolecta.

[0014] Examples of algae in the genus Nannochloropsis include Nannochloropsis gaditana, Nannochloropsis oceanica, and Nannochloropsis oculata.

[0015] As Nannochloropsis oceanica, a particular example is Nannochloropsis oceanica NIES-2146 strain.

[0016] In addition, the following strains are particularly exemplified as Nannochloropsis gaditana. 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) These strains have been internationally deposited under the above accession number at the National Institute of Technology and Evaluation, Patent Organism Depositary (IPOD), which is a depository institution under Article 27-2 and 3 of the Enforcement Regulations of the Patent Act and an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). The date of receipt is March 7, 2023.

[0017] The algae, in one embodiment, can be a related or derived strain of Nannochloropsis gaditana high oil producing strain.

[0018] The closely-related strains include algae that have an 18S rDNA sequence consisting of a base sequence having 98% or more sequence identity with any of the Nannochloropsis gaditana high oil-producing strains and exhibit oil-producing ability. The closely-related strains may belong to the genus Nannochloropsis, preferably Nannochloropsis gaditana. These closely related species are expected to have oil production capabilities similar to those of the high oil-producing Nannochloropsis gaditana strain.

[0019] In terms of maintaining oil production ability, the related strain has an 18S rDNA sequence consisting of a base sequence that shows sequence identity of 98% or more, preferably 99% or more, and more preferably 99.5% or more to the 18S rDNA sequence of any of the high oil-producing strains of Nannochloropsis gaditana. Closely related strains can be obtained by selecting candidate algae from a database based on sequence identity of the base sequence of the 18S rDNA sequence and evaluating the oil production ability of the candidate algae.

[0020] Derivative strains include genetically modified strains of Nannochloropsis gaditana high oil-producing strains. Genetic modification can be appropriately selected from methods conventionally known to those skilled in the art, such as culture using mutagens and gene introduction using vectors. Genetic modifications include modifications to improve sugar assimilation, modifications to improve light harvesting ability, modifications to improve CO 2 These include modifications to improve immobilization ability, modifications to improve nutrient uptake, modifications to accelerate cell division, modifications to promote synthesis of fats and oils, modifications to suppress decomposition of fats and oils, etc. Further examples include improvements in high proliferation, wide optimum culture temperature range, high temperature resistance, high photosynthetic efficiency, light damage resistance, high salt concentration resistance, high shear stress resistance, buoyancy, polar environment resistance, low oxygen adaptability, low respiration rate, colony formation ability, weak cell shell, low autoinhibitory ability, lipid composition, etc.

[0021] [Symbiotic bacteria] The symbiotic bacteria are not particularly limited as long as they have the ability to promote the growth of algae, and examples thereof include microorganisms belonging to the genera Croceibacter, Marinoscillum, Roseivirga, Parasphingorhabdus, and Marinobacter. One or more of these symbiotic bacteria may be used.

[0022] Examples of the genus Croceibacter include Croceibacter atlanticus, and in particular, Croceibacter atlanticus strain Na1 (accession number: NITE BP-03911). Examples of the genus Marinoscillum include Marinoscillum luteum, Marinoscillum furvescens, Marinoscillum pacificum, and the like, and in particular Marinoscillum luteum Na5 strain (Accession No.: NITE BP-03912). Examples include Examples of the genus Roseivirga include Roseivirga marina, Roseivirga echinicomitans, Roseivirga ehrenbergii, Roseivirga halotolerans, Roseivirga maritima, Roseivirga misakiensis, Roseivirga pacifica, Roseivirga seohaensis, Roseivirga spongicola, and Roseivirga thermotolerans. In particular, Roseivirga sp. Na8 strain (accession number: NITE BP-03913) is an example. Examples of the genus Parasphingorhabdus include Parasphingorhabdus flavimaris, Parasphingorhabdus cellanae, Parasphingorhabdus halotolerans, Parasphingorhabdus litoris, Parasphingorhabdus marina, and Parasphingorhabdus pacifica, and in particular, the Parasphingorhabdus flavimaris Na11 strain (accession number: NITE BP-03914). Examples of the genus Marinobacter include Marinobacter adhaerens, Marinobacter alkaliphilus, Marinobacter bacchus, Marinobacter excellens, Marinobacter halotolerans, Marinobacter mobilis, and Marinobacter vulgaris, and in particular, the Marinobacter adhaerens Nb5 strain (accession number: NITE BP-03915). These strains have been internationally deposited under the above accession number at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD), which is a depository institution under Article 27-2 and 3 of the Enforcement Regulations of the Patent Act and an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). The date of receipt is June 13, 2023.

