WO2010116611A1 - ナビクラ属に属する微細藻類、該微細藻類の培養による油分の製造方法、および該微細藻類から採取した油分 - Google Patents
ナビクラ属に属する微細藻類、該微細藻類の培養による油分の製造方法、および該微細藻類から採取した油分 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
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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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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
- C11B1/108—Production of fats or fatty oils from raw materials by extracting after-treatment, e.g. of miscellae
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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
- C12N1/125—Unicellular algae isolates
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- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Definitions
- the present invention relates to a microalga belonging to the genus Navicula, a method for producing an oil having a step of culturing the microalga, and an oil collected from the microalga. More specifically, a microalga belonging to the genus Navicula having an ability to produce an aliphatic hydrocarbon having 16 to 26 carbon atoms, a method for producing an oil having a step of culturing the microalga, and a sample collected from the microalgae
- the present invention relates to an oil component, a dry alga body obtained by drying the microalgae, a fuel obtained from the microalgae, and a carbon dioxide fixing method including a step of culturing the microalgae.
- microalga Botriococcus brownie As the algae having the ability to produce light oil hydrocarbons, the microalgae Nostocc muscorum, Trichodesmium erythaeum, Plectonema terebran et al.
- Non-Patent Document 1 having a carbon number of 17 can be used.
- Microalgae Coccochoris elabens et al. having the ability to produce various hydrocarbons
- Microalgae Pseudocollistis ellipsoidia MBIC11204 strain having the ability to produce hydrocarbons having 17, 18, 19, and 20 carbon atoms
- Patent Document 2 a microalgae colitissis minor SAG17.98 strain (see Patent Document 2) having the ability to produce hydrocarbons having 17, 19, 21, and 23 carbon atoms is known.
- Light oil-based hydrocarbons that can be produced by microalgae are industrially useful as diesel fuel, and are also expected as carbon-neutral fuels aimed at preventing global warming.
- the content of light oil-based hydrocarbons is usually about 0.025 to 0.12% by mass (see Non-Patent Document 1), and the hydrocarbons. Productivity is not always sufficient.
- the present invention has been made in view of the above circumstances, and is a microalgae having a high ability to produce an aliphatic hydrocarbon having 16 to 26 carbon atoms, a method for producing an oil having a step of culturing the microalgae, and the microalgae. It is an object of the present invention to provide a method for fixing carbon dioxide having a collected oil, a dried algal body obtained by drying the microalgae, a fuel obtained from the microalgae, and a step of culturing the microalgae.
- the present invention has the following configuration.
- a microalga belonging to the genus Navicula having an ability to produce an aliphatic hydrocarbon having 16 to 26 carbon atoms.
- a species of the microalgae Navicula sp. Having the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.
- a method for producing an oil component comprising a step of culturing the microalgae according to any one of (1) to (3).
- the method includes a step of extracting oil from the culture with an organic solvent, wherein the organic solvent is a solvent composed of n-hexane, a solvent composed of n-hexane and methanol, or n
- the organic solvent is a solvent composed of n-hexane, a solvent composed of n-hexane and methanol, or n
- the method for producing an oil according to any one of the above (4) to (7) which is any one of solvents consisting of hexane and ethanol.
- a dry alga obtained by drying the microalgae according to any one of (1) to (3).
- (11) A fuel obtained from the microalgae according to any one of (1) to (3).
- a carbon dioxide fixing method comprising a step of culturing the microalgae according to any one of (1) to (3).
- “having the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms” mainly means fats having 16, 18, 20, 22, 24 and 26 carbon atoms. It has the ability to produce a group hydrocarbon.
- the “oil” means a liquid component mainly composed of a hydrophobic organic compound. Examples of the hydrophobic organic compound include aliphatic hydrocarbons and neutral fats.
- a microalgae having high ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms a method for producing oil having a step of culturing the microalgae, an oil collected from the microalgae, and the microalgae It is possible to provide a carbon dioxide fixing method including a dry alga body obtained by drying, a fuel obtained from the microalgae, and a step of culturing the microalgae.
- FIG. 1 It is a figure which shows the molecular phylogenetic tree obtained using the 18S rDNA base sequence of JPCC DA0580 stock. It is a growth curve of JPCC DA0580 strain. It is the growth curve in the culture which changed the seawater component density
- the present invention will be described in detail below.
- the microalgae belonging to the genus Navicula in the present invention have the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.
- the aliphatic hydrocarbons having 16 to 26 carbon atoms are mainly aliphatic hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms.
- microalgae in particular, from the viewpoint of high content of aliphatic hydrocarbons having 16 to 26 carbon atoms in the algal bodies and easy culturing, species of the microalgae Navicula genus oilicus are preferable.
- the microalgae Navicula genus oiliticus sp. JPCC DA0580 (FERM BP-11201) (hereinafter abbreviated as JPCC DA0580) is more preferred.
- JPCC DA0580 strain is more preferable as the microalgae of the present invention from the viewpoint of having a high ability to produce neutral lipids and squalene in addition to the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.
- the neutral lipid will be described later.
- JPCC DA0580 strain is a new strain of a new species of marine microalgae belonging to the family of yellow plant diatoms, diatom wings, arachnids, and flute, isolated from brackish water.
- the method for isolating the microalgae and the background of determining the JPCC DA0580 strain of the microalgae as a new type of new strain will be described.
- the f / 2 trace metal solution was Na 2 EDTA dihydrate 4.36 g / l, ferric chloride hexahydrate 3.15 g / l, manganese (II) chloride tetrahydrate 180 mg / l, zinc sulfate 7hydrate 22.0 mg / l, cobalt (II) chloride hexahydrate 10.0 mg / l, disodium molybdate (VI) dihydrate 6.3 mg / l, copper (II) sulfate pentahydrate
- the product was prepared as an aqueous solution having a composition of 9.8 mg / l.
- the f / 2 vitamin solution was prepared as an aqueous solution having a composition of 200.0 mg / l thiamine, 0.1 mg / l biotin, and 1.0 mg / l vitamin (B12). Further, in addition to the composition of the f / 2 medium, an agar f / 2 medium was further added to which agar was further added to a concentration of 1.2% (w / v).
- the artificial seawater is a salt imitating a salt contained in natural seawater. By dissolving a predetermined amount (37 g / l) of artificial seawater in distilled water, an aqueous solution having seawater components simulating natural seawater can be produced.
- FIG. 4 shows an electron microscope image of JPCC DA0580 strain.
- JPCC DA0580 performs both sexual reproduction and asexual reproduction.
- Asexual reproduction grows asexually by bisection.
- Cell division proceeds in the putamen, and the two daughter cells form a new semi-putamen inside the outer and inner shells of the parent cell.
- Each parent cell half-putamen is distributed to daughter cells.
- one of the daughter cells is the same size (a putamen) as the parent cell, while the other uses the inner shell as the outer shell.
- the cell becomes one size smaller than the parent cell, and sexual reproduction is performed on the cell that has become smaller to some extent.
- the cells undergo meiosis and form isomorphic gametes. This zygote comes close through the putamen, changes the protoplasm into an amoeba, joins, forms an enlarged spore, grows and increases its volume, and returns to a normal cell of the appropriate size.
- ⁇ Culture medium Grows in a culture medium based on seawater. It does not grow in fresh water.
- Photosynthesis Photoautotrophic growth is possible by photosynthesis. Heterotrophic growth is not confirmed.
- -Contained pigment Chlorophyll a, c1, c2, carotenoid pigments mainly composed of fucoxanthin and fucoxanthin derivatives-Storage material: starch-Growth temperature: 20 ° C to 35 ° C (optimum temperature 25 ° C) -Growth pH: 7.0 to 9.0 (optimum pH 8.0) Accumulate large amounts of oil, including aliphatic hydrocarbons with 16 to 26 carbon atoms, neutral lipids and squalene, stained intracellularly with Nile red.
- -Growth period 1 week ⁇ period required for turbidity (OD750) to reach 0.05 to 1.2 ⁇
- the presence of oil colored with Nile red is confirmed as a bright fluorescent color development region in the algae in the fluorescent field.
- the oil can accumulate as oil droplets in algal cells. Further, the oil contains an aliphatic hydrocarbon having 16 to 26 carbon atoms, a neutral lipid, and squalene.
- JPCC DA0580 strain was presumed to be an algae belonging to the family Navicraaceae from the yellow plant gate, diatom lepidoptera, anterofluids, from the morphological properties, reproductive modes, and physiological / biochemical properties. Furthermore, according to a conventionally known method, DNA was extracted from the JPCC DA0580 strain using a DNA extraction kit ⁇ Product name: QIAampaDNA Broad Mini Kit 50 (manufactured by Qiagen) ⁇ , and 18S rDNA The region was amplified and sequence analysis was performed to determine the base sequence of the 18S rDNA region. The base sequence of the obtained 18S rDNA is shown in SEQ ID NO: 1 in the sequence listing.
- the obtained 18S rDNA base sequence was collated with the public DNA database of Japan DNA Data Bank (DDBJ) to search for homology (Blast search), and phylogenetic analysis was performed using analysis software ClustalW and display software Treeview. .
- the resulting system diagram is shown in FIG.
