WO2017141318A1 - Procédé de production de matière grasse et d'huile - Google Patents

Procédé de production de matière grasse et d'huile Download PDF

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WO2017141318A1
WO2017141318A1 PCT/JP2016/054301 JP2016054301W WO2017141318A1 WO 2017141318 A1 WO2017141318 A1 WO 2017141318A1 JP 2016054301 W JP2016054301 W JP 2016054301W WO 2017141318 A1 WO2017141318 A1 WO 2017141318A1
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algae
salt
light
culture
fat
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PCT/JP2016/054301
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English (en)
Japanese (ja)
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近藤　昭彦
誠久 蓮沼
悠一 加藤
純 皆川
西江　晴男
太郎田　博之
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国立大学法人神戸大学
大学共同利用機関法人自然科学研究機構
Dic株式会社
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Priority to PCT/JP2016/054301 priority Critical patent/WO2017141318A1/fr
Publication of WO2017141318A1 publication Critical patent/WO2017141318A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • This invention relates to the manufacturing method of fats and oils.
  • a photosynthetic organism is a general term for organisms that fix CO 2 using light energy.
  • algae are a kind of photosynthetic organisms with high photosynthetic efficiency if the culture conditions are good.
  • Industrial cultivation of algae has been carried out for more than half a century, and there is a demand for industrial raw materials, fuels, feeds, health foods, etc., and industrial cultivation of algae will continue to occupy an important industrial position in the future.
  • Patent Document 1 describes a method for growing algae in order to produce algae products. More specifically, (1) growing the algae under the first heterotrophic or photoheterotrophic growth conditions so as to increase the rate of algal cell division and the number of algal cells; Growing the algae under a second growth condition to produce a product, wherein a method is described in which the algal cell number does not increase significantly under the second growth condition. It is also described that the algal product is oil or lipid.
  • Patent Document 1 cell division and algal growth (growth stage) are promoted under the first growth condition, and grown alga cells are produced (produced) under the second growth condition. To focus on the stage). Furthermore, in the second growth condition, algal cell growth is described as spending most of the energy and resources on producing the desired algal product rather than further cell division / proliferation. Patent Document 1 describes that the first growth condition is performed in a first bioreactor, and the second growth condition is performed in a second bioreactor.
  • an object of the present invention is to provide a technique for suitably producing desired oils and fats even when applied to outdoor culture.
  • the present inventors have found that the problem of the present invention can be solved by irradiating light at night in a light-dark cycle that inevitably occurs during outdoor culture, and the present invention is completed. It came to. That is, the present invention includes the following aspects.
  • a method for producing fats and oils comprising a step of culturing salt-tolerant algae in a medium containing salt, wherein the culturing is performed outdoors, and a total of 50 to 2500 mmol photons / m 2 is irradiated at night.
  • Production method (2) The production method according to (1), wherein the light irradiation is a continuous irradiation of 2 to 50 ⁇ mol photons / m 2 / sec at night.
  • fats and oils can be suitably produced even in outdoor culture.
  • the time-dependent change of the biomass production amount in Experimental example 1 is shown.
  • the time-dependent change of the fat content in Experimental Example 1 is shown.
  • the time-dependent change of the fat production amount in Experimental example 1 is shown.
  • the time-dependent change of the starch content rate in Experimental example 1 is shown.
  • the time-dependent change of the biomass production amount in Experimental example 1 is shown.
  • the time-dependent change of the fat content in Experimental Example 2 is shown.
  • the time-dependent change of the fat production amount in Experimental Example 2 is shown. It is a graph which shows the time-dependent change of the biomass production in Experimental example 3.
  • the present invention is a method for producing fats and oils comprising the step of culturing salt-tolerant algae in a medium containing salt, wherein the culturing is performed outdoors and a total of 50 to 2500 mmol photons / m 2 at night.
  • a manufacturing method is provided that irradiates light.
  • the inventors further cited that one of the causes is the presence of a dark period (night) in the culture, which reduces the starch production by irradiating light of a predetermined intensity at night, It was clarified that the production efficiency of fats and oils could be improved, and the present invention was completed.
  • culturing salt-tolerant algae outdoors means culturing salt-tolerant algae outdoors or in an environment equivalent to the outdoors. More specifically, when culturing salt-tolerant algae with sunlight, it means culturing in a situation where a light-dark cycle occurs.
