JP5481876B2 - Microalgae belonging to the genus Seddesmus, a method for producing an oil having a step of culturing the microalgae, and an oil collected from the microalgae - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a microalga belonging to the genus Scenedesmus, a method for producing an oil having a step of culturing the microalga, and an oil collected from the microalgae. More specifically, a microalga belonging to the genus Senedesmus having an ability to produce an aliphatic hydrocarbon having 14 to 22 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.

Several methods for producing heavy or light oil hydrocarbons called biofuels by culturing microalgae have been reported.
Examples of algae having the ability to produce heavy oil-based hydrocarbons include the microalga Botriococcus brownie (see Non-Patent Document 1) capable of producing hydrocarbons having 36 carbon atoms and the ability to produce hydrocarbons having 33 carbon atoms. There is known a microalga Botriococcus brownie A race (see Patent Document 1).
As the algae having the ability to produce light oil-based hydrocarbons, the microalgae Nostocc muscorum, Trichodesmium erythaeum, Plectonema terebran et al. Microalgae Coccochoris elabens et al. (See Non-Patent Document 1) 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 (See 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.
However, in the dry algae obtained by drying the microalgae, the light oil-based hydrocarbon content is usually about 0.025 to 0.12% by mass (see Non-Patent Document 1), and the hydrocarbons. Productivity is not always sufficient.

Japanese Patent Laid-Open No. 9-234055 International Publication No. 2006/109588 Pamphlet

R. Raja, S .; Hemaiswaya, N .; Ashok Kumar, S .; Sridhar and R.K. Rengasamy (2008) A Perspective on the Biotechnological Potential of Microalgae. Critical Reviews in Microbiology, 34: 77-88.

  The present invention has been made in view of the above circumstances, and is a microalgae having high productivity of aliphatic hydrocarbons having 14 to 22 carbon atoms, a method for producing an oil component 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.

In order to solve the above problems, the present invention employs the following configuration.
The invention described in claim 1 is a microalga Senedesmus genus rubescens type JPCC GA0024 strain (FERM P-2174).
9).
Invention of Claim 2 is a manufacturing method of the oil component which has the process of culture | cultivating the micro algae of Claim 1.
The invention according to claim 3 is the method for producing an oil according to claim 2, wherein the oil contains an aliphatic hydrocarbon having 14 to 22 carbon atoms .
請 Motomeko 4 invention described is a dry algal cells obtained by drying the microalgae according to claim 1.
請 Motomeko 5 the described invention, is carbon dioxide fixing method having a step of culturing a microalgae according to claim 1.

  In the claims and the specification, “having the ability to produce an aliphatic hydrocarbon having 14 to 22 carbon atoms” mainly means an aliphatic hydrocarbon having 14, 16, 20, and 22 carbon atoms. It has the ability to produce The “oil” refers to a liquid component composed of a hydrophobic organic compound. Examples of the hydrophobic organic compound include aliphatic hydrocarbons and neutral fats.

  According to the present invention, a microalgae having high ability to produce aliphatic hydrocarbons having 14 to 22 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.

It is a figure which shows the molecular phylogenetic tree obtained using the 18S rDNA base sequence of JPCC GA0024 strain | stump | stock. It is a growth curve of JPCC GA0024 strain. It is the growth curve in the culture which changed the seawater component density | concentration of JPCC GA0024 strain | stump | stock. It is a growth curve in the nutrient-restricted culture of JPCC GA0024 strain.

The present invention will be described in detail below.
<Microalgae belonging to the genus Senedesmus>
The microalgae belonging to the genus Scenedesmus in the present invention has an ability to produce aliphatic hydrocarbons having 14 to 22 carbon atoms.
As the microalgae, the microalga Senedesmus rubescens is preferable. Particularly, the content of the aliphatic hydrocarbons having 14 to 22 carbon atoms in the algal body is high, and the microalgae can be easily cultured. Senedesmus rubescens JPCC GA0024 strain (FERM P-21749) (hereinafter abbreviated as JPCC GA0024 strain) is more preferred.

The JPCC GA0024 strain is a novel strain of marine microalgae belonging to the chlorococcum saccharides rubescens species isolated from brackish water by the inventors.
Hereinafter, the method for isolating the microalgae and the background of determining the JPCC GA0024 strain of the microalgae as a new strain will be described.