- the isolated JPCC DA0580 strain was classified in the Navikura family in the phylogenetic tree and formed clusters with diatoms of the genus Navicula. However, since the known species and branching of the Navikura genus confirmed at the gene level were confirmed, the JPCC DA0580 strain isolated by the present inventor is a new species of algae of the Navicula genus. It was judged. Therefore, the strain was named Navicula genus oilicus species JPCC DA0580 strain.
- JPCC DA0580 shares, under the accession number FERM BP-11201, dated March 16, 2009, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan) No. 305-866)) (transferred from FERM P-21788, deposited on March 16, 2009).
- JPCC DA0580 strain can accumulate oil containing 16 to 26 carbons, aliphatic hydrocarbons, neutral lipids, and squalene in algal cells. Further, the content of the aliphatic hydrocarbon having 16 to 26 carbon atoms in the dry alga body of JPCC DA0580 can reach 0.2% by mass or more. Moreover, the content rate of this neutral lipid in the dry alga body of JPCC DA0580 strain can reach 36 mass% or more. Furthermore, the content of the squalene in the dry algal bodies of JPCC DA0580 can reach 0.3% by mass or more.
- the oil components include aliphatic hydrocarbons having 27 or more carbon atoms, phospholipids, free fatty acids, steroid compounds, and fucoxanthin and It may also contain photosynthetic pigments such as carotenoids containing derivatives of fucoxanthin.
- the aliphatic hydrocarbon having 16 to 26 carbon atoms is mainly an aliphatic hydrocarbon having 16, 18, 20, 22, 24 and 26 carbon atoms, and more specifically, mainly having 16, 16, 20, carbon atoms. 22, 24 and 26 straight chain aliphatic saturated hydrocarbons.
- the neutral lipid is mainly composed of tetradecanoyl group (myristoyl group), hexadecanoyl group (palmitoyl group), hexadecenoyl group (palmitreoyl group), octadecenoyl group (oleoyl group), and eico.
- It is a neutral lipid having a sapentanoyl group as an acyl group, and is mainly a triglyceride having these acyl groups.
- the squalene is 2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene.
- the aliphatic hydrocarbon having 27 or more carbon atoms is an aliphatic hydrocarbon other than squalene.
- the oil contained in the JPCC DA0580 strain is extracted using n-hexane as a solvent
- about 80% by mass of the extracted oil is usually a neutral lipid such as the triglyceride.
- This is the JPCC DA0580 strain. It can reach about 36% by mass or more of the dry algal cells. However, these values are calculated when the triglyceride is oleic acid triglyceride.
- the amount of the aliphatic hydrocarbon having 16 to 26 carbon atoms and the amount of squalene contained in the extracted oil are about 0.2% by mass or more and about 0% of the dry alga body of JPCC DA0580 strain. Each can reach 3% by mass or more.
- the composition of the extracted oil is usually obtained when JPCC DA0580 strain is cultured by the aeration culture method described later using the f / 2 medium. If the method is changed, the composition of the extracted oil may vary.
- Fatty acids constituting neutral lipids are mainly tetradecanoic acid ⁇ myristic acid (14 carbon atoms, 0 double bonds) ⁇ , hexadecanoic acid ⁇ palmitic acid (16 carbon atoms, double bonds) Is 0) ⁇ , hexadecenoic acid ⁇ palmitoleic acid (16 carbon atoms, 1 double bond number) ⁇ , octadecenoic acid ⁇ oleic acid (18 carbon atoms, 1 double bond number) ⁇ , and eicosapentaenoic acid ⁇ EPA (20 carbon atoms, 5 double bonds) ⁇ .
- the content of these fatty acids constituting neutral lipids such as triglycerides per dry algal body of JPCC DA0580 strain is about 1.0% by mass, about 12.7% by mass, and about 17.0% by mass in this order. %, About 0.8% by weight, and about 1.4% by weight. That is, about 32.9% by mass of the dry algal bodies are occupied by fatty acids constituting neutral lipids such as triglycerides.
- the content rate in the dry alga body of the fatty acid which comprises neutral lipids, such as the said triglyceride is the ratio for which the mass of the fatty acid methyl ester accounts in the said dry alga body when the said fatty acid is methyl-esterified. .
- the method for producing oil according to the present invention includes the step of culturing the microalgae belonging to the genus Navicula according to the present invention. It is possible to produce the oil by culturing the microalgae according to the present invention, collecting the microalgae grown in the culture step from the medium, and collecting the oil contained in the obtained microalgae. it can.
- the content of aliphatic hydrocarbons having 16 to 26 carbon atoms in the alga body is high, and from the viewpoint of easy culturing, the microalgae Navicula genus Eulycus Species are preferred, and JPCC DA0580 strain is more preferred.
- the JPCC DA0580 strain is more preferable as the microalgae of the present invention from the viewpoint of having a high ability to produce neutral lipids and squalene in addition to the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.
- the microalgae As the step of culturing the microalgae, a known method that can culture microalgae belonging to the genus Navikura can be applied.
- the microalgae may be inoculated into a culture vessel such as a flat flask containing a liquid medium, and aeration culture may be performed under light irradiation while gently stirring to such an extent that algal bodies do not precipitate.
- the liquid medium is not particularly limited as long as it is a liquid medium capable of culturing the microalgae, and a known medium can be used.
- a liquid medium capable of culturing the microalgae a liquid medium capable of culturing the microalgae
- a known medium can be used.
- the f / 2 medium used when isolating the aforementioned JPCC DA0580 strain is preferable from the viewpoint of good growth.
- the concentration of artificial seawater ⁇ product name: Marine Art SF-1 (manufactured by Senju Pharmaceutical Co., Ltd.) ⁇ in the liquid medium is 30 to 30% when 37 g / l is added as 100% (w / v).
- 100% (w / v) is preferable, 50 to 100% (w / v) is more preferable, 70 to 100% (w / v) is further preferable, 90 to 100 (w / v) is particularly preferable, and 95 to 100% (w / v) is most preferred.
- an aqueous solution in which a predetermined amount (37 g / l) of the artificial seawater is dissolved in distilled water is assumed to be substantially equal to the salt concentration of natural seawater.
- the light irradiation conditions may be appropriately adjusted depending on the algal body concentration in the culture solution. For example, 500 lux or more (lx) is preferable, 1000 to 30000 lux (lx) is more preferable, and 1000 to 10000 lux (lx) is preferable. More preferred is 1000 to 6000 lux (lx), and most preferred is 1000 to 3000 lux (lx).
- a known aeration gas suitable for the growth of the microalgae can be used.
- normal air, air to which CO 2 is added, or the like can be used.
- CO 2 concentration in air with the addition of CO 2 0.05 ⁇ 10% ( v / v) are preferred, more preferably 0.05 ⁇ 5.0% (v / v ), 1.0 ⁇ 3 0.0% (v / v) is most preferred.
- the culture temperature in the aeration culture may be a known culture temperature suitable for the growth of the microalgae, and is usually preferably 20 to 35 ° C, more preferably 25 to 30 ° C.
- the culture can be continued as long as the microalgae grow.
- the culture is preferably performed for 1 to 4 weeks, more preferably 1 to 3 weeks, and more preferably 1 to 2 weeks. More preferably,
- the microalgae may be further cultured under nutrient restriction after the aeration culture period.
- the content of oil contained in the algal bodies mass% of oil contained in the dried alga bodies
- the microalgae are grown in a medium without nutrient restriction (nutrient medium), cultured to a desired amount, and then switched to culture under nutrition restriction to be contained in the alga body.
- the oil content can be increased to increase the production efficiency of the oil.
- the culture under the above-mentioned nutrient restriction means that the culture is carried out in a medium (nutrient-restricted medium) in which nutrient salts containing vitamins and the like contained in the medium are less than usual.
- a medium nutrient-restricted medium
- nutrient salts sodium nitrate, disodium hydrogen phosphate, sodium metasilicate, f / 2 trace metal
- the f / 2 medium nutrient liquid medium
- the content of nutrient salts such as vitamins contained in the nutrient-restricted liquid medium is preferably 0 to 60%, more preferably 0 to 30%, from the viewpoint of increasing the oil content in the microalgae. -20% is more preferred, 0-10% is particularly preferred, and 0% is most preferred.
- the method for switching from the nutrient liquid medium to the nutrient-restricted liquid medium described above is not particularly limited, and may be switched to the nutrient-restricted medium at once or gradually.
- Examples of the method of switching at once include a method of precipitating algal bodies by centrifugation, removing the nutrient liquid medium that is a supernatant, and then adding a nutrient-restricted liquid medium.
- a nutrient liquid medium containing algal bodies is placed on one side separated by a semipermeable membrane, and a nutrient-restricted liquid medium is placed on the other side. Examples include a method of gradually reducing the content of nutrient salts containing vitamins and the like in the liquid medium to make a nutrient-restricted liquid medium.
- the period of culturing under the nutrient limitation is not particularly limited as long as the effect of the present invention is not impaired, and may be a period in which the oil content of the microalgae can be increased.
- the period is preferably 3 to 20 days, more preferably 3 to 10 days, and further preferably 3 to 7 days.
- a method for collecting the cultured microalgae it can be performed by a known method, for example, a method of precipitating algal bodies by centrifuging the culture solution and collecting them as pellets, or a filter having pores through which the microalgae cannot pass. Examples include a method of allowing the culture medium to pass through and collecting algal bodies remaining on the filter.
- the method for collecting the oil contained in the algal bodies obtained by the culture method and the recovery method is not particularly limited as long as the effect of the present invention is not impaired.
