JP2021126044A - Method for producing organic compound derived from microalga - Google Patents

Abstract

To effectively release microalga cells and intercellular matrices and obtain an easy-to-use organic compound derived from microalga.SOLUTION: Provided is a method for producing an organic compound, comprising: a releasing step in which microalga cells included in a microalga slurry are released to obtain released cells and intercellular matrices; an intercellular matrix recovery step in which the released cells and the intercellular matrices are separated to obtain the intercellular matrices; and an organic compound obtaining step in which the intercellular matrices obtained in the intercellular matrix recovery step are used to obtain an organic compound derived from microalga.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing an organic compound derived from microalgae.

Microalgae produce organic compounds such as arginan. Organic compounds derived from microalgae are used as raw materials for biofuels and the like. Therefore, it is useful to obtain organic compounds from microalgae.

For example, the international publication WO2013-121509 pamphlet (Patent Document 1) describes that the single cells of Botryococcus brownie are separated from the extracellular matrix by using hypochlorous acid. When hydrocarbons are obtained using an extracellular matrix mixed with hypochlorous acid, it is difficult to purify the obtained hydrocarbons.

Japanese Patent No. 6033551 describes a method for obtaining a hydrocarbon from a water-soluble slurry. This method heats an aqueous slurry containing hydrocarbon-producing microorganisms and liberates the hydrocarbons accumulated in the cytoplasm and / or the intercellular matrix into the aqueous slurry to improve the efficiency of hydrocarbon extraction.

Japanese Unexamined Patent Publication No. 2017-38536 describes a method for producing an arginan composition, which comprises performing planktonic separation treatment of arginane from a slurry containing microalgae-derived biomass.

International Publication WO2013-121509 Pamphlet Japanese Patent No. 6033551 JP-A-2017-38536

It is desired to obtain an organic compound derived from microalgae that effectively releases microalgae cells and extracellular matrix and is easy to use.

This specification is easy to use if, basically, a microalgae slurry is used to release microalgae cells and an intercellular matrix, and then the released cells are removed to obtain an intercellular matrix. Based on the finding that organic compounds derived from microalgae can be obtained. That is, one of the features of the invention in this specification is that an extracellular matrix is once obtained, and an organic compound is obtained using the extracellular matrix.

The method for producing this organic compound includes a release step, an extracellular matrix recovery step, and an organic compound acquisition step.
The release step is a step for causing cell release of microalgae contained in the microalgae slurry to obtain free cells and an intercellular matrix. The extracellular matrix recovery step is a step for separating the free cells obtained in the free step and the extracellular matrix to obtain an extracellular matrix. The organic compound acquisition step is a step for obtaining an organic compound derived from microalgae by using the cell-cell matrix obtained in the cell-cell matrix recovery step. This process is also called a fat extraction process.
The above-mentioned organic compounds derived from microalgae include hydrocarbons and arginane. This production method may be a method for obtaining a hydrocarbon derived from microalgae, or a method for obtaining arginane derived from microalgae.
The microalgae is preferably a microalgae belonging to the genus Botryococcus, and is preferably a Botryococcus brownie strain derived from Botryococcus brownie or Botryococcus brownie.
Further, the microalgae slurry has a water content of 75 to 99.9% by weight, and the liberation step may include a step of freezing the microalgae slurry.
The microalgae slurry has a water content of 75 to 99.9% by weight, and the liberation step includes a step of heating the microalgae slurry at 50 ° C. or higher and 70 ° C. or lower for 5 minutes or longer and 5 days or shorter. May be good.
Further, the microalgae slurry has a water content of 75 to 99.9% by weight, and the liberation step may include a step of adding either or both of sodium chloride and glycerol to the microalgae slurry.

According to the invention described herein, by adopting predetermined release conditions using a microalgae slurry, the cells of the microalgae and the intercellular matrix are effectively released, and then the intercellular matrix is obtained. Therefore, an organic compound derived from microalgae that is easy to use can be obtained.