[0023] In one embodiment, the symbiotic bacteria may be a closely related or derived strain of the above strains.

[0024] Examples of closely related strains include microorganisms that have a 16S rDNA sequence consisting of a base sequence that has 90% or more sequence identity with any of the above-mentioned symbiotic strains and that promote the growth of algae. Such closely related species are expected to have the same algae growth-promoting ability as the above strains.

[0025] In terms of retaining the ability to promote algal growth, a closely related strain has a 16S rDNA sequence consisting of a base sequence that shows sequence identity of 98% or more, more preferably 99% or more, and even more preferably 99.5% or more to the 16S rDNA sequence of any of the above-mentioned symbiotic strains. The closely related strains can be obtained by selecting candidate microorganisms from a database based on sequence identity of the 16S rDNA base sequence and evaluating the ability of the candidate microorganisms to promote algae growth.

[0026] The derivative strain may be a genetically modified strain of the above-mentioned symbiotic strain. The genetic modification may be appropriately selected from methods conventionally known to those skilled in the art, such as culture using a mutagenic substance or a gene introduction method using a vector.

[0027] [Culture process] Cultivation of algae in the presence of symbiotic bacteria may be carried out by adding the symbiotic bacteria to a culture medium for algae cultured according to a conventional method. The amount of symbiotic bacteria added to the culture solution is not particularly limited as long as the algae growth promoting ability is exhibited. For example, the OD 600 The value is 0.001-10, preferably 0.002-5. The effect of the symbiotic bacteria in promoting algae growth can be maintained in the culture medium after the symbiotic bacteria are added to the culture medium of the algae and cultured for an appropriate period of time, and then a part of the culture medium (including the algae and the symbiotic bacteria) is seeded in a new culture medium for subculture. Therefore, the symbiotic bacteria may be added to the culture medium only at the start of the culture of the algae, or each time the algae are subcultured, or at any timing.

[0028] Culture conditions other than the nitrogen concentration can be set according to conventional methods. CO 2 The conditions are, for example, 1% to 5%, preferably 1.5% to 2.5%, and more preferably 2%. The temperature conditions are, for example, 15°C to 40°C, preferably 20°C to 35°C, and more preferably 25°C to 30°C. The light conditions are, for example, 50 to 1000 μmol photons / m 2 The day-night cycle conditions are assumed to be 12 to 20 hours of light and 4 to 12 hours of darkness using natural white fluorescent lighting for approximately 10 seconds. The culture period is, for example, 2 to 12 days, preferably 5 to 9 days, and more preferably about 7 days of pre-culture, and when the cells have grown smoothly and the number of cells is sufficient, expansion culture is performed, and main culture is performed for, for example, 10 to 20 days, preferably 12 to 16 days, and more preferably about 14 days. The culture method may be a stationary culture method, a shaking culture method, or a deep aeration agitation culture method. The shaking culture may be a reciprocal shaking culture or a rotary shaking culture.

[0029] The medium is not particularly limited as long as it is a medium in which algae can grow. For example, basal media include Modified Bold 12N (MB12N) medium, TAP medium, HSM medium, BG-11 medium, BBM medium, and the like. The medium may contain sea salt, seawater, concentrated seawater, or artificial seawater. For example, the concentration of sea salt added to the basal medium is 0.5 to 5% by weight, preferably 2 to 5% by weight, and more preferably 3 to 4% by weight. When envisioning mass cultivation of algae, convenient seawater can also be used as the base of the medium. Alternatively, for example, Daigo IMK medium, f / 2 medium, ESM medium, MNK medium, etc. can be used as media for marine and brackish algae.

