WO2016181902A1 - ユーグレナを用いた有機酸の生産方法 - Google Patents
ユーグレナを用いた有機酸の生産方法 Download PDFInfo
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- WO2016181902A1 WO2016181902A1 PCT/JP2016/063656 JP2016063656W WO2016181902A1 WO 2016181902 A1 WO2016181902 A1 WO 2016181902A1 JP 2016063656 W JP2016063656 W JP 2016063656W WO 2016181902 A1 WO2016181902 A1 WO 2016181902A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
Definitions
- the present invention relates to a method for producing an organic acid typified by succinic acid that is useful as a raw material for polybutylene succinate (PBS) using Euglena, and a Euglena culture product obtained by the method.
- PBS polybutylene succinate
- biodegradable biomass plastics having the properties of both biomass plastic and biodegradable plastic include polylactic acid (PLA), starch resin, polyhydroxyalkanoate (PHA), and polybutylene succinate (PBS).
- PLA polylactic acid
- PHA polyhydroxyalkanoate
- PBS polybutylene succinate
- PBS is synthesized by a condensation reaction from succinic acid and 1,4-butanediol, and is known as a highly useful biodegradable biomass plastic.
- the raw material succinic acid has been synthesized using petroleum as a raw material, but recently, saccharides derived from corn, sugarcane, etc. are used to produce heterotrophic bacteria by fermentation.
- succinic acid is preferable to produce succinic acid using as a raw material biomass that does not compete with existing food production.
- Patent Document 1 discloses a method for producing Euglena containing a high amount of wax ester as a raw material for biofuel.
- An object of this invention is to provide the method of producing organic acids, such as a succinic acid, by algae.
- the gist of the present invention is as follows. (1) Using Euglena, including a nitrogen-deficient culture step in which Euglena is cultured aerobically under a nitrogen-deficient condition, and an anaerobic culture step in which a culture product obtained in the nitrogen-deficient culture step is incubated under anaerobic conditions Organic acid production method.
- (11) By a method comprising a nitrogen-deficient culture step in which Euglena is cultured aerobically for 3 days or more under nitrogen-deficient conditions, and an anaerobic culture step in which the culture product obtained in the nitrogen-deficient culture step is incubated under anaerobic conditions.
- a heterotrophic culture step in which Euglena is cultured aerobically using a medium containing a carbon source an anaerobic culture step in which a culture product obtained in the heterotrophic culture step is incubated under anaerobic conditions, and anaerobic culture Euglena cell bodies obtained by a method comprising a step of collecting Euglena cell bodies from the culture product obtained in the step.
- an organic acid typified by succinic acid that is useful as a raw material for PBS or the like using Euglena, which is an algal biomass.
- useful Euglena cell bodies can be obtained simultaneously with the production of the organic acid.
- the method of the present invention includes either a nitrogen-deficient culture step in which Euglena is cultured aerobically under nitrogen-deficient conditions or a heterotrophic culture step in which a culture containing a carbon source is aerobically cultured. And an anaerobic culture step of incubating the culture product obtained in (4) under anaerobic conditions.
- An organic acid such as succinic acid, which is the target compound, is contained in the culture product obtained in the anaerobic culture process.
- “cultured product” is a concept including all products obtained by culturing Euglena.
- the concept when euglena is cultured in a culture solution in a culture vessel, the concept includes all of the culture solution containing Euglena cell bodies and substances produced by euglena, that is, the entire contents of the culture vessel. It is.
- Euglena Japanese name: Euglena
- Euglena is a general term for microorganisms belonging to the genus Euglena.
- Euglena is an alga having motility, and most of them are known to have both an animal property of flagellar movement and a plant property of having chloroplasts for photosynthesis.
- Euglena examples include Euglena acus, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena intermedia, Euglena mutabilis, Euglena prox, Euglena prolen, Among these, in the method of the present invention, Euglena gracilis that has been widely used in research can be suitably used.
- Euglena uses wild-type Euglena that has not undergone genetic recombination, so it is not necessary to consider genetic hybridization with the natural world during cultivation, so it can be cultured outdoors and the cost of cultivation can be reduced. Therefore, it is preferable.