FIG. 1 is a flowchart for explaining an example of a processing process. FIG. 2 is a photomicrograph that replaces the drawing showing the state of cell release under condition 3 of Example 1. FIG. 3 is a photomicrograph instead of a drawing showing the state of cell release under condition 6 of Example 1. FIG. 4 is a photomicrograph that replaces the drawing showing the state of cell release under condition 8 of Example 1. FIG. 5 is a diagram showing a micrograph and an evaluation thereof in place of the drawing showing the state of cell release in Example 4. FIG. 6 is a diagram showing a micrograph and an evaluation thereof in place of the drawing showing the state of cell release in Example 4. FIG. 7 is a photomicrograph instead of a drawing showing the state of cell release under condition 2 of Example 6. FIG. 8 is a photomicrograph instead of a drawing showing the state of cell release under condition 4 of Example 6. FIG. 9 is a photomicrograph instead of a drawing showing the state of cell release under condition 6 of Example 6. FIG. 10 is a diagram showing a micrograph and an evaluation thereof in place of the drawing showing the state of cell release in Example 7. FIG. 11 is a graph that replaces the drawing showing the GC / MS results of the crude oil obtained in Example 9. FIG. 12 is a graph that replaces the drawing showing the TG / DTA results of Arginan obtained in Example 9. FIG. 13 shows the GC / MS results of the crude oil obtained in Comparative Example 1. FIG. 14 shows the TG / DTA results of the dried algae obtained in Comparative Example 1. FIG. 15 shows the GC / MS results of the crude oil obtained in Example 10. FIG. 16 shows the TG / DTA results of Arginan obtained in Example 10.

Hereinafter, embodiments for carrying out the present invention will be described. The present invention is not limited to the forms described below, but also includes those which are appropriately modified by those skilled in the art from the following forms to the extent obvious to those skilled in the art.

The invention described herein relates to a method for obtaining an organic compound derived from microalgae.

Microalgae means microalgae whose individual existence cannot be identified by the human eye.
Examples of microalgae are indigo plants, gray plants, crimson plants, green plants, cryptophyta, haptophyta, unequal hair plants, whirlpool plants, euglena plants, chloralakunion plants. It is a microalga belonging to the phylum. These may be used alone or in combination of two or more. Among these, as the microalgae, the phylum Green algae is preferable, and green algae are more preferable. In terms of producing biomass, as microalgaes, the genus Haematococcus sp., The genus Chlamydomonas sp., The genus Chlamydomonas sp., The genus Botryococcus sp.

Botryococcus microalgae form an extracellular matrix mainly composed of arginane, which is a polymerization compound of organic compounds such as hydrocarbons, outside the cells, and several to several hundred cells with a size of 2-10 μm. It maintains a colony (40-300 μm) consisting of and forms a living area. In addition, 95% of the hydrocarbon produced in the cell is accumulated in the extracellular matrix. Organic compounds such as hydrocarbons and arginane accumulated in this way are expected as raw materials for biofuels and biorefineries. Examples of microalgaes belonging to the genus Botryococcus are Botryococcus brownie and Botryococcus prochubalance. Examples of Botryococcus brownie are the Botryococcus brownie A variety (Race-A) and the Botryococcus brownie B variety (Race-B), as well as their derivatives and mutants. _{Other examples of Botryococcus brownie include L varieties and C 18-} C ₂₀ alkanes (eg, n-alkanes and epoxy n-alkanes) that produce the tetraterpene lycopazine, (Lycopageene) as the major organic compound. It is an S variety to be produced. In the present specification, "Botryococcus brownie A variety" means a variety of Botryococcus brownie that produces a linear organic compound derived from a fatty acid, and specifically, C25-31 linear alkaziene. Alternatively, it means a Botryococcus brownie variety having the property of producing alkatriene (here, the number of carbon atoms is an odd integer in the above range). Further, in the present specification, "Botryococcus brownie B variety" means a variety of Botryococcus brownie that produces an organic compound which is a terpene, and specifically, C30-37 linear or linear or Branched triterpenes, especially those of Botryococcus brownie, which have the property of producing methylated triterpenes represented by the _{formula: C n} H _{2n-10 (where n is an integer between 30 and 37).} Means a variety. Botryococcus brownie A and B varieties can be identified based on cultivar characteristics known in the art (Metzger, P. et al., Phytochemistry, Vol. 24, No. 10, p. 2305-2312). , 1985).

The method for obtaining microalgae is not particularly limited and may be appropriately selected according to the purpose. For example, a method for collecting microalgae from the natural world, a method using a commercially available product, or a method for obtaining microalgae may be obtained from a storage organization or a depository organization. ， In addition, these may be cultivated. The microalgae are preferably those that have undergone a purification step. The purification step is a step performed for the purpose of making microalgae into a single type, and does not necessarily mean making only a single type of microalgae.