[0030] In addition to the nitrogen source, the medium may contain a carbon source, inorganic substances, and other nutrients. As the carbon source, sugars, sugar alcohols, acidic sugars, or biomass containing these can be used without any particular limitation. Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, ferrous chloride, manganese sulfate, calcium chloride, calcium carbonate, zinc sulfate, copper sulfate, ammonium borate / molybdate, and potassium iodide. The medium may contain antibiotics and the like that do not affect the growth of the algae.

[0031] [Extraction process] The cultured algae are separated from the medium, dried as necessary, and then oils and fats are extracted. The separation is preferably performed by using a 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, drying by heating, etc. By drying, a solid powder of the algae can be obtained. The algae separated from the medium may be subjected to oil extraction without drying, or the culture solution containing the algae may be directly subjected to oil extraction following the culture step without separation.

[0033] The fat extraction can be performed using a conventional fat extraction method, in particular, a general extraction method using an organic solvent such as a chloroform / methanol system, as represented by the Folch method or the Bligh-Dyer method, but is not limited to these. EXAMPLES

[0034] [Example 1: Isolation of candidate strains of algal symbiotic bacteria] (1) Acquisition of algae-symbiotic bacteria from nature Seawater collected from the beach in Ota Ward, Tokyo, Japan was filtered through a filter with a pore size of 1.6 μm to remove protozoa and algae, and then further filtered through a filter with a pore size of 0.2 μm to collect bacterial groups on the filter. The bacteria were suspended in a culture medium of Nannochloropsis oceanica NIES-2146 strain purchased from the National Institute for Environmental Studies (NIES), and the effect of the bacteria in promoting algae growth was confirmed. ESM medium, light intensity 150μmol / m 2 The incubation was carried out for 14 days at 28°C and 120 rpm for 16 h light / 8 h dark / s. Under the bacterial coexistence conditions, algae growth was observed to be approximately 1.8 times higher than in the control (Nannochloropsis oceanica NIES-2146 strain only) (the culture medium after this co-cultivation was designated Nannochloropsis-marine bacteria culture medium 1 and used for the following strain isolation). In addition, a similar study was performed again using seawater collected on a different day, and the results showed that under the bacterial coexistence conditions, algae growth was approximately 1.9 times higher than in the control (the culture medium after this co-cultivation was designated Nannochloropsis-marine bacteria culture medium 2 and used for the following strain isolation).

[0035] (2) Isolation of candidate strains of algal symbiotic bacteria Nannochloropsis-marine bacteria culture solutions 1 and 2 were each filtered using a 1.6 μm filter. The filtrate was spread on R2A-seawater agar medium (R2A + 2% NaCl + 1 / 3 concentration ESM medium + 1.5% concentration agar) and cultured at 28°C for 2 weeks. Colonies on the agar medium were picked up and pure cultures of each strain were performed. The 16S rDNA sequences of these isolated strains were analyzed and the bacterial species were identified by BLAST search. Seventeen and fifteen strains were obtained from Nannochloropsis - marine bacteria cultures 1 and 2, respectively.

[0036] [Example 2: Identification of symbiotic bacteria for Nannochloropsis oceanica NIES-2146 strain] (1) Identification of symbiotic bacteria The 32 strains isolated in Example 1 were each measured at OD 600 The value of the α-methyltransferase was 0.05 when the culture medium (ESM medium) of Nannochloropsis oceanica NIES-2146 was added to the medium. The light intensity was 150 μmol / m 2 The algae were cultured at 28°C and 120 rpm for 14 days, with a light / dark ratio of 16 hours / 8 hours / s and a light / dark ratio of 16 hours / 8 hours. The dry weight of the algae on the 7th and 14th days of culture was compared with that of the control (Nannochloropsis oceanica NIES-2146 strain only) to confirm whether the added bacteria had any effect on promoting the growth of the algae.

