HK1167160B - Micro-alga belonging to genus navicula, process for production of oil by culture of the micro-alga, and oil collected from the micro-alga - Google Patents

Description

Microalgae belonging to genus navicula, method for producing oil by culturing the microalgae, and oil collected from the microalgae

Technical Field

The present invention relates to a microalgae belonging to genus Navicula (Navicula), a method for producing an oil fraction including a step of culturing the microalgae, and an oil fraction collected from the microalgae. In particular, the present invention relates to: microalgae belonging to genus Navicula (Navicula) having an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms; an oil production method comprising a step of culturing the microalgae; oil collected from the microalgae; drying the microalgae to obtain dried microalgae; a fuel derived from the microalgae; and a carbon dioxide fixation method comprising the step of culturing the microalgae.

Background

Several methods have been reported for producing heavy oil-type or light oil-type hydrocarbons called "biofuels" by culturing microalgae.

As algae having an ability to produce heavy oil-based hydrocarbons, there are known microalgae Botryococcus braunii (see non-patent document 1) having an ability to produce hydrocarbons having a carbon number of 36 and microalgae Botryococcus braunii a race (see patent document 1) having an ability to produce hydrocarbons having a carbon number of 33.

Further, as algae having an ability to produce light oil hydrocarbons, there are known microalgae candida bryantii (Nostoc muscorum), trichodinum rubrum (Trichodesmium erythraeum), synechocystis sanguinea (plectomes terebans) and the like having an ability to produce hydrocarbons having 17 carbon atoms (see non-patent document 1), microalgae cocochloris elabes and the like having an ability to produce hydrocarbons having 19 carbon atoms (see non-patent document 1), microalgae pseudocytocystis albumsoidea ic11204 strain having an ability to produce hydrocarbons having 17 carbon atoms, 18 carbon atoms, 19 carbon atoms and 20 carbon atoms (see patent document 2), and microalgae chlorocystis minoxidi 17.98 strain having an ability to produce hydrocarbons having 17 carbon atoms, 19 carbon atoms, 21 carbon atoms and 23 carbon atoms (see patent document 2).

Light oil hydrocarbons that microalgae can produce are industrially useful as diesel fuel, and are expected as carbon-neutral (carbon-neutral) fuels intended to prevent global warming.

However, the content of light oil hydrocarbons in the dried algal bodies obtained by drying the microalgae is usually about 0.025 to 0.12% by mass (see non-patent document 1), and the ability to produce hydrocarbons is not necessarily sufficient.

Documents of the prior art

Patent document 1: japanese patent laid-open No. 9-234055

Patent document 2: international publication No. 2006/109588 pamphlet

Non-patent document 1: r.raja, s.heawshira, n.ashok Kumar, s.sridhar and r.rengnamy (2008) a selective on the biotechnology patent of micro-algae.critical Reviews in Microbiology, 34: 77-88.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above circumstances. The present invention addresses the problem of providing a microalgae having a high production capacity for aliphatic hydrocarbons having 16 to 26 carbon atoms, a method for producing an oil component comprising a step of culturing the microalgae, an oil component collected from the microalgae, a dried algal body obtained by drying the microalgae, a fuel obtained from the microalgae, and a method for fixing carbon dioxide comprising a step of culturing the microalgae.

Means for solving the problems

In order to solve the above problem, the present invention adopts the following aspects.

(1) A microalgae belonging to genus Navicula (Navicula) has an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.

(2) A microalgae genus oiliticus species of navicula having an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms.

(3) The microalgae Navicula genus Alliticus species JPCCDA0580 strain (FERM BP-11201).

(4) An oil production method comprising the step of culturing the microalgae according to any one of (1) to (3).

(5) The method for producing an oil according to item (4) above, which comprises, after the culturing step, further culturing the microalgae using a medium having a lower nutrient salt concentration than the medium used in the culturing step.

(6) The method for producing an oil component according to (4) or (5), wherein the oil component contains a neutral lipid.

(7) The method for producing an oil component according to any one of (4) to (6), wherein the oil component contains squalene.

(8) The oil production method according to any one of (4) to (7), which comprises a step of extracting the oil with an organic solvent selected from a solvent consisting of n-hexane, a solvent consisting of n-hexane and methanol, or a solvent consisting of n-hexane and ethanol after the culturing step.

(9) An oil component produced by the oil component production method according to any one of (4) to (8).

(10) And (3) drying the microalgae in any one of (1) to (3) to obtain a dried algal body.

(11) A fuel obtained from the microalgae according to any one of (1) to (3).

(12) A carbon dioxide fixation method comprising a step of culturing the microalgae according to any one of (1) to (3).

Further, in the claims and the specification of the present application, "having an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms" means having an ability to produce mainly aliphatic hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms. The "oil component" refers to a liquid component mainly composed of a hydrophobic organic compound. Examples of the hydrophobic organic compound include aliphatic hydrocarbons and neutral fats.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, there can be provided a microalgae having an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms, an oil production method having a culture step of the microalgae, an oil fraction collected from the microalgae, a dried algal body obtained by drying the microalgae, a fuel obtained from the microalgae, and a carbon dioxide fixation method having a culture step of the microalgae.

Drawings

FIG. 1 is a diagram of a molecular phylogenetic tree obtained using the 18S rDNA base sequence of JPCC DA0580 strain.

FIG. 2 is a growth curve of strain JPCC DA 0580.

FIG. 3 is a growth curve of JPCC DA0580 strain cultured while changing the concentration of seawater components.

FIG. 4 is an electron micrograph of a strain of Navicula (Navicula) oiticiticus JPCC DA0580 (FERM BP-11201) (10000 times magnification on the left and 15000 times magnification on the right).

FIG. 5 is a fluorescence micrograph of a strain JPCCDA0580 (FERM BP-11201) of Navicula (Navicula) oiiticus (Navicula) stained with Nile red (Nile red) (the region showing bright fluorescence emission is oil and fat inside the algal body emitting color by Nile red, and the slightly dark region is photosynthetic pigment emitting color by autofluorescence, etc.).

Detailed Description

The present invention will be described in detail below.

(microalgae belonging to genus Navicula)

The microalgae belonging to the genus Navicula (Navicula) in the present invention has an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms. Here, the aliphatic hydrocarbon having 16 to 26 carbon atoms mainly means aliphatic hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms.

As the microalgae, in particular, from the viewpoint of high content of aliphatic hydrocarbons having 16 to 26 carbon atoms in the algal body and easy cultivation, the microalgae is preferably a species of Navicula (Navicula) oiticiticus, and more preferably a strain JPCC DA0580 (FERM BP-11201) (hereinafter, abbreviated as JPCC DA0580 strain) of Navicula (Navicula) oiticicus.

