JP7307435B2 - Production method and production system for euglena with high wax ester content, production method and production system for wax ester or biofuel composition, and wax ester fermentation accelerator - Google Patents

Description

The present invention provides Euglena having a wax ester (wax ester: hereinafter also referred to as "WE") content increased compared to the prepared Euglena (hereinafter referred to as "wax ester high content Euglena", also referred to as "WE high content Euglena" ), a method and system for producing a WE or a biofuel composition, and an accelerator for wax ester fermentation (hereinafter also referred to as “WE fermentation”).

Biofuels are fuels derived from biological sources such as animals or plants. Among the various components that can be used as biofuels, WE derived from Euglena can be mentioned as a component with a large number of carbon atoms in the molecule and a relatively large amount of heat generated during combustion (Patent Documents 1 and 2). ). Generally, WE is an ester of a fatty acid having 10 or more carbon atoms and a fatty alcohol having 8 or more carbon atoms, that is, an ester having 18 or more carbon atoms in the molecule. Euglena (genus name: Euglena, Japanese name: Euglena) is a unique single-celled organism that combines the animal-like properties of flagellar movement and the plant-like properties of photosynthesis. Euglena has the characteristic of being able to grow under various nutritional conditions by changing the intracellular storage substances according to the nutritional conditions. In order to take advantage of this characteristic, various research and development have been carried out.

For example, in Patent Document 2, Euglena is cultured under aerobic conditions at a liquid temperature of about 27° C. to 35° C., cultured at about 16° C. with hypoxia treatment, and then WE is extracted from the cultured Euglena. A method for manufacturing a WE is described. By culturing under aerobic conditions, paramylon (β-1,3-glucan), which is a kind of polysaccharide, is produced and stored in Euglena cells. Thereafter, by culturing with hypoxia treatment, metabolism occurs in the Euglena cells to degrade paramylon and biosynthesize ATP (adenosine triphosphate). Patent document 2 explains that WE having 24 or more and 26 or less carbon atoms in the molecule can be produced in cells as a metabolite. Thus, the reaction in which paramylon is metabolized in an oxygen-independent manner in Euglena cells under anaerobic conditions and the by-product of ATP production is stored in the cell in the form of WE is also called wax ester fermentation (WE fermentation). This phenomenon is known to be unique to Euglena cells. Patent Document 2 also explains that it is preferable to use KAT1 knockdown Euglena in which the expression of the 3-ketoacyl-CoA thiolase (KAT)1 gene is suppressed.

In Patent Document 1, a biofuel production method is provided in which Euglena is cultured by giving sugar under aerobic conditions, then propionic acid is given in an acidic environment, left for a certain period of time, and then WE is recovered from the left Euglena. Are listed. Since the recovered WE contains a large amount of myristic acid (14 carbon atoms) and myristyl alcohol (14 carbon atoms), that is, myristyl myristate (28 carbon atoms), it can be used as an alternative fuel for jet fuel, for example. Patent document 1 explains that utilization is expected. In addition, propionic acid inhibits ATP biosynthesis by aerobic respiration in Euglena cells, decomposes paramylon and induces a state in which ATP biosynthesis is forced, so it is patented that WE fermentation is promoted in cells. Reference 1 explains.

WO2020/162502 JP 2017-148005 A Patent No. 6329440 Patent No. 6019305

M. Cramer, and one others, Arch. Mikrobiol., 1952, volume 17, pp.384-402 J. A. Schiff, et al., Methods Enzymol., 1971, volume 23, pp.143-162 L. E. Koren, et al., The Journal of Protozoology, 1967, volume 14, supplement, p.17

Compared with the manufacturing method described in Patent Document 2, the manufacturing method described in Patent Document 1 is considered to have the following advantages. First, since sunlight is not essential for culturing Euglena because sugar is provided, it has the advantage of being able to be cultivated all year round if the temperature is controlled without being affected by weather or sunshine hours. Secondly, since the low-oxygen treatment and the low-temperature treatment are not essential, there is an advantage that the cost and labor for these treatments can be reduced. Third, since even wild-type Euglena has a high WE yield, there is an advantage that the Euglena to be used is not limited to a specific mutant strain or the like.

However, in order to expand the scale of WE production and commercialize it in the future, it would be desirable to discover a method capable of promoting WE fermentation more efficiently. Furthermore, if possible, it would be preferable to develop a manufacturing method that saves labor and reduces manufacturing costs compared to conventional manufacturing methods. Propionic acid used in Patent Literature 1 is designated as a specific offensive odor substance under the Ordinance for Enforcement of the Offensive Odor Control Law. For this reason, if possible, it would be preferable if the WE production method could be improved so that even if propionic acid is used, the WE fermentation can be efficiently promoted even if the amount used is somewhat reduced.

Therefore, the object of the present invention is to provide a production method and production system for Euglena with a high wax ester content, and a wax ester that has been improved so that wax ester fermentation can be promoted more efficiently than conventional wax ester production methods using Euglena. Alternatively, it is to provide a method and system for producing a biofuel composition, and a wax ester fermentation accelerator.

In order to solve the above problems, the method for producing Euglena with a high wax ester content according to the present invention includes the steps of preparing Euglena, culturing the Euglena under aerobic conditions in the presence of sugar, and culturing providing a wax ester fermentation-promoting substance to the Euglena in an acidic environment; and maintaining the Euglena to which the wax ester fermentation-promoting substance has been given at room temperature for a predetermined period of time, wherein the wax ester fermentation-promoting substance is , a monovalent carboxylic acid having at least one of a keto group, an aldehyde group and a hydroxy group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and a salt thereof. It is a method for producing Euglena with a high wax ester content, which is one or more compounds.

In the method for producing Euglena with a high wax ester content according to the present invention, the wax ester fermentation-promoting substance is one or more compounds selected from the group consisting of glyoxylic acid (also referred to as glyoxylic acid), glycolic acid, and salts thereof. can be

The wax ester fermentation promoting substance may further contain a fatty acid having 1 to 4 carbon atoms in the molecule. In this case, the molar ratio (molar amount of fatty acid/total molar amount of the entire wax ester fermentation-promoting substance) of the fatty acid content relative to the wax ester fermentation-promoting substance is preferably 0.5 or less. preferable.

The method for producing a wax ester or biofuel composition according to the present invention includes the steps of preparing Euglena, culturing the Euglena under aerobic conditions in the presence of sugar, and culturing the cultured Euglena in an acidic environment. a step of providing a wax ester fermentation-promoting substance; a step of keeping the Euglena to which the wax ester fermentation-promoting substance has been given at room temperature for a predetermined time; and extracting wax ester from the Euglena kept at room temperature for a predetermined time. wherein the wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group and a hydroxy group in its molecule and has 2 or more and 4 or less carbon atoms in its molecule. A method for producing a wax ester or a biofuel composition, which is one or more compounds selected from the group consisting of carboxylic acids and salts thereof.