The organic compound derived from microalgae means, for example, a hydrocarbon or arginane that can be obtained from microalgae. In the present specification, "hydrocarbon" means an organic compound composed of carbon and hydrogen, and specifically, a linear or branched aliphatic or alicyclic compound containing a specific number of carbon atoms. Or it means an aromatic hydrocarbon. For example, "C _20-40 hydrocarbon" means a linear or branched aliphatic, alicyclic or aromatic hydrocarbon containing at least 20 and at most 40 carbon atoms. It is preferably a linear or branched aliphatic or alicyclic hydrocarbon in the range of 20-40 carbons (C _{20-40 ), preferably C 25-31} linear alkaziene or alkatorien (here). , The number of carbon atoms is an odd integer in the above range), or C _30-37 linear or branched triterpenes are more preferred. In the present specification, "alkaziene" means a hydrocarbon defined above, in which two CC single bonds are replaced with a double bond, and "alkatorien" is defined above. It means a hydrocarbon in which three CC single bonds are replaced with double bonds. Further, in the present specification, "triterpene" means a hydrocarbon derived from _{C 30} hydrocarbon biosynthesized using presqualene diphosphate produced by dimerization of farnesyl diphosphate (FPP) as a precursor. ..

The organic compounds produced by Botryococcus microalgae are as described above. For example, it is known that an organic compound highly produced by a strain of the B cultivar of the genus Botryococcus produces a high amount of an organic compound having a triterpene structure of C30 to C37 (carbon number 30 to 37). This organic compound is expected as a biofuel and a biorefinery raw material. On the other hand, the structure of arginan has been estimated by instrumental analysis. The arginan produced by cultivar B is a polymer polymer derived from terpenoids, and since there are double bonds in the hydrocarbon chain, it is necessary. It is expected to have rubber-like and sponge-like elasticity, and is expected to be used in various industrial fields.

Ardinane means, for example, a polymerization compound (biopolymer) such as organic compounds produced when microalgae form an aggregate (colony) of several to several hundred cells in the process of growth. (Japanese Patent Laid-Open No. 2017-38536).

The method for producing an organic compound includes a release step, an extracellular matrix recovery step, and an organic compound acquisition step. This method may further include known steps relating to carbohydrates, such as a culturing step of culturing microalgae, a slurry step of obtaining a slurry, and a purification step of purifying the obtained organic compound.

Culturing process For example, microalgae can be cultivated according to the method described in Japanese Patent No. 6033551. Hereinafter, an example using Botryococcus brownie will be described, but the microalgae described in the present specification is not limited to Botryococcus brownie. In the culturing process, Botryococcus brownie is cultivated. In this step, a known culture method may be appropriately adopted according to the characteristics of Botryococcus brownie. In addition, if a sufficient amount of Botryococcus brownie is present, the culture step may be omitted as appropriate. Examples of the composition of the culture medium of Botryococcus brownie can include general inorganic media such as AF6 medium, Chu13 medium, BG11 medium, and MDM medium, and modified media thereof. The mineral medium, usually as a nitrogen source, the _{Ca (NO 3) 2 · 4H} 2 O and _KNO 3, NaNO _3, include _KH 2 PO ₄ and MgSO ₄ · 7H ₂ O as other key nutrients .. Each composition may be appropriately replaced with a compound having the same properties as that, or may contain a compound other than the above. The culture form may be an open pond type or raceway type open system culture form, or a tube type or PBR type closed system culture form. If necessary, carbon dioxide can be supplied, the culture solution can be circulated, and light can be irradiated with an LED or the like for culturing. As a method for recovering Botryococcus brownie, the culture solution may be recovered as it is, or microalgae including Botryococcus brownie may be separated and concentrated by centrifugation or a filter cloth having an appropriate pore size and recovered. .. In addition, since Botryococcus brownie has the property of floating after standing, the culture solution may be allowed to stand after culturing, and the upper layer containing the floating Botryococcus brownie may be scraped off and collected. The left is an example of culture, and a known medium and culture form for culturing Botryococcus brownie can be appropriately used. The culture environment may be outdoors or indoors. When culturing indoors, it is preferable that a light source such as an LED can be used to irradiate light of a specific wavelength with a specific intensity. An example of the light irradiation time is 1 hour or more and 24 hours or less per day, 6 hours or more and 18 hours or less, or 9 hours or more and 15 hours or less. Examples of the culture period are 1 day or more and 6 months or less, 3 days or more and 3 months or less, 7 days or more and 1 month or less, and may be appropriately adjusted according to the characteristics of Botryococcus brownie. The culture temperature is preferably room temperature. An example of the culture temperature is 15 ° C. or higher and 40 ° C. or lower, more preferably 20 ° C. or higher and 35 ° C. or lower. A gas such as carbon dioxide may be aerated in the medium. In this case, the carbon dioxide concentration may be 1% by volume or more and 100% by volume or less, or 5% by volume or more and 20% by volume or less. Further, the air may be continuously ventilated or may be ventilated intermittently. The medium may be appropriately stirred or allowed to stand.