The JPCC DA0580 strain is a further preferable microalga in the present invention from the viewpoint of having a high ability to produce neutral lipids and squalene, in addition to having an ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms. The description of neutral lipids is as follows.

The JPCC DA0580 strain was isolated from seawater in the brackish water (blackish water) field by the inventors, and is a novel strain belonging to a novel species of marine microalgae belonging to the family naviridae of the order canaliculus of the phylum chrysophyceae (chrysophhyta) order, lagomorpha.

Hereinafter, a method for isolating the microalgae and a process for judging the JPCC DA0580 strain of the algae as a new strain of a new species will be described.

(method for preparing culture Medium)

The medium was prepared as f/2 as follows. 75.0mg/l of sodium nitrate, 5.0mg/l of disodium hydrogenphosphate, 30.0mg/l of sodium metasilicate nonahydrate, 1.0ml of an f/2 trace metal solution, 0.5ml of an f/2 vitamin solution, and 37g/l of artificial seawater (artificial sea water) (product name: MARINE ART SF-1, manufactured by Kakkiso K.K.) were dissolved in distilled water at the predetermined concentration to obtain a liquid medium.

The f/2 trace metal solution is Na dihydrate₂4.36g/l of EDTA, 3.15g/l of ferric trichloride hexahydrate, 180mg/l of manganese (II) chloride tetrahydrate, 22.0mg/l of zinc sulfate heptahydrate, 10.0mg/l of cobalt (II) chloride hexahydrate, 6.3mg/l of sodium molybdenum (VI) dihydrate and 9.8mg/l of copper (II) sulfate pentahydrate.

The f/2 vitamin solution is an aqueous solution prepared from 200.0mg/l of thiamine, 0.1mg/l of biotin and 1.0mg/l of vitamin (B12).

In addition to the above-mentioned components of the f/2 medium, agar was further added so that the concentration thereof became 1.2% (w/v), to prepare an agar f/2 medium.

In addition, the artificial seawater is a salt simulating a salt contained in natural seawater. An aqueous solution of a seawater component that simulates natural seawater can be prepared by dissolving a predetermined amount (37g/l) of the artificial seawater in distilled water.

(separation method)

To a 24-well Microtiter plate (microtiterplate) containing 2ml of the f/2 medium, a suitable amount of a sediment sample was added, which was taken from Mangrove (Mangrove) forest located at the junction of the living and the living victory, expressage, japan deer island county, 7 months in 2005. Subsequently, the culture solution was subjected to static culture under irradiation with 1000 lux (lx) light, and a part of the culture solution was taken out from the wells where the growth of microalgae was confirmed. Inoculating the culture solution to the f/2 agar culture medium, and culturing under the illumination condition to obtain unicellular (isolated) microalgae colony (colony) of brown unicellular algae JPCC DA 0580.

(morphological Properties)

The culture was carried out on the agar medium at 25 ℃ for 7 days to obtain brown colonies of JPCC DA0580 having a diameter of about 2.0mm to 5.0 mm. The shape of the colony is punctiform and semi-lenticular without bulge. The outer edge is smooth, and the surface is in a smooth shape. In addition, no colony morphology change due to the mutation was observed, and no colony morphology change due to the culture conditions and physiological state was observed.

The marine microalgae in the colony is unicellular algae with the average size of about 10-20 μm, sometimes forms a colony, and vegetative cells do not have eyespots and shrinkage bubbles and are rhombic. Has no floating property, has a vitreous shell, has a longitudinal groove on the shell (putamen), and has a smooth cell surface. The vegetative cells do not have flagella and do not exhibit motility. An electron microscope image of JPCC DA0580 strain is shown in fig. 4.

(propagation means)

The JPCC DA0580 strain is propagated in two ways of sexual propagation and asexual propagation. Asexual propagation is asexual propagation by binary division. Cell division takes place inside the shell, and the two daughter cells form new half-shells inside the outer and inner shells of the mother cell, respectively. The half-shells of the mother cells are each assigned one by one to the daughter cells. As a result, one of the daughter cells has the same size as the mother cell (has a shell of the same size), and the other daughter cell uses the inner shell as the outer shell. As a result, the size of the cells is smaller than that of the mother cells. Cells that have become small to some extent undergo sexual reproduction. In this cell, homogametes (isogametes) are formed by meiosis. The zygotes approach each other through the intermediary of the shell, and the protoplasts are transformed into amoeba shapes and then combined with each other to form complex micelles (auxospore), which grow and increase in volume to respond to normal cells of a certain size.

(physiological and biochemical Properties)

Culture solution: the culture medium is grown in a culture medium with seawater as a basic composition. Cannot grow in fresh water.

Photosynthesis: photoautotrophic growth can be performed by photosynthesis. Heterotrophic growth was not confirmed.

The pigment contained: chlorophyll a, c1, c2, fucoxanthin and carotenoid pigments centered on fucoxanthin derivatives

Storage material: starch

Growth temperature: 20 ℃ to 35 ℃ (most suitable temperature is 25 ℃)

Growth pH: 7.0 to 9.0 (most preferably pH8.0)

A large amount of oil accumulates in the cell and can be stained with nile red, including aliphatic hydrocarbons with 16 to 26 carbon atoms, neutral lipids, and squalene.

During the growth period: one week (time required for turbidity (OD750) to reach 1.2 from 0.05)

As shown in fig. 5, when JPCC DA0580 strain stained with nile red was observed with a fluorescence microscope, the presence of a bright region that fluoresces was observed in the algal body under the fluorescent field, that is, oil components that developed color by nile red was observed. The oil component can be accumulated in algal cells in the form of oil droplets. Further, the oil component includes an aliphatic hydrocarbon having 16 to 26 carbon atoms, a neutral lipid, and squalene.

From the above morphological properties, propagation pattern, and physiological and biochemical characteristics, the strain JPCCDA0580 is presumed to be an alga belonging to the naviridae family of the subgenus canaliculata of the phylum chrysophyceae, class lupidophyceae of the phylum chrysophyceae. Then, according to a method known so far, DNA was extracted from this JPCC DA0580 strain using a DNA extraction kit (product name: QIAampDNA Probe Mini kit 50 (manufactured by QIAGEN, Japan Co., Ltd.), the 18S rDNA region was amplified by a PCR method and subjected to sequence analysis, and the base sequence of the 18S rDNA region was determined, the obtained 18S rDNA base sequence was represented in SEQ ID NO. 1 of the sequence Listing, the obtained 18S rDNA base sequence was aligned with a public database, namely, Japanese DNA database (DNA data bank of Japan, DDBJ), and subjected to homology search (Blast search), systematic analysis was performed using analysis software ClustalW and representation software Treeview, and the resulting phylogenetic tree was shown in FIG. 1.

