WO2015025553A1 - 油脂成分を産生する方法、高級不飽和脂肪酸の製造方法、及びクラミドモナス・スピーシーズjsc4株 - Google Patents
油脂成分を産生する方法、高級不飽和脂肪酸の製造方法、及びクラミドモナス・スピーシーズjsc4株 Download PDFInfo
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- WO2015025553A1 WO2015025553A1 PCT/JP2014/057185 JP2014057185W WO2015025553A1 WO 2015025553 A1 WO2015025553 A1 WO 2015025553A1 JP 2014057185 W JP2014057185 W JP 2014057185W WO 2015025553 A1 WO2015025553 A1 WO 2015025553A1
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- chlamydomonas
- oil
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- algae
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Images
Classifications
-
- 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
- C12P7/6409—Fatty acids
-
- 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
-
- 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
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
-
- 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
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
Definitions
- the present invention relates to a method for producing a fat component useful as a fuel or a chemical raw material, and more particularly to a method for producing a fat component characterized by culturing algae of the genus Chlamydomonas in a medium containing sea salt.
- Photosynthetic organisms are a general term for organisms that fix CO 2 using light energy.
- algae are a kind of photosynthetic organisms that have high photosynthetic efficiency under good culture conditions.
- Industrial culture of algae has been carried out for more than half a century, and there is a demand as a raw material for industrial raw materials, fuel, feed and fine chemicals, and as a health food. Algal production will continue to occupy an important position in the industry. Conceivable.
- Patent Document 1 discloses that starch grows at a salt concentration of seawater and has starch in cells.
- the microalga Chlamydomonas sp. which belongs to the genus Chlamydomonas, accumulates and produces ethanol from intracellular starch by keeping it dark and anaerobic.
- MT-JE-SH-1 There is a description of MT-JE-SH-1.
- Patent Document 2 describes a 4,7,10,13,16-docosapentaenoic acid producing bacterium L59 strain (FERM P-18987) belonging to the genus Labyrinthula of Labyrinthula family. After culturing this microorganism and accumulating fats and oils containing 4,7,10,13,16-docosapentaenoic acid as a constituent fatty acid in the microbial cell, the microbial cell is separated and the solvent is separated from the separated microbial cell. Describes a method of extracting the oil and fat and then hydrolyzing the extract.
- Non-Patent Document 1 examines the relationship between fat production using marine algae and the salt concentration during culture, and when the salt concentration exceeds 1.5M at the initial concentration, the growth of algae is suppressed, In the case of 0.5 to 1.0 M, it is described that fats and oils are produced with a high content.
- the present inventors have studied in detail the search for algae and the method for culturing the algae, and have solved the problems. That is, in this invention, the said subject is solved by providing the method of producing the fats and oils component described below, and a novel microalgae.
- a method for producing an oil and fat component by culturing algae comprising culturing an algae of the marine Chlamydomonas genus in a medium containing sea salt.
- [6] A method for producing a higher unsaturated fatty acid, comprising hydrolyzing an oil component obtained by the method for producing an oil component according to any one of [1] to [5]. [7] The method for producing a higher unsaturated fatty acid according to [6], wherein the higher unsaturated fatty acid is oleic acid or linolenic acid. [8] Chlamydomonas sp. JSC4 having the ability to produce oil and fat components.
- a CO 2 fixing ability analysis of Chlamydomonas sp JSC4 strain was cultured at different sea salt concentrations.
- a CO 2 fixing ability analysis of Chlamydomonas sp JSC4 strain was cultured at different sea salt concentrations.
- a CO 2 fixing ability analysis of Chlamydomonas sp JSC4 strain was cultured at different sea salt concentrations.
- a CO 2 fixing ability analysis of Chlamydomonas sp JSC4 strain was cultured at different sea salt concentrations.
- the algae used in the present invention are characterized by being algae of the genus Chlamydomonas.
- Chlamydomonas is a genus consisting of single-celled flagellates belonging to the order of the green alga Chlamydomonas (or Mongolia). Many Chlamydomonas are freshwater products, but some grow in seawater.
- the marine Chlamydomonas algae according to the present invention refers to algae of the genus Chlamydomonas that can grow on a medium containing marine, brackish and marine products.
- the algae of the genus Chlamydomonas used in the present invention is not particularly limited as long as it is marine. Since there is a nutrient source in seawater, it is not necessary to add a nutrient source to the medium separately. Moreover, it is not necessary to use pure water. Furthermore, no sugar source is required for algae culture.