[0037] Five strains of symbiotic bacteria were identified that showed a growth-promoting effect on algae. The 16S rDNA sequences of the five strains are shown in SEQ ID NOs: 1-5. These strains have been internationally deposited under the following accession numbers at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD), which is a depository institution under Article 27-2 and 3 of the Enforcement Regulations of the Patent Act and an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). The date of receipt is June 13, 2023. Croceibacter atlanticus Na1 strain: Accession number: NITE BP-03911 Marinoscillum luteum Na5 strain: Accession number: NITE BP-03912 Roseivirga sp. Na8 strain: Accession number: NITE BP-03913 Parasphingorhabdus flavimaris Na11 strain: Accession number: NITE BP-03914 Marinobacter adhaerens Nb5 strain: Accession number: NITE BP-03915

[0038] (2) Confirmation of the promotion of growth of Nannochloropsis oceanica NIES-2146 strain by symbiotic bacteria OD of each of the five strains 600 The NIES-2146 strain was cultured at a cell density of 0.2 g / L at the start of cultivation, and the light intensity was 150 μmol / m 2 The culture was performed for 15 days at 28°C and 120 rpm, with a light / dark setting of 16 hours / 8 hours / s. Sampling was performed on days 0, 5, 10, and 15 of culture, and the dry weight of the algae was measured to confirm the growth progress.

[0039] The results are shown in Figures 1-5. In the case of adding Croceibacter atlanticus Na1 strain, OD 600 On the 15th day of culture under the =0.1 addition condition, a 1.3-fold increase in biomass (dry weight) was observed compared to the control (Nannochloropsis oceanica NIES-2146 strain only) (Figure 1). Under conditions where the Na5 strain was added, the OD 600 On the 15th day of culture under the =0.1 addition condition, a 2.1-fold increase in biomass (dry weight) was observed compared to the control (Figure 2). roseivirga sp. The OD 600 On the 10th day of culture under the =0.1 addition condition, a 1.9-fold increase in biomass (dry weight) was observed compared to the control (Figure 3). Under conditions where Parasphingorhabdos flavimaris Na11 strain was added, OD 600 On the 15th day of culture under the =1 addition condition, a 1.4-fold increase in biomass (dry weight) was observed compared to the control (Figure 4). Under conditions where Marinobacter adherens Nb5 strain was added, OD 600 On the 15th day of culture under the =1 addition condition, a 2.0-fold increase in biomass (dry weight) was observed compared to the germ-free control (Figure 5). All five symbiotic bacteria exhibited growth-promoting effects on Nannochloropsis oceanica NIES-2146 strain.

[0040] [Example 3: Discovery of new microalgae] We searched for new microalgae in seawater collected from Amami Oshima, Kagoshima Prefecture, and isolated five strains. The five isolated strains have been internationally deposited under the following accession numbers at the National Institute of Technology and Evaluation, Patent Organism Depositary (IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), which is a depository institution under Article 27-2 and 3 of the Enforcement Regulations of the Patent Act and an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. The date of receipt of the five strains is 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

[0041] The 18S rDNA sequences of the five strains were determined (SEQ ID NO:6-10). Sequence analysis suggested that the five strains were novel microalgae belonging to Nannochloropsis gaditana.

[0042] [Example 4: Examination of the effect of symbiotic fungi on Nannochloropsis gaditana MCNa1 strain] (1) Evaluation of the growth-promoting effect of symbiotic fungi on Nannochloropsis gaditana MCNa1 strain The growth-promoting effects of five symbiotic fungal strains on Nannochloropsis gaditana MCNa1 strain were evaluated in the same manner as in Example 2(2). OD of each of the five strains 600 The light intensity was 148 μmol / m 2 The medium was incubated at 120 rpm for 14 days under the conditions of 16 hours light / 8 hours dark, 28°C, and 160 rpm. 3 400mg / L (100% seawater concentration, NaNO 3 The final concentration was 800 mg / L. Sampling was performed on days 0, 3, 7, and 14 of culture, and the OD 750 The growth progression was confirmed by measuring the OD 750 Values ​​were converted to dry weight values).