The isolated JPCC DA0580 strain was classified in the phylogenetic tree as naviculaceae (Navicula), forming clusters (cluster) with diatoms of Navicula (Navicula). However, we confirmed that there was a divergence at the gene level between the JPCC DA0580 strain and the known species of Navicula (Navicula), and thus identified the JPCC DA0580 strain isolated by the present inventors as an alga belonging to a new species of Navicula (Navicula). Here, we named this strain of Navicula (Navicula) oiticicus species JPCC DA0580 strain.

JPCC DA0580 strain was deposited at 16.3.2009 with accession number FERM BP-11201 at the Japan national institute of independent Engineers' Integrated Industrial science and technology Collection (center 6, postal code 305-8566, 1, east 1 Diway 1, Bubo, Tokyo, Ipomoea, Ichc, Japan). (handed over from Japanese national deposit number FERM P-21788, deposited on 16.3.2009.)

The JPCC DA0580 strain can accumulate oil components including aliphatic hydrocarbons having 16 to 26 carbon atoms, neutral lipids, and squalene in algal cells. The content of the aliphatic hydrocarbon having 16 to 26 carbon atoms in the dried algal cells of the JPCC DA0580 strain may be 0.2 mass% or more. The content of neutral lipids in the dried algal cells of JPCC DA0580 strain can be 36 mass% or more. The content of squalene in the dried algal cells of the JPCC DA0580 strain can be 0.3% by mass or more.

The oil component may contain, in addition to the aliphatic hydrocarbon having 16 to 26 carbon atoms, neutral lipid, and squalene, an aliphatic hydrocarbon having 27 or more carbon atoms, a phospholipid, a free fatty acid, a steroid compound, and a photosynthetic dye such as fucoxanthin and a carotenoid containing a fucoxanthin derivative.

The aliphatic hydrocarbon having 16 to 26 carbon atoms mainly refers to aliphatic hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms, and specifically mainly refers to linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms.

As described later, the neutral lipid mainly refers to neutral lipids having tetradecanoyl (myristoyl), hexadecanoyl (palmitoyl), hexadecanoyl (hexadecanoyl) group (palmitoleyl), octadecanoyl (oleanoyl) group (oleoyl), and eicosapentaoyl (eicosapentaenoyl) groups as acyl groups, and is mainly a triglyceride (trigycede) having these acyl groups.

The squalene is 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene.

The aliphatic hydrocarbon having 27 or more carbon atoms is an aliphatic hydrocarbon other than squalene.

When the oil of JPCC DA0580 strain is extracted using n-hexane as a solvent, about 80 mass% of the extracted oil is generally neutral lipid such as the triglyceride, and this can be about 36 mass% or more of the dried algal cells of JPCCDA0580 strain. However, these values were obtained when the triglyceride was calculated as the oleic triglyceride.

The amount of the aliphatic hydrocarbon having 16 to 26 carbon atoms and the amount of squalene contained in the extracted oil component may be about 0.2 mass% or more and about 0.3 mass% or more, respectively, of the dried algal cells of the JPCC DA0580 strain.

However, the composition of the extracted oil is generally obtained when the JPCC DA0580 strain is cultured by the aeration culture method described later using the f/2 medium described above, and when the culture method of the microalgae is changed, the composition of the extracted oil may change.

The fatty acids constituting the neutral lipids such as triglycerides are mainly myristic acid (carbon number 14, double bond number 0), palmitic acid { palmitic acid (carbon number 16, double bond number 0), hexadecenoic acid { palmitoleic acid (carbon number 16, double bond number 1) }, octadecenoic acid { oleic acid (carbon number 18, double bond number 1) }, and eicosapentaenoic acid { EPA (carbon number 20, double bond number 5) }. the content of the fatty acids constituting the neutral lipids such as triglycerides in the unit dried algal cell of JPCC DA0580 strain is, in this order, about 1.0 mass%, about 12.7 mass%, about 17.0 mass%, about 0.8 mass%, and about 1.4 mass%, that is, the content of the fatty acids constituting the neutral lipids such as triglycerides is about 32.9 mass% of the dried algal cell.

The content of fatty acids constituting neutral lipids such as triglycerides in the dried algal cells is a ratio of the mass of fatty acid methyl esters in the dried algal cells when the fatty acids are methyl-esterified.

(method for producing oil component)

The oil production method of the present invention includes the step of culturing the microalgae belonging to genus navicula according to the present invention. The oil component can be produced by culturing the microalgae according to the present invention, recovering the microalgae grown in the culturing step from the culture medium, and collecting the oil component contained in the obtained microalgae.

The microalgae belonging to the genus navicula is preferably a microalgae belonging to the genus oiliticus species, more preferably strain JPCC DA0580, from the viewpoint that the content of aliphatic hydrocarbons having 16 to 26 carbon atoms in the algal body is high and the algal body can be easily cultured.

In addition to the high production capacity of the strain JPCC DA0580 for said aliphatic hydrocarbons having 16 to 26 carbon atoms, the strain JPCC DA0580 is also more preferred as a microalgae according to the invention from the viewpoint of having a high production capacity for neutral lipids and squalene as well.

As the step of culturing the microalgae, a known method capable of culturing microalgae belonging to the genus navicula can be applied. For example, the microalgae may be inoculated into a culture vessel such as a flat flask containing a liquid culture medium, and aerated culture may be performed under light irradiation while slowly stirring the microalgae to such an extent that the microalgae 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. For example, the f/2 medium used for isolating the JPCC DA0580 strain may be a preferable medium from the viewpoint that algae can grow well.

The concentration of artificial seawater (product name: MARINE ART-SF-1 (manufactured by Kakkiso Kagaku K.K.) in the liquid medium is preferably 30 to 100% (w/v), more preferably 50 to 100% (w/v), still more preferably 70 to 100% (w/v), particularly preferably 90 to 100(w/v), most preferably 95 to 100% (w/v) when 37g/l is added as 100% (w/v). Here, a predetermined amount (37g/l) of the artificial seawater is dissolved in an aqueous solution in distilled water, and it can be considered that the salt concentration thereof is substantially equal to that of natural seawater.

The conditions of the light irradiation may be appropriately adjusted according to the concentration of the algal cells in the culture medium, and for example, the concentration is preferably 500 lux or more (lx), more preferably 1000 to 30000 lux (lx), still more preferably 1000 to 10000 lux (lx), particularly preferably 1000 to 6000 lux (lx), and most preferably 1000 to 3000 lux (lx).