  • the salt-resistant algae is cultured outdoors and irradiated with light of a predetermined intensity at night, thereby reducing starch production and improving the production efficiency of fats and oils.
  • night means the total time of the dark period per day.
  • the dark period in this specification means that the amount of light to be irradiated is less than 2 ⁇ mol photons / m 2 / sec.
  • the nighttime outdoors is usually a continuous time once a day.
  • the amount of light irradiated at night is minimal.
  • Light irradiation amount of the total in the nighttime 50mmol photons / m 2 or more, for example 100 mmol photons / m 2 or more, for example 500 mmol photons / m 2 or more, for example if 1000 mmol photons / m 2 or more, a culture of salt tolerance algae outdoors Even in the case of carrying out, the production efficiency of fats and oils can be improved.
  • the upper limit of the total amount of light irradiation at night is about 2500 mmol photons / m 2 .
  • the light irradiation at night is not particularly limited as long as the total irradiation amount is 50 to 2500 mmol photons / m 2 , and for example, light having a certain intensity may be continuously irradiated. Specifically, light of 2 to 50 ⁇ mol photons / m 2 / sec may be continuously irradiated at night.
  • “continuously” means irradiating light so that the dark period does not occur at night.
  • intermittently means that the dark period occurs for a predetermined time.
  • an irradiation pattern in which light of 30 ⁇ mol photons / m 2 / sec is irradiated for a predetermined time, then is dark for a predetermined time, and light of 30 ⁇ mol photons / m 2 / sec is irradiated again for a predetermined time.
  • the dark period is preferably as short as possible.
  • the length of the dark period may be, for example, 10 minutes or less, for example, 1 minute or less, for example, 30 seconds or less, for example, 10 seconds or less.
  • the light source of the light irradiated at night may be, for example, a fluorescent lamp or an LED. Further, the light to be irradiated may be white light or light having a wavelength of 400 to 700 nm.
  • the salt-tolerant algae used in the method for producing fats and oils of the present embodiment are not particularly limited as long as they are salt-tolerant.
  • Algae algae of the genus Nannochloropsis, algae of the genus Botryococcus, algae of the genus Chaetoceros, genus Chlorecoccum, algae of the genus Euglena ) Algae, Isochrysis Algae, Naviculaa Algae, Neochloris Genus , Porphyridium algae, Prymnesium algae, Senedes algae, Spirulina algae, Spirogyra algae o cc And algae of the genus Tetraselmis.
  • Chlamydomonas algae, Chlorella algae, Donariella algae, Nannochloropsis algae are preferable.
  • Chlamydomonas is a genus consisting of single-celled flagellates that belong to the green algae Chlamydomonas (or Mongolia). Many Chlamydomonas are freshwater products, but some grow in seawater. Marine algae of the genus Chlamydomonas refers to algae of the genus Chlamydomonas that can grow on media containing marine, brackish and marine products. Examples of the algae of the genus Chlamydomonas include Chlamydomonas reinhardtii, Chlamydomonas sp. JSC4 strain (Chlamydomonas sp. JSC4, FERM BP-22266) and the like.
  • Examples of algae belonging to the genus Chlorella include Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella sorokiniana, and the like. Among these, chlorella sorokiniana is preferable.
  • Examples of chlorella solokiniana include chlorella solokiniana NIES-2168.
  • Examples of algae belonging to the genus Donariella include Donaliella bioclata, Dunaliella salina, Dunaliella teriolecta, and the like.
  • An example of Donariella salina is Donariella salina NIES-2168.
  • An example of the Donariella biocrater is Donaliella biocrater NIES-2253.
  • An example of the Donariella terciolecteta is Donariella terciorecta NIES-2258.
  • Nannochloropsis oculata examples include Nannochloropsis oculata.
  • Nannochloropsis oculata includes Nannochloropsis oculata NIES-2146.
  • Chlamydomonas sp. JSC4 strain As the salt-tolerant algae used in the method for producing fats and oils of this embodiment, among them, algae belonging to the genus Chlamydomonas is preferable, and Chlamydomonas sp. JSC4 strain is particularly preferable.