- the present invention is not limited to this, and Euglena that has been genetically modified to produce a large amount of organic acid such as succinic acid may be used.
- Euglena is cultured aerobically using, for example, a nitrogen-deficient medium under nitrogen-deficient conditions.
- a nitrogen-deficient medium under nitrogen-deficient conditions.
- the nitrogen-deficient culture period is 3 days or longer, particularly 4 days or longer, especially 5 days or longer, and further 6 days or longer, because it is considered that more organic acid is accumulated.
- the aerobic culture conditions using Euglena's nitrogen-deficient medium may be those known in conventional Euglena culture methods.
- the culture temperature is usually preferably in the range of 20 to 34 ° C. for efficient growth.
- bubbling air into the culture medium in particular Euglena to assimilate CO 2, more preferably bubbling air containing 1-5% of CO 2 in the medium.
- the aerobic culture is preferably performed under light irradiation (light conditions), and more preferably performed under a light / dark cycle condition, particularly a light / dark cycle condition (for example, a 12 hour light / dark cycle) according to the circadian rhythm.
- the light intensity under bright conditions is preferably in the range of 30 to 200 ⁇ mol m ⁇ 2 s ⁇ 1 .
- the pH of the medium is preferably in the range of 3-8.
- Euglena can be cultured using, for example, a modified medium obtained by changing a part of the composition of a known medium such as Cramer-Myers medium (CM medium), Hutner medium, and Koren-Hutner medium, AY medium.
- Modification of the medium includes removal of the nitrogen source to achieve nitrogen deficiency conditions.
- CM medium Cramer-Myers medium
- Hutner medium Hutner medium
- Koren-Hutner medium AY medium.
- Modification of the medium includes removal of the nitrogen source to achieve nitrogen deficiency conditions.
- a nitrogen deficient condition can be obtained by excluding ammonium phosphate ((NH 4 ) 2 HPO 4 ) from the composition.
- the nitrogen deficient condition means a state where the nitrogen concentration in the medium is 0.1 mmol / L or less, particularly 0.01 mmol / L or less in terms of N atom.
- the modification of the medium used for euglena culture may include the addition of a nutrient source that promotes the growth of euglena or a carbon source that serves as a biosynthetic raw material for organic acids.
- Euglena culture can be performed using a medium supplemented with a carbon source, under fully heterotrophic conditions, or under conditions where photosynthesis and a carbon source added to the medium can be used together (this photosynthesis is referred to herein).
- the conditions to be used in combination may also be included in the concept of heterotrophic conditions). By culturing Euglena under heterotrophic conditions, more organic acids are accumulated compared to when cultivated under autotrophic conditions.
- the nitrogen-deficient culture process described above can be replaced with a heterotrophic culture process in which culture is performed under heterotrophic conditions.
- the heterotrophic culture step may be performed under nitrogen-satisfying conditions where the nitrogen source is not removed from the medium.
- the heterotrophic culture process can be performed in the same manner as the nitrogen-deficient culture process except that a medium having a different configuration is used.
- Examples of the carbon source added to the medium include sugars such as glucose and fructose, alcohols such as ethanol, carboxylic acids such as acetic acid and malic acid, organic acids such as amino acids such as glutamic acid, and salts thereof, and sodium hydrogen carbonate. And inorganic carbon compounds. For example, adding 0.5 to 1.0% by volume of ethanol in the medium leads to promotion of Euglena growth.
- an alkali metal or alkaline earth metal salt such as potassium chloride or magnesium chloride may be added to the medium.
- saccharides and organic acids are preferable, and glucose and acetic acid and salts thereof are particularly preferable.
- the carbon source added to the medium is preferably added at a concentration in the range of 1 to 200 mM, particularly 1 to 100 mM, particularly 1 to 20 mM, from the viewpoint of balance with the other components of the medium.
- Euglena used in the nitrogen-deficient culture process or heterotrophic culture process is pre-cultured using a medium that is not normally nitrogen-deficient.
- a culture solution in which Euglena pre-cultured for 1 day or more, particularly 2 days or more, particularly 3 days or more is collected, centrifuged, washed with deionized water as necessary, and then added to the medium. Place under nitrogen deficiency or heterotrophic conditions.