The slurrying step is a step for obtaining a microalgae slurry containing microalgae.
Microalgae slurry is a general term for a fluid mixture containing microalgae.
Examples of the microalgae slurry are the microalgae aqueous slurry described in Japanese Patent No. 6033551 and JP-A-2017-38536.
The state of the microalgae contained in the microalgae slurry is, for example, a culture solution obtained by culturing Botryococcus brownie using a known culture method, and an example of the microalgae slurry is a microalgae containing Botryococcus brownie. It is a concentrated solution in which the above is separated and concentrated, or a mixed solution in which water is added to adjust the water content.
The components other than the microalgae contained in the microalgae slurry are the medium components used for culturing. In the case of the open culture form, impurities mixed during the culture such as dust, earth and sand, and germs may be contained, or the impurities may be removed.

The release step is a step for causing cell release of microalgae contained in the microalgae slurry to obtain free cells and an intercellular matrix. The cell release of microalgae is, for example, from the state in which several to several hundred cells of Botryococcus brownie contained in the microalgae slurry form a colony based on the intercellular matrix, and from the intercellular matrix. It is the transition to a state in which several to several hundred cells are suspended alone. A method of confirming the cell release of microalgae is, for example, to observe the colony of Botryococcus brownie and the proportion of free cells using a microscope. In addition, micrographs can be taken and image analysis software can be used to calculate the free cell release rate. A free cell is a cell that exists independently of an aggregate formed by directly or indirectly joining a plurality of cells. An example of a free cell is a cell that floats alone away from the extracellular matrix of Botryococcus brownie contained in a microalgae slurry. The extracellular matrix is a polymer structure that functions as a base for indirectly joining cells that exist outside the cell. An example of the extracellular matrix is that in plants, polysaccharides such as cellulose and hemicellulose are formed, and in vertebrates, glycoproteins such as collagen and proteoglycan are the main components. In microalgae, in addition to polysaccharides such as cellulose and agar, there is an intercellular matrix whose main component is arginane, which is a polymerized compound of organic compounds such as hydrocarbons.

The cell release step is a step in which a microalgae slurry containing Botryococcus brownie is exposed to conditions that promote cell release, and Botryococcus brownie cells are released from the intercellular matrix. In this step, the characteristics of Botryococcus brownie may be utilized and cell release conditions may be appropriately adopted.

Known cell release conditions include methods for promoting cell release by changing osmotic pressure, such as glycerol, xylitol, erythritol, sorbitol, and sodium chloride. For these, one kind or two or more kinds may be added to the slurry. These are 1% by mass or more and 30% by mass or less, 3% by mass or more and 20% by mass or less, 3% by mass or more and 10% by mass or less, 5% by mass or more and 20% by mass or less, or 5% by mass or more and 10% by mass or less with respect to the slurry. It may be added below. Further, these may be added so as to be 0.05 regulation (M) or more and 10 regulation (M) or less, 0.1M or more and 5M or less, 0.3M or more and 4M or less, 0.4M or more and 2M or less. In addition, there is a method of destroying and sterilizing cells by adding hypochlorous acid, and cell liberation also occurs at this time. Since removal of additives from the purified product may be a problem in these chemical treatments, a method of promoting cell release without adding a chemical product is preferable.

As a physical treatment, high temperature stress is achieved by exposing the microalgae slurry to heating conditions of 45 ° C or higher and 70 ° C or lower (or 45 ° C or higher and lower than 70 ° C, 50 ° C or higher and 65 ° C or lower, 55 ° C or higher and 60 ° C or lower). It is possible to promote cell release. We also found that cell release occurs by freezing and thawing, so these methods may be used. In order to induce cell liberation under heating conditions, equipment that can keep the temperature at a constant temperature should be used, and in order to induce cell liberation by freezing and thawing, the microalgae slurry is frozen using a freezing equipment and at a temperature above room temperature. Just unzip it.

In addition to this, conditions under which cell release occurs with high efficiency may be appropriately adopted. An example of such a condition is a condition in which cells are released from the extracellular matrix by using ultrasonic treatment, UV irradiation, or the like. The efficiency of cell release is preferably 60% or more and 100% or less, more preferably 90% or more and 100% or less, 95% or more and 99% or less, or 96% or more and 98% or less).