- the method for producing the oil and fat component of the present invention is also excellent in cost. Furthermore, since the salt concentration in the medium is high, there is no risk of contamination of the culture solution.
- the present invention is excellent in that algae of the genus Chlamydomonas can be cultivated easily, can be cultured in large quantities, and can produce oil and fat components on a large scale.
- the present inventors have searched for algae that produce the target oil and fat component with high efficiency, and found that algae belonging to the genus Chlamydomonas is preferable as the algae. Furthermore, among the genus Chlamydomonas, Chlamydomonas sp. JSC4 was found to be particularly preferable because it produces oil and fat components with high efficiency, and the present invention was completed.
- Chlamydomona sp. Separation and purification of Chlamydomonas sp. JSC4 used in the present invention was performed by the following procedure. That is, only one cell was isolated and sterilized by a conventional method from a brackish water sample collected on the coast in the midwestern part of Taiwan. This is cultured in an HSM agar medium having the following composition at 20 ° C. under light conditions of 8 to 15 ⁇ mol photons / m 2 / sec, 12 hours light period, 12 hours dark period, and planted once every two weeks. Thus, an algae strain was established, identified as a green algae belonging to the genus Chlamydomonas by morphological observation and others, and named JSC4 strain.
- FIG. 1 shows a photomicrograph of Chlamydomonas sp. JSC4 strain vegetative cells (preferably growing environment, cells proliferating vigorously under abundant nutritional conditions).
- the vegetative cell has an elliptical shape and a size of about 10 ⁇ m.
- a vegetative cell has two flagella about the same length as the cell length. Vegetative cells have motility.
- the vegetative cell is surrounded by a cell wall, and has a nucleus and a chloroplast inside. In addition, mitochondria, Golgi apparatus, vacuole, oil droplets, etc. are observed. It has a pyrenoid at the base of the chloroplast.
- Culture solution It can grow in a culture solution containing marine products, brackish fishery products and sea salt.
- Photosynthesis ability Photoautotrophic growth by photosynthesis is possible.
- Containing pigment chlorophyll a, chlorophyll b, and other carotenoids.
- Assimilation storage material starch.
- Growth temperature range 15 ° C. to 35 ° C. (optimum temperature 25 ° C.).
- Growth pH range pH 6.0 to 10.0 (optimum pH is 7.0).
- Chlamydomonas sp. JSC4 strain was identified as a green algae belonging to the genus Chlamydomonas by morphological observation and others.
- the base sequence of the 18S rDNA gene of Chlamydomonas sp. JSC4 strain is shown in SEQ ID NO: 1 in the sequence listing.
- Figures 2-4 compare the 18S rDNA sequences of closely related Chlamydomonas species. Shading is a molecular marker sequence of Chlamydomonas sp. JSC4 strain. The latest related species of Chlamydomonas sp.
- JSC4 strain is Chlamydomonas debaryana, but it is clear that it is not the same species when focusing on the molecular marker sequence. Thus, Chlamydomonas sp. JSC4 strain was judged as a novel microalgae strain from the point of comparison of 18S rDNA sequences. Chlamydomonas sp. It is deposited internationally as ABP-22266.
- a medium for culturing algae belonging to the genus Chlamydomonas is not limited as long as algae belonging to the genus Chlamydomonas grows, but the medium containing sea salt improves sea fat, concentrated sea water, or artificial sea water improves oil production capacity. Particularly preferred.
- a modified Bold 3N medium can be preferably used as such a medium.
- Other media that can be used include a modified Basal medium, a modified Bristol medium, a BG-11 medium, a modified HSM (High Salt medium) medium, and the like. Since the oil component can be produced with high efficiency, the modified Bold 3N Medium is particularly preferred.
- culture under a low nitrogen content can be mentioned.
- the culture under a low nitrogen content may be a culture in a nitrogen deficient state due to nitrogen consumption accompanying growth, or a culture by transplanting algal cells to a medium with a low nitrogen content.
- the nitrogen content contained in the medium can be evaluated by measuring the content of nitrate contained in the medium at a wavelength of 220 nm.
- the nitrogen content contained in the medium is not limited to this method, and can also be evaluated by measuring the content of nitrate or ammonium salt, for example, by measuring the absorbance with an ion sensor or a coloring reagent. .
- the measurement was performed by modifying the method reported by Collos et al. In 1999 (Reference: Journal of Applied Phicology, 11, 179-184 (1999) (Journal of Applied Physology, Volume 11, P179). -184 (1999)). Detailed measurement methods are described in the following examples.
- composition of the modified Bold 3N medium used in the present invention is described below.