  • the Chlamydomonas sp. JSC4 strain was transferred to the Patent Biological Deposit Center of the National Institute of Technology and Evaluation (2-5-8, Kazusa-Kamazu, Kisarazu City, Chiba Prefecture) on March 5, 2014. It is deposited internationally as BP-22266.
  • salt The salt has a function of suppressing or preventing the growth of salt-tolerant algae and promoting the production of fats and oils. For this reason, the salt may be added to the medium from the beginning of cultivation of the salt-tolerant algae, or may be added to the medium after the salt-tolerant algae are grown. Alternatively, the salt-tolerant algae may be grown using a medium that does not contain a salt, and then transferred to a medium containing a salt and cultured.
  • the salt examples include sea salt, sea water, sea water concentrate, artificial sea water, and the like.
  • the sea salt may be one obtained by evaporating and drying sea water.
  • Artificial seawater is a powder or concentrate that has been artificially adjusted to mimic the composition of seawater, and is excellent in availability, reproducibility, and low cost in breeding and culturing organisms that require seawater. It can be used as a substitute for natural seawater.
  • Commercial artificial seawater contains sodium chloride as a main component and includes various inorganic salts, pH adjusters, and the like, and becomes a component close to seawater when diluted with tap water or distilled water.
  • the salt is not limited to the sea salt and artificial sea water described above, and an appropriate salt may be prepared and used.
  • an appropriate salt may be prepared and used.
  • seawater when a large-scale culture of algae is assumed, it is convenient to use seawater, but even if sodium chloride is used, the same effect can be exerted on the production amount of fats and oils.
  • the salt may be any of the above-mentioned salts, but seawater salt, artificial seawater and the like are easy to use because the salt concentration in the medium can be easily adjusted.
  • the amount of production of fats and oils can be further improved by irradiating with light at night and increasing the salt concentration in the medium stepwise. More specifically, in the initial stage, the salt concentration is low, so that salt-tolerant algae grow favorably. And by increasing the salt concentration in stages, the growth of salt-tolerant algae is suppressed or prevented, while the salt-tolerant algae grown in the initial stage produce fats and oils. Thereby, fats and oils can be produced efficiently and in large quantities.
  • the salt concentration in the medium used when culturing salt-tolerant algae is started may be referred to as “first stage salt concentration”.
  • first stage salt concentration When the salt concentration in the medium is increased only once, the salt concentration in the medium at the time of algal body recovery is the “second stage salt concentration”.
  • second stage salt concentration When the salt concentration is increased in multiple steps, for example, when the salt concentration in the medium is increased N times (N is an integer of 2 or more), the salt concentration in the medium at the time of algal body recovery is “N + 1 stage salt concentration”.
  • the salt concentration at the first stage in the medium is preferably 0.01 to 5% (w / v), and preferably 0.01 to 3% (w / v). Is more preferable, and 0.01 to 2% (w / v) is still more preferable.
  • the culture period of the first stage varies depending on the growth rate of the salt-tolerant algae used, but is preferably 1 to 3 days.
  • the salt concentration in the medium may be increased stepwise by 0.5 to 5% (v / w).
  • a stepwise control method of the salt concentration a “two-stage culture method” in which the salt concentration in the medium is increased only once, and the salt concentration in the medium is increased N times (N represents an integer of 2 or more).
  • Multi-stage culture method also referred to as gradient method
  • a culture method with a constant salt concentration may be referred to as a “batch method”.
  • the final salt concentration in the medium is preferably 2% (v / w) or more, more preferably 3% (v / w) or more. More preferably, it is 5% (v / w) or more.
  • the salt concentration to be increased is preferably 0.5 to 5% (v / w), more preferably 1 to 5% (v / w).
  • the culture period of the second stage varies depending on the growth rate of the salt-tolerant algae used, and may be 3 to 8 days.
  • the salt concentration to be increased at each stage is preferably 0.5 to 2% (v / w), more preferably 0.5% to 1.5%.
  • the salt concentration to be increased at each stage may be different, but is preferably the same from the viewpoint of easy culture.
  • the culture period in each stage may be different, but is preferably the same.
  • the culture period in each stage is preferably 1 to 3 days, more preferably 1 day.
  • the number of steps (N described above) is preferably 2 to 7, and more preferably 3 to 5.