The wax ester fermentation accelerator according to the present invention is selected from the group consisting of monovalent carboxylic acids having a keto group or a hydroxy group in the molecule and having 2 to 4 carbon atoms in the molecule, and salts thereof. A wax ester fermentation promoter, which is a composition containing one or more compounds described above and is a composition to be given in an acidic environment to Euglena cultured under aerobic conditions in the presence of sugar.

The production system for Euglena with a high wax ester content according to the present invention includes means for culturing Euglena under aerobic conditions in the presence of sugar, means for providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment, means for recovering Euglena kept at room temperature for a predetermined time after being given the wax ester fermentation promoting substance, wherein the wax ester fermentation promoting substance contains a keto group, an aldehyde group and a hydroxy group in the molecule. and at least one compound selected from the group consisting of monovalent carboxylic acids having 2 or more and 4 or less carbon atoms in the molecule, and salts thereof, having a high wax ester content. Euglena production system.

The wax ester or biofuel production system according to the present invention includes means for culturing Euglena under aerobic conditions in the presence of sugar, means for providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment, means for recovering Euglena kept at room temperature for a predetermined time after being given the wax ester fermentation promoting substance; and means for extracting the wax ester from the recovered Euglena, wherein the wax ester fermentation promoting substance is provided. is selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group and a hydroxy group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and salts thereof A system for the production of wax esters or biofuels, which are one or more compounds that have

According to the method for producing Euglena with a high wax ester content according to the present invention, the wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group and a hydroxy group in the molecule, and a carbon atom in the molecule Since one or more compounds selected from the group consisting of monovalent carboxylic acids having a number of 2 or more and 4 or less and salts thereof are used, the yield of the wax ester can be further increased compared to conventional methods. rice field. In addition, according to the method for producing Euglena with a high wax ester content according to the present invention, Euglena with a high wax ester content can be produced by reducing the amount of expensive propionic acid used compared to the conventional method using propionic acid. cost can be reduced more than before.

Furthermore, according to the method for producing a wax ester or biofuel according to the present invention, it is possible to shorten the culture period compared to the conventional method, so the culture cost can be reduced, and the culture period can be shortened. Regardless, wax ester fermentation can be promoted to increase the yield of wax esters. The same applies to the production system for Euglena with a high wax ester content according to the present invention and the production system for wax ester or biofuel according to the present invention.

The wax ester fermentation-promoting substance in the method for producing Euglena with a high wax ester content according to the present invention is selected from the group consisting of, for example, glyoxylic acid, glycolic acid, and salts thereof, from the viewpoint of facilitating a further increase in the yield of wax esters. One or more compounds are preferred. Since the wax ester fermentation promoter according to the present invention is a composition containing one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof, it can efficiently promote wax ester fermentation. be.

1 is a flowchart illustrating an example of a method for producing Euglena with a high wax ester content according to the present invention. The flowchart explaining an example of the wax-ester manufacturing method which concerns on this invention. 1 is a flow chart explaining an example of a method for producing a biofuel composition according to the present invention. Measurement results of glucose content (paramylon content) quantified by the phenol-sulfuric acid method for each group containing Euglena gracilis Z strain aerobically cultured to mid-logarithmic growth phase in the presence of sugar in Experimental Example 1 Graph showing . n=3. Mean ± standard deviation. The vertical axis of the graph indicates the sugar content (μg/mL) in terms of glucose per 50 mL medium containing Euglena cells, and this is the same in FIGS. 6 and 8 described below. For each group containing the Euglena gracilis Z strain aerobically cultured to mid-logarithmic growth phase in the presence of sugar in Experimental Example 1, the content of the compound (C ₂₈ ) having 28 carbon atoms in the molecule , that is, a graph showing the measurement results of the myristyl myristate content. n=3. Mean ± standard deviation. The vertical axis of the graph shows the _C28 content (μg/1.0×10 ⁶ cells) per 1.0×10 ⁶ Euglena cells in 50 mL medium containing Euglena cells, which will be explained below. The same applies to FIGS. 7 and 9 as well. ** means significant difference at p<0.01 in Welch's t-test (two-tailed), and this is the same for FIGS. 7 and 9 described below. For each group containing the SM-ZK strain that was aerobically cultured to mid-logarithmic growth phase in the presence of sugar in Experimental Example 2, the glucose content (paramylon content) measured by the phenol-sulfuric acid method was measured. Graph showing. n=3. Mean ± standard deviation. 4 is a graph showing measurement results of _C28 content for each group containing Euglena gracilis Z strain aerobically cultured to mid-logarithmic growth phase in the presence of sugar in Experimental Example 2. FIG. n=3. Mean ± standard deviation. * means significant difference at p<0.05 in Welch's t-test (two-tailed), and this is the same for FIG. 9 described below. For each group containing the Euglena gracilis Z strain that was aerobically cultured in the presence of sugar until reaching the stationary phase in the Reference Experimental Example, the measurement results of the glucose content (paramylon content) quantified by the phenol-sulfuric acid method were obtained. Graph showing. n=3. Mean ± standard deviation. Graph showing measurement results of _C28 content for each group containing Euglena gracilis Z strain aerobically cultured in the presence of sugar until reaching stationary phase in Reference Experimental Example. n=3. Mean ± standard deviation.

<Production method of Euglena with high wax ester content>
The method for producing Euglena with a high wax ester content (Euglena with a high WE content) according to the present invention includes, for example, a preparation step S1, an aerobic culture step S2, a wax ester fermentation promoting substance (WE fermentation promoting substance ) including a mixing step S3, a wax ester fermentation (WE fermentation) step S4, and a Euglena recovery step S5.

In the preparation step S1, live Euglena cells are prepared. Euglena to be prepared is one or more kinds of protozoa selected from the group consisting of species belonging to the genus Euglena (genus Euglena) in zoological classification and variants thereof. Mutants herein also include cell lines (mutants) obtained by genetic methods such as recombination, transduction, or transformation. Examples of Euglena to be prepared include Euglena acus, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena gracilis (hereinafter referred to as "Euglena gracilis"), Euglena granulata, Euglena intermedia, Euglena mutabilis, Euglena oxyuris, Euglena piride, Euglena proxima, Euglena Species such as spirogyra, Euglena vermiformis, or Euglena viridis. Regardless of whether the Euglena to be prepared is a wild strain or a mutant strain, it is sufficient that the Euglena is capable of WE fermentation in its cells. For example, the Euglena to be prepared is good because even SM-ZK strain, which is a chloroplast-deficient mutant strain obtained by treating Euglena gracilis Z strain as a parent strain with streptomycin, can be WE fermented.

In the preparation step S1, wild Euglena widely distributed in freshwater such as ponds and marshes may be collected, but it is efficient to obtain any already isolated Euglena cell line. The Euglena to be prepared is preferably one or more protozoa selected from the group consisting of Euglena gracilis and its mutant strains from the viewpoint that suitable culture conditions have been studied in detail and are known and easy to culture. For example, Euglena gracilis, Euglena gracilis Z strain, Euglena gracilis var. One or more Euglena selected from the group consisting of strain Kishu gracilis (see Patent Document 4, accession number FERM P-22300) is preferred. From the same point of view, the Euglena to be prepared is more preferably Euglena gracilis Z strain.