- the oil contained in the alga can be extracted into the organic solvent by suspending the collected alga in the organic solvent.
- the organic solvent is not particularly limited as long as it can dissolve the oil contained in the alga body without impairing the effects of the present invention.
- n-hexane hereinafter referred to as hexane
- acetone acetonitrile
- Methanol ethanol
- ethanol butanol
- chloroform chloroform and the like.
- hexane is preferable from the viewpoint of oil extraction efficiency.
- these organic solvents may be used individually by 1 type, and may be used in combination of 2 or more type. Further, in order to increase the extraction efficiency of the oil, it is preferable to physically destroy the alga body by applying an organic solvent in which the alga body is suspended to an ultrasonic homogenizer or the like.
- hexane cited as a preferred solvent for extracting the oil from the algal bodies of the microalgae may be used as a single one-phase solvent, or as a two-phase solvent mixed with methanol and / or ethanol. Also good.
- a ratio (volume ratio) of hexane: methanol 10: 0 to 5: 5
- a ratio of hexane: methanol 10: 0.5 to 8: 2
- It is more preferable to mix them at a ratio of hexane: methanol 10: 1 to 9: 1.
- ethanol and methanol are used as a one-phase solvent mixed at an arbitrary ratio (volume ratio).
- hexane: ethanol and methanol are preferably mixed at a ratio (volume ratio) of 10: 0 to 5: 5, and hexane: ethanol and methanol are mixed at a ratio of 10: 0.5 to 8: 2. More preferably, hexane: ethanol and methanol are more preferably mixed at a ratio of 10: 1 to 9: 1. In addition, it is more preferable to use ethanol than methanol from the viewpoint of increasing the safety of the solvent and the amount of oil extracted.
- the oil may be extracted with an organic solvent after the alga is dried. Oil extraction may be performed.
- it is preferable to extract the oil from the algal body in an undried state without drying the algal body in advance. Can be increased, which is preferable.
- oil is extracted from an algal body of a known microalga using an organic solvent, it is generally preferable to dry the alga body in advance and extract the oil from the dried algal body using an organic solvent. The amount can be increased.
- drying algal bodies in advance is disadvantageous in terms of time and energy costs for the drying process.
- the algal bodies of microalgae according to the present invention have very excellent properties that can efficiently extract oil from undried algal bodies. Due to this advantage, it is possible to reduce the energy of the production process of biofuel using the microalgae of the present invention.
- the amount of extraction can be increased by using a two-phase solvent composed of hexane and methanol and / or ethanol.
- higher extraction efficiency can be obtained by using a one-phase solvent composed of hexane.
- the organic solvent Since the organic solvent has a boiling point lower than that of the oil, it can be removed by blowing a nitrogen stream, distilling off under reduced pressure, or the like. It can also be reused.
- the oil extracted with the organic solvent can be further purified as necessary.
- a known method may be used, and for example, a method of fractionating each component contained in the oil component by solid phase extraction using silica gel, liquid chromatography, distillation or the like can be mentioned.
- the oil produced by the method for producing oil according to the present invention includes an oil that is observed when the microalgae are stained with Nile red.
- the oil contains an aliphatic hydrocarbon having 16 to 26 carbon atoms.
- the aliphatic hydrocarbons having 16 to 26 carbon atoms are mainly aliphatic hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms.
- the oil component includes photosynthesis of neutral lipids, squalene, aliphatic hydrocarbons having 27 or more carbon atoms, phospholipids, free fatty acids, steroid compounds, and carotenoids.
- dye etc. may also be included.
- the oil in the present invention is produced by the oil production method according to the present invention.
- the description of the oil content is the same as the description of the oil content in the above-described oil content manufacturing method.
- aliphatic hydrocarbons having 16 to 26 carbon atoms can be purified from the oil.
- the oil contains neutral lipid and / or squalene
- the neutral lipid and / or squalene can be purified from the oil.
- the purification method is not particularly limited and can be performed by a known method.
- an oil component extracted from hexane from the microalgae is dissolved in an organic solvent such as hexane, and silica gel is added to the solution to adsorb a substance other than the aliphatic hydrocarbon onto the silica gel. It can be purified by elution. Further, it can be purified by eluting neutral lipid adsorbed on silica gel with an organic solvent or the like. Furthermore, the aliphatic hydrocarbon having 16 to 26 carbon atoms and squalene can be separated by subjecting the eluted aliphatic hydrocarbon to a known purification method such as distillation and chromatography. In addition, as an organic solvent which melt
- the dry alga body in the present invention is obtained by drying the microalga belonging to the genus Navikura according to the present invention.
- the method for drying the microalgae is not particularly limited as long as it is a method capable of removing moisture in the algal bodies.
- the drying method by freeze-drying is preferable from the viewpoint of suppressing the decomposition of the components contained in the algal cells.
- the fuel in the present invention is obtained from the microalga belonging to the genus Navikura according to the present invention.
- the said micro algae the thing similar to what was mentioned by description of the micro algae which belongs to the said Navikura genus concerning this invention is mentioned.
- a method of using the microalgae as a fuel a method of burning the microalgae, a method of burning oil collected from the microalgae, an aliphatic carbonization having 16 to 26 carbon refined from the oil collected from the microalgae Examples thereof include a method of burning hydrogen, neutral lipid, and / or squalene.
- the microalgae When burning the microalgae, it is preferable to use a dry alga body obtained by drying the microalgae from the viewpoint of enhancing the combustion efficiency.
- the dry algal bodies are the same as the dry algal bodies according to the present invention.
- the calorific value of the dried alga body can reach the same or higher value than the calorific value of coal (approximately 6000 kcal / kg).
- Oil collected from the microalgae When burning the oil collected from the microalgae, the oil is the same as the oil according to the present invention. Oil collected from the microalgae is flammable and can be used as fuel for boilers, for example. When the microalgae is JPCC DA0580 strain, the amount of heat of the hexane extract (oil) can reach 8700 kcal / kg or more.
- the aliphatic hydrocarbon having 16 to 26 carbon atoms, neutral lipid, and / or squalene are the same as the aliphatic hydrocarbon having 16 to 26 carbon atoms, neutral lipid, and squalene in the oil according to the present invention.
- the aliphatic hydrocarbon having 16 to 26 carbon atoms can be used as a fuel for a diesel engine.
- the neutral lipid can be made into diesel fuel (so-called biodiesel fuel) by a known transesterification method or the like.
- the carbon dioxide fixing method in the present invention includes a step of culturing the microalgae belonging to the genus Navikura according to the present invention.
- the photosynthesis performed when the microalgae grow has the effect of assimilating carbon dioxide in the culture solution (in the atmosphere). That is, carbon dioxide can be fixed by culturing the microalgae.
- the step of culturing the microalgae is the same as the method for culturing the microalgae in the oil production method according to the present invention.
- a 24-well microtiter plate containing 2 ml of the f / 2 medium was inoculated with JPCC DA0580 strain, and subjected to static culture for 12 weeks under light irradiation of 1000 lux (lx). Subsequently, the culture solution was transferred to a 1.5 ml microtube and centrifuged at 13000 rpm to collect algal bodies as pellets. In order to remove the medium contained in the pellet, the suspension was suspended in 0.5 ml of physiological saline, and then centrifuged at 13,000 rpm for 5 minutes to collect algal bodies as pellets.
- the pellet was resuspended in 450 ⁇ l of physiological saline, further mixed with 50 ⁇ l of Nile red solution, and then incubated at room temperature for 10 minutes. Thereafter, the pellet was collected by centrifuging at 13,000 rpm for 5 minutes. In order to wash away the excess Nile red solution, the pellet was suspended in 0.5 ml of physiological saline and centrifuged again to collect alga bodies as a pellet. A suspension of the obtained algal bodies in 50 ⁇ l of physiological saline was observed with a fluorescence microscope. As a result, it was possible to confirm a region emitting yellow fluorescence indicating an oil existing region stained with Nile red in the algal cells.
- the Nile red solution is a solution prepared by dissolving 1 mg of Nile red in 10 ml of acetone and further diluting it four times with physiological saline.
- JPCC DA0580 strain was inoculated into a 500 ml flat flask containing 500 ml of the f / 2 medium, and aerated culture was carried out for 1 week at an aeration rate of 1 vvm under irradiation of 3000 lux (lx). Thereafter, the algal body pellet collected by centrifugation was freeze-dried overnight. 6 ml of hexane was added to and suspended in 0.1 g of the obtained dry alga bodies, and extraction was performed for 30 minutes while crushing the alga bodies at room temperature using an ultrasonic homogenizer.
- the hexane extract was centrifuged to precipitate alga bodies, and about 6 ml of the supernatant hexane extract was recovered. The remaining algal cell residue as a precipitate was suspended again by adding 6 ml of new hexane, and the same extraction operation was further performed twice (a total of three extraction operations). Thereafter, about 18 ml of the collected hexane extract was removed using a Sep-pak cartridge (6 cc / 1 g) (manufactured by Waters Corporation) silica gel column to remove neutral lipids, free fatty acids, pigments, etc. 16 ml containing minutes were obtained. The sample was dried in a nitrogen stream and redissolved in 0.5 ml of hexane. The sample was analyzed by gas chromatograph mass spectrometry (GCMS) to identify hydrocarbons contained in the sample.