- Euglena cell body concentration at the start of the culture process is 10 to 20 g / L
- Euglena cell body concentration at the completion of the culture process is 20 to 50 g / L.
- the weight of Euglena cell body means dry weight unless otherwise specified.
- the dry weight of Euglena cell bodies is the weight obtained by sampling a predetermined amount of the culture solution in which Euglena is dispersed, measuring the Euglena cell bodies after removing water through steps such as centrifugation, washing and freeze-drying. is there. Therefore, the concentration of Euglena cell bodies expressed in units of g / L means the dry weight of Euglena cell bodies contained per liter of culture solution.
- the culture product obtained in the above nitrogen-deficient culture process or heterotrophic culture process is incubated under anaerobic conditions.
- Incubation under anaerobic conditions is preferably performed under dark conditions.
- the “dark condition” specifically refers to a weak light condition with a light intensity of 1 ⁇ mol m ⁇ 2 s ⁇ 1 or less or a dark condition without light.
- Anaerobic culture is a process that can be called anaerobic fermentation.
- Euglena In order for Euglena to gain energy under anaerobic conditions, it catalyzes the production of organic acids such as succinic acid and carbon dioxide by oxidizing organic matter by anaerobic respiration. Means metabolism.
- the “anaerobic condition” is a condition in which Euglena is less than the minimum oxygen amount at which aerobic respiration can be performed and substantially no oxygen is present, and the oxygen concentration is usually 1% or less, preferably 0. This means a condition of 5% or less, particularly 0.2% or less, and completely oxygen-free (the amount of oxygen is below the detection limit).
- Euglena accumulates organic acids such as succinic acid in cells when cultured under aerobic and nitrogen-deficient or heterotrophic conditions, but when it is cultured under anaerobic and dark conditions, organic acids such as succinic acid are accumulated. It is thought that biosynthesis is further promoted and organic acids are released to the outside of cells.
- Anaerobic conditions can be achieved by adjusting the oxygen concentration in the gas phase in the culture tank within the above range. For example, it can be achieved by replacing the gas in the gas phase with an inert gas such as nitrogen or argon.
- the culture product subjected to anaerobic culture is centrifuged, and then the supernatant is discarded.
- the Euglena cell bodies are concentrated in advance by dispersing the precipitated Euglena cell bodies in a small amount of new medium. It is preferable because the organic acid is easily released into the culture solution and the organic acid content in the culture solution after the anaerobic culture step is increased. Concentration is such that the volume of the Euglena cell body dispersion subjected to the anaerobic culturing step relative to the volume of the culture product is 1/10 or less, particularly 1/20 or less, especially 1/50 or less, and further 1/75 or less.
- the method for concentration is not limited to centrifugation, and may be performed by other known methods, for example, a method of accumulating dispersoids by filter filtration.
- the medium used in the anaerobic culture process may be the same as that used in the nitrogen-deficient culture process and heterotrophic culture process described above, or may be a buffer that does not have a nutrient source that promotes Euglena growth.
- Examples of the buffer that can be used for the culture include HEPES buffer, Tris buffer, PBS buffer, MOPS buffer, and MES buffer.
- the culture conditions in the anaerobic culture process may be the same as those in the nitrogen-deficient culture process and the heterotrophic culture process except that air bubbling is not performed. This is preferable because the acid content becomes higher.
- Anaerobic culture is preferably performed for at least 1 day, more preferably for 2 days or more, and particularly preferably for 3 days or more.
- Euglena releases organic acids such as succinic acid into the culture.
- the culture solution after the anaerobic culture contains succinic acid of 100 mg / L or more, preferably 200 mg / L or more, particularly preferably 300 mg / L or more in a non-concentrated state immediately after the end of the culture.
- the succinic acid content in the culture solution after anaerobic culture can be set to 800 mg / L or more, 1000 mg / L or more, and further 1500 mg / L or more by optimizing the culture conditions.
- the organic acid content in the culture solution after anaerobic culture can be determined by analyzing a sample obtained by drying the supernatant collected by centrifuging the culture product, for example by HPLC.
- the weight ratio of the organic acid contained in the supernatant to the dry weight of Euglena cell bodies after anaerobic culture is preferably 0.5% by weight or more, particularly 1% by weight or more, and more preferably 1.5% by weight or more. It is more preferable.