As the gas for aeration used in the aeration culture, a known gas for aeration suitable for the growth of the microalgae can be used, and for example, ordinary air or CO-added gas can be used₂Air, etc. Among these, it is preferable to use a microalgae which is added with CO in order to grow the microalgae better and shorten the growth period₂Of the air of (2).

In the presence of CO₂CO in the air of₂The concentration is preferably 0.05 to 10% (v/v), more preferably 0.05 to 5.0% (v/v), and most preferably 1.0 to 3.0% (v/v).

As the aeration rate in the aeration culture, a known aeration rate suitable for the growth of the algae can be applied, and for example, it is preferably 0.5 to 5vvm, more preferably 0.5 to 3.0vvm, and still more preferably 0.5 to 2.0 vvm.

The culture temperature in the aeration culture may be a known culture temperature suitable for the growth of the algae, and is usually preferably 20 to 35 ℃ and more preferably 25 to 30 ℃.

The length of the aeration culture period is preferably 1 to 4 weeks, more preferably 1 to 3 weeks, and still more preferably 1 to 2 weeks, as long as the algae can be continuously cultured as long as they grow.

In the oil production method of the present invention, the microalgae may be further cultured under nutrient-limiting conditions after the aeration culture period. By culturing under nutrient-limiting conditions, the content of oil contained in the algal cells (mass% of oil contained in dried algal cells) can be increased. Therefore, in the aeration culture period, the microalgae can be cultured in a nutrient-free medium (nutrient medium) to grow a desired amount, and then the culture is switched to nutrient-limited conditions, so that the oil content in the algae can be increased, thereby improving the oil production efficiency.

The term "culture under nutrient-limiting conditions" as used herein refers to a culture in a medium containing less nutrient salts such as vitamins than usual (nutrient-limiting medium). For example, when the content of nutrient salts containing vitamins (sodium nitrate, disodium hydrogen phosphate, sodium metasilicate, f/2 trace metal solution, and f/2 vitamin solution) in the f/2 medium (nutrient broth) used for isolation of the JPCC DA05800 strain is defined as 100%, culturing using the f/2 medium (nutrient-limiting broth) having a content of less than 100% corresponds to culturing under nutrient-limiting conditions.

The content of nutrient salts such as vitamins contained in the nutrient-limiting liquid medium is preferably 0 to 60%, more preferably 0 to 30%, even more preferably 0 to 20%, particularly preferably 0 to 10%, most preferably 0%, from the viewpoint of increasing the oil content in the microalgae.

The method for switching from the nutrient medium to the nutrient-restricted medium is not particularly limited, and the nutrient-restricted medium may be switched to the nutrient-restricted medium at one time or gradually. The method of single-pass conversion includes, for example, a method of precipitating algal cells by centrifugation or the like, removing the nutrient broth as a supernatant, and adding a nutrient-limiting broth. In addition, as a method of gradually changing the culture medium, for example, a nutrient-limited liquid culture medium can be prepared by placing a nutrient-limited liquid culture medium containing algae on one side separated by a semipermeable membrane and placing a nutrient-limited liquid culture medium on the other side, and gradually decreasing the concentration of nutrient salts containing vitamins and the like in the nutrient-limited liquid culture medium by the principle of osmotic pressure.

The period of culturing under nutrient limitation is not particularly limited as long as the oil content of the microalgae can be increased within a range that does not impair the effects of the present invention. The period is preferably 3 to 20 days, more preferably 3 to 10 days, and still more preferably 3 to 7 days.

As a method for recovering the cultured microalgae, known methods can be applied, and examples thereof include a method in which the culture solution is centrifuged to precipitate the algal bodies and the algal bodies are recovered as a centrifugal precipitate, and a method in which the nutrient solution is passed through a filter having holes through which the microalgae cannot pass, and the algal bodies remaining on the filter are recovered.

The method for extracting the oil contained in the algal cells obtained by the above-mentioned culture method and recovery method is not particularly limited as long as the effect of the present invention is not impaired. For example, the recovered algal cells may be suspended (suspension) in an organic solvent to extract oil contained in the algal cells into the organic solvent.

The organic solvent is not particularly limited as long as it can dissolve the oil contained in the algal cells without impairing the effects of the present invention. Examples thereof include n-hexane (hereinafter referred to as "hexane"), acetone, acetonitrile, methanol, ethanol, butanol, chloroform, and the like. Among these, hexane is preferable from the viewpoint of the extraction efficiency of the oil component. These organic solvents may be used alone or in combination of two or more.

In order to improve the efficiency of extraction of the oil component, it is preferable that the algal cells are physically disrupted by treating the organic solvent in which the algal cells are suspended with an ultrasonic homogenizer (ultrasonic homogenizer) or the like.

The hexane mentioned above as a preferred solvent for extracting the oil component from the algal cells of the microalgae may be used as a single-phase solvent or as a two-phase solvent mixed with methanol and/or ethanol.

When the solvent is mixed with methanol and used as a two-phase solvent, the solvent is preferably mixed at a ratio of hexane/methanol of 10: 0 to 5: 5 (volume ratio), more preferably at a ratio of hexane/methanol of 10: 0.5 to 8: 2, and still more preferably at a ratio of hexane/methanol of 10: 1 to 9: 1.

When the mixture is mixed with ethanol and used as a two-phase solvent, the mixture is preferably mixed at a ratio of 10: 0 to 5: 5 (volume ratio) of hexane to ethanol, more preferably at a ratio of 10: 0.5 to 8: 2 of hexane to ethanol, and still more preferably at a ratio of 10: 1 to 9: 1 of hexane to ethanol.

When ethanol and methanol are mixed into hexane to form a two-phase solvent, ethanol and methanol may be mixed at an arbitrary ratio (volume ratio) and used as a single-phase solvent. In this case, the mixture is preferably in a ratio of hexane to methanol to ethanol of 10: 0 to 5: 5 (volume ratio), more preferably in a ratio of hexane to methanol to ethanol of 10: 0.5 to 8: 2, and even more preferably in a ratio of hexane to methanol to ethanol of 10: 1 to 9: 1.

In addition, from the viewpoint of improving the safety of the solvent and the amount of oil extracted, ethanol is more preferably used than methanol.

When the oil is extracted from the algal cells with an organic solvent, the oil may be extracted with an organic solvent after drying the algal cells, or the oil may be extracted with an organic solvent in an undried state in which the algal cells contain water.

When the oil is extracted from the microalgae of the present invention with an organic solvent, the oil extraction amount can be increased when the oil is extracted from the undried microalgae with an organic solvent without drying the microalgae beforehand, which is preferable.