  • the total culture period of the 2nd to Nth stages varies depending on the growth rate of the salt-resistant algae used, but is preferably 3 to 8 days, more preferably 4 to 6 days.
  • the salt-resistant algae culture method can be carried out by a known and commonly used method.
  • the above-mentioned medium can be used.
  • a culture method a stationary culture method can be used, but considering the productivity of fats and oils, a shaking culture method or a deep aeration stirring culture method is preferable.
  • the shaking culture may be reciprocal shaking or rotational shaking.
  • the culture temperature is 15 to 40 ° C.
  • the algal bodies can be collected from the culture solution by a general method such as a centrifugal separation method or a filtration method using a filter paper or a glass filter.
  • the alga bodies collected in this way may be used as they are, or may be made into dry alga bodies by a freeze drying method, a hot air drying method, or the like.
  • the fat and oil component can be extracted from the obtained algal body or dried algal body.
  • a method for supplying the carbon dioxide gas a known and commonly used method may be mentioned.
  • aeration in the culture solution can be mentioned.
  • the fats and oils manufactured by the manufacturing method of fats and oils of this embodiment are triglycerides.
  • Triglycerides are acyl forms of glycerin and can be used as biodiesel fuel by alkyl esterification.
  • the triglyceride produced by the method for producing fats and oils according to the present embodiment is an ester of glycerin and a fatty acid, and the fatty acid is a saturated or unsaturated fatty acid having 10 to 30 carbon atoms.
  • Higher unsaturated fatty acids with high combustion efficiency can be produced by hydrolyzing the above triglycerides.
  • Examples of higher unsaturated fatty acids with high combustion efficiency include oleic acid and linolenic acid. Of these, oleic acid is preferred because of its particularly high combustion efficiency.
  • the normal oil-fat extraction method can be used. Specifically, a general extraction method using an organic solvent such as a chloroform / methanol system represented by the Folch method or the Bligh-Dyer method can be used.
  • biomass production A liquid sample collected from the bioreactor was filtered through a 0.45 ⁇ m pore size filter precisely weighed in advance, and this was freeze-dried to a constant weight and precisely weighed. The difference in filter mass before and after filtration was divided by the amount of filtered liquid sample to determine the algae concentration.
  • biomass production A liquid sample collected from the bioreactor was filtered through a 0.45 ⁇ m pore size filter precisely weighed in advance, and this was freeze-dried to a constant weight and precisely weighed. The difference in filter mass before and after filtration was divided by the amount of filtered liquid sample to determine the algae concentration.
  • biomass production the algae concentration may be referred to as “biomass production”.
  • a GC-23 analyzer column (GCMS-QP2010Plus, Shimadzu Corporation) was equipped with a DB-23 capillary column (0.25 mm ⁇ ⁇ 60 m, 0.15 ⁇ m film thickness, Agilent Technologies), and 2.3 mL of helium gas was flowed per minute. .
  • the injector, ion source, and interface temperature were set to 230, 230, and 250 ° C., respectively, and the column temperature was maintained at 50 ° C. for 1 minute after sample injection, then increased to 175 ° C. at 25 ° C. per minute, and further 4 per minute. The temperature was raised to 230 ° C. and kept for 5 minutes.
  • Example 1 In addition to culturing salt-tolerant algae under a light-dark cycle, the salt concentration in the medium was changed to study the production efficiency of fats and oils. Specifically, 0.1% (w / v) of sea salt was added to 1 L of MB6N medium having the composition shown in Table 1, and the mixture was placed in a plurality of bioreactors and sterilized by autoclave. Subsequently, Chlamydomonas sp. JSC4 strain was inoculated to the medium in each bioreactor so that the algal concentration was about 100 mg / L.
  • the bioreactors were divided into two groups and cultured for about 2 days under the conditions of “without low light” and “with low light”, respectively (hereinafter referred to as “the group without low light” and “the group with low light”). ").
  • seawater salt was added to each bioreactor so as to have a final concentration of 2% (w / v), and further culturing was continued under the conditions of “no low light” and “with low light” shown below. Samples in the bioreactor were sampled over time, and biomass production, fat content, fat production, and starch content were measured.
  • FIG. 1A to 1D are graphs showing measurement results.