(Isolation method)
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 0.0024 mg / l, selenious acid pentahydrate 0.0016 mg / l}, and artificial seawater (Mari, manufactured by Senju Pharmaceutical Co., Ltd.) Art · SF-1) was prepared liquid medium dissolving the above predetermined concentration of 37 g / l distilled water. Also, an agar medium containing nutrient salts containing vitamins and the like and artificial seawater in the same composition as the liquid medium and further adding agar to a concentration of 1.2% (w / v) was prepared.
To a 24-well microtiter plate containing 2 ml of the liquid medium, an appropriate amount of sand collected from the Onagagawa Kawaguchi tidal flat in Ashiya-gun, Fukuoka Prefecture in November 2002 was added. Subsequently, stationary culture was performed under light irradiation of 1000 lux (lx), and a part of the culture solution in the well in which the growth of microalgae was confirmed was collected. By inoculating the sorted culture solution on the agar medium and culturing under the light irradiation conditions, the green unicellular alga JPCC GA0024 strain becomes a unicellular (isolated) microalgae colony. Obtained.

(Morphological properties)
As a result of culturing on the agar medium at 25 ° C. for 14 days, green JPCCC GA0024 strain colonies having a diameter of about 2.0 to 5.0 mm were obtained. The shape of the colony was punctiform and a semi-lens shape with no bulge. The peripheral edge was the whole edge, and the surface was a smooth shape. Moreover, the change of the colony form by a mutation was not seen, and the change of the colony form by a culture condition or a physiological state was not seen. It was confirmed that the colony color changed from green to brown as the culture days increased.
The marine microalgae in the colony are green unicellular algae with an average size of about 10-20 μm, some of which are over 100 μm, do not form colonies, and vegetative cells do not have eye drops or contractile vesicles The cell shape was circular. There is no buoyancy and the cell surface is smooth. Vegetative cells do not have flagella and do not show motility. In addition, the outer wall is surrounded by a cell wall, one nucleus and a plurality of chloroplasts are present inside, and other mitochondria, Golgi apparatus, vacuole, oil droplets and the like are observed. Pyrenoids are found in chloroplasts.

(Reproductive style)
The JPCC GA0024 strain does not form an inner cell and proliferates by two divisions.

(Physiological and biochemical properties)
Culture medium: Grows in a known culture medium based on seawater. In fresh water, the growth is extremely slow.
・ Photosynthesis: Photoautotrophic growth is possible by photosynthesis. Heterotrophic growth is not confirmed.
-Contained pigment: Chlorophyll a, chlorophyll b, and other carotenoid pigments-Storage material: Oil containing starch and aliphatic hydrocarbons having 14 to 22 carbon atoms-Growth temperature: 20 ° C to 35 ° C (optimum temperature 25 ° C) )
-Growth pH: 7.0-9.0 (optimum pH 8.0)
Accumulate oil that is stained with Nile red in the cells.

  When the JPCC GA0024 strain stained with Nile red is observed with a fluorescence microscope, the presence of oil colored with Nile red is confirmed as a bright fluorescent color development region in the algal cells in the fluorescence field. The oil can accumulate in a wide area within algal cells. Moreover, this oil component contains a C14-C22 aliphatic hydrocarbon.

  The JPCC GA0024 strain was presumed to be an algae belonging to the existing green algae chlorococcum from the above-mentioned morphological characteristics, reproductive mode, and physiological and biochemical properties. Furthermore, in accordance with a conventionally known method, DNA is extracted from the JPCC GA0024 strain using QIAamp DNA Broad Mini Kit 50 (Qiagen Co., Ltd.), and the 18S rDNA region is amplified by PCR to perform sequence analysis. And the base sequence of the 18S rDNA region was determined. 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 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 system diagram obtained as a result is shown in FIG.

The isolated JPCC GA0024 strain was classified as Chlorococcum in the phylogenetic tree and formed a cluster with the Scenedesmus rubescens species. Since the cell morphology with the rubescens species is also similar, the JPCC GA0024 strain was determined to be an algae of the Scenedesmus rubescens species.
The JPCC GA0024 strain can accumulate an oil component containing an aliphatic hydrocarbon having 14 to 22 carbon atoms in the algal cells. Furthermore, the content of the aliphatic hydrocarbon having 14 to 22 carbon atoms in the dry alga body of JPCC GA0024 can reach 0.6% by mass or more.
On the other hand, a strain that accumulates oil containing the aliphatic hydrocarbon in the Senedesmus rubescens has not been known to date.
Therefore, the JPCC GA0024 strain was determined to be a new strain of the Senedesmus rubescens species.