When oil is extracted from algae of known microalgae with an organic solvent, the algae are dried in advance, and the oil is extracted from the dried algae with the organic solvent, and generally the oil extraction amount can be increased. However, previously drying the algal cells is disadvantageous in terms of time and energy consumption required for the drying process. In this regard, the microalgae according to the present invention can extract oil from the algal cells in an undried state with high efficiency, and thus is very excellent in properties. This advantage can reduce the energy consumption in the production process of biofuels and the like using the microalgae of the present invention.

When an organic solvent is used to extract oil from the undried algal cells, the amount of extraction can be increased by using a two-phase solvent composed of hexane, methanol and/or ethanol. This two-phase solvent is particularly preferably a two-phase solvent mixed at a ratio (volume ratio) of hexane to methanol of 10: 1 or a two-phase solvent mixed at a ratio (volume ratio) of hexane to ethanol of 10: 1.

When oil is extracted from the dried algal cells with an organic solvent, the extraction efficiency is higher by using a single-phase solvent composed of hexane.

The organic solvent has a lower boiling point than the oil component, and therefore can be removed by blowing a nitrogen gas stream, distillation under reduced pressure, or the like. In addition, the resin can be recycled.

Further, the oil extracted from the organic solvent may be further purified as necessary. The purification method may be a known method, and examples thereof include a method of fractionating each component contained in the oil component by a solid phase extraction method using silica gel, a liquid chromatography method, a distillation method, and the like.

The oil produced by the oil production method of the present invention includes oil observed when the microalgae are stained with nile red. The oil component contains an aliphatic hydrocarbon having 16 to 26 carbon atoms. The aliphatic hydrocarbon having 16 to 26 carbon atoms is mainly an aliphatic hydrocarbon having 16, 18, 20, 22, 24, and 26 carbon atoms.

The oil may contain, in addition to the aliphatic hydrocarbon having 16 to 26 carbon atoms, a neutral lipid, squalene, an aliphatic hydrocarbon having 27 or more carbon atoms, a phospholipid, a free fatty acid, a steroid compound, a photosynthetic dye such as a carotenoid, and the like.

In the method for producing an oil of the present invention, when JPCC DA0580 strain is used as the microalgae, the description of the oil produced therefrom is the same as the description of the oil that can be accumulated in the algal cells of the JPCC DA0580 strain.

(oil component)

The oil component of the present invention is produced by the oil component production method of the present invention.

The description of the oil component is the same as that of the oil component in the above-described oil component production method.

Further, an aliphatic hydrocarbon having 16 to 26 carbon atoms may be purified from the oil component. When the oil component contains a neutral lipid and/or squalene, the neutral lipid and/or squalene can be purified from the oil component.

The purification method is not particularly limited, and a known method can be used. For example, the oil extracted from the microalgae with hexane may be dissolved in an organic solvent such as hexane, and silica gel may be added to the solvent to adsorb substances other than aliphatic hydrocarbons by silica gel and elute only the aliphatic hydrocarbons. The neutral lipid adsorbed on silica gel may be purified by eluting with an organic solvent or the like. The eluted aliphatic hydrocarbon may be purified by a known purification method such as distillation or chromatography to separate the aliphatic hydrocarbon having 16 to 26 carbon atoms from squalene.

The organic solvent that dissolves the oil component may be the same as the organic solvent mentioned in the description of the organic solvent in the above-described oil component production method.

(dried algal bodies)

The dried algal cell of the present invention is obtained by drying the microalgae belonging to genus navicula of the present invention.

The microalgae can be exemplified by the same ones as those exemplified in the description of the above-mentioned microalgae belonging to the genus navicula of the present invention.

The method for drying the microalgae is not particularly limited as long as it can remove water in the algal cells. For example, a method of sun-drying the algal cells, a method of blowing dry air to the algal cells, a method of freeze-drying (freeze-drying) the algal cells, and the like can be cited. Among these, from the viewpoint of being able to suppress decomposition of components contained in algal cells, a drying method by freeze drying is preferably used.

(Fuel)

The fuel according to the present invention is obtained from the above-mentioned microalgae belonging to genus navicula of the present invention.

The microalgae can be the same ones as those described in the description of the above-mentioned microalgae belonging to the genus navicula of the present invention.

Examples of the method for using the microalgae as a fuel include a method of burning the microalgae, a method of burning an oil collected from the microalgae, and a method of burning an aliphatic hydrocarbon having 16 to 26 carbon atoms, a neutral lipid and/or squalene purified from the oil collected from the microalgae.

When burning the microalgae, it is preferable to use dried algal bodies obtained by drying the microalgae from the viewpoint of improving the combustion efficiency. The dried algal cells are the same as those described above in relation to the present invention. When the microalgae is JPCC DA0580, the heat of the dried algae can reach the same degree or more than the heat of coal (about 6000 kcal/kg).

When the oil extracted from the microalgae is burned, the oil is the same as the oil of the present invention. The oil extracted from the microalgae is combustible, and can be used as fuel for boilers and the like. When the algae is JPCC DA0580 strain, the amount of heat possessed by the hexane extract (oil component) may reach more than 8700 kcal/kg.

When aliphatic hydrocarbons, neutral lipids and/or squalene having 16 to 26 carbon atoms, which are purified from an oil component extracted from the microalgae, are burned, the aliphatic hydrocarbons, neutral lipids and squalene having 16 to 26 carbon atoms are the same as those of the aliphatic hydrocarbons, neutral lipids and squalene having 16 to 26 carbon atoms in the oil component described earlier in the present invention. The aliphatic hydrocarbon having 16 to 26 carbon atoms is suitably used as a fuel for a diesel engine. The neutral lipid can be converted into a diesel fuel (i.e., biodiesel fuel) by a known transesterification method or the like.

(method of fixing carbon dioxide)

The method for fixing carbon dioxide of the present invention includes a culturing step of culturing the microalgae belonging to genus navicula of the present invention.

Photosynthesis performed during the growth of the microalgae has the effect of assimilating carbon dioxide in the culture medium (in the atmosphere). That is, carbon dioxide can be fixed by culturing the microalgae.

The microalgae can be exemplified by the same microalgae as in the description of the above-mentioned microalgae belonging to the genus navicula of the present invention.

The step of culturing the microalgae is the same as the method of culturing the microalgae in the oil production method of the present invention.

The present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1

(hydrocarbon productivity of JPCC DA0580 Strain)

After culturing JPCC DA0580 strain in a 24-well microtiter plate for 12 weeks, the algal cells were stained with nile red, and it was confirmed that oil was produced and accumulated in the algal cells.