- GCMS gas chromatograph mass spectrometry
- the gas chromatograph instrument used was GC2010 manufactured by Shimadzu Corporation, the column used was DB-1 (column length: 30 m, column inner diameter: 0.25 mm), and the measurement conditions (temperature increase: 100 ° C. (0 min. ) To 330 ° C. (10 ° C./min, hold), injection temperature: 300 ° C., injection mode: splitless, carrier gas: He, injection amount: 1.0 ⁇ l).
- GCMS-QP5050A manufactured by Shimadzu Corporation that performs ionization by electron impact (EI) method was used.
- JPCC DA0580 strain produced an aliphatic hydrocarbon having 16 to 26 carbon atoms. More specifically, linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms were identified as the main components. Moreover, when the amount of hydrocarbons produced by the JPCC DA0580 strain was quantified from the area ratio indicating the hydrocarbons on the GCMS chart using a sample obtained by dissolving 0.1% light oil in hexane as a sample for quantification, drying of the JPCC DA0580 strain was determined. The content of the linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms in the algal cells was about 0.2% by mass.
- This content rate was higher than the content rate of algae producing light oil-based aliphatic hydrocarbons known so far.
- the known algae occupy 0.025 to 0.12% by mass of light oil-based aliphatic hydrocarbons in the dry alga body.
- the peak having the largest area in the GCMS chart was identified as squalene.
- the squalene content per dry alga body of the JPCC DA0580 strain calculated from the peak area was about 0.3% by mass.
- JPCC DA0580 algal cell pellet obtained by culturing in the same manner as in Example 2 was freeze-dried overnight.
- About 20 ml of hexane extract was obtained in the same manner as in Example 2 except that 20 mg of the dried alga body was used as a sample and hexane used for one extraction operation was changed to 7 ml.
- Hexane which is the solvent of the hexane extract, was removed by evaporation with a nitrogen stream to obtain 9.1 mg of hexane extract (oil).
- hexane extract (oil content) occupied 45.5% by mass of the dry algal cells.
- a 5% hydrochloric acid-methanol solution is added to the hexane extract (oil) and reacted in a sealed test tube at 90 ° C. for 2 hours to obtain a fatty acid constituting a neutral lipid such as triglyceride contained in the hexane extract.
- Methyl esterification was performed. Thereafter, extraction with chloroform was performed, and the chloroform phase was separated and used as a sample.
- the sample was analyzed by the following GCMS apparatus and quantified using oleic acid methyl ester as a standard substance.
- the gas chromatograph instrument used was GC6890N (model number) manufactured by Agilent Technologies International Co., Ltd., and the column used was DB-WAX manufactured by J & W (column length: 30 m, column inner diameter: 0.25 mm, film thickness: 0). Measurement conditions ⁇ temperature rise: 50 ° C. (0 min) to 250 ° C. (10 ° C./min, hold for 15 min), injection temperature: 230 ° C., injection mode: split injection (split ratio 10: 1) , Carrier gas: He (1.2 ml / min), detector: FID (250 ° C.), injection amount: 0.6 ⁇ l ⁇ .
- a JMS-GCmate-II type manufactured by JEOL Ltd., which performs ionization by an electron impact (EI) method was used.
- fatty acids constituting neutral lipids are mainly tetradecanoic acid ⁇ myristic acid (C14: 0) ⁇ , hexadecanoic acid ⁇ palmitic acid (C16: 0) ⁇ , hexadecenoic acid ⁇ palmitolein Acid (C16: 1) ⁇ , octadecenoic acid ⁇ oleic acid (C18: 1) ⁇ , and eicosapentaenoic acid ⁇ EPA (C20: 5) ⁇ .
- the content of these fatty acids per dry alga body of JPCC DA0580 is about 1.0% by mass, about 12.7% by mass, about 17.0% by mass, about 0.8% by mass, and about It was 1.4% by mass. That is, about 32.9% by mass of the dry algal bodies were occupied by fatty acids constituting neutral lipids such as triglycerides.
- neutral lipids such as triglycerides.
- all the fatty acids constituting the neutral lipid such as triglyceride are oleic acid
- it is calculated that about 3.8% by mass of the dry algal body is glycerin constituting the neutral lipid such as triglyceride. It was concluded that about 36.7% by mass of the dry algal cells were neutral lipids such as triglycerides.
- JPCC DA0580 strain was cultured in the same manner as in Example 2. Further, JPCC DA0580 strain was cultured in the same manner as in Example 2 except that the liquid medium was changed to the following IMK medium. In order to evaluate the growth of JPCC DA0580 strain in each medium, the turbidity (OD750) of each culture solution was measured over time. The result is shown in FIG.
- the IMK medium was prepared as follows. Nutrients containing vitamins ⁇ sodium nitrate 200 mg / l, disodium hydrogen phosphate 1.4 mg / l, sodium dihydrogen phosphate 5.0 mg / l, ammonium chloride 68 mg / l, thiamine 0.2 mg / l, biotin 0 .0015 mg / l, vitamin (B12) 0.0015 mg / l, Na 2 EDTA 37.2 mg / l, FeEDTA 5.2 mg / l, MnEDTA 0.3332 mg / l, manganese (II) chloride tetrahydrate 0.18 mg / l, Zinc sulfate heptahydrate 0.024 mg / l, cobalt (II) chloride hexahydrate 0.014 mg / l, disodium molybdate (VI) dihydrate 0.0072 mg / l, copper (II) sulfate penta Hydrate
- the JPCC DA0580 strain had an OD750 of 0.05 to 0.9 in the f / 2 medium and a steady growth in the culture period of 1 week.
- the alga bodies that reached the steady state were recovered, dried and frozen, and the recovered amount of the dried alga bodies was examined. As a result, it was 0.46 g per liter of the culture solution.
- oil productivity per week of JPCC DA0580 strain is It was confirmed that it reached 0.26 g or more per liter of culture scale.
- JPCC DA0580 strain did not reach a steady state in the IMK medium in the culture period of 1 week.
- ⁇ Required seawater of JPCC DA0580> Of the composition of the f / 2 medium used in Example 2, five types of nutrient liquid medium were prepared in which the concentration of artificial seawater ⁇ product name: Marine Art SF-1 (manufactured by Senju Pharmaceutical Co., Ltd.) ⁇ was changed. The concentration of the artificial seawater is 0% when added at 0 g / l, and in the same manner 11.1 g / l (30%), 18.5 g / l (50%), 29.8 g / l (80 %) And 37 g / l (100%).
- JPCC DA0580 strain was inoculated into a 500 ml flat flask containing 500 ml of these mediums, and static culture was performed for 1 week under the same culture conditions as in Example 2.
- the turbidity (OD750) of the culture solution was measured over time. The result is shown in FIG.
- JPCC DA0580 strain grows best under the condition containing 100% artificial seawater (seawater component) during the growth period of 1 week. Further, it was confirmed that the growth decreased as the artificial seawater (seawater component) concentration decreased, and that the artificial seawater (seawater component) was 0%, and the growth did not occur. From this, it was confirmed that JPCC DA0580 strain is a marine microalgae.
- the hexane extract obtained by the method similar to Example 2 was dried by nitrogen stream using the obtained dried alga body as a sample, and obtained as a hexane extract (oil content).
- the calorific value of the hexane extract (oil) was measured by the cylinder type calorimeter, and the calorific value of the hexane extract (oil content) was 8780 kcal / kg.
- Extraction efficiency when extracting oil from alga bodies of microalgae classified into other genera was cultivated by a known method to obtain algal body pellets. These three kinds of microalgae accumulate oil in their algal bodies.
- the oil contains at least a neutral lipid such as triglyceride.
- the microalgae according to the present invention has a high production capacity for aliphatic hydrocarbons having 16 to 26 carbon atoms, it can be used for the production of diesel fuel, and is expected as a carbon neutral fuel aimed at preventing global warming. it can.
- the JPCC DA0580 strain of the present invention is particularly promising from the viewpoint of having a high ability to produce neutral lipids and squalene in addition to the ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.
- the neutral lipid can be made into diesel fuel (so-called biodiesel fuel) by a known transesterification method or the like.