  The JPCC GA0024 strain was deposited at the Patent Microorganism Depositary, National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-21749.

<Oil content production method>
The method for producing oil according to the present invention includes a step of culturing the microalgae belonging to the genus Senedesmus 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 microalga belonging to the genus Seddesmus is preferably the microalga Senedesmus rubescens, especially from the viewpoint that the content of aliphatic hydrocarbons having 14 to 22 carbon atoms in the algal body is high and can be easily cultured. Senedesmus rubescens type JPCC GA0024 strain is more preferred.

  As the step of culturing the microalgae, a known method capable of culturing microalgae belonging to the genus Senedesmus can be applied. For example, 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 can cultivate the microalgae, and a known medium can be used. For example, a liquid medium containing nutrient salts containing vitamins and artificial seawater (Marine Art SF-1 manufactured by Senju Pharmaceutical Co., Ltd.) used when isolating the aforementioned JPCC GA0024 strain is preferable. The artificial seawater is a salt imitating the salt contained in natural seawater. By dissolving a predetermined amount of artificial seawater in distilled water, an aqueous solution having seawater components simulating natural seawater can be produced.

  The concentration of artificial seawater (Marin Art SF-1 manufactured by Senju Pharmaceutical Co., Ltd.) in the liquid medium is 100% (w / v) when 37 g / l is added (substantially equal to the salt concentration of natural seawater). 10 to 100% (w / v) is preferable, 30 to 100% (w / v) is more preferable, 50 to 100% (w / v) is more preferable, and 80 to 100% (w / v) ) Is most preferred.

What is necessary is just to adjust suitably according to the algal body density | concentration in a culture solution as said light irradiation conditions, For example, 500 lux or more (lx) is preferable, 1000-30000 lux (lx) is more preferable, 1000-10000 lux (lx) is preferable. More preferably, 1000 to 6000 lux (lx) is particularly preferable, and 1000 to 3000 lux (lx) is most preferable.
As the aeration amount in the aeration culture, a known aeration amount suitable for growing the microalgae can be applied, and for example, preferably 1 to 5 vvm, more preferably 2 to 4 vvm, and still more preferably 2.5 to 3 Aeration culture can be performed at .5 vvm.
The culture temperature in the aeration culture may be a known culture temperature suitable for growing the microalgae, and is usually preferably 20 to 35 ° C, more preferably 25 to 30 ° C.
As the period of the aeration culture, the culture can be continued as long as the microalgae grow, and it is usually preferably 1 to 4 weeks, more preferably 1 to 3 weeks, and more preferably 1 to 2 weeks. More preferably,

  In the method for producing oil according to the present invention, after the aeration culture period of the microalgae, it is preferable to further culture under nutrient limitation. By culturing under the restriction of nutrition, the content of oil contained in the algal bodies (mass% of oil contained in the dried alga bodies) can be increased. Therefore, during the aeration culture period, 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. It is preferable to increase the oil content to increase the production efficiency of the oil.

  Here, the culture under 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. For example, in the liquid medium (nutrient liquid medium) containing nutrient salts containing vitamins and artificial seawater (Marine Art SF-1 manufactured by Senju Pharmaceutical Co., Ltd.) used when isolating the aforementioned JPCC GA0024 strain When the content of nutrient salts including vitamins and the like is 100% (w / v), culturing using a liquid medium (nutrient-restricted liquid medium) having the content of less than 100% (w / v) It is equivalent to culturing under nutritional restrictions.

  The content of nutrient salts containing vitamins and the like contained in the nutrient-restricted liquid medium is preferably 0 to 60% (w / v), and preferably 0 to 30% from the viewpoint of increasing the content of oil in the microalgae. (W / v) is more preferable, 0 to 20% (w / v) is further preferable, 0 to 10% (w / v) is particularly preferable, and 0% (w / v) is most preferable.

  The method for switching from the nutrient liquid medium to the nutrient-restricted liquid medium is not particularly limited, and may be switched to the nutrient-restricted medium at a time or may be gradually switched. 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. In addition, as a method of gradually switching, for example, 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 the culture under the nutrition 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 30 days, more preferably 3 to 20 days, and even more preferably 3 to 7 days.