The extracellular matrix recovery step is a step for separating free cells and the extracellular matrix to obtain an extracellular matrix. The cell separation step is a step of separating the extracellular matrix from which Botryococcus brownie cells have been released and the free cells as described above. In this step, the characteristics of Botryococcus brownie may be utilized and cell separation conditions may be appropriately adopted. Utilizing the difference in size between the free cells and the extracellular matrix, the cells after cell release can be separated from the extracellular matrix by filtration cloth. In addition, since the extracellular matrix has the property of ascending and precipitating cells, the extracellular matrix may be recovered by centrifugation or static separation. Insufficient cell removal after cell release leads to a decrease in purity of the free cells. Therefore, after cell removal, the cells are exposed to alkaline conditions of pH 9 or more and 13 or less, pH 9 or more and 12 or less, or pH 10 or more and 12 or less). ， It is preferable to completely remove free cells. At this time, after removing free cells, it is preferable to wash and dehydrate on a filter cloth to collect the extracellular matrix.

The organic compound acquisition step is a step for obtaining an organic compound derived from microalgae by using the cell-cell matrix obtained in the cell-cell matrix recovery step. The fat extraction step is a step of extracting an organic compound from the intercellular matrix recovered as described above to separate and purify hydrocarbons and arginane. In this step, a known fat extraction method may be appropriately adopted.
As a general method, a squeezing method using an oil squeezing machine, an extraction method using an organic solvent as an extraction solvent, or a squeezing method using these in combination may be used. Specifically, the organic solvent of the extraction method is one or more from a group of organic solvents consisting of n-hexane, chloroform, methanol, ethanol, diethyl ether, acetone, n-heptadecene, and n-eicosaziene, or hexane / acetone. It is a mixture of the above-mentioned organic solvents exemplified in the mixed solvent of the above, the mixed solvent of chloroform / methanol, the mixed solvent of ethanol / diethyl ether, and the like. In the extraction method using n-hexane, when a microalgae slurry consisting of Botryococcus brownie is used, the polysaccharide layer on the cell surface and water gel, and the extraction efficiency decreases, so dry algae are used. Since the polysaccharide layer can be removed together with the cells through the cell removal step, gelation does not occur even in the state of containing water, and organic compounds can be efficiently extracted.
High-purity arginane can be obtained by drying the extracellular matrix extracted from fats and oils.

Purification Step Further, in order to increase the purity of an organic compound such as alginan, a known purification treatment using an organic solvent, an acid, and a base can be performed. The purification process itself is known. Examples of purification steps are reflux and extraction.

Applications of Microalgae-Derived Organic Compounds Microalgae-derived organic compounds can be used, for example, in polymer raw materials, cosmetic raw materials, lubricating oils, foods, biofuels, fermentation raw materials, feeds, feeds, and fertilizers.
[Example]

1. 1. Cell release evaluation Micrographs of various samples prepared in Examples and Comparative Examples were taken, and the cell release rate in the colony was calculated using image analysis software (ImageJ) and used as a stepwise index of cell release. .. Evaluation A: 90% or more, Evaluation B: 60 to less than 90%, Evaluation C: 40 to less than 60%, Evaluation D: 10 to less than 40%, Evaluation E: Less than 10%. It was assumed that A was the most cell-free and evaluation E was not cell-free.

2. Measurement of Moisture Content The moisture content of various samples prepared in Examples and Comparative Examples was measured using a heat-drying moisture meter (A & D MX-50). The measurement conditions are as follows.
<Measurement conditions>
Temperature rise start temperature; 25 ° C
Final temperature reached; 110 ° C
Heating rate; + 10 ° C / min Termination condition: 0.10% / min

3. 3. Measurement of crude oil content To 0.5 g of dry weight of various samples prepared in Examples and Comparative Examples, 10 ml of n-hexane was added, mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice. .. The recovered hexane phase was distilled under reduced pressure using a centrifugal evaporator to remove n-hexane, the residual liquid was used as crude oil, and the crude oil weight per dry weight of various samples was defined as the crude oil content.

4. Measurement of hydrocarbon composition ratio Using crude oil obtained from various samples prepared in Examples and Comparative Examples, component analysis was performed by GC / MS (Shimadzu Gas Chromatograph QP-5050), and hydrocarbons in the crude oil were used. The composition ratio of hydrogen and fatty acid was determined. The measurement conditions are as follows.