Specifically, JPCCDA0580 strain was inoculated into a 24-well microtiter plate containing 2ml of the f/2 medium, and the plate was subjected to static culture for 12 weeks under irradiation with 1000 lux (lx) light. Subsequently, the culture solution was transferred into a 1.5ml microtube (microtube), and centrifuged at 13000rpm to collect algal bodies as a centrifugal precipitate. To remove the medium contained in the centrifuged pellet, the medium was suspended in 0.5ml of physiological saline, centrifuged at 13000rpm for 5 minutes, and the algal bodies were collected as a centrifuged pellet.

Next, the centrifuged precipitate was resuspended in 450. mu.l of physiological saline, and 50. mu.l of Nile Red solution was added thereto and mixed, followed by incubation at room temperature for 10 minutes. After this, the mixture was centrifuged at 13000rpm for 5 minutes, and the centrifuged precipitate was recovered. In order to wash off the excess nile red solution, the centrifuged precipitate was suspended in 0.5ml of physiological saline, and then centrifuged again to recover the algal bodies as a centrifuged precipitate. The algal cells were suspended in 50. mu.l of physiological saline and observed under a fluorescence microscope. As a result, a region emitting yellow fluorescence indicating a region where oil stained with nile red exists in the algal cells was confirmed. In this fluorescence microscope image, a large amount of oil was accumulated in the form of oil droplets in the algal cells (fig. 5).

The nile red solution is obtained by dissolving 1mg of nile red in 10ml of acetone and diluting the solution 4-fold with physiological saline.

Example 2

(identification of hydrocarbons accumulated in strain JPCC DA 0580)

JPCCDA0580 strain was inoculated into a 500-ml flat flask containing 500ml of the f/2 medium, and subjected to aerobic culture for one week under irradiation with 3000 lux (lx) light and an aeration rate of 1 vvm. Thereafter, the algal bodies recovered by centrifugation were subjected to a centrifugal precipitation freeze-drying treatment overnight.

To 0.1g of the obtained dried algal cells, 6ml of hexane was added and suspended, and the algal cells were extracted for 30 minutes by an ultrasonic homogenizer at room temperature while being pulverized. Next, the hexane extract was centrifuged to precipitate algal cell residues, and about 6ml of the hexane extract was collected as a supernatant. 6ml of fresh hexane was added to the residue of algal cells remaining as precipitates, and the same extraction was carried out twice more (3 extraction operations in total). Next, about 18ml of the recovered hexane extract was passed through a silica gel column of Sep-pak cartridge (6cc/1g) (manufactured by Waters Corporation) to remove neutral lipids, free fatty acids, pigments and the like, thereby obtaining 16ml of an aliphatic hydrocarbon fraction. This was dried with a nitrogen stream and redissolved in 0.5ml of hexane, thereby obtaining a sample. The sample was analyzed by gas chromatography-mass spectrometry (GCMS) to identify the hydrocarbons contained in the sample.

The gas chromatography/mass spectrometry analyzer used was GC2010 manufactured by Shimadzu corporation, Japan, using a column of DB-1 (column length: 30m, column inner diameter: 0.25mm), and the gas chromatography was performed under the measurement conditions (temperature rise: 100 ℃ C. (0 min) to 330 ℃ C. (10 ℃ C./min, hold), sample introduction temperature: 300 ℃ C., sample introduction mode: splitless), carrier gas: He, sample introduction amount: 1.0. mu.l).

The mass analyzer used GCMS-QP5050A manufactured by Shimadzu of Japan and ionized by Electron Impact (EI) method.

The results of GCMS analysis confirmed that strain JPCC DA0580 produced aliphatic hydrocarbons having 16 to 26 carbon atoms. More specifically, it was confirmed that the main components thereof were linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms.

Further, a sample in which 0.1% of gas oil was dissolved in hexane was used as a quantitative sample, and the hydrocarbons produced by the JPCC DA0580 strain were quantified from the area ratio of the hydrocarbons in the GCMS chart, and it was found that the content of the linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms in the dried algal bodies of the JPCCDA0580 strain was about 0.2 mass%. The content is higher than that of algae known so far that can produce light-oil aliphatic hydrocarbons. In addition, as described in table 2 of non-patent document 1, the proportion of light oil-based aliphatic hydrocarbons in the dry algal bodies of the known algae is 0.025 to 0.12% by mass.

And, the peak with the largest area in the GCMS plot was identified as squalene. The squalene content per dry algal cell of the JPCC DA0580 strain was about 0.3 mass%, as calculated from the peak area.

A trace amount of the hexane extract before purification using the silica gel column was taken out of about 18ml, and analyzed by the GCMS under the same conditions.

As a result, it was confirmed that the oil content extracted from hexane was 45.5 mass% or more based on the dry algal cell of JPCC DA0580 strain. It was also confirmed that the hexane-extracted oil content of the dried algal cells of the strain JPCC DA0580 obtained from another culture batch (lot) cultured under the same conditions was 58% by mass at the maximum.

Example 3

(identification of fatty acids constituting triglyceride accumulated in JPCC DA0580 Strain)

Algal cells of JPCC DA0580 strain grown by the same method as in example 2 were subjected to centrifugal precipitation and freeze-drying overnight.

About 20ml of a hexane extract was obtained in the same manner as in example 2 except that 7ml of hexane was used for each extraction operation as a sample of 20mg of the dried algal cells. The hexane solvent of the hexane extract was evaporated and removed by a nitrogen stream to obtain 9.1mg of hexane extract (oil component). Therefore, it was confirmed that the ratio of the hexane extract (oil component) was 45.5% by mass of the dry algal cells.

Adding 5% hydrochloric acid-methanol solution into the hexane extract (oil component), and reacting in a sealed test tube at 90 deg.C for 2 hr to methyl-esterify fatty acid of neutral lipid such as triglyceride contained in the hexane extract. Thereafter, the mixture was extracted with chloroform, and a chloroform phase was taken out as a sample.

The sample was analyzed by the following GCMS apparatus, and quantified using methyl oleate as a standard substance.

The gas chromatograph used was a GC6890N (model number) produced by Agilent Technologies International, and the gas chromatograph used was a J & W DB-WAX column (column length: 30m, column inner diameter: 0.25mm, film thickness: 0.25mm), and was carried out under measurement conditions (temperature rise: 50 ℃ C. (0 min) to 250 ℃ C. (10 ℃ C./min, holding time: 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.), and injection amount: 0.6. mu.l).

The mass analyzer used a JMS-GCmate-II model manufactured by Nippon electronics Co., Ltd, which was ionized by an Electron Impact (EI) method.