  As 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. For example, 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 does not impair the effects of the present invention and can dissolve the oil contained in the algal cells. For example, 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 oil extraction efficiency. Moreover, 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.

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. The purification method may be carried out by a known method, 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.

The oil produced by the method includes an oil observed when the microalgae are stained with Nile red. The oil contains an aliphatic hydrocarbon having 14 to 22 carbon atoms. In addition to the aliphatic hydrocarbons having 14 to 22 carbon atoms, the oil component can also contain neutral lipids such as triglycerides, phospholipids, free fatty acids, photosynthetic pigments such as steroid compounds and carotenoids.
The aliphatic hydrocarbon having 14 to 22 carbon atoms is mainly a linear aliphatic hydrocarbon having 14, 16, 20, and 22 carbon atoms, and more specifically, tetradecane, hexadecane, icosane, and docosane. It is. The linear aliphatic hydrocarbon may contain an unsaturated bond.

<Oil content>
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.
In addition, an aliphatic hydrocarbon having 14 to 22 carbon atoms can be purified from the oil.
The purification method is not particularly limited and can be performed by a known method. For example, an oil component extracted from hexane from the microalgae is dissolved in an organic solvent such as hexane, and by introducing silica gel into the solution, a substance other than the aliphatic hydrocarbon having 14 to 22 carbon atoms is adsorbed on the silica gel, Only the aliphatic hydrocarbon having 14 to 22 carbon atoms can be eluted.
In addition, as an organic solvent which melt | dissolves the said oil component, the thing similar to what was mentioned by description of the organic solvent in the above-mentioned manufacturing method of an oil component is mentioned.

<Dried algae>
The dry alga body in the present invention is obtained by drying the microalga belonging to the genus Senedesmus according to the present invention.
Examples of the microalgae include the same microalgae as those described in the description of the microalgae belonging to the genus Senedesmus 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. For example, there are a method of drying the algae in the sun, a method of blowing dry air on the algae, a method of freeze-drying (freeze drying) the algae. Among these, the drying method by freeze-drying is preferable from the viewpoint of suppressing the decomposition of the components contained in the algal cells.

<Fuel>
The fuel in the present invention is obtained from the microalga belonging to the genus Senedesmus according to the present invention.
Examples of the microalgae include the same microalgae as those described in the description of the microalgae belonging to the genus Senedesmus according to the present invention.
As a method of using the microalgae as a fuel, a method of burning the microalgae, a method of burning oil collected from the microalgae, aliphatic carbonization of hydrocarbons 14 to 22 purified from oil collected from the microalgae Examples include a method of burning hydrogen.

  When burning the microalgae, it is preferable to use a dried alga body obtained by drying the microalgae from the viewpoint of increasing the combustion efficiency. The dry algal bodies are the same as the dry algal bodies according to the present invention. When the microalgae is JPCC GA0024 strain, the calorific value of the dried alga body can reach the same or more than the calorific value of coal (approximately 6000 kcal / kg).

  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 GA0024 strain, the amount of heat of the hexane extract (oil) can reach 8400 kcal / kg or more.

  When combusting an aliphatic hydrocarbon having 14 to 22 carbon atoms refined from an oil collected from the microalgae, the aliphatic hydrocarbon having 14 to 22 carbon atoms is the 14 to 22 carbon atom in the oil according to the present invention. Similar to 22 aliphatic hydrocarbons. The aliphatic hydrocarbon having 14 to 22 carbon atoms can be used as a fuel for a diesel engine.

<CO2 fixation method>
The carbon dioxide fixing method in the present invention includes a step of culturing the microalgae belonging to the genus Senedesmus 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.
Examples of the microalgae include the same microalgae as those described in the description of the microalgae belonging to the genus Senedesmus according to the present invention.
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.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
<Hydrocarbon production ability of JPCC GA0024>
After culturing JPCC GA0024 strain in a 24-well well for 12 weeks, the algal cells were stained with Nile red to confirm that oil was produced and accumulated in the algal cells.

Specifically, nutrient salts containing vitamins and the like {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, molybdate (VI) disodium dihydrate 0.0072 mg / l, sulfuric acid Copper (II) pentahydrate 0.0024 mg / l, selenite pentahydrate 0.0016 mg / l}, and artificial seawater (Senju Pharmaceutical) To prepare a nutrient liquid medium dissolving the above predetermined concentration Ltd.) Marine Art · SF-1) of 37 g / l distilled water. Next, the JPCC GA0024 strain was inoculated into a 24-well microtiter plate containing 2 ml of the medium, and statically cultured 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.
Next, the pellet was resuspended in 450 μl of physiological saline, further mixed with 50 μl 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 1.0 ml of physiological saline and centrifuged again to collect alga bodies as a pellet. A suspension of the obtained algal bodies in 50 μl 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. In the fluorescence microscope image, it was confirmed that oil was accumulated throughout 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.