Euglena can be cultured, for example, in pond water or tap water collected in a pool. From the viewpoint of culturing efficiently while avoiding various germs, it is preferable to also prepare a medium capable of culturing Euglena in the preparation step S1. The medium is not particularly limited as long as the seeded Euglena can be cultured and grown when the Euglena is seeded and kept under aerobic conditions at a medium temperature of 15° C. or higher and 35° C. or lower. The medium may be a solid medium such as an agar slant medium from the viewpoint of easiness to deal with contamination, but a liquid medium is preferable from the viewpoint of being inexpensive, easy to prepare, easy to stir, and easy to culture Euglena at high density.

Liquid media generally suitable for Euglena culture include vitamin _B12 , ammonium salts, and other inorganic salts (compounds containing phosphorus, potassium, iron, manganese, cobalt, zinc, copper, molybdenum, or nickel in the molecule ) and, if necessary, other nutrients such as sugars and amino acids. From the viewpoint that suitable culture conditions have been studied in detail and are known and easy to culture, it is more preferable to prepare a known liquid medium conventionally used for Euglena culture, or a liquid medium with a composition similar to this. As known liquid media, for example, Kramer-Meier medium (see Table 1, Non-Patent Document 1), Hattner medium (Table 2, see Non-Patent Document 2), or Koren-Hatner medium (hereinafter referred to as "KH medium See Table 3 and Non-Patent Document 3) and the like. When partially changing the formulation, it is preferable to prepare a medium with an increased amount of sugar blended from a known liquid medium from the viewpoint of saving the labor of adding sugar to the medium in the next aerobic culture step S2.

The medium preferably contains a chelating agent, a pH adjuster, or the like, as long as it does not contradict the object of the present invention. For example, EDTA-Na ₂ (ethylenediaminetetraacetic acid disodium salt) listed in Table 3 acts as a chelating agent. For example, in the formulations described in Tables 1-3, phosphate and citric acid act as pH modifiers. The medium may contain a peptide-containing composition or aqueous ammonia. Compositions containing peptides include, for example, peptone, casamino acids, yeast extract, corn steep liquor, and the like. The medium may contain one or more free amino acids or salts thereof. From the viewpoint of saving the trouble of adding sugar in the next aerobic culture step S2, the medium to be prepared preferably contains sugar. The sugar is not particularly limited as long as it is a water-soluble sugar that Euglena can take up into its cells and metabolize. Examples include glucose, fructose, sucrose, maltose, starch, and the like. Disaccharides and starch are decomposed into monosaccharides by enzymes and utilized by Euglena as nutrients. The sugar is preferably glucose from the viewpoint of promoting paramylon production in Euglena in the next aerobic culture step S2, or starch, molasses, or blackstrap molasses from the viewpoint of keeping production costs low.

In the aerobic culture step S2, the prepared Euglena is cultured under aerobic conditions in the presence of sugar in order to produce and store paramylon in the Euglena cells. The concentration of the sugar contained in the medium is preferably in the range of 1% by mass to 5% by mass, more preferably 1.5% by mass to 3% by mass during the aerobic culture step. . From the viewpoint of keeping the medium under an aerobic condition, it is preferable to culture while passing an oxygen-containing gas, such as air, through the medium. From the viewpoint of efficient aeration, shaking culture is preferable when the medium volume is 5 L or less, and aerobic culture with bubbling is preferable when the medium volume is greater than 5 L. As the medium and sugar, the sugars exemplified in the previous explanation of the preparation step S1 may be used. From the viewpoint of increasing the number of cells and promoting paramylon production, it is preferable to additionally add sugar to the medium according to the cell density of Euglena in the medium during the aerobic culture period. When a medium containing a sufficient amount of sugar is prepared and used, batch culture may be performed without adding sugar to this medium. When various conditions suitable for Euglena culture can be maintained, for example, when the dissolved oxygen concentration and the concentration of various nutrient components in the medium can be maintained within a certain range, continuous culture may be performed.

Euglena may be cultured in an environment where it is irradiated with sunlight so that Euglena can undergo photosynthesis, but since it is cultured in the presence of sugar in the aerobic culture step S2, Euglena can be cultured even in the dark. It is preferable to culture Euglena in an air-permeable culture tank from the viewpoint of cultivating it regardless of the weather, and in addition, from the viewpoint of avoiding contamination, culture in a culture tank with a closed space that allows aeration culture. more preferably. The medium temperature may be, for example, 15° C. or higher and 35° C. or lower, but is preferably 25° C. or higher and 30° C. or lower from the viewpoint of promoting efficient storage of paramylon in Euglena cells.

In the aerobic culture step S2, from the viewpoint of efficiently promoting the WE fermentation later in the WE fermentation step S4, the period for culturing Euglena under aerobic conditions in the presence of sugar is set to a steady state from the start of culture under these conditions. It is preferred to stay within the period to mid- or late-logarithmic growth phase that has not reached phase. Generally, the culture is started at the time when Euglena is seeded in the medium. The logarithmic growth phase is the period during which the cell number increases logarithmically in the growth curve of Euglena cells. The growth curve is, for example, a curve that can be created by seeding Euglena cells in a predetermined medium, measuring the cell number or cell density in the medium at regular intervals, and plotting the measured cell number or cell density on a graph. be. The stationary phase is the time after the logarithmic growth phase, the number of proliferating cells and the number of dying cells reach a general balance, and no increase in cell number is observed. Depending on the culture conditions such as the type of Euglena strain to be seeded, the type of medium, the type and amount of sugar, the temperature of the medium, and the amount of light irradiated to the medium, the cell density at the stationary phase, that is, the maximum number of cells that can be reached in the medium Density is different.

A person skilled in the art can use any known indicator or method to determine whether the growth is in the middle or late logarithmic growth phase, which has not yet reached the stationary phase. For example, turbidity or specific growth rate may be used as an index for determination. For example, when turbidity is used as an index, when Euglena cells are cultured under predetermined culture conditions, the turbidity of the medium that has not yet been seeded is set to 0%, and the turbidity of the medium that has reached the stationary phase is set to 100%. It is better to use the relative turbidity (%) of the medium as an indicator. When this relative turbidity is used as an index, the middle of the logarithmic growth phase is the period when the relative turbidity of the medium is greater than 35% and 70% or less, and the late logarithmic growth phase is the period when the relative turbidity of the medium is greater than 70%. It is a period of 95% or less. Turbidity is a measure of how cloudy the culture is. For example, light with a single wavelength of 660 nm is incident on the culture solution, and the transmitted light is measured with a spectrophotometer. Absorbance τ calculated by the formula "I = I ₀ Exp (-τL)", where I ₀ is the intensity of incident light, I is the intensity of transmitted light, L is the thickness of the transmission layer, and τ is the absorbance. is taken as the turbidity (OD) of the culture solution.