As a result of GCMS analysis, the fatty acids constituting the neutral lipids such as triglycerides are mainly: myristic acid { myristic acid (C14:0) }, palmitic acid { palmitic acid (C16:0) }, hexadecenoic acid { palmitoleic acid (C16:1) }, octadecenoic acid { oleic acid (C18:1) }, and eicosapentaenoic acid { EPA (C20:5) }. The content of these fatty acids in the dried algal cells of the JPCC DA0580 strain was about 1.0 mass%, about 12.7 mass%, about 17.0 mass%, about 0.8 mass%, and about 1.4 mass%, in this order. That is, the fatty acids constituting the neutral lipids such as triglycerides account for about 32.9% by mass of the dried algal cells.

Assuming that all the fatty acids constituting the neutral lipid such as triglyceride are oleic acid, it was calculated that about 3.8% by mass of the dried algal bodies were glycerin constituting the neutral lipid such as triglyceride, and it was concluded that about 36.7% by mass of the dried algal bodies were the neutral lipid such as triglyceride.

Example 4

(growth Rate of JPCC DA0580 Strain)

The strain JPCC DA0580 was cultured in the same manner as in example 2. Further, the strain JPCC DA0580 was cultured in the same manner as in example 2, except that the liquid medium was changed to the IMK medium described below. In order to evaluate the growth of strain JPCC DA0580 in each medium, the turbidity (OD750) of each culture broth was followed. The results are shown in FIG. 2.

The IMK medium was prepared as follows.

Nutritional salts including vitamins { sodium nitrate 200mg/l, disodium hydrogen phosphate 1.4mg/l, sodium dihydrogen phosphate 5.0mg/l, ammonium chloride 68mg/l, thiamine 0.2mg/l, biotin 0.0015mg/l, vitamin (B12)0.0015mg/l, Na₂EDTA 37.2mg/l, FeEDTA 5.2mg/l, MnEDTA0.3332l, manganese (II) chloride tetrahydrate 0.18mg/l, zinc sulfate heptahydrate 0.024mg/l, cobalt (II) chloride hexahydrate 0.014mg/l, sodium molybdenum (VI) dihydrate 0.0072mg/l, copper (II) sulfate pentahydrate 0.0024mg/l, selenious acid pentahydrate 0.0016mg/l } and artificial seawater (product name: MARINE ART SF-1 (manufactured by Qianshou pharmaceutical Co., Ltd.) 37g/l) were dissolved in distilled water at the above-specified concentrations to prepare a liquid medium as an IMK medium.

From the results obtained, it was confirmed that the strain JPCC DA0580 had an OD750 of 0.05 to 0.9 and had grown in a steady state in the f/2 medium over a one-week period of culture. After the algal cells in a stable state were collected, they were freeze-dried, and the amount of collected dried algal cells was examined, and as a result, the amount of collected algal cells was 0.46g per 1L of the culture medium.

As described in example 2 above, since the hexane extract (oil content) per unit dry algal cell of the JPCC DA0580 strain can be 55 mass% or more, it was confirmed that the oil content productivity per one week of the JPCC DA0580 strain was 0.26g or more per 1L culture scale.

On the other hand, the growth of strain JPCC DA0580 in the IMK medium did not reach a steady state over a 1-week cultivation period.

Example 5

(sea water requirement of JPCC DA0580 Strain)

5 kinds of nutrient liquid media were prepared by changing the concentration of artificial seawater (product name: MARINE ART. SF-1, manufactured by Kakkiso K.K.) in the composition of f/2 medium used in example 2. The concentration of this artificial seawater was 0% at 0g/l addition, and the concentrations were 11.1g/l (30%), 18.5g/l (50%), 29.8g/l (80%), and 37g/l (100%) in the following.

500ml of these media were placed in a flat flask having a volume of 500ml, and JPCCDA0580 strain was inoculated thereto and subjected to static culture for one week under the same culture conditions as in example 2. For the evaluation of growth, the turbidity (OD750) of the culture broth was followed. The results are shown in fig. 3.

From the obtained results, it was confirmed that the strain JPCC DA0580 grew best under the condition of containing 100% artificial seawater (seawater component) in the growth period of one week. It was also confirmed that the growth decreased with the decrease in the concentration of artificial seawater (seawater component), and the growth was not able to be achieved when the concentration of artificial seawater (seawater component) was 0%.

From this, it was confirmed that the strain JPCC DA0580 is a marine microalga.

Example 6

(calorific value of dried algal cells of JPCC DA0580 Strain)

The strain JPCC DA0580 was cultured for one week in the same manner as in example 2. Thereafter, the culture solution was centrifuged, and the collected algal cells were centrifuged and precipitated to be subjected to freeze-drying treatment.

The resulting dried algal cells were used as samples, and the calorific value was measured with a Bomb-type calorimeter (model 1013-J, manufactured by GmbH, Japan, heat measuring range: 4000-33500J) to obtain 6730 kcal/kg. That is, it was confirmed that the dried algal cells of the JPCC DA0580 strain have a calorific value equal to or higher than that of coal.

Further, using the obtained dried algal cells as samples, the hexane extract was dried with a nitrogen gas flow in the same manner as in example 2 to obtain hexane extract (oil component). As a sample, the calorific value of the hexane extract (oil content) was 8780kcal/kg as measured by the aforementioned bomb calorimeter.

Example 7

(examination of organic solvent for extraction of oil from JPCC DA0580 algal cells 1)

The strain JPCC DA0580 was cultivated in the same manner as in example 2 for one week. Thereafter, the culture solution was centrifuged to collect algal bodies and the algal bodies were centrifuged and precipitated.

The undried algal cells were subjected to centrifugal precipitation (mass at the time of drying was 0.1g), 6ml of a two-phase solvent (mixing ratio (volume ratio) of hexane to methanol was 10: 1) composed of hexane/methanol was added to suspend the algal cells, and the algal cells were extracted for 30 minutes while being pulverized by an ultrasonic homogenizer at room temperature. Then, the extract was centrifuged to precipitate algal residue, and about 6ml of the supernatant extract was recovered. 6ml of the biphasic solvent was added again to the algal cell residue remaining as a precipitate to suspend the same, and the same extraction was performed twice (3 extraction operations in total). The resulting extract (total 18ml) was dried with a nitrogen gas flow, and quantitatively analyzed by GCMS in the same manner as in example 2.

As a result, it was confirmed that the oil content extracted with the two-phase solvent was 51.8 mass% based on the dried algal cells of JPCC DA0580 strain. The results are also written in table 1.

Then, 0.1g of dried algal cells obtained by freeze-drying overnight were precipitated by centrifugation from algal cells recovered by centrifugation in the culture solution, and oil was extracted by the same method using a single-phase solvent composed of hexane instead of the two-phase solvent. The resulting extract (total 18ml) was dried with a nitrogen stream and quantitatively analyzed by GCMS in the same manner as in example 2.