- sea salt In this invention, it discovered that the density
- sea salt that can be used in the present invention include known and commonly used sea salt.
- the sea salt used in the present invention may be obtained by evaporating and drying sea water, or sea water or sea water concentrate may be used, but in order to adjust the concentration contained in the medium, It is more preferable to use sea salt which is the solid content of sea water.
- Artificial seawater can also be used. Artificial seawater used in the present invention is a powder or concentrate that has been artificially adjusted to imitate the composition of seawater, and is available, reproducible, and inexpensive in breeding and culturing organisms that require seawater. It is a substitute for natural seawater for reasons such as sex. Commercial artificial seawater can be used, but commercially available artificial seawater contains sodium chloride as a main component and contains various inorganic salts and pH adjusters. By diluting with tap water or distilled water depending on the application, It becomes a component close to seawater. In addition, even if it is not said sea salt etc., the salt which can be used as a culture medium suitable for the objective of this invention can be adjusted and used.
- seawater salt concentration greatly affects the oil production efficiency.
- preferable seawater salt concentration can be 0.5 to 5% by mass, and the range of 2.0 to 5.0% by mass is particularly desirable. Since the content of the oil and fat component is also high, it is particularly preferable.
- mass culture of algae it is convenient to use seawater, but even if sodium chloride is used, it has the same effect on oil production and can be preferably used.
- the method for culturing algae belonging to the genus Chlamydomonas can be carried out by a known and commonly used method.
- the medium can be used in the present invention.
- the culture method used in the present invention it is possible to use a static culture method, but considering the algal body productivity of algae and the productivity of fat and oil components, culture by shaking culture method or deep aeration stirring culture method is possible. preferable.
- the shaking culture may be reciprocal shaking or rotary shaking.
- the culture temperature can usually be 15 to 40 ° C. to produce algal bodies.
- Chlamydomonas algae having not only stable growth but also a high proportion of oil and fat components are obtained.
- the light condition is not particularly limited as long as it is a condition capable of photosynthesis, but continuous light is preferable.
- the algal bodies can be collected from the culture solution as a method for obtaining the crude oil and fat by a general method such as a centrifugal separation method or a filtration method using a filter paper and a glass filter.
- the alga bodies collected in this way can be used as they are, or can be converted into dry alga bodies by freeze drying, hot air drying, or the like. It is possible to extract an oil and fat component from the obtained algal body or the dried algal body.
- the carbon dioxide gas can be supplied by a publicly known and commonly used method.
- the carbon dioxide gas can be suitably supplied by aeration in the culture solution.
- the fat component produced in the present invention is characterized by being a triglyceride.
- Triglycerides are acyl forms of glycerin and are expected to be used as biodiesel fuel by alkyl esterification.
- the compound exemplified as the triglyceride is an ester of glycerin and a fatty acid, and the fatty acid is a higher saturated or unsaturated fatty acid having 10 to 30 carbon atoms.
- the present invention also provides a method for producing higher unsaturated fatty acids useful as biodiesel fuel. That is, a higher unsaturated fatty acid with high combustion efficiency can be produced by hydrolyzing the fat and oil component obtained in the method of the present invention.
- higher unsaturated fatty acids having high combustion efficiency include oleic acid and linolenic acid, and oleic acid is particularly preferable because of its high combustion efficiency.
- the liquid sample from the photobioreactor 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.
- the liquid sample from the photobioreactor was filtered through a 0.22 ⁇ m pore size filter and diluted 20-fold with distilled water.
- the nitrate content was determined by optical density at a wavelength of 220 nm (OD 220 ) using a UV / VIS spectrophotometer. That is, the value at OD 220, using the calibration curve of OD 220 and nitrate content shown in FIG. 5, by converting the nitrate concentration.
- 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 allowed to flow 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, and then increased to 175 ° C. at 25 ° C./min. The temperature was raised to 230 ° C. at 4 ° C. and held for 5 minutes.
- Inject 1 ⁇ L of the supernatant separate the column at a split ratio of 5: 1, detect each component of fatty acid methyl ester in a full scan mode of 50 to 500 m / z, and quantify based on the amount of internal standard added. This was defined as the amount of oil.
- ⁇ X represents the amount of change in biomass concentration (mg / L) during the culture time ⁇ t (d).
- CO 2 immobilization rate P CO2 ; mg / L / d
- CO 0.48 H 1.83 N 0.11 P 0.01 was used as a typical molecular formula for algal biomass.
- the CO 2 immobilization rate (%) is obtained by the following formula.