In the aerobic culture step S2, from the viewpoint of efficiently promoting the WE fermentation later in the WE fermentation step S4, in order to stop the aerobic culture step of Euglena at the middle or late logarithmic growth phase, Euglena is kept in the presence of sugar. The period of culturing under aerobic conditions should be 0.5 times or more and 0.8 times or less than the length of the period from the start of culturing to the stationary phase under these conditions. preferable. For example, if there are culture conditions that require 120 hours from the start of Euglena cell culture to reach the stationary phase, in the aerobic culture step S2, the Euglena cell culture period is kept within the range of 60 hours or more and 96 hours or less. is preferred.

In the WE fermentation-promoting substance mixing step S3, a WE fermentation-promoting substance is given to the aerobically cultured Euglena storing paramylon in the cells in an acidic environment. By providing the WE fermentation-promoting substance in an acidic environment, paramylon is decomposed in the Euglena cells to promote the production of a compound having 28 carbon atoms in the molecule (hereinafter also referred to as “C ₂₈ ”), that is, the production of WE. . From the viewpoint of promoting WE fermentation, the acidic environment is sufficient if the pH of the medium is less than 7.0, preferably 6.0 or less, more preferably 3.5 or less. It is preferred that the pH is 2.5 or higher. In the WE fermentation-promoting substance mixing step S3, for example, a medium containing Euglena that has undergone the previous aerobic culture step S2 is added with a WE fermentation-promoting substance and a medium pH of 2.5 or more and 3.5 or less. It is preferable to mix with a pH adjuster.

The WE fermentation-promoting substance in the present invention includes a monovalent carboxylic acid having at least one of a keto group, an aldehyde group and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and One or more compounds (A) selected from the group consisting of salts. The compound (A) has a carboxyl group in addition to the keto group, the aldehyde group, or the hydroxy group. Examples of this compound (A) include hydroxycarboxylic acids and monovalent carboxylic acids having an aldehyde group or a keto group in the molecule. Examples of the hydroxycarboxylic acid include compounds such as glycolic acid, lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, or γ-hydroxybutyric acid. Examples of monovalent carboxylic acids having an aldehyde group or keto group in the molecule include compounds such as glyoxylic acid, pyruvic acid, 3-oxopropanoic acid, α-ketobutyric acid, and acetoacetic acid. Among these, it is particularly preferable to use one or more compounds selected from the group consisting of glycolic acid, glyoxylic acid, and salts thereof as the compound (A). Such low-molecular-weight organic compounds are considered to easily permeate Euglena cells through cell membranes and the like. When Euglena cells are supplied with an excessive amount of low-molecular-weight organic compounds that are easily permeable into the cells, aerobic respiration is inhibited (e.g., feedback inhibition) within the cells, and WE is used to acquire ATP instead of aerobic respiration. It is considered that fermentation is likely to be promoted.

As a WE fermentation promoting substance, in addition to the compound (A) described above, a fatty acid (B) having 1 or more and 4 or less carbon atoms in the molecule may be further added. Examples of the fatty acid (B) include compounds such as acetic acid, propionic acid, butyric acid, isobutyric acid, and formic acid. The fatty acid (B) preferably contains a compound having a linear saturated hydrocarbon group having 1 or more and 3 or less carbon atoms in its molecule. The WE fermentation-promoting substance may be a combination of two or more compounds from the group of compounds (A) exemplified here, or a combination of two or more compounds from the group of fatty acids (B). good. Moreover, the salt is not particularly limited as long as it is ionizable in the aqueous solution of the medium as long as it does not contradict the purpose of the present invention. The salt is preferably a sodium salt or a potassium salt from the viewpoint of not easily precipitating in the medium.

When the compound (A) and the fatty acid (B) are used in combination, the WE fermentation-promoting substance is a combination of glyoxylic acid and glycolic acid, a combination of glyoxylic acid and propionic acid, from the viewpoint of easily promoting WE fermentation. Alternatively, a combination of glycolic acid and propionic acid is preferred. In the case of a combination of glyoxylic acid and propionic acid, or in the case of a combination of glycolic acid and propionic acid, compared to the case of using only propionic acid, the amount of propionic acid used is reduced while promoting WE fermentation. It is also preferable from the viewpoint of being possible. From a similar point of view, when two or more compounds containing propionic acid are used as WE fermentation promoting substances, the molar ratio of propionic acid to the WE fermentation promoting substance (molar amount of fatty acid/total molar amount of all wax ester fermentation promoting substances) is preferably 0.5 or less, more preferably 0.4 or less.

Alternatively, from the viewpoint of avoiding the problem of odor caused by propionic acid, in the promoting substance mixing step S3, the cultured Euglena is given a WE fermentation promoting substance in an acidic environment, but propionic acid is not substantially given. good. The term "substantially not provided" means that even if a trace amount of a component that is supposed not to be provided is contained in the medium or in the cells, it is a very small amount that is recognized as not contributing to WE fermentation in the content and essence of the present invention. is acceptable. For example, a small amount of propionic acid may be produced in Euglena cells during metabolism, but it is allowed in the present invention because it is only a small amount that does not contribute to WE fermentation. “Substantially not provided” means that the concentration of the compound that is not provided in the medium is, for example, 100 μmol/L or less, preferably 10 μmol/L or less, and more preferably 1.0 μmol/L or less.

In the WE fermentation promoting substance mixing step S3, from the viewpoint of efficiently promoting WE fermentation in Euglena cells, the content of the WE fermentation promoting substance in the medium containing Euglena is, for example, 0.5 mmol/L or more, preferably 1.0 mmol/L. It is desirable to provide Euglena with the WE fermentation-promoting substance by mixing it with the culture medium so that the amount of the WE fermentation promoting substance is 2.0 mmol/L or more, preferably 2.0 mmol/L or more. From the viewpoint of avoiding death of Euglena cells, the content of the WE fermentation promoter in the medium containing Euglena is, for example, less than 10 mmol/L, preferably less than 8.0 mmol/L, more preferably less than 6.0 mmol/L. It is preferable to give Euglena a WE fermentation-promoting substance by mixing it with the medium. When there are two or more corresponding compounds, for example, when glyoxylic acid and glycolic acid are used as WE fermentation promoting substances, the content means the total content of two or more compounds. It is preferable to mix the WE fermentation-promoting substance and the pH adjuster in the medium so that the composition of the medium containing Euglena has the above molar concentration ratio.

In the WE fermentation step S4, the Euglena fed with the WE fermentation promoting substance is kept at room temperature for a predetermined period of time for intracellular WE fermentation. In the natural world, Euglena cells that have photosynthesized on the surface of the water and have stored paramylon sink to the bottom of the water during winter when the sunlight is weak, and overwinter while performing WE fermentation that decomposes paramylon. However, WE fermentation progresses over time. On the other hand, at high temperatures over 35°C, metabolism is likely to be disturbed in Euglena cells. The liquid temperature of the Euglena-containing medium may be adjusted in order to maintain normal temperature. From the viewpoint of cost saving, it is preferable to reduce the cost of adjusting the medium temperature by performing the WE fermentation step S4 in a season or region where the outside temperature is 5°C or higher and 35°C or lower. From the viewpoint of promoting WE fermentation, the "normal temperature" is, for example, 5°C or higher and 35°C or lower, preferably 15°C or higher and 35°C or lower, and more preferably 20°C or higher and 30°C or lower.