[0043] The results are shown in Figures 6-10. In the case of adding Croceibacter atlanticus Na1 strain, OD 600 On the 14th day of culture under the =0.1 addition condition, a 3.3-fold increase in biomass (dry weight) was observed compared to the control (Nannochloropsis gaditana MCNa1 strain only) (Figure 6). Under conditions where Marinocirum rutheum Na5 strain was added, the OD 600On the 7th day of culture under the =0.1 addition condition, a 2.0-fold increase in biomass (dry weight) was observed compared to the control (Figure 7). roseivirga sp. The OD 600 On the 14th day of culture under the =0.1 addition condition, a 1.8-fold increase in biomass (dry weight) was observed compared to the control (Figure 8). Under conditions where Parasphingorhabdos flavimaris Na11 strain was added, OD 600 On the 14th day of culture under the addition of β-lactamase, a 3.9-fold increase in biomass (dry weight) was observed compared to the control (Figure 9). Under conditions where Marinobacter adherens Nb5 strain was added, OD 600 On the 7th day of culture under the addition of =0.1, a 1.8-fold increase in biomass (dry weight) was observed compared to the control (Figure 10). All five symbiotic fungi exhibited growth-promoting effects on Nannochloropsis gaditana MCNa1. [Sequence List Free Text]

[0044] SEQ ID NO: 1: Base sequence of 16S rDNA of Croceibacter atlanticus Na1 strain SEQ ID NO: 2: Base sequence of 16S rDNA of Marinoscillum luteum Na5 strain SEQ ID NO: 3: Base sequence of 16S rDNA of Roseivirga sp. Na8 strain SEQ ID NO: 4: Base sequence of 16S rDNA of Parasphingorhabdus flavimaris Na11 strain SEQ ID NO: 5: 16S rDNA sequence of Marinobacter adhaerens Nb5 strain SEQ ID NO: 6: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNa1 SEQ ID NO: 7: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNb1 SEQ ID NO: 8: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNc1 SEQ ID NO: 9: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNd1 SEQ ID NO: 10: Nucleotide sequence of 18S rDNA of Nannochloropsis gaditana MCNe1

Claims

1. A method for producing fats and oils, comprising culturing algae and extracting fats and oils from the algae, The production method, wherein the culture is carried out in the presence of a symbiotic bacterium.

2. The method according to claim 1, wherein the symbiotic bacteria is at least one microorganism selected from the group consisting of microorganisms belonging to the genus Croceibacter, microorganisms belonging to the genus Marinoscillum, microorganisms belonging to the genus Roseivirga, microorganisms belonging to the genus Parasphingorhabdus, and microorganisms belonging to the genus Marinobacter.

3. The method according to claim 2, wherein the symbiotic bacteria is at least one microorganism selected from the group consisting of microorganisms belonging to Croceibacter atlanticus, microorganisms belonging to Marinoscillum luteum, microorganisms belonging to Roseivirga sp., microorganisms belonging to Parasphingorhabdus flavimaris, and microorganisms belonging to Marinobacter adhaerens.

4. The production method described in claim 3, wherein the symbiotic fungus is any of the following strains or has a 16S rDNA sequence consisting of a base sequence that has 98% or more sequence identity between these strains and promotes the growth of algae. Croceibacter atlanticus Na1 strain (Accession number: NITE BP-03911) Marinoscillum luteum Na5 strain (Accession number: NITE BP-03912) Roseivirga sp. Na8 strain (Accession number: NITE BP-03913) Parasphingorhabdus flavimaris Na11 strain (Accession number: NITE BP-03914) Marinobacter adhaerens Nb5 strain (Accession number: NITE BP-03915)

5. The method according to any one of claims 1 to 4, wherein the algae belong to the genus Nannochloropsis.

6. The method according to claim 5 , wherein the algae belongs to Nannochloropsis gaditana.

7. The method according to claim 6, wherein the algae is any one of the following strains, or has an 18S rDNA sequence consisting of a base sequence having 98% or more sequence identity between the algae and any of the following strains, and has the ability to produce oils and fats. 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)

8. The method according to claim 5, wherein the algae belong to the genus Nannochloropsis oceanica.

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