- C CO2, influx and C CO2, effect indicate the inflow concentration and the outflow concentration of CO 2 , respectively.
- Example 1 (Comparison of culture media) A modified Basal medium, a modified Bristol medium, a BG-11 medium, a modified Bold 3N medium, and a modified HSM (High Salt medium) medium having the composition shown in Table 3 were prepared in 1 liter each and charged into a 1 liter photobioreactor. Sterilized with an autoclave. Each photobioreactor is inoculated with Chlamydomonas sp. JSC4 so that the algal concentration is about 100 mg / L, and continuously fluorescent light with an intensity of 200 ⁇ mol photons / m 2 / sec for 24 hours.
- Chlamydomonas sp. JSC4 Chlamydomonasp. JSC4 so that the algal concentration is about 100 mg / L, and continuously fluorescent light with an intensity of 200 ⁇ mol photons / m 2 / sec for 24 hours.
- the culture was performed for 5.7 days under the conditions of irradiation, 50 mL of air containing 2% carbon dioxide gas per minute, and stirring at 200 rpm with a stirrer.
- Table 4 shows the results of analyzing the fat and oil components of each culture solution.
- the modified Bold 3N medium had the highest oil and fat content in the algal bodies and the oil and fat production rate per culture solution.
- Example 2 (Additional effect of sea salt) Prepare 1 liter of each of the modified Bold 3N media shown in Table 3 with Sea Salt addition amounts of 0.5%, 2%, 3.5%, and 5% (w / v), respectively. The mixture was charged into a 1-liter photobioreactor and sterilized by autoclave. Each photobioreactor is inoculated with Chlamydomonas sp. JSC4 so that the algal concentration is about 100 mg / L, and continuously fluorescent light with an intensity of 200 ⁇ mol photons / m 2 / sec for 24 hours. Culturing was performed for 10 days under the conditions of irradiation, 50 mL of air containing 2% carbon dioxide gas per minute, and stirring at 200 rpm with a stirrer.
- the nitrate content in the culture broth decreased with the growth of the algal bodies, and became 10 mg / L or less in 1.9 to 2.7 days. Thereafter, the fat content and fat production rate in the algal bodies increased significantly.
- the addition amount of sea salt is 2%, 3.5%, and 5%
- the fat content in the alga body reaches a high content of 50% or more, and the maximum fat production rate is 140 mg / L / d or more. It was very high.
- Example 3 (Effect of culture under nitrogen-deficient conditions on biodiesel quality) Biodiesel quality is assessed by the ratio of unsaturated fatty acids to saturated fatty acids. Saturated fatty acid content in biodiesel affects oxidation inhibition at high temperatures. On the other hand, the unsaturated fatty acid content affects the fluidity at low temperatures. Equal amounts of saturated and unsaturated fatty acids in biodiesel are important for imparting good properties to biodiesel at low and high temperatures. Fatty acid profiles affect environmental stresses resulting from nutrients in the medium, ambient temperature, and light intensity. Of these stresses, nitrogen deficiency conditions are the most important factor affecting algae fat metabolism. FIG. 6 shows the fatty acid composition of Chlamydomonas sp.
- FIG. 6 it is compared with the composition of fatty acid derived from soybean oil as a control.
- the culture conditions for Chlamydomonas sp. JSC4 strain are the same as in Example 2.
- fat accumulation in Chlamydomonas sp. JSC4 strain tends to increase for oleic acid (C18: 1) and to decrease for linolenic acid (C18: 3).
- CFPP clogging point
- Chlamydomonas sp. JSC4 strain is an appropriate strain for biofuel production.
- Example 4 (Effects of control of sea salt and nitrogen source on CO 2 fixation of Chlamydomonas sp. JSC4)
- the CO 2 fixation ability of Chlamydomonas sp. JSC4 strain cultured at different seawater salt concentrations was examined at regular intervals. The results are shown in FIGS. As shown in FIGS. 7 to 10, the CO 2 fixation rate and the CO 2 fixation rate at different seawater salt concentrations showed the same tendency over time. That is, it showed a bell-shaped curve that gradually decreased after reaching the maximum value after 2-3 days of culture.
- FIGS. 7 to 10 the CO 2 fixation rate and the CO 2 fixation rate at different seawater salt concentrations showed the same tendency over time. That is, it showed a bell-shaped curve that gradually decreased after reaching the maximum value after 2-3 days of culture.
- useful carbon components using algae can be produced with high efficiency.