From the viewpoint of sufficient WE fermentation in individual Euglena cells, the "predetermined time" in the WE fermentation step S4 is, for example, 6 hours or longer, preferably 12 hours or longer, and more preferably 24 hours or longer. From the viewpoint of avoiding an unnecessarily long period of time, the "predetermined time" is, for example, 96 hours or less, preferably 72 hours or less, and still more preferably 48 hours or less. Euglena fed with a WE fermentation-promoting substance inhibits aerobic respiration in the cell by the WE fermentation-promoting substance and performs WE fermentation. produces WE even when kept at From the viewpoint of reducing the labor and cost of the treatment to maintain the hypoxic condition, it is preferable to keep the Euglena to which the WE fermentation promoting substance has been given at room temperature in the presence of air for a predetermined period of time.
Furthermore, in this WE fermentation step S4, the medium containing Euglena may be bubbled with nitrogen using, for example, a bubbling device for aerobic culture. Nitrogen bubbling may be performed throughout the WE fermentation step, but may be performed only for a predetermined period of time, such as the first three hours, in an environment in which it is difficult for oxygen to enter the medium from the outside.
It is presumed that similar effects can be obtained by reducing the pressure in the container containing the culture medium to reduce the amount of oxygen instead of nitrogen bubbling.

In the Euglena recovery step S5, Euglena that has been kept at room temperature for a predetermined time after being given the WE fermentation promoting substance is recovered. For example, the medium may be centrifuged with a centrifuge to collect the precipitate (precipitated Euglena cells). The recovered Euglena has a clearly increased WE content compared to the Euglena prepared in the previous preparation step S1, that is, Euglena with a high WE content.

According to the method for producing Euglena with a high WE content according to the present invention, Euglena is cultured under aerobic conditions in the presence of sugar, then given a WE fermentation promoting substance in an acidic environment and kept at room temperature for a predetermined time. , promoting WE fermentation in Euglena cells. By limiting the period of culturing Euglena under aerobic conditions in the presence of sugar from the start of the culture to the middle or late period of the logarithmic growth phase of Euglena, Euglena kept at room temperature for a predetermined period of time after that unexpectedly Also, the yield of WE is enhanced. Thus, the method for producing Euglena with a high WE content according to the present invention includes a step of culturing from the start of the culture to the stationary phase when Euglena is cultured under aerobic conditions in the presence of sugar as in the conventional method. Compared to the method, the culture cost can be reduced because the culture period is shortened, and the WE fermentation can be promoted to increase the yield of WE despite the shortened culture period.

The mechanism by which WE fermentation is further promoted when glyoxylic acid or glycolic acid is given to Euglena compared to when only propionic acid is given as a WE fermentation promoting substance to Euglena is unknown. Considering that both glyoxylic acid and glycolic acid are intermediate products in the glycolic acid cycle, it is possible that their addition more efficiently inhibits aerobic respiration in Euglena cells and promotes WE fermentation. gender can be considered.

<Method for producing wax ester>
The wax ester production method (WE production method) according to the present invention includes, for example, as shown in FIG. It includes a step S5 and a wax ester extraction (WE extraction) step S6. Steps S1 to S5 are as described with reference to FIG. 1 in the method for producing Euglena with a high WE content according to the present invention. In the WE extraction step S6 shown in FIG. 2, WE is extracted from the collected Euglena, that is, the WE-rich Euglena. A specific method for this is not particularly limited as long as it is possible to extract the WE, as long as it does not contradict the purpose of the present invention. For example, WE-rich Euglena cells may be disrupted, centrifuged, and the hydrophobic supernatant (WE layer derived from Euglena) may be collected. If necessary, the collected supernatant may be purified, for example, by the WE extraction method described below. According to the method for producing WE according to the present invention, the method for producing Euglena with a high WE content according to the present invention is carried out in the course of its implementation. The cost can be reduced, and the WE fermentation can be promoted and the yield of WE can be increased despite the shortened culture period. WE derived from Euglena can be used not only as a raw material for biofuel compositions, but also as raw materials for cosmetics, soap, livestock feed, or fish feed.
In addition, although a method for extracting WE from Euglena was described here, it is also possible to use the dried body of WE-rich Euglena collected from the medium as it is as a fuel without extracting WE from Euglena with a high WE content.

<Method for producing biofuel composition>
The method for producing a biofuel composition according to the present invention includes, for example, as shown in FIG. , a WE extraction step S6, and a biofuel preparation step S7. Steps S1 to S5 are as described above with reference to FIG. 1, and step S6 is as described with reference to FIG. In the biofuel preparation step S7, a biofuel composition is prepared using WE derived from Euglena. For example, known techniques for producing biofuels using WE may be implemented. According to the method for producing a biofuel composition according to the present invention, in order to implement the method for producing Euglena with a high WE content according to the present invention in the process of implementation, compared to the conventional biofuel production method using Euglena, the culture period can be shortened, the culture cost can be reduced, and despite the shortened culture period, WE fermentation can be promoted and the yield of WE can be increased.

<Wax ester fermentation accelerator>
The wax ester fermentation promoter (WE fermentation promoter) according to the present invention is selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof from the viewpoint of being a compound that promotes WE fermentation more easily than propionic acid. Compositions containing one or more compounds. Moreover, the WE fermentation promoter according to the present invention is a composition to be given in an acidic environment to Euglena cultured under aerobic conditions in the presence of sugar. The WE fermentation promoter may be a composition to be given in an acidic environment to Euglena cultured under aerobic conditions until reaching the stationary phase in the presence of sugar. Furthermore, from the viewpoint of promoting WE fermentation, the WE fermentation promoter promotes the cultivation of Euglena under aerobic conditions in the presence of sugar until the middle or late logarithmic growth phase, which has not yet reached the stationary phase. It is preferably a composition intended to be given in the environment. The WE fermentation promoter may be a liquid formulation or a solid formulation. The WE fermentation promoter when it is a solid formulation can take forms such as tablets, tablets, granules, suspensions, syrups, or dry powders so as to be easily dissolved in a liquid medium. The WE fermentation promoter may optionally contain known additives such as excipients, antioxidants, diluents, buffers, flavoring agents, or coloring agents together with the WE fermentation promoter. good. The WE fermentation promoter is used, for example, in the WE fermentation promoter mixing step S3 ( 1 to 3).

<Production system for Euglena with high wax ester content>
A production system for Euglena with a high WE content according to the present invention comprises a culture means, a means for supplying a WE fermentation-promoting substance, and a recovery means. The culture means are means for culturing Euglena under aerobic conditions in the presence of sugar. Examples of culture means include a combination of a culture tank for culturing Euglena and a medium suitable for culturing Euglena so that the preparation step S1 and the aerobic culture step S2 described with reference to FIG. 1 can be performed. be done. As a culturing means, a combination of a nutrient such as sugar, which is preferably mixed with the medium, and a bubbling device for aerobic culture is preferred as required. The period of aerobic culture is limited from the start of the culture to the mid- or late-logarithmic growth phase of Euglena. The WE fermentation-promoting substance supplying means is a means for supplying the above-described WE fermentation-promoting substance in an acidic environment to Euglena that has been cultured under aerobic conditions in the presence of sugar and has paramylon-imbued intracellularly. The recovering means is a means for recovering Euglena kept at room temperature for a predetermined period of time after being given the WE fermentation-promoting substance.