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Abstract
Description
重質油系炭化水素の産生能を有する藻類としては、炭素数36の炭化水素の産生能をもつ微細藻類ボツリオコッカス ブラウニー(非特許文献1参照。)や炭素数33の炭化水素の産生能をもつ微細藻類ボツリオコッカス・ブラウニーAレース(特許文献1参照。)が知られる。
また、軽質油系炭化水素の産生能を有する藻類としては、炭素数17の炭化水素の産生能をもつ微細藻類Nostoc muscorum、Trichodesmium erythaeum、Plectonema terebrans等(非特許文献1参照。)、炭素数19の炭化水素の産生能をもつ微細藻類Coccochloris elabens等(非特許文献1参照。)、炭素数17、18、19、および20の炭化水素の産生能をもつ微細藻類シュードコリシスチス エリプソイディア MBIC11204株(特許文献2参照。)、炭素数17、19、21、および23の炭化水素の産生能をもつ微細藻類コリシスチス マイナー SAG17.98株(特許文献2参照。)が知られる。
しかしながら、前記微細藻類を乾燥して得られる乾燥藻体において、軽質油系炭化水素の含有率は通常0.025~0.12質量%程度であり(非特許文献1参照。)、その炭化水素産生能は必ずしも十分ではない。
(1)炭素数16~26の脂肪族炭化水素の産生能を有するナビクラ(Navicula)属に属する微細藻類。
(2)炭素数16~26の脂肪族炭化水素の産生能を有する微細藻類ナビクラ(Navicula)属オイリティカス(oiliticus)種。
(3)微細藻類ナビクラ(Navicula)属 オイリティカス (oiliticus)種 JPCC DA0580株(FERM BP-11201)。
(4)前記(1)~(3)のいずれか一つに記載の微細藻類を培養する工程を有する油分の製造方法。
(5)前記培養する工程の後に、当該培養に用いた培地中の栄養塩類の濃度を低減した別の培地で前記微細藻類を更に培養する工程を有する、前記(4)に記載の油分の製造方法。
(6)前記油分が中性脂質を含む、前記(4)又は(5)記載の油分の製造方法。
(7)前記油分がスクアレンを含む、前記(4)~(6)の何れか一つに記載の油分の製造方法。
(8)前記培養する工程の後、培養物から有機溶媒で油分を抽出する工程を有し、当該有機溶媒が、n-へキサンからなる溶媒、n-へキサン及びメタノールからなる溶媒、又はn-へキサン及びエタノールからなる溶媒の何れかである前記(4)~(7)のいずれか一つに記載の油分の製造方法。
(9)前記(4)~(8)のいずれか一つに記載の油分の製造方法によって製造された油分。
(10)前記(1)~(3)のいずれか一つに記載の微細藻類を乾燥して得られる乾燥藻体。
(11)前記(1)~(3)のいずれか一項に記載の微細藻類から得られる燃料。
(12)前記(1)~(3)のいずれか一つに記載の微細藻類を培養する工程を有する二酸化炭素固定方法。
<ナビクラ属に属する微細藻類>
本発明におけるナビクラ(Navicula)属に属する微細藻類は、炭素数16~26の脂肪族炭化水素の産生能を有するものである。ここで、前記炭素数16~26の脂肪族炭化水素は、主に炭素数16、18、20、22、24、および26の脂肪族炭化水素である。
JPCC DA0580株は、前記炭素数16~26の脂肪族炭化水素の産生能に加えて、中性脂質およびスクアレンの高い産生能を有する観点からも、本発明の微細藻類として、より好ましい。該中性脂質の説明は、後述する。
以下に、該微細藻類の単離方法および該微細藻類のJPCC DA0580株を新種の新規株と判定するに至った経緯を説明する。
硝酸ナトリウム75.0mg/l、リン酸水素二ナトリウム5.0mg/l、メタケイ酸ナトリウム9水和物30.0mg/l、f/2微量金属溶液1.0ml、f/2ビタミン溶液0.5ml、および人工海水{製品名:マリンアート・SF-1(千寿製薬株式会社製)}37g/lを蒸留水に上記所定の濃度で溶解した液体培地を、f/2培地として調製した。
前記f/2微量金属溶液は、Na2EDTA2水和物4.36g/l、塩化第二鉄6水和物3.15g/l、塩化マンガン(II)4水和物180mg/l、硫酸亜鉛7水和物22.0mg/l、塩化コバルト(II)6水和物10.0mg/l、モリブテン(VI)酸二ナトリウム2水和物6.3mg/l、硫酸銅(II)5水和物9.8mg/lの組成をもつ水溶液として調製した。
前記f/2ビタミン溶液は、チアミン200.0mg/l、ビオチン0.1mg/l、ビタミン(B12)1.0mg/lの組成をもつ水溶液として調製した。
また、前記f/2培地の組成に加えて、さらに寒天を1.2%(w/v)の濃度となるように添加した寒天f/2培地を調製した。
なお、前記人工海水は、天然海水に含まれる塩類を模した塩類である。前記人口海水の所定量(37g/l)を蒸留水に溶解することで、天然海水を模した海水成分をもつ水溶液を作製することができる。
前記f/2培地2mlを含む24穴のマイクロタイタープレートに、2005年7月鹿児島県奄美市住用川と役勝川の合流地点にあるマングローブ林より採取した砂泥サンプルを適量添加した。つづいて、1000ルクス(lx)の光照射下で静置培養を行い、微細藻類の生育が確認できたウェル中の培養液の一部を分取した。その分取した培養液を前記f/2寒天培地上に接種して、前記光照射条件下で培養することによって、茶色の単細胞藻類JPCC DA0580株が、単菌化(単離)された微細藻類のコロニーとして得られた。
前記寒天培地上で、25℃、7日間培養した結果、直径2.0~5.0mm程度の茶色のJPCC DA0580株のコロニーが得られた。コロニーの形状は点状で、隆起の無い半レンズ状であった。周縁は全縁であり、表面はスムーズな形状であった。また、変異によるコロニーの形態の変化は見られず、培養条件や生理的状態によるコロニー形態の変化も見られなかった。
前記コロニー中の海洋微細藻類は、大きさが平均して10~20μm程度の単細胞藻類で、時に群体を形成し、栄養細胞には点眼や収縮胞は無く、細胞形は菱形であった。浮遊性は無く、ガラス質の殻を有し、被殻に縦溝があり、細胞表面は滑らかである。栄養細胞は鞭毛を持たず運動性を示さない。
JPCC DA0580株の電子顕微鏡像を図4に示す。
JPCC DA0580株は、有性生殖と無性生殖の両方を行う。無性生殖は二分裂により無性的に増殖する。細胞分裂は被殻の中で進行し、二つの娘細胞は親細胞の外殻と内殻の内側にそれぞれ新たな半被殻を形成する。親細胞の半被殻はそれぞれ一つずつ娘細胞に分配される。その結果、娘細胞の一つは親細胞と同じ大きさ(の被殻)であるが、もう一方は内殻を外殻として利用する。その結果、親細胞より一回り小さくなるため、ある程度小さくなった細胞では有性生殖が行われる。その細胞では、減数分裂を行って同形配偶子が形成される。この接合子が被殻を介して近接し、原形質をアメーバ状に変化させて接合、増大胞子を形成し、成長して体積を増し、然るべき大きさの通常細胞に戻る様式を取っている。
・ 培養液:海水を基調とする培養液中で生育する。淡水では生育しない。
・ 光合成:光合成による光独立栄養生育ができる。従属栄養生育は確認されない。
・ 含有色素:クロロフィルa、c1、c2、フコキサンチン及びフコキサンチン誘導体を中心とするカロテノイド色素類
・ 貯蔵物質:デンプン
・ 生育温度:20℃~35℃(至適温度25℃)
・ 生育pH:7.0~9.0(至適pH8.0)
・ 細胞内にNile redで染色される、炭素数16~26の脂肪族炭化水素、中性脂質およびスクアレンを含む大量の油分を蓄積する。
・ 生育期間:1週間{濁度(OD750)が0.05から1.2に達するのに要する期間}
前記炭素数16~26の脂肪族炭化水素は、主に炭素数16、18、20、22,24および26の脂肪族炭化水素であり、より詳しくは、主に炭素数16、18、20、22,24および26の直鎖脂肪族飽和炭化水素である。
前記中性脂質は、後述するように、主にテトラデカノイル基(ミリストイル基)、ヘキサデカノイル基(パルミトイル基)、ヘキサデセノイル基(パルミトレオイル基)、オクタデセノイル基(オレオイル基)、およびエイコサペンタエノイル基を、アシル基として有する中性脂質であり、主にこれらのアシル基を有するトリグリセライドである。
前記スクアレンは、2,6,10,15,19,23-ヘキサメチルテトラコサ-2,6,10,14,18,22-ヘキサエンである。
前記炭素数27以上の脂肪族炭化水素は、スクアレン以外の脂肪族炭化水素である。
また、当該抽出された油分に含まれる、前記炭素数16~26の脂肪族炭化水素の量および前記スクアレンの量は、JPCC DA0580株の乾燥藻体の、約0.2質量%以上および約0.3質量%以上に、それぞれ達しうる。
ただし、当該抽出された油分のこれらの組成は、前記f/2培地を用いて、後述する通気培養の方法によって、JPCC DA0580株を培養した際に通常得られるものであり、当該微細藻類の培養方法を変更した場合には、当該抽出された油分の組成は変動しうる。
なお、前記トリグリセライド等の中性脂質を構成する脂肪酸の乾燥藻体中の含有率は、当該脂肪酸をメチルエステル化した場合に、その脂肪酸メチルエステルの質量が前記乾燥藻体中に占める割合である。
本発明における油分の製造方法は、本発明にかかる前記ナビクラ(Navicula)属に属する微細藻類を培養する工程を有するものである。本発明にかかる微細藻類を培養し、当該培養工程によって生育させた当該微細藻類を培地中から回収し、得られた微細藻類中に含まれる油分を採取することによって、当該油分を製造することができる。
JPCC DA0580株は、前記炭素数16~26の脂肪族炭化水素の産生能に加えて、中性脂質およびスクアレンの高い産生能を有する観点からも、本発明の微細藻類として、より好ましい。
前記CO2を添加した空気におけるCO2濃度としては、0.05~10%(v/v)が好ましく、0.05~5.0%(v/v)がより好ましく、1.0~3.0%(v/v)が最も好ましい。
前記通気培養における培養温度としては、当該微細藻類が生育するのに適した公知の培養温度でよく、通常、20~35℃で行うことが好ましく、25~30℃で行うことがより好ましい。
前記通気培養の期間としては、当該微細藻類が生育する限り培養を継続することができ、通常、1~4週間で行うことが好ましく、1~3週間で行うことがより好ましく、1~2週間で行うことがさらに好ましい。