- Examples of organic acids contained in the culture solution after anaerobic culture include citric acid, malic acid, and lactic acid in addition to succinic acid.
- the content of organic acids other than citric acid is often very low compared to succinic acid.
- the amount of malic acid and lactic acid contained in the culture solution after anaerobic culture may be 1/5 or less, particularly 1/6 or less, particularly 1/7, or even 1/10 or less of the succinic acid content, respectively. Good.
- a small amount of malic acid and lactic acid is preferable from the viewpoint of separation and purification of succinic acid, which is more useful.
- the culture solution after anaerobic culture may contain other useful compounds such as the following: 3-hydroxypropionic acid, malonic acid, propionic acid, serine, acetoin, aspartic acid, fumaric acid, 3- Hydroxybutyrolactone, threonine, arabinitol, furfural, glutamic acid, itaconic acid, levulinic acid, proline, xylitol, xylonic acid, aconitic acid, citric acid, 2,5-furandicarboxylic acid, glucaric acid, lysine, levoglucosan, sorbitol.
- Their content may be 1/5 or less, especially 1/6 or less, especially 1/7, or even 1/10 or less of the succinic acid content.
- the culture product after anaerobic culture contains organic acids not only in the culture solution but also in Euglena cell bodies.
- the organic acid contained in the Euglena cell body can be taken out by extraction with water or an organic solvent.
- the cell membrane may be destroyed by applying a mechanical force with an ultrasonic crushing device or adding an enzyme such as lysozyme.
- the Euglena cell body may contain not only organic acids but also useful compounds as described above.
- a culture product containing an organic acid obtained after anaerobic culture it is possible to purify a high purity organic acid by a conventionally known method.
- the product obtained by drying the supernatant collected by centrifuging the culture product is desalted with an ion exchange resin or the like, and converted into high-purity succinic acid by a technique such as crystallization or column chromatography. Can be purified.
- Euglena cell bodies in the culture product obtained after anaerobic culture have usefulness not only in the contained organic acid but also in the cell bodies themselves.
- Euglena cell bodies are known to contain many vitamins, minerals, amino acids, unsaturated fatty acids, and the like, and are useful as foods, dietary supplements, or feeds.
- carbohydrates contained in the cell bodies are reduced during anaerobic culture. It is possible to provide a low-sugar Euglena cell body that is valuable.
- the present invention in another aspect, includes the above-described nitrogen-deficient culture process or heterotrophic culture process and anaerobic culture process, and further, from the culture product obtained in the anaerobic culture process, euglena cell bodies by centrifugation or the like. It also relates to Euglena cell bodies obtained by a method comprising a step of collecting.
- the culture solution after the anaerobic culture step in the method of the present invention is 100 mg / L or more, preferably 200 mg / L or more, particularly preferably 300 mg / L or more in a non-concentrated state immediately after the end of the culture.
- the culture solution after the anaerobic culture step contains 100 mg / L or more, preferably 200 mg / L or more, particularly preferably 300 mg / L or more of succinic acid in a non-concentrated state immediately after the end of the culture.
- this invention relates to the culture product obtained by culture
- the dry weight of Euglena cell bodies before anaerobic culture was separately determined by collecting 50 mL of the culture solution obtained in (1) above, placing it in a tube, discarding the supernatant after centrifugation, and further removing the cell body with sterile water. The salt was removed by washing twice, and the weight of the product freeze-dried at minus 80 ° C. for one day or more was measured.
- Test result (1) Test 1 Euglena cell bodies obtained by centrifuging 800 mL of a culture solution obtained by culturing for 11 days under nitrogen sufficiency or under nitrogen deficiency were suspended in 10 mL of buffer and anaerobically cultured for 3 days. Table 2 shows the amount of organic acid contained in the culture solution after anaerobic culture. Even when Euglena cultured under nitrogen sufficiency is subjected to anaerobic culture, the obtained supernatant hardly contains succinic acid, and the amount of succinic acid relative to the dry weight of the cell body before anaerobic culture is 0. Only 0.08% by weight.