<Manufacturing system for wax ester or biofuel composition>
The WE production system according to the present invention further includes extraction means for extracting WE from the collected Euglena in addition to the above-described production system for Euglena with a high WE content according to the present invention. Examples of extraction means include a combination of a centrifuge and a cell disruption device. When WE is purified, a combination further containing a solvent suitable for WE extraction is preferred as an extraction means. Suitable solvents for WE extraction include, for example, acetone. The biofuel composition production system according to the present invention includes, in addition to the WE production apparatus according to the present invention, a means for mixing WE derived from Euglena with other fuel raw materials to prepare a biofuel composition. Prepare.

The present invention can also be implemented in aspects with various improvements, modifications, or variations based on the knowledge of those skilled in the art without departing from the spirit of the invention. The present invention may be implemented in a form in which any of the matters specifying the invention is replaced with another technique within the scope of producing the same action or effect. EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

<Method for quantitative determination of paramylon>
In Experimental Example 1, Experimental Example 2, and Reference Experimental Example, which will be described later, paramylon was extracted from Euglena cells and quantified by the method described below. A liquid medium containing Euglena cells (e.g., KH medium) is centrifuged, a precipitate (precipitated Euglena cells) is collected, 100% by mass of acetone is added to the precipitate, and the mixture is vigorously stirred with a vortex mixer or sonicated. A suspension was prepared by processing with a crusher. This suspension is centrifuged (12,000 rpm, 4 ° C., 10 minutes), the supernatant is removed, 100% by mass of acetone is added to the precipitate, and the mixture is vigorously stirred with a vortex mixer or sonicated. The operation of preparing a suspension by treating with was repeated until the precipitate became white. Add 1.0 mL of 1.0% by mass SDS (sodium dodecyl sulfate) solution to the white precipitate obtained by removing the supernatant, stir with a vortex mixer to suspend, boil for 5 minutes, It was left on ice to cool sufficiently. This ice-cooled SDS solution is centrifuged (12,000 rpm, 4°C, 10 minutes), the supernatant is removed, the precipitate is washed with ultrapure water, and centrifuged again (12,000 rpm, 4°C). , 10 min), the supernatant was removed, 1.0 mL of 1.0 N aqueous sodium hydroxide solution was added to the precipitate, and the suspension for quantitation of paramylon was added by shaking overnight until the precipitate was no longer visible. A turbid liquid was obtained.

The paramylon content in the suspension was determined by the phenol-sulfuric acid method described below. 0.2 mL of diluted paramylon quantitative suspension was collected, poured into a test tube, stirred with a vortex mixer, mixed with 0.2 mL of 5% by mass phenol solution, and mixed with Eppendorf's Multipette (registered trademark) M4. 1.0 mL of conc. After this mixture was kept at 30° C. for 30 minutes, 200 μL of each was dispensed into a 96-well microplate, and the absorbance at a wavelength of 490 nm was measured using a microplate reader. For the calibration curve by the phenol-sulfuric acid method, a 10 mg/mL glucose aqueous solution was diluted with ultrapure water to give 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, and 31.25 µg/mL, respectively. , and 15.625 μg/mL glucose aqueous solutions were prepared, each glucose aqueous solution was treated in the same manner as the paramylon quantification suspension, and the absorbance at a wavelength of 490 nm was measured to create a calibration curve.

<Wax ester quantitative method>
In Experimental Example 1, Experimental Example 2, and Reference Experimental Example, which will be described later, WE was extracted from Euglena cells and quantified by the method described below. Liquid medium containing Euglena cells (eg, KH medium) is centrifuged to collect the precipitate (precipitated Euglena cells), chloroform: methanol: ultrapure water (eg Milli-Q (registered trademark) water) = 10: Add the mixed solution at a volume ratio of 20:8, stir vigorously with a vortex mixer or treat with an ultrasonicator for 15 minutes, and centrifuge (13,000 rpm, 4 ° C., 3 minutes) to obtain the supernatant. was operated to collect The supernatant obtained by repeating this operation three times became a white lipid solution containing a high concentration of WE. For example, 1.0 mL of chloroform and 1.0 mL of ultrapure water are added to 1.8 mL of lipid solution, stirred with a vortex mixer for 30 seconds, and centrifuged (13,000 rpm, 4 ° C., 3 minutes ), the upper mixed layer of water and methanol was removed, and the lower chloroform layer was collected. The collected chloroform layer was dried in a vacuum dryer, the dried solid was dissolved in 500 μL of hexane, diluted to prepare a sample solution for analysis, and the gas chromatograph mass spectrometer (hereinafter "GC-MS ) was used to quantify a compound (C ₂₈ ) having 28 carbon atoms in the molecule (C 28 ), that is, myristyl myristate, which is a type of WE.

As GC-MS, GCMS-QP2010 Ultra manufactured by Shimadzu Corporation was used. As a column for separation, Agilent J&W GC column-DB-5ms (column length 30 cm, inner diameter 0.25 mm, membrane pore 0.25 μm) manufactured by Agilent Technologies, Inc. was used. A sample solution for analysis of 1.0 μL was injected into the separation column, and helium as a carrier gas was injected into the separation column at a flow rate of 1.16 mL/min. In separating the components contained in the sample solution for analysis, the column temperature was set to 100°C for the first minute, then the column temperature was raised to 280°C at a rate of 10°C/min, and then the column temperature was raised to 10°C. It was held at 280° C. for minutes. The interface and ion source were set at 250°C. Myristyl myristate was ionized at 70 eV and was detected and quantified in SIM mode at m/z = 229.2 and m/z = 57.1. As a standard sample solution, the content of myristyl myristate manufactured by Sigma-Aldrich was dissolved in hexane so that the content was 3.0 μg/mL, 1.0 μg/mL, 0.3 μg/mL, or 0.1 μg/mL. solution was used.

<Experimental example 1>
As Euglena, an experimental Euglena gracilis Z strain, which was distributed from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, was prepared. In order to culture this Euglena, a KH medium (see Table 3 above) was prepared in a flask. The neck of the flask was stuffed with a cotton stopper, and autoclaved at 2 atmospheres and 121° C. for 15 minutes for autoclave sterilization. The sterilized medium is placed in a clean bench, and after the medium has cooled, about 1.0 × 10 ⁴ cells or more and 3.0 × 10 ⁴ cells or less of Euglena cells are seeded in the medium so as not to be contaminated with various bacteria. A small amount of cell suspension was added as such. The culture was started when the cell suspension was added. From the start of the culture, in a culture room maintained at 28 ° C. or higher and 30 ° C. or lower, the medium in which the cells were seeded is continuously shaken at about 80 rpm with a shaker in the dark, so that Euglena is added to 2% by mass of sugar. was cultured under aerobic conditions in the presence of When cultured under these conditions, a culture period of 6 days or more (144 hours or more) is required for Euglena cells to reach the stationary phase. In Experimental Example 1, the aerobic culture under these conditions was terminated after 96 hours (4 days) from the start of the culture. That is, in Experimental Example 1, the aerobic culture period was limited to the mid-logarithmic growth phase, which had not yet reached the stationary phase.