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Abstract
Description
今後、化石燃料の枯渇化が懸念されることから、代替燃料の早期の探索の必要性が高まり、また、需要者の健康志向の向上から、健康維持・向上に好ましい機能性化学品への需要が増加し、益々藻類の産生する有用成分への関心が高まってきている。
そこで、本発明の課題は、上記背景技術に鑑み、藻類を用いた有用炭素成分の高効率の産生方法を提供することである。
即ち、本発明では、以下に記載の油脂成分を産生する方法、及び新規微細藻類を提供することにより、上記課題を解決するものである。
海生のクラミドモナス(Chlamydomonas)属の藻類を、海水塩を含む培地で培養することを特徴とする油脂成分を産生する方法。
[2]クラミドモナス(Chlamydomonas)属に属する藻類が、クラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)である請求項1に記載の油脂成分を産生する方法。
[3]海水塩を含む培地が、220nmの波長において硝酸塩含有量を測定した場合、含有量が、10mg/L以下である[1]又は[2]に記載の油脂成分を産生する方法。
[4]培地における海水塩の質量%が、0.5~5質量%の範囲である[1]~[3]のいずれか一つに記載の油脂成分を産生する方法。
[5]海水塩を含む培地が、海水、濃縮海水、又は人工海水を含むものである[1]~[4]のいずれか一つに記載の油脂成分を産生する方法。
[7]高級不飽和脂肪酸が、オレイン酸、又はリノレン酸である、[6]に記載の高級不飽和脂肪酸の製造方法。
[8]油脂成分生産能を有するクラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)。
本発明で用いられる藻類は、クラミドモナス(Chlamydomonas)属の藻類であることに特徴を有する。
クラミドモナスは、緑藻綱クラミドモナス目(もしくはオオヒゲマワリ目)に属する単細胞の鞭毛虫からなる属である。クラミドモナスの多くは淡水産であるが、海水中に生育するものもある。本発明の海生のクラミドモナス(Chlamydomonas)属の藻類とは、海産や汽水産及び海水塩を含む培地で生育可能なクラミドモナス(Chlamydomonas)属の藻類を言う。
海水中には栄養源が存在するため、別途培地に栄養源を添加する必要がない。また、純水を用いる必要もない。更に、藻類の培養には糖源を必要としない。本発明の油脂成分を産生する方法は、コスト面においても優れている。
更に、培地中の塩濃度が高いため、培養液のコンタミネーションのおそれがない。本発明は、簡易にクラミドモナス(Chlamydomonas)属の藻類を培養でき、大量培養が可能で、大規模に油脂成分を産生できる点でも優れている。
更に、クラミドモナス属のなかでも、高効率に油脂成分を産生することから、クラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)が特に好ましいことを見出し、本発明を完成させた。
本発明で用いられるクラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)の分離精製は以下の手順により行った。
即ち、台湾中西部の海岸で採取した汽水試料から、常法により1細胞だけを単離し、無菌化した。これを、以下に組成を示すHSM寒天培地を用いて、20℃、8~15μmol photons/m2/sec、12時間明期12時間暗期の光条件で培養し、2週間に1度植え継ぐことで藻株を確立し、形態観察その他よりクラミドモナス属の緑藻と同定して、JSC4株と名づけた。
(1)栄養型細胞は、楕円形であり、大きさは、約10μmである。栄養型細胞において、細胞長の約等倍の鞭毛を2本有する。栄養型細胞は、運動性を有する。
(2)栄養型細胞は外囲を細胞壁に囲まれ、内部に核、葉緑体が一個存在し、その他、ミトコンドリア、ゴルジ体、液胞、油滴等が認められる。葉緑体内の基底部にピレノイドを有する。
(1)内生胞子は栄養細胞内に二~八個形成され、細胞内に均等に分布する。内生胞子はその細胞内に核、葉緑体を一個有する。
(2)二分裂による増殖も行う。
(1)培養液:海産や汽水産及び海水塩を含む培養液中で生育できる。
(2)光合成能:光合成による光独立栄養生育ができる。
(3)含有色素:クロロフィルa、クロロフィルb、及び他のカロテノイド類。
(4)同化貯蔵物質:澱粉。
(5)生育温度域:15℃~35℃(至適温度25℃)。
(6)生育pH域:pH6.0~10.0(至適pHは7.0)。