- the obtained supernatant contained a large amount of succinic acid, which amount was the dry weight of the cell body before anaerobic culture. It was 1.9% by weight, and reached 20 times or more the succinic acid amount in the sample cultured under nitrogen sufficiency (based on the dry weight of the cell body before anaerobic culture).
- Citric acid could not be quantified because the peak in the HPLC chart overlapped with other components, but it was inferred from the chart that citric acid was produced in the same amount as succinic acid.
- Test 2 Euglena cell bodies obtained by centrifuging 800 mL of the culture solution obtained by culturing for 11 days or 14 days under nitrogen deficiency were suspended in 10 mL of buffer and anaerobically cultured for 3 days. Table 3 shows the amount of organic acid contained in the culture solution after anaerobic culture. In any culture days, a large amount of succinic acid is contained in the obtained supernatant, and the amount is 7.5% by weight and 3% respectively based on the dry weight of the cell body before anaerobic culture. 1% by weight.
- Test 3 Euglena cell bodies obtained by centrifuging 800 mL of a culture solution obtained by culturing for 14 days under nitrogen deficiency were suspended in 10 mL of buffer and cultured anaerobically for 6 days. Table 4 shows the amount of organic acid contained in the culture solution after anaerobic culture. Compared with the results of Test 2, no significant change was observed in the succinic acid content even when the number of days of anaerobic culture was changed from 3 days to 6 days. The content of succinic acid relative to the dry weight of the cell body before anaerobic culture was 4.8% by weight. In addition, it was speculated that the variation in the succinic acid content in the results of Tests 1 to 3 was caused by the difference in the position of the culture vessel in the incubator and the degree of light irradiation associated therewith.
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Abstract
Description
(1)ユーグレナを、窒素欠乏条件下で好気的に培養する窒素欠乏培養工程、および窒素欠乏培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む、ユーグレナを用いた有機酸の生産方法。
(2)窒素欠乏培養工程を3日間以上行う、(1)に記載の方法。
(3)ユーグレナを、炭素源を含む培地を用いて好気的に培養する従属栄養培養工程、および従属栄養培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む、ユーグレナを用いた有機酸の生産方法。
(4)嫌気培養工程の培養産物から有機酸を含有する培養液を取り出す工程をさらに含む、(1)~(3)のいずれかに記載の方法。