Immediately after the end of the aerobic culture period, the medium was stirred, a small amount was collected, dropped onto a hemocytometer, and a cover glass was attached on the drop. Observed under a microscope, the number of Euglena cells in a section of 1.0 mm long×1.0 mm wide was counted on a hemocytometer. The number of cells N _C per 1.0 mL of medium was calculated according to the formula "N _C =N _N ×10 ⁴ ", where _NN is the "average number of cells per 1.0 mm ² ". “10 ⁴ ” in this formula is a conversion value of capacitance for 1.0 mm ² . The number of Euglena cells in the KH medium at the end of the aerobic culture in Experimental Example 1 was 5.37×10 ⁶ cells/mL. In addition, a large number of flasks were prepared, and KH medium containing Euglena gracilis Z strain that had been aerobically cultured to mid-logarithmic growth phase, which had not yet reached the stationary phase, was added to each flask immediately after the end of the aerobic culture period. 50 mL each was dispensed into each. The Euglena in each dispensed flask is described below as Time 0 Group A, Nitrogenated Group A, Propionic Acid Group A, Glycolic Acid Group A, Glyoxylic Acid Group A, Glycolic Acid & Propionic Acid Group A, and , glyoxylic acid & propionic acid group A.

In the 0-hour group A, the paramylon content and the WE content of the Euglena cells were quantified by the above-described quantification method immediately after completing the aerobic culture. In the nitrogen treatment group A, the medium was allowed to stand for 24 hours in a culture chamber maintained at a room temperature of 28° C. or higher and 30° C. or lower, without adding a WE fermentation promoting substance to the medium while supplying nitrogen gas to the medium and bubbling it. After that, the paramylon content and WE content of Euglena cells were quantified in the same manner. In the propionic acid group A, propionic acid was added so that the propionic acid concentration in the medium was 4.0 mmol/L to make the medium weakly acidic, and the medium was allowed to stand still for 24 hours while being aerated through a cotton plug. After placement, the paramylon content and WE content of the Euglena cells were quantified in the same manner. In the glycolic acid group A, the procedure was the same as in the propionic acid group A, except that propionic acid was added to the medium so that the glycolic acid concentration in the medium was 4.0 mmol/L. In the glyoxylic acid group A, the procedure was the same as in the propionic acid group A, except that glyoxylic acid was added to the medium so that the glyoxylic acid concentration in the medium was 4.0 mmol/L. In the glycolic acid & propionic acid group A, glycolic acid and propionic acid were added to the medium so that the glycolic acid concentration was 2.0 mmol/L and the propionic acid concentration was 2.0 mmol/L. Same as acid group A. In the glyoxylic acid & propionic acid group A, glyoxylic acid and propionic acid were added to the medium so that the glyoxylic acid concentration was 2.0 mmol/L and the propionic acid concentration was 2.0 mmol/L. Same as acid group A. The quantitative results of the paramylon content are shown in FIG. 4 and Table 4 below, and the quantitative results of the _C28 content are shown in FIG. 5 and Table 4 below.

As shown in FIG. 4 and Table 4, the propionic acid group A showed no reduction in paramylon content compared to the 0 hour group A. On the other hand, compared with the propionic acid group A, the nitrogen-treated group A, the glycolic acid group A, and the glyoxylic acid group A each had a clearly lower paramylon content. Compared to each of the nitrogen-treated group A, the glycolic acid group A, and the glyoxylic acid group A, the paramylon content was clearly lower in the glycolic acid & propionic acid group A and the glyoxylic acid & propionic acid group A, respectively. . As shown in FIG. 5 and Table 4, _C28 content was higher in other groups than in 0 hour group A. Also, compared to propionic acid group A, each of nitrogen treatment group A, glycolic acid group A, glyoxylic acid group A, glycolic acid & propionic acid group A, and glyoxylic acid & propionic acid group A had a _C28 content of There were many

From the experimental results shown in FIGS. 4 and 5 and Table 4, each of glycolic acid and glyoxylic acid metabolizes paramylon implied in cells more than propionic acid, which is preferred in Patent Document 1. It was suggested that it is a compound that is likely to efficiently promote the WE fermentation that produces myristyl myristate. In addition, as the WE fermentation promoting substance, when glycolic acid and propionic acid are used together so that the total is 4 mmol/L, or when glyoxylic acid and propionic acid are used together so that the total is 4 mmol/L , it was suggested that WE fermentation can be efficiently promoted while reducing the amount of propionic acid used, compared to the case of using only 4 mmol/L of propionic acid.

<Experimental example 2>
Compared to Experimental Example 1 using the Euglena gracilis Z strain, in Experimental Example 2, as Euglena, the SM-ZK strain distributed from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University was changed to use. , and other conditions were the same as in Experimental Example 1. The number of Euglena cells in the KH medium at the end of the aerobic culture in Experimental Example 2 was 7.05×10 ⁶ cells/mL. Immediately after the end of the aerobic culture period, 50 mL of KH medium containing the SM-ZK strain that had been aerobically cultured to mid-logarithmic growth phase, which had not yet reached the stationary phase, was dispensed into each flask. In Experimental Example 1 described above, each of 0 hour group A, nitrogen treatment group A, propionic acid group A, glycolic acid group A, glyoxylic acid group A, glycolic acid & propionic acid group A, and glyoxylic acid & propionic acid group A was prepared in this order by the same operation in Experimental Example 2, 0 hour group B, nitrogen treatment group B, propionic acid group B, glycolic acid group B, glyoxylic acid group B, glycolic acid & propionic acid group B, and each of glyoxylic acid & propionic acid group B was prepared. For each group prepared in Experimental Example 2, the quantitative results of paramylon content are shown in FIG. 6 and Table 5 below, and the _C28 content is shown in FIG. 7 and Table 5 below.

As shown in FIGS. 6 and 7 and Table 5, Experimental Example 2 using the SM-ZK strain showed the same experimental results as Experimental Example 1 using the Euglena gracilis Z strain. Therefore, not only Euglena gracilis Z strain and SM-ZK strain, but also glycolic acid and glyoxylic acid are considered to be compounds that promote WE fermentation more efficiently than propionic acid.