クラミドモナス・スピーシーズJSC4株の18S rDNA遺伝子の塩基配列を配列表の配列番号1に示す。図2~図4は、近縁クラミドモナス種の18S rDNA配列を比較したものである。網掛けは、クラミドモナス・スピーシーズJSC4株の分子マーカー配列である。クラミドモナス・スピーシーズJSC4株の最近縁種は、Chlamydomonas debaryanaであるが、分子マーカー配列に着目すれば同一種でないことは明らかである。このように、18S rDNA配列の比較の点から、クラミドモナス・スピーシーズJSC4株を新規の微細藻類株と判断した。
クラミドモナス・スピーシーズJSC4株は、2014年3月5日付で独立行政法人製品評価技術基盤機構特許生物寄託センター(千葉県木更津市かずさ鎌足2-5-8)にプタベスト条約の規定下で受領番号FERM ABP-22266として国際寄託されている。
本発明では、クラミドモナス属に属する藻類を培養するにあたり培地を用いることが好ましい。
用いられる培地は、クラミドモナス属に属する藻類が生育する条件であれば制限はないが、海水塩を含む培地が、海水、濃縮海水、又は人工海水を含むものが油脂産生能を向上させることから、特に好ましい。
例えば、このような培地として、特に改変Bold 3N培地を好ましく用いることができる。
その他用いることのできる培地として、改変Basal培地、改変Bristol培地、BG-11培地、改変HSM(High Salt medium)培地などを挙げることができるが、高効率で油脂成分を産生できることから、改変Bold 3N培地が特に好ましい。
窒素含有量が低い条件下での培養は、増殖に伴う窒素消費による窒素欠乏状態下における培養であっても、藻体を窒素含有量が低い培地に移植させる等による培養であってもよい。
本発明において、培地中に含まれる窒素含有量は、培地中に含まれる硝酸塩の含有量を220nmの波長で測定することにより評価することができる。
培地中に含まれる窒素含有量は、この方法に限定されるものではなく、 例えばイオンセンサーや発色試薬による吸光度測定などで、硝酸塩やアン モニウム塩の含有量を測定することにより評価することもできる。
測定法は、1999年にCollosらによって報告された方法を改変して行った(文献:ジャーナル オブ アプライド ファイコロジー,11巻、179-184ページ(1999年)(Journal of Applied Phycology,Volume 11, P179-184 (1999))。
詳細な測定法は、下記実施例に記載する。
本発明では、培地中の海水塩の濃度(培地全体に占める質量%)が、油脂成分の産生能に大きく影響を及ぼすことを見出した。よって、上記培地に最適濃度の海水塩を添加することで、油脂成分の産生効率を向上させることができる。
本発明で使用が可能な海水塩は、公知慣用の海水塩を挙げることができる。本発明で用いられる海水塩は、海水を蒸発乾固させて得られたものであっても、海水や海水の濃縮液を用いてもよいが、培地中に含まれる濃度を調整するためには、海水の固形分である海水塩を用いる方がより好ましい。
その他、上記の海水塩等でなくても、本発明の目的に適う培地として使用が可能な塩を調整して用いることができる。
藻類の油脂産生能(mg/L/day)で評価すると、好ましい海水塩濃度として、0.5~5質量%を挙げることができ、中でも2.0~5.0質量%の範囲が、目的とする油脂成分の含有量も高いことから、特に好ましい。
なお、藻類の大量培養を想定した場合には、海水を用いることに利便性があるが、塩化ナトリウムを用いても、油脂産生に対して同様の効果を有し、好ましく用いることができる。
本発明において、クラミドモナス属に属する藻類の培養方法は、公知慣用の方法で行うことができる。
培養においては、本発明では前記培地を用いることができる。
本発明に用いる培養方法としては、静置培養法を用いることも可能であるが、藻類の藻体生産性と油脂成分の生産性を考えると、振盪培養法又は深部通気撹拌培養法による培養が好ましい。振盪培養は、往復振盪であっても、回転振盪であってもよい。培養温度としては、通常15~40℃で藻体産生を行なうことが可能である。
上述のように海洋性微細藻類を、本発明の培養方法で培養すると、安定した増殖を示すばかりでなく、油脂成分の割合が高度に高いクラミドモナス藻類が得られる。
また、光条件は、光合成可能な条件下であれば特に制限はないが、連続光とすることが好ましい。
炭酸ガスの供給法として、公知慣用の方法で行うことができ、例えば、培養液中に通気することにより、好適に炭酸ガスの供給を行うことができる。
トリグリセリドは、グリセリンのアシル体であり、アルキルエステル化することにより、バイオディーゼル燃料としての利用が期待されている。