[Example 2]
<Identification of hydrocarbons accumulated in JPCC GA0024>
The JPCC GA0024 strain was inoculated into a 500 ml flat flask containing 500 ml of the liquid medium having the same composition as the liquid medium prepared in Example 1, and irradiated with light at 3000 lux (lx) for 3 weeks at an aeration rate of 3 vvm. Culture was performed. 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. This was done three times. Next, the hexane extract is centrifuged, and about 18 ml of the hexane extract solution from which the algal cell residue is removed is separated using a Sep-pak cartridge (6 cc / 1 g) (Waters Corporation) silica gel column. (Including fatty acid and pigment) was removed, and 16 ml containing an aliphatic hydrocarbon fraction was 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.

The gas chromatograph apparatus 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 μl).
As the mass spectrometer, GCMS-QP5050A manufactured by Shimadzu Corporation that performs ionization by an electron impact (EI) method was used.

As a result of GCMS analysis, it was revealed that JPCC GA0024 strain produced an aliphatic hydrocarbon having 14 to 22 carbon atoms. More specifically, linear aliphatic hydrocarbons having 14, 16, 20, and 22 carbon atoms were identified as the main components. In addition, although it was suggested that what has an unsaturated bond is contained in this linear aliphatic hydrocarbon, it did not come to pinpoint the position in the molecule | numerator of the unsaturated bond.
Moreover, when the amount of hydrocarbons produced by the JPCC GA0024 strain was determined from the area ratio of 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 GA0024 strain The content rate of the said C14-C22 aliphatic hydrocarbon in an algal body was 0.6 mass%. This content is the content of algae producing light oil-based aliphatic hydrocarbons known so far (in the dry alga body, 0.025 to 0.12% by mass; see TABLE 2 in Non-Patent Document 1). )) And higher than that.

[Example 3]
<Growth rate of JPCC GA0024>
JPCC GA0024 strain was cultured in the same manner as in Example 2, and the turbidity (OD750) of the culture solution was measured over time in order to evaluate the growth. The result is shown in FIG.
From the obtained results, it was confirmed that the JPCC GA0024 strain reached a steady growth in a culture period of about 2 weeks. In addition, the algal bodies were collected after 2 weeks from the start of the culture, dried and frozen, and the recovered amount of the dried algal bodies was examined.

[Example 4]
<Seawater requirement of JPCC GA0024>
Of the composition of the liquid medium prepared in Example 1, the composition of nutrient salts containing vitamins and the like is the same, and the concentration of artificial seawater (Marine Art SF-1 manufactured by Senju Pharmaceutical Co., Ltd.) is changed. 5 types were produced. The concentration of the artificial seawater is 0% when added at 0 g / l, and similarly, 11.1 g / l (30%), 18.5 g / l (50%), 29.8 g / l (80 %) And 37 g / l (100%).
The JPCC GA0024 strain was inoculated into a 500 ml flat flask containing 500 ml of these nutrient liquid media, and aerated culture was performed for 2 weeks under an irradiation of 3000 lux (lx) at an aeration rate of 3 vvm. In order to evaluate the growth, the turbidity (OD750) of the culture solution was measured over time. The result is shown in FIG.
From the obtained results, it was confirmed that the JPCC GA0024 strain grew best under the condition containing 80 to 100% artificial seawater (seawater component) during the growth period of 2 weeks. It was also confirmed that the growth decreased as the artificial seawater (seawater component) concentration decreased.
From this, it was confirmed that JPCC GA0024 strain is a marine microalgae.