  • FIG. 1A shows changes in biomass production over time.
  • FIG. 1B shows the change over time in the fat content.
  • FIG. 1C shows the change over time in the production of fats and oils.
  • FIG. 1D shows the time course of starch content.
  • Example 2 The initial value of the salt concentration in the medium was set to 2% (w / v) and increased by 0.5% (w / v) every day, and the conditions of “no light” and “with light”
  • the Chlamydomonas sp. JSC4 strain was cultured in the same manner as in Experimental Example 1 except that was changed to the following conditions. Samples in the bioreactor were sampled over time, and biomass production and fat content were measured. Moreover, based on biomass production and fat content, the fat production was calculated in the same manner as in Experimental Example 1.
  • FIG. 2A to 2C are graphs showing the measurement results.
  • FIG. 2A shows the change in biomass production over time.
  • FIG. 2B shows the change over time in the fat content.
  • FIG. 2C shows the change over time in the production of fats and oils.
  • the amount of increase in the fat content is larger in the condition with low light than in the condition without low light, and the biomass production and the fat production tend to be higher. Admitted.
  • the fat content at the time of a measurement start was not able to arrange
  • Example 3 The Chlamydomonas sp. JSC4 strain is cultured in the same manner as in Experimental Example 2, except that the initial salt concentration in the medium is 2% (w / v) and is increased by 1% (w / v) every day. did. Samples in the bioreactor were sampled over time, and biomass production and fat content were measured. Moreover, based on biomass production and fat content, the fat production was calculated in the same manner as in Experimental Example 1.
  • FIG. 3A is a graph showing a change in biomass production with time
  • FIG. 3B is a graph showing a change in fat content with time.
  • Example 4 Chlamydomonas sp. JSC4 strain in the same manner as in Experimental Example 2, except that the initial salt concentration in the medium was 2% (w / v) and increased by 1.5% (w / v) every day. Was cultured. Samples in the bioreactor were sampled over time, and biomass production and fat content were measured. Moreover, based on biomass production and fat content, the fat production was calculated in the same manner as in Experimental Example 1.
  • FIG. 4A is a graph showing the change over time in biomass production
  • FIG. 4B is a graph showing the change over time in the fat content.
  • fats and oils can be suitably produced even in outdoor culture.

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Abstract

La présente invention concerne un procédé de production de matière grasse et d'huile comprenant une étape d'incubation d'une algue halotolérante dans un milieu de culture qui comprend du sel, où l'incubation est exécutée à l'extérieur et où l'algue halotolérante est exposée à un rayonnement avec un total de 50 à 2 500 mmol de photons/m2 de lumière durant la nuit.
PCT/JP2016/054301 2016-02-15 2016-02-15 Procédé de production de matière grasse et d'huile WO2017141318A1 (fr)

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JP2020195344A (ja) * 2019-06-04 2020-12-10 国立大学法人神戸大学 オイル高蓄積有用藻類株の育種方法、藻類のオイル高蓄積変異株及びそれを用いた油脂の製造方法

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WO2020071444A1 (fr) * 2018-10-02 2020-04-09 国立研究開発法人科学技術振興機構 Procédé de culture de microalgue d'eau douce
CN113015791A (zh) * 2018-10-02 2021-06-22 国立研究开发法人科学技术振兴机构 淡水微藻的培养方法
JPWO2020071444A1 (ja) * 2018-10-02 2021-11-25 国立研究開発法人科学技術振興機構 淡水産微細藻類の培養方法
AU2019355498B2 (en) * 2018-10-02 2023-02-02 Japan Science And Technology Agency Method for culturing fresh water microalga
JP7455386B2 (ja) 2018-10-02 2024-03-26 国立研究開発法人科学技術振興機構 淡水産微細藻類の培養方法
JP2020195344A (ja) * 2019-06-04 2020-12-10 国立大学法人神戸大学 オイル高蓄積有用藻類株の育種方法、藻類のオイル高蓄積変異株及びそれを用いた油脂の製造方法
JP7486725B2 (ja) 2019-06-04 2024-05-20 国立大学法人神戸大学 オイル高蓄積有用藻類株の育種方法、藻類のオイル高蓄積変異株及びそれを用いた油脂の製造方法

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