本発明においてトリグリセリドとして挙げられる化合物としては、グリセリンと脂肪酸とのエステル体であり、脂肪酸としては炭素数10~30の高級飽和或いは不飽和脂肪酸である。
即ち、本発明の方法において得られる油脂成分を加水分解することにより、燃焼効率の高い高級不飽和脂肪酸を製造することができる。
燃焼効率の高い高級不飽和脂肪酸として、オレイン酸、又はリノレン酸を挙げることができ、燃焼効率が特に高いことから特にオレイン酸が好ましい。
上記高級不飽和脂肪酸を産生するための最適海水塩濃度の検討の結果、0.5~5質量%が好ましく、特に好ましくは2.0~5質量%の範囲を挙げることができる。
藻体から油脂成分を抽出する方法としては、通常の油脂の抽出方法を用いることができ、特に、Folch法やBligh-Dyer法に代表されるクロロホルム/メタノール系等の有機溶媒による一般的な抽出方法を用いることが可能であるが、これらに限らない。
フォトバイオリアクターからの液体試料を、予め精密に秤量した0.45μm孔径のフィルターでろ過し、これを恒量になるまで凍結乾燥して精密に秤量した。ろ過前後のフィルター質量の差を、ろ過した液体試料量で割り、藻濃度を決定した。
フォトバイオリアクターからの液体試料を、0.22μm孔径のフィルターでろ過し、蒸留水で20倍に希釈した。硝酸塩含有量はUV/VIS分光光度計を用いて、220nm(OD220)の波長における光学濃度によって決定した。
即ち、OD220における値を、図5に示すOD220と硝酸塩含有量の検量線を用いて、硝酸塩濃度を換算した。
凍結乾燥した藻体15mgを、直径0.5mmのガラスビーズ0.5gが入ったマイクロバイアルに取り、これに1mLの0.5M濃度KOH溶液を加えて、ビーズビーター式ホモジナイザーで40分破砕処理した。処理液を7mLの0.5M濃度KOH溶液で共洗いしながら50mL容耐熱ガラスビンに移して密栓し、ウオーターバス中で100℃15分間処理した。室温まで冷却後、8mLの0.7M濃度HClメタノール溶液と10mLの14%(v/v)3フッ化ホウ素メタノール溶液(シグマ・アルドリッチ社)を加えて、再度ウオーターバス中で100℃15分間処理した。室温まで冷却後、4mLの飽和食塩水と3mLのn-へキサンを加えて、ボルテックスミキサーで5分間撹拌した。撹拌した液を50mL容プラスチック遠沈管に移して7,000rpmで2分間遠心分離した。上清100μLをエッペンドルフチューブに取り、890μLのn-へキサンと10μLの内部標準物質(ペンタデカン酸メチル、シグマ社)を加えた後、10,000rpmで3分間遠心分離し、上清をGCMS分析装置で分析した。
バイオマスの濃度(g/L)の時間経過プロファイルを用いて、乾燥藻体重量当たりの時間プロットに対する増殖率を計算した。
バイオマス生産速度(Pbiomass;mg/L/d)は、以下の式で求められる。
更に、CO2固定化速度(PCO2;mg/L/d)は、以下の式で求められる。
CO2固定化率(%)は、以下の式で求められる。
(培地の比較)
表3に組成を示した改変Basal培地、改変Bristol培地、BG-11培地、改変Bold 3N培地、改変HSM(High Salt medium)培地を各1リッター調製し、それぞれ容量1リッターのフォトバイオリアクターに仕込んでオートクレーブ滅菌した。各々のフォトバイオリアクターに、クラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)を藻濃度が約100mg/Lとなるよう接種し、室温、200μmol photons/m2/secの強度の蛍光灯光を24時間連続照射、2%炭酸ガス含有空気を毎分50mL通気、スターラーで200rpm撹拌、の条件で5.7日間培養した。
各培養液の油脂成分を分析した結果を、表4に示した。藻体中の油脂含量、培養液当りの油脂生産速度とも、改変Bold 3N培地が最も高かった。
(海水塩の添加効果)
表3に組成を示した改変Bold 3N培地のSea Salt添加量を、0.5%、2%、3.5%、及び5%(w/v)とした培地を各1リッター調製し、それぞれ容量1リッターのフォトバイオリアクターに仕込んでオートクレーブ滅菌した。各々のフォトバイオリアクターに、クラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)を藻濃度が約100mg/Lとなるよう接種し、室温、200μmol photons/m2/secの強度の蛍光灯光を24時間連続照射、2%炭酸ガス含有空気を毎分50mL通気、スターラーで200rpm撹拌、の条件で10日間培養した。