(5)嫌気培養工程の前に、窒素欠乏培養工程または従属栄養培養工程の培養産物に含まれるユーグレナ細胞体を濃縮する工程をさらに含む、(1)~(4)のいずれかに記載の方法。
(6)ユーグレナが野生型ユーグレナである、(1)~(5)のいずれかに記載の方法。
(7)有機酸がコハク酸である、(1)~(6)のいずれかに記載の方法。
(8)ユーグレナが産生した有機酸を100mg/L以上の濃度で含有する、ユーグレナを培養して得られる培養産物。
(9)有機酸がコハク酸である、(8)に記載の培養産物
(10)ユーグレナを培養した後に得られる培養液である、(8)または(9)に記載の培養産物。
(11)ユーグレナを、窒素欠乏条件下で3日間以上好気的に培養する窒素欠乏培養工程、および窒素欠乏培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む方法によりユーグレナを培養して得られる、(8)~(10)のいずれかに記載の培養産物。
(12)ユーグレナを、炭素源を含む培地を用いて好気的に培養する従属栄養培養工程、および従属栄養培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む方法によりユーグレナを培養して得られる、(8)~(10)のいずれかに記載の培養産物。
(13)前記ユーグレナが野生型である、(8)~(12)のいずれかに記載の培養産物。
(14)ユーグレナを、窒素欠乏条件下で3日間以上好気的に培養する窒素欠乏培養工程、窒素欠乏培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程、および嫌気培養工程で得られた培養産物からユーグレナ細胞体を採取する工程を含む方法により得られる、ユーグレナ細胞体。
(15)ユーグレナを、炭素源を含む培地を用いて好気的に培養する従属栄養培養工程、および従属栄養培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程、および嫌気培養工程で得られた培養産物からユーグレナ細胞体を採取する工程を含む方法により得られる、ユーグレナ細胞体。
窒素欠乏培養工程では、窒素欠乏条件下で、例えば窒素欠乏培地を用いて、ユーグレナを好気的に培養する。窒素欠乏条件下で培養すると、当初はユーグレナ細胞体中に蓄積された窒素源が利用されるが、それを全て資化してしまった後は、ユーグレナは窒素欠乏状態に陥る。ユーグレナはそのストレスに応じてコハク酸などの有機酸を産生し細胞体内に蓄積するものと考えられる。窒素欠乏培養期間は3日間以上であり、特に4日間以上、とりわけ5日間以上、さらに6日間以上とすると、有機酸をより多く蓄積すると考えられるため好ましい。
嫌気培養工程では、上記の窒素欠乏培養工程または従属栄養培養工程で得られた培養産物を嫌気条件下でインキュベートする。嫌気条件下のインキュベートは、好ましくは暗条件で行われる。ここで、「暗条件」とは、具体的には、光強度が1μmol m-2s-1以下の弱光条件または光のない暗黒条件をいう。
(1)ユーグレナの培養(窒素充足または欠乏条件下)
野生型Euglena gracilis(ユーグレナ社より取得、窒素充足下の通常の培養環境で1~2週間程度の前培養を行ったもの)は、初期ユーグレナ細胞体濃度を10~20g/Lとし、表1に示す組成を有する改変Cramer-Myers培地(改変CM培地、pH3.5)を用いて、25℃、12時間明暗サイクルで、1%のCO2を含む空気をバブリングしながらインキュベーター中で好気的に静置培養した。光強度は50~100μmol m-2s-1とした。培養は1Lの培養容器を用いて行った。なお、培養を窒素欠乏下で行う場合は、改変CM培地の組成から窒素源である(NH4)2HPO4を除外した。
上記(1)で得られた培養液を所定量採取して遠心分離(5000rpm×5分)し、上清を捨てた。得られたユーグレナ細胞体を10mLの20mM HEPES-KOHバッファ(pH7.8)に懸濁し、20mL容量のバイアル瓶に移した。バイアル瓶にはゴム栓をして注射針を2本刺し、その片方から窒素ガスを1時間導入した。なお、その間、バイアル瓶は30℃の光照射環境下に置かれた。1時間後に注射針を取り除き、アルミホイルで遮光したバイアル瓶を25℃のインキュベーターに入れ、振盪培養を行った。3日後または6日後に開栓し、内容物を15mLチューブに移してすぐに遠心分離した(10,000rpm×2分)。得られた上清は、他のチューブに移し、凍結乾燥させた。
ブロモチモールブルーを用いたポストラベル法により分析を行った。上記(2)で得られた上清の凍結乾燥物を、濾過済みの100μLの3mM過塩素酸水溶液に溶解させ、フォトダイオードアレイ検出器および2本のカラム(RSpak KC-811、昭和電工社製)を備えた高速液体クロマトグラフ(LC-2000Plus、日本分光社製)により分析した。0.2mMブロモチモールブルー溶液(15mMリン酸ナトリウムバッファ中)を用いて有機酸を定量し、波長445nmでピークを検出した。カラム温度は60℃とし、3mM過塩素酸水溶液および0.2mMブロモチモールブルー溶液の流速はそれぞれ1.0mL/分および1.5mL/分とした。
(1)試験1