<Analysis preprocessing>
(1) After weighing 100 mg of the crude oil sample, add 5 mL of n-hexane and dissolve well.
(2) 50 μl of this hexane solution, 50 μl of trimethylsilylating agent (BSTFA), and 100 μl of n-hexane are added and heated at 80 ° C. for 1 h, and this sample is used as a TMS-ized sample.
<Measurement conditions>
Column SHIMADZU-GLC ZB-1
(Φ0.25 mm x 60 m, film thickness 0.25 μm)
Column temperature rise 120 ° C (1 min) -15 ° C / min-230 ° C (1 min)
-5 ° C / min-320 ° C-10 ° C / min-340 ° C (12 min)
Measurement time 40.33 min
Equipment Shimadzu Gas Chromatograph Mass Spectrometer QP-5050
Carrier 0.5 mL / min He (constant flow control)
Pressure 80 kPa
Total flow 27.7 mL / min
Linear velocity 18.8 cm / sec
Purge flow rate 3 mL / min
Sample inlet Split, 330 ° C
Slit ratio 1:50
Detector interface temperature 260 ° C, ion source 250 ° C
SCAN mode m / z 35-700
Sample injection volume 1 μl

<Hydrocarbon composition ratio calculation method>
A calibration curve was prepared using dodecane and squalene as hydrocarbon preparations, and the hydrocarbon composition ratio was calculated.

5. Measurement of arginane content The arginane content was measured according to the measurement method described in JP-A-2015-124091. The details were determined from the weight loss rate at 360 ° C. or higher under the following temperature conditions using a differential thermal / thermogravimetric simultaneous measuring device for various samples prepared in Examples and Comparative Examples. The measurement conditions are as follows.
<Measurement conditions>
Temperature rise start temperature; 25 ° C
Final temperature reached; 800 ° C
Temperature rise rate; + 10 ° C / min (maintain for 30 minutes at 100 ° C)
Temperature calibration standard sample (indium (156.6 ° C), zinc (419.4 ° C))

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 90% by weight, and 20 ml each was dispensed into eight centrifuge tubes. Each centrifuge tube was allowed to stand for 24 hours or 48 hours under the conditions of room temperature (24 ° C.), refrigeration (4 ° C.), warming (60 ° C.), and freezing (-20 ° C.).

After standing, the state of cell release was evaluated by microscopic observation, free cells were removed using a filter cloth with a pore size of 40 μm, a 0.1N potassium hydroxide solution was added, and the cells were exposed to alkaline conditions for 1 day. The cell-cell matrix from which the cells had been removed was washed with a filter cloth having a pore size of 40 μm, concentrated and collected, and then dried by air drying. To 0.5 g of the dry weight of the intercellular matrix obtained under each condition, 10 ml of n-hexane was added and mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. The crude oil content of various samples was calculated from the amount of crude oil recovered.

In addition, the extracellular matrix after extraction was dried, and the arginane content was measured as purified arginane. It was found that cell release occurs with high efficiency under warming or freezing conditions, the crude oil content in the obtained extracellular matrix is high, and high-purity arginane of 80% or more can be purified.

Table 1 shows the cell release evaluation, crude oil content, and arginane content under each condition of Example 1.

FIG. 2 shows a micrograph showing the state of cell release under condition 3 of Example 1. FIG. 3 shows a micrograph showing the state of cell release under condition 6 of Example 1. FIG. 4 shows a micrograph showing the state of cell release under condition 8 of Example 1. Micrographs can be identified as black for cells and free cells and gray for arginan by image processing. When cell release does not occur, it can be observed as a black colony, and when cells are released, it can be observed as a gray colony.

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 98% by weight, and 100 μl each was dispensed into a centrifuge tube. Each centrifuge tube was allowed to stand for 1 hour, 2 hours, and 4 hours under the conditions of 40 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C., 70 ° C., and 80 ° C. After standing, the state of cell release was evaluated by microscopic observation. Table 2 shows the cell release evaluation in Example 2.

It was found that the heating temperature range in the cell release treatment by heating is preferably 50 ° C. or higher and lower than 70 ° C., and more preferably 55 ° C. or higher and 65 ° C. or lower. Furthermore, it was found that if the treatment time is 1 hour or more, it is effective, and preferably 2 hours or more should be treated.

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 90% by weight, and 100 μl each was dispensed into a centrifuge tube. Each centrifuge tube was frozen under the conditions of -4 ° C, -20 ° C, and -80 ° C, and after thawing, the state of cell release was evaluated by microscopic observation.
It was found that in the cell release treatment by freezing, cell release occurs by freezing the slurry regardless of the freezing temperature.
Table 3 shows the cell release evaluation in Example 3.