該有機溶媒としては、本発明の効果を損なわず、藻体中に含まれる油分を溶解できるものであれば特に制限されず、例えば、n-ヘキサン(以下では、ヘキサンという。)、アセトン、アセトニトリル、メタノール、エタノール、ブタノール、クロロホルム等が挙げられる。これらのなかでも、油分の抽出効率の観点から、ヘキサンが好ましい。また、これらの有機溶媒は、1種を単独で用いてもよく、2種以上を組み合わせて用いてもよい。
また、該油分の抽出効率を高めるために、該藻体を懸濁した有機溶媒を超音波ホモジナイザー等にかけて、該藻体を物理的に破壊することが好ましい。
メタノールを混合した2相溶媒とした場合、ヘキサン:メタノール=10:0~5:5の割合(体積比)で混合することが好ましく、ヘキサン:メタノール=10:0.5~8:2の割合で混合することがより好ましく、ヘキサン:メタノール=10:1~9:1の割合で混合することがさらに好ましい。
エタノールを混合した2相溶媒とした場合、ヘキサン:エタノール=10:0~5:5の割合(体積比)で混合することが好ましく、ヘキサン:エタノール=10:0.5~8:2の割合で混合することがより好ましく、ヘキサン:エタノール=10:1~9:1の割合で混合することがさらに好ましい。
エタノール及びメタノールをヘキサンに混合した2相溶媒の場合、エタノール及びメタノールは任意の割合(体積比)で混合した1相の溶媒として用いられる。この場合、ヘキサン:エタノール及びメタノール=10:0~5:5の割合(体積比)で混合することが好ましく、ヘキサン:エタノール及びメタノール=10:0.5~8:2の割合で混合することがより好ましく、ヘキサン:エタノール及びメタノール=10:1~9:1の割合で混合することがさらに好ましい。
なお、溶媒の安全性及び油分抽出量を高める観点から、メタノールよりもエタノールを用いる方がより好ましい。
本発明にかかる微細藻類の藻体から有機溶媒を用いて油分を抽出する場合、該藻体を予め乾燥させずに未乾燥状態の藻体から有機溶媒による油分抽出を行う方が、油分抽出量を高めることができるので、好ましい。
公知の微細藻類の藻体から有機溶媒を用いて油分を抽出する場合には、該藻体を予め乾燥させておき、乾燥状態の藻体から有機溶媒による油分抽出を行う方が、一般に油分抽出量を高めることができる。しかし、予め藻体を乾燥することは、乾燥処理にかかる時間・エネルギーコストの面から不利である。この点において、本発明にかかる微細藻類の藻体は、未乾燥状態の藻体から効率的に油分を抽出できる、非常に優れた性質を持つといえる。この利点によって、本発明の微細藻類を用いたバイオ燃料等の製造プロセスの低エネルギー化を図ることができる。
乾燥状態の前記藻体から油分を有機溶媒により抽出する場合は、ヘキサンからなる1相溶媒を用いると、より高い抽出効率が得られる。
また、前記有機溶媒によって抽出した油分は、必要に応じてさらに精製することができる。その精製方法としては、公知の方法で行えばよく、例えばシリカゲルを用いた固相抽出、液体クロマトグラフィー、蒸留等によって、当該油分に含まれる成分ごとに分取する方法が挙げられる。
また、該油分は前記炭素数16~26の脂肪族炭化水素の他に、中性脂質、スクアレン、炭素数27以上の脂肪族炭化水素、リン脂質、遊離脂肪酸、ステロイド化合物、およびカロテノイド等の光合成色素等も含みうる。
本発明における油分は、本発明にかかる油分の製造方法によって製造されたものである。
当該油分の説明は、前述の油分の製造方法における油分の説明と同様である。
また、当該油分から炭素数16~26の脂肪族炭化水素を精製することもできる。さらに、当該油分が中性脂質及び/又はスクアレンを含む場合は、その中性脂質及び/又はスクアレンを該油分から精製することもできる。
該精製方法としては、特に制限されず、公知の方法で行うことができる。例えば、当該微細藻類からヘキサン抽出した油分をヘキサン等の有機溶媒に溶解し、該溶液にシリカゲルを投入することにより、脂肪族炭化水素以外の物質をシリカゲルに吸着させ、該脂肪族炭化水素のみを溶出させることで精製できる。また、シリカゲルに吸着した中性脂質を有機溶媒等で溶出させることで精製できる。さらに、溶出した脂肪族炭化水素を蒸留、クロマトグラフィー等の公知の精製方法にかけることにより、炭素数16~26の脂肪族炭化水素とスクアレンとを分離できる。
なお、当該油分を溶解する有機溶媒としては、前述の油分の製造方法における有機溶媒の説明で挙げたものと同様のものが挙げられる。
本発明における乾燥藻体は、本発明にかかる前記ナビクラ属に属する微細藻類を乾燥させたものである。
当該微細藻類としては、本発明にかかる前記ナビクラ属に属する微細藻類の説明で挙げたものと同様のものが挙げられる。
当該微細藻類を乾燥させる方法としては、藻体中の水分を除去できる方法であれば特に制限されない。例えば、藻体を天日干しにする方法、藻体に乾燥空気を吹き付ける方法、藻体を凍結乾燥(フリーズドライ)する方法等が挙げられる。これらのうち、藻体に含まれる成分の分解を抑制できる観点から、凍結乾燥による乾燥方法が好ましい。
本発明における燃料は、本発明にかかる前記ナビクラ属に属する微細藻類から得られたものである。
当該微細藻類としては、本発明にかかる前記ナビクラ属に属する微細藻類の説明で挙げたものと同様のものが挙げられる。
当該微細藻類を燃料として用いる方法としては、当該微細藻類を燃焼させる方法、当該微細藻類から採取した油分を燃焼させる方法、当該微細藻類から採取した油分から精製した炭素数16~26の脂肪族炭化水素、中性脂質、及び/又はスクアレンを燃焼させる方法等が例示できる。
本発明における二酸化炭素固定方法は、本発明にかかる前記ナビクラ属に属する微細藻類を培養する工程を有するものである。
当該微細藻類が生育する際に行う光合成は、培養液中(大気中)の二酸化炭素を同化する作用がある。すなわち、当該微細藻類を培養することによって、二酸化炭素を固定することができる。
当該微細藻類としては、本発明にかかる前記ナビクラ属に属する微細藻類の説明で挙げたものと同様のものが挙げられる。
当該微細藻類を培養する工程は、本発明にかかる前記油分の製造方法における当該微細藻類の培養方法と同様である。
JPCC DA0580株を24穴ウェルで12週間培養した後、その藻体をNile redで染色することにより、藻体中に油分が産生および蓄積されていることを確認した。
つぎに、そのペレットを450μlの生理食塩水に再懸濁して、さらに50μlのNile red溶液を加えて混合した後、室温で10分間のインキュベーションを行った。その後、13000rpmの遠心を5分行ってペレットを回収し、余分なNile red溶液を洗い流すために該ペレットを0.5mlの生理食塩水へ懸濁してから再度遠心してペレットとして藻体を回収した。得られた藻体を50μlの生理食塩水へ懸濁したものを、蛍光顕微鏡によって観察した。その結果、藻体中にNile redで染色された油分の存在領域を示す黄色の蛍光を発する領域を確認することができた。該蛍光顕微鏡像では、藻体内に大量の油分が油滴状に蓄積されている様子が確認された(図5)。
なお、前記Nile red溶液は、1mgのNile redを10mlのアセトンに溶解し、さらに生理食塩水で4倍に希釈した溶液である。
前記f/2培地500mlを入れた500ml容の偏平フラスコにJPCC DA0580株を接種して、3000ルクス(lx)の光照射下、通気量1vvmで1週間、通気培養を行った。その後、遠心で回収した藻体ペレットを、一晩凍結乾燥した。
得られた乾燥藻体0.1gにヘキサン6mlを加えて懸濁し、超音波ホモジナイザーを用いて室温で藻体を破砕しながら30分間の抽出を行った。つぎに、そのヘキサン抽出液を遠心し、藻体の残渣を沈殿させ、上清のヘキサン抽出溶液を約6ml回収した。沈殿として残った藻体の残渣に対して、再び新しいヘキサン6mlを加えて懸濁し、同じ抽出操作をさらに2回(合計3回の抽出操作)を行った。その後、回収したヘキサン抽出液約18mlをSep-pak cartridge(6 cc/1g)(Waters Corporation社製)シリカゲルカラムを用いて、中性脂質、遊離脂肪酸、色素等を除去し、脂肪族炭化水素画分を含む16ml得た。窒素気流によって乾燥させ、ヘキサン0.5mlに再溶解させたものを試料とした。その試料をガスクロマトグラフ質量分析(GCMS)によって分析し、試料に含まれる炭化水素を同定した。
質量分析機器は、電子衝撃(EI)法によるイオン化を行う、株式会社島津製作所製のGCMS-QP5050Aを使用した。
また、軽油0.1%をヘキサンに溶解した試料を定量用の試料として、GCMSチャートにおける当該炭化水素を示す面積比からJPCC DA0580株が産生する炭化水素量を定量したところ、JPCC DA0580株の乾燥藻体における当該炭素数16、18、20、22、24および26の直鎖状脂肪族飽和炭化水素の含有率は約0.2質量%であった。この含有率は、これまでに公知の、軽質油系の脂肪族炭化水素を産生する藻類の含有率よりも高いものであった。なお、当該公知の藻類は、非特許文献1のTABLE2に記載されているように、その乾燥藻体において、0.025~0.12質量%を軽質油系脂肪族炭化水素が占める。
さらに、前記GCMSチャートにおいて最大の面積をもつピークは、スクアレンであると同定された。そのピーク面積から計算した、当該JPCC DA0580株の乾燥藻体当たりのスクアレン含有率は約0.3質量%であった。
その結果、ヘキサンによって抽出された油分は、JPCC DA0580株の乾燥藻体のうち、45.5質量%以上を占めることが確認された。なお、同一条件で培養して得られた、別の培養ロットから得られたJPCC DA0580株の藻体では、その乾燥藻体のうち、最大で58質量%を前記へキサンによって抽出された油分が占めることも確認された。
実施例2と同様の方法で培養して得たJPCC DA0580株の藻体ペレットを、一晩凍結乾燥した。
当該乾燥藻体20mgを試料として、一回の抽出操作に用いたヘキサンを7mlとした以外は実施例2と同様の方法で、ヘキサン抽出液約20mlを得た。該ヘキサン抽出液の溶媒であるヘキサンを窒素気流によって蒸発させて除き、9.1mgのヘキサン抽出物(油分)を得た。したがって、当該乾燥藻体の45.5質量%をヘキサン抽出物(油分)が占めることを確認した。
前記ヘキサン抽出物(油分)に5%塩酸-メタノール溶液を加え、封管した試験管中で90℃、2時間反応させ、前記ヘキサン抽出物に含まれるトリグリセライド等の中性脂質を構成する脂肪酸のメチルエステル化を行った。その後、クロロフォルムで抽出し、クロロフォルム相を分取し試料とした。