窒素充足下、または窒素欠乏下で11日間培養して得られた培養液800mLを遠心分離して得られたユーグレナ細胞体を10mLのバッファに懸濁し、3日間嫌気培養した。嫌気培養後の培養液に含まれていた有機酸の量を表2に示す。窒素充足下で培養したユーグレナを嫌気培養に付しても、得られた上清中にはコハク酸はほとんど含まれておらず、嫌気培養前の細胞体の乾燥重量に対するコハク酸の量は0.08重量%に過ぎなかった。一方、窒素欠乏下で11日間培養してユーグレナを嫌気培養に付したところ、得られた上清中には多量のコハク酸が含まれており、その量は嫌気培養前の細胞体の乾燥重量に対して1.9重量%であり、窒素充足下で培養したサンプルにおけるコハク酸量の20倍以上(嫌気培養前の細胞体の乾燥重量基準)にも及んだ。なお、クエン酸はHPLCチャートにおけるピークが他の成分と重なったため定量ができなかったが、チャートからコハク酸と同程度の量のクエン酸が産生されていることが推察された。
窒素欠乏下で11日間または14日間培養して得られた培養液800mLを遠心分離して得られたユーグレナ細胞体を10mLのバッファに懸濁し、3日間嫌気培養した。嫌気培養後の培養液に含まれていた有機酸の量を表3に示す。いずれの培養日数の場合においても、得られた上清中には多量のコハク酸が含まれており、その量は嫌気培養前の細胞体の乾燥重量に対してそれぞれ7.5重量%および3.1重量%であった。
窒素欠乏下で14日間培養して得られた培養液800mLを遠心分離して得られたユーグレナ細胞体を10mLのバッファに懸濁し、6日間嫌気培養した。嫌気培養後の培養液に含まれていた有機酸の量を表4に示す。試験2の結果と比較すると、嫌気培養の日数を3日間から6日間としてもコハク酸の含有量に大きな変化はみられなかった。嫌気培養前の細胞体の乾燥重量に対するコハク酸の含有量は4.8重量%であった。なお、試験1~3の結果におけるコハク酸含有量のバラつきは、培養容器のインキュベーター中の位置およびそれに伴う光の照射程度等の相違に起因したものと推察された。
窒素充足下、10mMの酢酸ナトリウムを加えたCM培地、あるいは対照として添加物を加えないCM培地を用いて、14日間培養して得られた培養液20~100mLを遠心分離し、得られたユーグレナ細胞体を10mLのバッファに懸濁し、3日間嫌気培養した。なお、窒素充足下での培養は、光強度を200μmol m-2s-1に設定して行った。嫌気培養前の細胞体の乾燥重量に対するコハク酸の含有量は、対照で1.1重量%、培地に酢酸ナトリウムを添加した場合で1.5重量%であった。なお、他の試験例と比較してコハク酸生産量が少ないのは、用いたユーグレナ細胞体の量が少なかったためと考えられる。
Claims (7)
- ユーグレナを、窒素欠乏条件下で好気的に培養する窒素欠乏培養工程、および窒素欠乏培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む、ユーグレナを用いた有機酸の生産方法。
- 窒素欠乏培養工程を3日間以上行う、請求項1に記載の方法。
- ユーグレナを、炭素源を含む培地を用いて好気的に培養する従属栄養培養工程、および従属栄養培養工程で得られた培養産物を嫌気条件下でインキュベートする嫌気培養工程を含む、ユーグレナを用いた有機酸の生産方法。
- 嫌気培養工程の培養産物から有機酸を含有する培養液を取り出す工程をさらに含む、請求項1~3のいずれか1項に記載の方法。
- 嫌気培養工程の前に、窒素欠乏培養工程または従属栄養培養工程の培養産物に含まれるユーグレナ細胞体を濃縮する工程をさらに含む、請求項1~4のいずれか1項に記載の方法。
- ユーグレナが野生型ユーグレナである、請求項1~5のいずれか1項に記載の方法。
- 有機酸がコハク酸である、請求項1~6のいずれか1項に記載の方法。
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US20190054127A1 (en) * | 2016-03-04 | 2019-02-21 | Euglena Co., Ltd. | Antiviral agent and antiviral food |
CN108148763A (zh) * | 2018-03-08 | 2018-06-12 | 青岛旭能生物工程有限责任公司 | 一种通过流加方法提高摇瓶发酵纤细裸藻产量的方法 |
KR102089449B1 (ko) | 2018-11-27 | 2020-03-16 | 고려대학교 산학협력단 | 유글레나 그라실리스 및 박테리움 비브리오 나트리에젠스 공동배양을 통한 파라밀론의 생산방법 |
CA3143895A1 (en) * | 2019-06-28 | 2020-12-30 | Noblegen Inc. | Methods of optimized euglena fermentation using engineered tank design |
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JP2019110894A (ja) * | 2017-12-21 | 2019-07-11 | 学校法人明治大学 | アミノ酸の製造方法及びユーグレナの培養方法 |
JP7274851B2 (ja) | 2017-12-21 | 2023-05-17 | 学校法人明治大学 | グルタミン酸の製造方法 |
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