[Example 5]
<Change in amount of hexane extract by nutrient-limited culture of JPCC GA0024>
Among the composition of the liquid medium prepared in Example 1, the concentration of artificial seawater (Marin Art SF-1 manufactured by Senju Pharmaceutical Co., Ltd.) is the same, and the composition ratio of nutrient salts containing vitamins and the like remains the same. Five types of nutrient-restricted liquid media with different addition concentrations were prepared. Concentrations of the nutrients containing the vitamins are {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, molybdate (VI) disodium dihydrate 0.0072 mg / l, sulfuric acid Copper (II) pentahydrate 0.0024 mg / l, selenite pentahydrate 0.0016 mg / l} The case of adding the 0.2-fold amount of 20%, and the case of adding the 0.1-fold amount of 10%, the case was not added as 0%.
The JPCC GA0024 strain was inoculated into each of 500 ml flat flasks containing 500 ml of these nutrient-restricted liquid media, and aerated culture was performed for 11 days with an aeration rate of 3 vvm under irradiation of 3000 lux (lx). In order to evaluate the growth, the turbidity (OD750) of the culture solution was measured over time. The result is shown in FIG.

  After the culturing period, the amounts of the culture solutions shown in Table 1 were collected and centrifuged to collect algal bodies. The mass of the dried alga body obtained by freeze-drying the alga body was measured. 6.0 ml of hexane was added to 0.1 g of the obtained dry algal cells, suspended, and extraction was performed for 30 minutes while crushing the algal cells at room temperature using an ultrasonic homogenizer. Next, the hexane extract was centrifuged to recover a hexane extract solution from which the algal residue was removed, and then the solvent hexane was distilled off under reduced pressure, and the mass of the remaining hexane extract (oil) was measured. These results are shown in Table 1.

From the obtained results, it was confirmed that the JPCC GA0024 strain grew best under the condition containing 100% of nutrient salts containing vitamins and the like during the growth period of 11 days. It was also confirmed that growth decreased as the concentration of nutrients including vitamins decreased. Naturally, the recovered amount of alga bodies (mass of dry alga bodies) also showed a similar tendency.
On the other hand, the amount of hexane extract (oil content) per unit mass of the recovered dry alga body (oil content of dry alga body) is the highest when the concentration of nutrients including vitamins is 0%, and the concentration is It was confirmed that the amount of the hexane extract (oil) decreased as the value increased. The results are shown in Table 1.

[Example 6]
<The calorific value of the dried alga body of JPCC GA0024>
The JPCC GA0024 strain was inoculated into a 500 ml flat flask containing 500 ml of a liquid medium having the same composition as the liquid medium prepared in Example 1, and aerated for 3 weeks under an irradiation of 3000 lux (lx) with an aeration rate of 3 vvm. Culture was performed. Thereafter, the algal pellet recovered by centrifugation was freeze-dried overnight to obtain 0.8 g of dried algal bodies.
When the calorific value was measured with a cylinder-type calorimeter (manufactured by Yoshida Seisakusho Co., Ltd., model: 1013-J, measurement range of calorific value: 4000 to 33500J) using the obtained dry algal body as a sample, it was 6160 kcal / kg. . That is, it was confirmed that the dry alga body of JPCC GA0024 strain has a calorific value equivalent to that of coal.

<The calorific value of oil collected from JPCC GA0024>
In the same manner as in Example 6, 4.0 g of dried alga bodies of JPCC GA0024 strain was obtained. 6.0 ml of hexane was added to 0.1 g of the obtained dry alga body to suspend it, and extraction was performed for 30 minutes while crushing the alga body at room temperature using an ultrasonic homogenizer. Next, the hexane extract was centrifuged to recover a hexane extract solution from which algal residue was removed, and then hexane as a solvent was distilled off under reduced pressure to obtain 0.360 g of a hexane extract (oil).
When the obtained hexane extract (oil content) was used as a sample and the calorific value was measured with a cylinder-type calorimeter (manufactured by Yoshida Seisakusho Co., Ltd., model: 1013-J, calorie measurement range: 4000 to 33500 J), 8420 kcal / kg. Met.

  Since the microalgae according to the present invention has a high productivity for producing aliphatic hydrocarbons having 14 to 22 carbon atoms, it can be used for the production of diesel fuel and is expected to be a carbon neutral fuel aimed at preventing global warming. it can.

FERM P-21749

Claims (5)

  The microalga Senedesmus genus rubescens JPCC GA0024 strain (FERM P-21749).   The manufacturing method of the oil component which has the process of culture | cultivating the micro algae of Claim 1.   The method for producing an oil according to claim 2, wherein the oil contains an aliphatic hydrocarbon having 14 to 22 carbon atoms.   A dry algal body obtained by drying the microalgae according to claim 1.   A carbon dioxide fixing method comprising a step of culturing the microalgae according to claim 1.

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