<Experimental example 3>
Compared to Experimental Example 1 in which the aerobic culture period was limited to the middle of the logarithmic growth phase, which had not yet reached the stationary phase, in Experimental Example 3, aerobic culture was performed until the stationary phase was reached. , and other conditions were the same as in Experimental Example 1. That is, in Experimental Example 3, Euglena gracilis Z strain was cultured in KH medium (in the presence of sugar) under aerobic conditions for 168 hours (7 days) from the start of culture. The number of Euglena cells in the KH medium at the end of the aerobic culture in Experimental Example 3 was 2.04×10 ⁷ cells/mL. Therefore, in Experimental Example 3, the number of Euglena cells increased by about 3.8 times as compared with Experimental Example 1 due to aerobic culture until reaching the stationary phase. Immediately after the end of the aerobic culture period, 50 mL of KH medium containing the Euglena gracilis Z strain that had been aerobically cultured until reaching the stationary phase was dispensed into each flask. In Experimental Example 1 described above, each of the 0-hour group A, the nitrogen treatment group A, the propionic acid group A, the glycolic acid group A, and the glyoxylic acid group A was prepared. A time 0 group C, a nitrogen treatment group C, a propionic acid group C, a glycolic acid group C, and a glyoxylic acid group C were each prepared. For each group prepared in Experimental Example 3, the quantitative results of paramylon content are shown in FIG. 8 and Table 6 below, and the quantitative results of C ₂₈ content are shown in FIG. 9 and Table 6 below.

As shown in FIG. 9 and Table 6, even in the Euglena gracilis Z strain cultured under aerobic conditions in KH medium (in the presence of sugar) until reaching the stationary phase, each of glycolic acid and glyoxylic acid It was suggested that it is a compound that promotes WE fermentation more efficiently than propionic acid. As is clear from a comparison of Table 6 (Experimental Example 3) and Table 4 (Experimental Example 1) described above, or from a comparison of FIG. 8 (Experimental Example 3) and FIG. 4 (Experimental Example 1), In addition, compared to each group in Experimental Example 1, in which the aerobic culture was stopped until the middle of the logarithmic growth phase, each group in Experimental Example 3, in which the aerobic culture was performed until reaching the stationary phase, was quantified by the phenol-sulfuric acid method. The resulting glucose content (paramylon content) was about 100 times higher. Therefore, the number of digits on the vertical axis of the graph differs between FIG. 8 (reference experiment example) and FIG. 4 (experiment example 1). Thus, it was confirmed that when aerobic culture is continued until reaching the stationary phase, more paramylon is stored in individual Euglena cells than when the aerobic culture is stopped until the mid-logarithmic growth phase. .

Nevertheless, unexpectedly, as can be seen by comparing Table 6 (Experimental Example 3) with Table 4 (Experimental Example 1) above, or FIG. 9 (Experimental Example 3) and FIG. 5 (Experimental Example 1) ), compared with each group in Experimental Example 1 in which the aerobic culture was stopped until the middle of the logarithmic growth phase, each group in Experimental Example 3 in which the aerobic culture was performed until the stationary phase was reached. In the group, the _C28 content (myristyl myristate content) was around 0.1 times. In other words, from the comparison between each group of Experimental Example 3, in which the aerobic culture was performed until reaching the stationary phase, and Experimental Example 1, in which the aerobic culture was limited to the middle of the logarithmic growth phase, the period of the aerobic culture was It can be seen that the _C28 content (myristyl myristate content) in the body of Euglena is sufficiently high even when it is limited to the middle of the logarithmic growth phase. From this result, it was found that when glycolic acid and glyoxylic acid were given, Euglena with a sufficiently high WE content could be obtained even when the aerobic culture period was shortened.

Although the mechanism by which the WE content per individual Euglena increases compared to the conventional aerobic culture up to the stationary phase is unknown at the time of filing the present application, it is speculated as follows. Euglena has the characteristic of being able to grow under various nutritional conditions by changing the intracellular storage substances according to the nutritional conditions. This characteristic has evolved in the process of evolution as a characteristic that guarantees the survival of Euglena under a starvation environment by encapsulating nutrients that can be produced in the cell depending on the environment that the Euglena cell is exposed to. It is thought that Euglena cells are equipped with this through repetition of mutation and natural selection. In addition, in Euglena cells in the middle of the logarithmic growth phase, considering that the cells are actively dividing or proliferating, the metabolic pathways that contribute to the increase in cell number and cell survival are not substantially suppressed. It is considered that the WE fermentation ability inherent in Euglena cells is likely to be sufficiently exhibited.

INDUSTRIAL APPLICABILITY According to the present invention, a method and system for producing Euglena with a high WE content, and a method and system for producing a WE or biofuel composition, which have been improved so as to promote wax ester (WE) fermentation more efficiently than before. and a WE fermentation promoter.

Claims (10)

A step of preparing Euglena;
a step of culturing the Euglena under aerobic conditions in the presence of sugar;
A step of providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
a step of keeping the Euglena to which the wax ester fermentation promoting substance has been given at room temperature for a predetermined period of time;
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
A method for producing Euglena with a high wax ester content. 2. The method for producing a wax ester-rich Euglena according to claim 1, wherein the wax ester fermentation promoting substance further contains a fatty acid having 1 or more and 4 or less carbon atoms in the molecule. 3. The wax ester-rich Euglena according to claim 2, wherein the molar ratio of the fatty acid in the wax ester fermentation-promoting substance (molar amount of fatty acid/total molar amount of the entire wax ester fermentation-promoting substance) is 0.5 or less. production method. The method for producing Euglena with a high wax ester content according to any one of claims 1 to 3, wherein nitrogen bubbling is performed in the step of maintaining the room temperature. A step of preparing Euglena;
a step of culturing the Euglena under aerobic conditions in the presence of sugar;
A step of providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
a step of keeping the Euglena to which the wax ester fermentation promoting substance has been given at room temperature for a predetermined time;
a step of extracting the wax ester from the Euglena kept at room temperature for a predetermined time,
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
A method for producing a wax ester. Wax ester fermentation promotion for feeding Euglena cultured under aerobic conditions in the presence of sugar in an acidic environment, containing one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof agent. A means for culturing Euglena under aerobic conditions in the presence of sugar;
means for providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
and means for recovering Euglena kept at room temperature for a predetermined time after being given the wax ester fermentation promoting substance,
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
Production system for Euglena with high wax ester content. A means for culturing Euglena under aerobic conditions in the presence of sugar;
means for providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
means for recovering Euglena kept at room temperature for a predetermined time after being given the wax ester fermentation promoting substance;
and means for extracting wax esters from the collected Euglena,
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
Wax ester manufacturing system. A step of preparing Euglena;
a step of culturing the Euglena under aerobic conditions in the presence of sugar;
A step of providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
a step of keeping the Euglena to which the wax ester fermentation promoting substance has been given at room temperature for a predetermined time;
A step of extracting a wax ester from the Euglena kept at room temperature for a predetermined time;
mixing the extracted wax ester with other fuel raw materials to prepare a biofuel,
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
A method for producing biofuel. A means for culturing Euglena under aerobic conditions in the presence of sugar;
means for providing a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment;
means for recovering Euglena kept at room temperature for a predetermined time after being given the wax ester fermentation promoting substance;
means for extracting wax esters from the recovered Euglena;
means for mixing the extracted wax ester with other fuel raw materials to prepare a biofuel,
The wax ester fermentation promoting substance has at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule, and has 2 to 4 carbon atoms in the molecule, and a monovalent carboxylic acid and a salt thereof. One or more compounds selected from the group consisting of
The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof.
Biofuel production system.

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