(窒素欠乏条件下での培養がバイオディーゼルの品質にもたらす効果)
バイオディーゼルの品質は、飽和脂肪酸に対する不飽和脂肪酸の割合で評価される。バイオディーゼル中の飽和脂肪酸含量は、高温下での酸化抑制に影響する。その一方、不飽和脂肪酸含量は、低温下での流動性に影響する。バイオディーゼル中の飽和脂肪酸と不飽和脂肪酸が等量であることが、バイオディーゼルに、低温下及び高温下における良好な特性を付与するために重要である。脂肪酸のプロファイルは、培地中の栄養分、外気温、及び光強度から生じる環境ストレスに影響する。これらのストレスのうち、窒素欠乏条件は、藻類の脂肪代謝に影響する最も重要な因子である。
窒素十分条件下、及び窒素欠乏条件下で培養したクラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)の脂肪酸の組成を図6に示す。図6において、対照として、大豆油由来の脂肪酸の組成と比較している。クラミドモナス・スピーシーズJSC4株の培養条件は、実施例2と同様である。
図6に示すように、窒素欠乏条件下では、クラミドモナス・スピーシーズJSC4株における脂肪の蓄積は、オレイン酸(C18:1)が増加傾向にあり、リノレン酸(C18:3)が減少傾向にあることが確認された。バイオディーゼルの特性によると、オレイン酸を高い割合で含有することにより、より良い酸化安定性と低い外気温下における適切な目詰まり点(CFPP)を備える。更に、欧州バイオ燃料規格(EN14214)に基づくと、リノレン酸(C18:3)の含有量は、最大12%(m/m)に限定されている。よって、クラミドモナス・スピーシーズJSC4株により生産される油脂は、バイオ燃料を製造するために適切な品質を有していることが確認された。
更に、図6に示すように、大豆油由来の脂肪酸の組成と比較して、クラミドモナス・スピーシーズJSC4株は、高い飽和脂肪酸含有量と低い多価不飽和脂肪酸(n≧2)含有量示した。一般的に油脂における飽和脂肪酸の高含有は、バイオ燃料に優れた流動性と密度をもたらす。一方、多価不飽和脂肪酸の低含有は、低い外気温下での酸化安定性の増加のみならず、適切な目詰まり点の付与をもたらす。よって、油脂において適切な脂肪酸のプロファイルを有するという観点から、クラミドモナス・スピーシーズJSC4株は、バイオ燃料の製造に適切な株であることが確認された。
(海水塩と窒素源の制御が、クラミドモナス・スピーシーズJSC4株のCO2固定にもたらす効果)
異なる海水塩濃度で培養したクラミドモナス・スピーシーズJSC4株のCO2固定化能を一定時間ごとに調べた。結果を図7~図10に示す。図7~図10に示すように、異なる海水塩濃度におけるCO2固定化率とCO2固定速度は、時間経過全般にわたり同様の傾向を示した。即ち、培養2-3日後に最高値に達した後、徐々に減少するベル型のカーブを示した。
図7~図10において、CO2固定化率及びCO2固定速度の最大値は、海水塩の添加量が2%の条件下で得られ、それぞれ54.9%及び1319.0mg/L/dであった。この優れたCO2固定能からクラミドモナス・スピーシーズJSC4株が、工業ガスを用いたCO2固定への実用的応用に対応しうる株であることが確認された。
Claims (8)
- 藻類を培養することにより油脂成分を産生する方法において、
海生のクラミドモナス(Chlamydomonas)属の藻類を、海水塩を含む培地で培養することを特徴とする油脂成分を産生する方法。 - クラミドモナス(Chlamydomonas)属に属する藻類が、クラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)である請求項1に記載の油脂成分を産生する方法。
- 海水塩を含む培地が、220nmの波長において硝酸塩含有量を測定した場合、含有量が、10mg/L以下である請求項1又は2に記載の油脂成分を産生する方法。
- 培地における海水塩の質量%が、0.5~5質量%の範囲である請求項1~3のいずれか一項に記載の油脂成分を産生する方法。
- 海水塩を含む培地が、海水、濃縮海水、又は人工海水を含むものである請求項1~4のいずれか一項に記載の油脂成分を産生する方法。
- 請求項1~5のいずれか一項に記載の油脂成分を産生する方法により得られる油脂成分を加水分解することを特徴とする高級不飽和脂肪酸の製造方法。
- 高級不飽和脂肪酸が、オレイン酸、又はリノレン酸である、請求項6に記載の高級不飽和脂肪酸の製造方法。
- 油脂成分生産能を有するクラミドモナス・スピーシーズJSC4株(Chlamydomonas sp. JSC4)。
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