As a result, it was confirmed that the oil extracted with the single-phase solvent composed of hexane accounted for 46.2 mass% of the dried algal cells of JPCCDA0580 strain. The results are also written in table 1.

Furthermore, 0.1g of dried algal cells obtained by freeze-drying overnight by centrifugation and precipitation of algal cells recovered from the culture solution by centrifugation were extracted with the same method using the two-phase solvent (hexane: methanol (mixing ratio (volume ratio) 10: 1), and oil was extracted, and the obtained extract (total 18ml) was dried by nitrogen gas flow and quantitatively analyzed by GCMS in the same manner as in example 2.

As a result, it was confirmed that the oil extracted with the single-phase solvent composed of hexane accounted for 45.3 mass% of the mass of the dried algal cells of JPCCDA0580 strain. The results are collectively registered in table 1.

(Table 1)

As described above, when the oil was extracted from undried algal cells of JPCC DA0580 strain with an organic solvent, a two-phase solvent consisting of hexane and methanol was used, and a high extraction efficiency was obtained. As can be seen from comparison of the results, when oil was extracted from JPCC DA0580 strain, it was preferable to perform extraction using a two-phase solvent composed of hexane/methanol for centrifugal precipitation of undried algal cells.

Example 8

(examination of organic solvent used for extraction of oil from algal cells of JPCC DA0580 Strain: 2)

The strain JPCC DA0580 was cultivated for one week using the same method as in example 2. Thereafter, the algal cells were recovered from the culture by centrifugation and precipitated by centrifugation.

The algal cells in an undried state were subjected to centrifugal precipitation (mass at the time of drying was 0.1g), 6ml of a two-phase solvent (mixing ratio (volume ratio) hexane: ethanol: 10: 1) composed of hexane/ethanol was added to suspend the algal cells, and the algal cells were extracted for 30 minutes by an ultrasonic homogenizer at room temperature while being pulverized. Then, the extract was centrifuged to precipitate algal residue, and 6ml of the supernatant extract was collected. The obtained extract (total 6ml) was dried by a nitrogen gas flow and quantified by GCMS in the same manner as in example 2.

As a result, it was confirmed that the oil extracted with the two-phase solvent accounted for 31.5 mass% of the dried algal cells of JPCC DA0580 strain. The results are also shown in Table 2.

Next, the oil content in the undried algal cells precipitated by centrifugation (mass at drying: 0.1g) was extracted by using a two-phase solvent composed of hexane/methanol (mixing ratio (volume ratio) hexane: methanol: 10: 1) in place of the two-phase solvent composed of hexane/ethanol, and the amount was determined by GCMS.

As a result, it was confirmed that the oil component extracted from the two-phase solvent was 30.7 mass% of the dried algal cells of JPCC DA0580 strain. The results are also written in table 2.

Further, the reason why the extraction amount is small in example 8 compared with the oil extraction amount in example 7 is because the number of times of organic solvent extraction is reduced from 3 times (example 7) to 1 time (example 8).

(Table 2)

Extraction efficiency in extracting oil from algal bodies classified into microalgae of other genera

Three kinds of microalgae, Scenedesmus rubescens JPCC GA0024 strain (accession number: FERM P-21749), Synechocystis sp and Tetraselmis strain, were cultured by a known method to obtain algal bodies, which were then centrifuged and precipitated. These three kinds of algae are algae that accumulate oil in their algal cells. The oil component contains neutral lipid such as triglyceride.

An oil fraction was extracted from each algal cell by centrifugation using a two-phase solvent (hexane/methanol (mixing ratio (volume ratio)) 10: 1) and a dry weight of 0.1g as a sample in the same manner as in example 7.

Next, using a sample of 0.1g of the dried mass of each algal cell, oil was extracted from the algal cells in a dried state by centrifugation using a single-phase solvent (hexane) in the same manner as in example 7.

Furthermore, a sample was prepared by using a dry mass of 0.1g of each algal cell as a centrifugation sediment, and an oil component was extracted from the dried algal cell by centrifugation sediment using a two-phase solvent (hexane/methanol (mixing ratio (volume ratio)) 10: 1 in the same manner as in example 7.

The resulting extract solutions (total 18ml) were dried by a nitrogen stream and quantified by GCMS in the same manner as in example 2. The results are also shown in Table 3.

(Table 3)

As can be seen from table 3, when the oil is extracted from the algal cells of known microalgae using an organic solvent, the amount of oil extracted becomes extremely small if the oil is extracted from the algal cells in a non-dried state. That is, a step of drying the algal cells in advance is necessary. The algae drying process requires considerable time and cost.

On the other hand, the algal cells of the microalgae of the present invention have very excellent properties that oil can be efficiently extracted from the undried algal cells, and the energy consumption of the production process of biofuel or the like using the microalgae of the present invention can be reduced.

Industrial applicability

The microalgae of the present invention can be used for the production of diesel fuel because of its high ability to produce aliphatic hydrocarbons having 16 to 26 carbon atoms, and is expected to be a carbon-neutralized fuel capable of preventing global warming. The JPCC DA0580 strain of the present invention is also expected from the viewpoint that it has a high production ability for neutral lipids and squalene, in addition to the ability to produce the aliphatic hydrocarbon having 16 to 26 carbon atoms.

The neutral lipid can be converted into a diesel fuel (so-called biodiesel fuel) by a known transesterification method or the like.

Claims (6)

1. The microalgae Navicula genus Alliticus species JPCCDA0580 strain, FERM BP-11201.

2. An oil production method comprising culturing the microalgae according to claim 1, wherein the oil is at least one selected from the group consisting of:

linear aliphatic saturated hydrocarbons having 16, 18, 20, 22, 24, and 26 carbon atoms;

triglycerides with a fatty acid moiety of tetradecanoic acid, hexadecanoic acid, hexadecenoic acid, octadecenoic acid, or eicosapentaenoic acid;

squalene; and

selected from carotenoid pigments, and pigments in chlorophyll.

3. The oil production method according to claim 2, further comprising extracting the oil from the culture product after the culturing with an organic solvent selected from the group consisting of a solvent consisting of n-hexane, a solvent consisting of n-hexane and methanol, and a solvent consisting of n-hexane and ethanol.

4. The oil production method according to claim 2, wherein the carotenoid pigment is fucoxanthin or a fucoxanthin derivative.

5. A dried algal cell obtained by drying the microalgae according to claim 1.

6. A method of carbon dioxide fixation comprising culturing the microalgae of claim 1.