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 85% by weight, 90% by weight, and 98% by weight. Each microalgae slurry was allowed to stand at 60 ° C. for 4 hours, and the state of cell release was evaluated by microscopic observation.
It was found that the efficiency of cell release does not decrease even if the algae concentration becomes high in the cell release process by heating. FIG. 5 shows the cell release evaluation in Example 4.

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 82% by weight, 91% by weight, and 96% by weight. Each microalgae slurry was frozen under the condition of −20 ° C., and the state of cell release was evaluated by microscopic observation.
It was found that in the cell release treatment by freezing, the efficiency of cell release does not decrease even if the algae concentration becomes high. FIG. 6 shows the cell release evaluation in Example 5.

The Botryococcus brownie NIES-836 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain a microalgae slurry having a water content of 85.6% by weight. 20 ml of the above microalgae slurry was dispensed into a centrifuge tube, and each solution was added so that the water content was 90% by weight. Each solution was a glucose solution, a glycerol solution, and a sodium chloride solution, and was adjusted to have a concentration of 5% glucose, 10% glucose, 5% glycerol, 10% glycerol, 0.5 M sodium chloride, and 1 M sodium chloride. It was allowed to stand at room temperature for 48 hours under each solvent condition. After standing, free cells were removed using a filter cloth with a pore size of 40 μm, the state of cell release was evaluated by microscopic observation, a 0.1N potassium hydroxide solution was added, and the cells were exposed to alkaline conditions for 1 day. The cell-cell matrix from which the cells had been removed was washed with a filter cloth having a pore size of 40 μm, concentrated and collected, and then dried by air drying. To 0.5 g of the dry weight of the intercellular matrix obtained under each condition, 10 ml of n-hexane was added and mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. The crude oil content of various samples was calculated from the amount of crude oil recovered. In addition, the extracellular matrix after extraction was dried, and the arginane content was measured as purified arginane.

It was found that cell release occurs under the condition of addition of glycerol or sodium chloride, and the purity of arginane increases according to the rate of cell release.
Table 4 shows the cell release evaluation, crude oil content, and arginane content under each condition of Example 6. FIG. 7 shows a photomicrograph under Condition 2 of Example 6. FIG. 8 shows a photomicrograph under condition 4 of Example 6. FIG. 9 shows a photomicrograph under condition 6 of Example 6.

The Botryococcus brownie UTEX572 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 20 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 98% by weight, dispensed into a 100 μl centrifuge tube, frozen under the condition of −20 ° C. for 1 hour, thawed, and then the state of cell release was evaluated by microscopic observation. .. FIG. 10 shows the cell release evaluation in Example 7.

The Botryococcus prochubalance ACOI249 strain was cultured by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 20 μm to obtain a microalgae slurry. The water content of the microalgae slurry was adjusted to 98% by weight, dispensed into a 100 μl centrifuge tube, frozen under the condition of −20 ° C. for 1 hour, and after thawing, it was confirmed by microscopic observation that the cells were released. ..

Alginan production method under heating conditions Botryococcus brownie NIES-836 strain was cultivated by an existing culture method, collected and concentrated using a filter cloth having a pore size of 40 μm, and 10 L of microalgae slurry having a water content of 98% by weight was obtained. Obtained.
5 L of the above microalgae slurry was exposed to heating conditions at 60 ° C. for 1 day to promote cell release. After cell release, free cells are removed using a filter cloth with a pore size of 40 μm, a 0.1N potassium hydroxide solution is added, the cells are exposed to alkaline conditions for 1 day, and the cell-removed intercellular matrix is washed with a filter cloth having a pore size of 40 μm. Concentrated and recovered. Then, it was dried by air drying, and 53 g of extracellular matrix was obtained.
To the dry weight of the intercellular matrix obtained above, 20 times the amount of n-hexane was added, mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. 35 g of crude oil was obtained. The crude oil content in this extracellular matrix was 65%, and the hydrocarbon composition ratio of the extracted crude oil was 100%.
The solid content after crude oil extraction was recovered and dried to obtain 15 g of prepared arginan. The arginane content of this prepared arginan was 87%.
FIG. 11 shows the GC / MS (gas chromatograph mass spectrometry) results of the crude oil obtained in Example 9.
FIG. 12 shows the TG / DTA (thermogravimetric analysis and differential thermal analysis) results of arginan obtained in Example 9.
[Comparative Example 1]