前記試料を下記のGCMS装置によって分析し、オレイン酸メチルエステルを標準物質として用いて定量した。
質量分析機器は、電子衝撃(EI)法によるイオン化を行う、日本電子株式会社製のJMS-GCmate-II型を使用した。
前記トリグリセライド等の中性脂質を構成する脂肪酸が、全てオレイン酸であると仮定した場合、当該乾燥藻体の約3.8質量%が前記トリグリセライド等の中性脂質を構成するグリセリンであると計算され、当該乾燥藻体の約36.7質量%が前記トリグリセライド等の中性脂質であると結論した。
実施例2と同様の方法でJPCC DA0580株を培養した。また、液体培地を下記IMK培地に変えた以外は実施例2と同様の方法でJPCC DA0580株を培養した。それぞれの培地におけるJPCC DA0580株の生育を評価するために、各培養液の濁度(OD750)を経時的に測定した。その結果を図2に示す。
ビタミン等を含む栄養塩類{硝酸ナトリウム200mg/l、リン酸水素二ナトリウム1.4mg/l、リン酸二水素ナトリウム5.0mg/l、塩化アンモニウム68mg/l、チアミン0.2mg/l、ビオチン0.0015mg/l、ビタミン(B12)0.0015mg/l、Na2EDTA37.2mg/l、FeEDTA5.2mg/l、MnEDTA0.3332mg/l、塩化マンガン(II)四水和物0.18mg/l、硫酸亜鉛七水和物0.024mg/l、塩化コバルト(II)六水和物0.014mg/l、モリブテン(VI)酸二ナトリウム二水和物0.0072mg/l、硫酸銅(II)五水和物0.0024mg/l、亜セレン酸五水和物0.0016mg/l}、および人工海水{製品名:マリンアート・SF-1(千寿製薬株式会社製)}37g/lを蒸留水に上記所定の濃度で溶解した液体培地を、IMK培地として作製した。
実施例2に前述したとおり、JPCC DA0580株の乾燥藻体当たりのヘキサン抽出物(油分)の含量は55質量%以上を占めるまでに達しうるので、JPCC DA0580株の1週間当たりの油分生産性は、培養スケール1L当たりで0.26g以上に達することが確認された。
実施例2で使用したf/2培地の組成のうち、人工海水{製品名:マリンアート・SF-1(千寿製薬株式会社製)}の濃度を変化させた栄養液体培地を5種類作製した。当該人口海水の濃度は、0g/lで添加した場合を0%とし、以下同様に、11.1g/l(30%)、18.5g/l(50%)、29.8g/l(80%)、37g/l(100%)とした。
これらの培地500mlを入れた500ml容の偏平フラスコにJPCC DA0580株を接種して、実施例2と同様の培養条件で、1週間の静置培養を行った。生育を評価するために培養液の濁度(OD750)を経時的に測定した。その結果を図3に示す。
このことから、JPCC DA0580株は、海洋性の微細藻類であることが確認された。
実施例2と同様の培養方法でJPCC DA0580株を、1週間培養した。その後、培養液から遠心で回収した藻体ペレットを、凍結乾燥した。
得られた乾燥藻体を試料として、ボンベ型熱量計(株式会社吉田製作所製、型式:1013-J、熱量の計測範囲:4000~33500J)で発熱量を測定したところ、6730kcal/kgであった。すなわち、JPCC DA0580株の乾燥藻体は、石炭と同等以上の発熱量をもつことが確認された。
また、得られた乾燥藻体を試料として、実施例2と同様の方法で得られたヘキサン抽出液を窒素気流によって乾燥させ、ヘキサン抽出物(油分)として得た。これを試料として、前記ボンベ型熱量計で発熱量を測定したところ、該ヘキサン抽出物(油分)の発熱量は、8780kcal/kgであった。
実施例2と同様の方法でJPCC DA0580株を、1週間培養した。その後、培養液から遠心によって藻体ペレットを回収した。
未乾燥状態の前記藻体ペレット(乾燥時の質量は0.1g)に対して、ヘキサン/メタノールからなる2相溶媒(混合比(体積比)はヘキサン:メタノール=10:1)を6ml加えて懸濁し、超音波ホモジナイザーを用いて室温で藻体を破砕しながら30分間の抽出を行った。つぎに、その抽出液を遠心し、藻体の残渣を沈殿させ、上清の抽出溶液を約6ml回収した。沈殿として残った藻体の残渣に対して、再び新しい前記2相溶媒6mlを加えて懸濁し、同じ抽出操作をさらに2回(合計3回の抽出操作)を行った。得られた抽出液(合計18ml)を窒素気流によって乾燥させ、実施例2と同様の方法でGCMSによって定量した。
その結果、前記2相溶媒によって抽出された油分は、JPCC DA0580株の藻体の乾燥時質量のうち、51.8質量%を占めることが確認された。この結果を表1に併記する。
その結果、前記へキサンからなる1相溶媒によって抽出された油分は、JPCC DA0580株の藻体の乾燥時質量のうち、46.2質量%を占めることが確認された。この結果を表1に併記する。
その結果、前記へキサンからなる1相溶媒によって抽出された油分は、JPCC DA0580株の藻体の乾燥時質量のうち、45.3質量%を占めることが確認された。この結果を表1に併記する。
実施例2と同様の方法でJPCC DA0580株を、1週間培養した。その後、培養液から遠心によって藻体ペレットを回収した。
未乾燥状態の前記藻体ペレット(乾燥時の質量は0.1g)に対して、ヘキサン/エタノールからなる2相溶媒(混合比(体積比)はヘキサン:エタノール=10:1)を6ml加えて懸濁し、超音波ホモジナイザーを用いて室温で藻体を破砕しながら30分間の抽出を行った。つぎに、その抽出液を遠心し、藻体の残渣を沈殿させ、上清の抽出溶液を約6ml回収した。得られた抽出液(合計6ml)を窒素気流によって乾燥させ、実施例2と同様の方法でGCMSによって定量した。
その結果、前記2相溶媒によって抽出された油分は、JPCC DA0580株の藻体の乾燥時質量のうち、31.5質量%を占めることが確認された。この結果を表2に併記する。
その結果、前記2相溶媒によって抽出された油分は、JPCC DA0580株の藻体の乾燥時質量のうち、30.7質量%を占めることが確認された。この結果を表2に併記する。
なお、実施例7における油分抽出量よりも、実施例8における油分抽出量が少ない理由は、有機溶媒による抽出回数を3回(実施例7)から1回(実施例8)に減じたためである。
セネデスムス(Scenedesmus rubescens) JPCC GA0024株(受託番号:FERM P-21749)、シアノバクテリア(Synechocystis sp.)、テトラセルミス(Tetraselmis striata)の3種の微細藻類を公知の方法で培養して、それぞれ藻体ペレットを得た。これら3種の微細藻類は、その藻体中に油分を蓄積するものである。該油分は、少なくともトリグリセライド等の中性脂質を含有する。
つぎに、各藻体ペレットの乾燥時質量0.1gを試料として、乾燥状態の藻体ペレットに対して1相溶媒(ヘキサン)を用いて、実施例7と同様の方法で油分を抽出した。
また、各藻体ペレットの乾燥時質量0.1gを試料として、乾燥状態の藻体ペレットに対して2相溶媒《ヘキサン/メタノール(混合比(体積比)10:1)》を用いて、実施例7と同様の方法で油分を抽出した。
得られた各抽出液(合計18ml)を窒素気流によって乾燥させ、実施例2と同様の方法でGCMSによって定量した。その結果を表3に併記する。
一方、本発明にかかる微細藻類の藻体は、未乾燥状態の藻体から効率的に油分を抽出できる非常に優れた性質を持つため、本発明の微細藻類を用いたバイオ燃料等の製造プロセスの低エネルギー化を図ることができる。
なお、該中性脂質は、公知のエステル交換法等によってディーゼル燃料(いわゆるバイオディーゼル燃料)とすることができる。
Claims (12)
- 炭素数16~26の脂肪族炭化水素の産生能を有するナビクラ(Navicula)属に属する微細藻類。
- 炭素数16~26の脂肪族炭化水素の産生能を有する微細藻類ナビクラ(Navicula)属オイリティカス(oiliticus)種。
- 微細藻類ナビクラ(Navicula)属 オイリティカス(oiliticus)種 JPCCDA0580株(FERM BP-11201)。
- 請求項1~3のいずれか一項に記載の微細藻類を培養する工程を有する油分の製造方法。
- 前記培養する工程の後に、当該培養に用いた培地中の栄養塩類の濃度を低減した別の培地で前記微細藻類を更に培養する工程を有する、請求項4に記載の油分の製造方法。
- 前記油分が中性脂質を含む、請求項4又は5に記載の油分の製造方法。
- 前記油分がスクアレンを含む、請求項4~6の何れか一項に記載の油分の製造方法。
- 前記培養する工程の後、培養物から有機溶媒で油分を抽出する工程を有し、当該有機溶媒が、n-へキサンからなる溶媒、n-へキサン及びメタノールからなる溶媒、又はn-へキサン及びエタノールからなる溶媒の何れかである、請求項4~7のいずれか一項に記載の油分の製造方法。
- 請求項4~8のいずれか一項に記載の油分の製造方法によって製造された油分。
- 請求項1~3のいずれか一項に記載の微細藻類を乾燥して得られる乾燥藻体。
- 請求項1~3のいずれか一項に記載の微細藻類から得られる燃料。
- 請求項1~3のいずれか一項に記載の微細藻類を培養する工程を有する二酸化炭素固定方法。
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EP10761319.2A EP2418270B1 (en) | 2009-04-10 | 2010-03-10 | Micro-alga belonging to genus navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga |
JP2011508201A JP5608640B2 (ja) | 2009-04-10 | 2010-03-10 | ナビクラ属に属する微細藻類、該微細藻類の培養による油分の製造方法、該微細藻類の乾燥藻体、および該微細藻類を培養する工程を有する二酸化炭素固定方法 |
US13/263,005 US8927285B2 (en) | 2009-04-10 | 2010-03-10 | Micro-alga belonging to genus Navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga |
CN2010800154211A CN102388126B (zh) | 2009-04-10 | 2010-03-10 | 属于舟形藻属的微藻、通过培养该微藻而制造油分的方法以及从该微藻中所采集的油分 |
AU2010233307A AU2010233307B2 (en) | 2009-04-10 | 2010-03-10 | Micro-alga belonging to genus Navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga |
ZA2011/07726A ZA201107726B (en) | 2009-04-10 | 2011-10-21 | Micro-alga belonging to genus navicula,process for production of oil by culture of the micro-alga,and oil collection from the micro-alga |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102533430A (zh) * | 2010-12-28 | 2012-07-04 | 中国科学院大连化学物理研究所 | 微藻油脂的提取方法 |
JP2015015918A (ja) * | 2013-07-10 | 2015-01-29 | 株式会社デンソー | 新規微細藻類 |
WO2015041351A1 (ja) * | 2013-09-20 | 2015-03-26 | 富士フイルム株式会社 | オイル含有率を向上させた微細藻類の培養方法、藻類バイオマスの製造方法、及び新規微細藻類 |
JP2016034925A (ja) * | 2014-08-04 | 2016-03-17 | 国立大学法人東京農工大学 | 新規珪藻タンパク質及びその利用 |
JP2020074725A (ja) * | 2018-11-09 | 2020-05-21 | 国立大学法人東京農工大学 | 珪藻類の回収方法及び回収装置 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6108331B2 (ja) * | 2012-05-08 | 2017-04-05 | 国立大学法人 東京大学 | 炭化水素高収率性藻体とその製造方法 |
NL2011472C2 (en) | 2013-09-19 | 2015-03-23 | Univ Delft Tech | Storage compound production by phototrophic diatoms. |
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05505726A (ja) * | 1990-03-21 | 1993-08-26 | マーテック・コーポレイション | エイコサペンタエン酸とその製法 |
JPH09234055A (ja) | 1996-03-01 | 1997-09-09 | Chikyu Kankyo Sangyo Gijutsu Kenkyu Kiko | ボツリオコッカス属に属する新規な微細藻類及びその培養方法 |
WO2006109588A1 (ja) | 2005-04-12 | 2006-10-19 | Denso Corporation | 新規微細藻類及び炭化水素の生産方法 |
JP2008519129A (ja) * | 2004-11-04 | 2008-06-05 | モンサント テクノロジー エルエルシー | 高pufa油組成物 |
US20080160593A1 (en) * | 2006-12-29 | 2008-07-03 | Oyler James R | Two-stage process for producing oil from microalgae |
WO2008151149A2 (en) * | 2007-06-01 | 2008-12-11 | Solazyme, Inc. | Production of oil in microorganisms |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0965871A (ja) * | 1995-09-04 | 1997-03-11 | Kawasaki Steel Corp | 海洋性微細藻類の培養方法 |
CN100392061C (zh) * | 2004-06-18 | 2008-06-04 | 徐州师范大学 | 舟形藻的一种开放式培养方法及其专用培养基 |
US9637714B2 (en) * | 2006-12-28 | 2017-05-02 | Colorado State University Research Foundation | Diffuse light extended surface area water-supported photobioreactor |
AU2008247252B2 (en) | 2007-05-02 | 2012-08-30 | Ouro Fino Participacoes E Empreendimentos S.A. | Process to produce biodiesel and/or fuel oil |
WO2009039015A2 (en) * | 2007-09-18 | 2009-03-26 | Sapphire Energy, Inc. | Methods for refining hydrocarbon feedstocks |
-
2010
- 2010-03-10 EP EP10761319.2A patent/EP2418270B1/en active Active
- 2010-03-10 ES ES10761319.2T patent/ES2482592T3/es active Active
- 2010-03-10 US US13/263,005 patent/US8927285B2/en active Active
- 2010-03-10 WO PCT/JP2010/001704 patent/WO2010116611A1/ja active Application Filing
- 2010-03-10 AU AU2010233307A patent/AU2010233307B2/en active Active
- 2010-03-10 PT PT107613192T patent/PT2418270E/pt unknown
- 2010-03-10 JP JP2011508201A patent/JP5608640B2/ja active Active
- 2010-03-10 KR KR1020117025551A patent/KR101615409B1/ko active IP Right Grant
- 2010-03-10 CN CN2010800154211A patent/CN102388126B/zh active Active
-
2011
- 2011-10-21 ZA ZA2011/07726A patent/ZA201107726B/en unknown
-
2012
- 2012-08-06 HK HK12107707.5A patent/HK1167160A1/xx unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05505726A (ja) * | 1990-03-21 | 1993-08-26 | マーテック・コーポレイション | エイコサペンタエン酸とその製法 |
JPH09234055A (ja) | 1996-03-01 | 1997-09-09 | Chikyu Kankyo Sangyo Gijutsu Kenkyu Kiko | ボツリオコッカス属に属する新規な微細藻類及びその培養方法 |
JP2008519129A (ja) * | 2004-11-04 | 2008-06-05 | モンサント テクノロジー エルエルシー | 高pufa油組成物 |
WO2006109588A1 (ja) | 2005-04-12 | 2006-10-19 | Denso Corporation | 新規微細藻類及び炭化水素の生産方法 |
US20080160593A1 (en) * | 2006-12-29 | 2008-07-03 | Oyler James R | Two-stage process for producing oil from microalgae |
WO2008151149A2 (en) * | 2007-06-01 | 2008-12-11 | Solazyme, Inc. | Production of oil in microorganisms |
Non-Patent Citations (6)
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102533430A (zh) * | 2010-12-28 | 2012-07-04 | 中国科学院大连化学物理研究所 | 微藻油脂的提取方法 |
CN102533430B (zh) * | 2010-12-28 | 2013-09-18 | 中国科学院大连化学物理研究所 | 微藻油脂的提取方法 |
JP2015015918A (ja) * | 2013-07-10 | 2015-01-29 | 株式会社デンソー | 新規微細藻類 |
WO2015041351A1 (ja) * | 2013-09-20 | 2015-03-26 | 富士フイルム株式会社 | オイル含有率を向上させた微細藻類の培養方法、藻類バイオマスの製造方法、及び新規微細藻類 |
JP2015192649A (ja) * | 2013-09-20 | 2015-11-05 | 富士フイルム株式会社 | オイル含有率を向上させた微細藻類の培養方法、藻類バイオマスの製造方法、及び新規微細藻類 |
JP2016034925A (ja) * | 2014-08-04 | 2016-03-17 | 国立大学法人東京農工大学 | 新規珪藻タンパク質及びその利用 |
JP2020074725A (ja) * | 2018-11-09 | 2020-05-21 | 国立大学法人東京農工大学 | 珪藻類の回収方法及び回収装置 |
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