4 L of the microalgae slurry obtained in Example 9 was concentrated using a filter cloth having a pore size of 40 μm and dried in a constant temperature dryer at 60 ° C. to obtain 80 g of dried algae. To the dry weight of the dried algae obtained above, 20 times the amount of n-hexane was added and mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. 46 g of crude oil was obtained. The crude oil content in the dried algae was 57%, and the hydrocarbon composition ratio of the extracted crude oil was 85%. The alginan content of the dried algae was 16%. FIG. 13 shows the GC / MS results of the crude oil obtained in Comparative Example 1.
FIG. 14 shows the TG / DTA results of the dried algae obtained in Comparative Example 1.

The hydrocarbon obtained by the production method of Example 9 had a hydrocarbon composition ratio of 100%, and a high-purity hydrocarbon could be recovered as compared with Comparative Example 1. The arginan obtained by the production method of Example 9 had a high purity of 87%, and the recovery rate of arginane was 80%.

Alginan production method under freezing conditions Botryococcus brownie NIES-836 strain was cultivated by an existing culture method, and collected and concentrated using a filter cloth having a pore size of 40 μm to obtain 800 mL of a microalgae slurry having a water content of 82% by weight. rice field. 500 mL of the microalgae slurry was frozen in a refrigerator and thawed at room temperature to promote cell release. After cell release, free cells were removed using a filter cloth with a pore size of 40 μm, a 0.1N potassium hydroxide solution was added, the cells were exposed to alkaline conditions for 1 day, and the intercellular matrix from which the cells had been removed was washed with a filter cloth having a pore size of 40 μm. The cells were concentrated and recovered to obtain 150 ml of a wet cell matrix (water content 65%). Half of it was dried by air drying to obtain 26 g of extracellular matrix.

To the dry weight of the intercellular matrix obtained above, 20 times the amount of n-hexane was added, mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. 19 g of crude oil was obtained. The crude oil content in this extracellular matrix was 72%, and the hydrocarbon composition ratio of the extracted crude oil was 100%. The solid content after crude oil extraction was recovered and dried to obtain 14 g of prepared arginane. The arginane content of this prepared arginan was 80%.

FIG. 15 shows the GC / MS results of the crude oil obtained in Example 10.
FIG. 16 shows the TG / DTA results of Arginan obtained in Example 10.
[Comparative Example 2]

200 mL of the microalgae slurry obtained in Example 10 was dried in a constant temperature dryer at 60 ° C. to obtain 36 g of dried algae. To the dry weight of the dried algae obtained above, 20 times the amount of n-hexane was added and mixed by shaking for 1 hour, and then the operation of recovering the hexane phase was repeated twice to extract crude oil from the hexane phase. 20 g of crude oil was obtained. The crude oil content in the dried algae was 55%, and the hydrocarbon composition ratio of the extracted crude oil was 86%. The alginan content of the dried algae was 15%.

The hydrocarbon obtained by the production method of Example 10 had a hydrocarbon composition ratio of 100%, and the recovery rate of the hydrocarbon in the algae was 88%. In addition, high-purity arginane with a purity of 80% could be adjusted, and the recovery rate of arginane was 88%. It was shown that the method of Example 10 has a higher recovery rate than the method of Comparative Example 2 and can prepare high-purity arginane.

Extraction of crude oil from an intercellular matrix containing an aqueous phase To the weight of 75 ml of the wet intercellular matrix (water content 65%) obtained in Example 7, 20 times the amount of n-hexane was added and shaken for 1 hour. , The operation of recovering the hexane phase was repeated twice, and crude oil was extracted from the hexane phase to obtain 15 g of crude oil. The crude oil content in this extracellular matrix was 72%, and the hydrocarbon composition ratio of the extracted crude oil was 100%. The hydrocarbon recovery rate was 88%.
It was shown that the extraction method using an organic solvent can be used without drying treatment by going through the cell removal step.

Since the present invention relates to a method for producing an organic compound derived from microalgae such as hydrocarbons and alginans, it is used in industries such as polymer raw materials, cosmetic raw materials, lubricating oils, foods, biofuels, fermentation raw materials, feeds, feeds, and fertilizers. sell.

Claims (7)

A release process that causes cell release of microalgae contained in the microalgae slurry to obtain free cells and an extracellular matrix, and
An intercellular matrix recovery step of separating the free cells and the extracellular matrix to obtain the extracellular matrix, and
Using the cell-cell matrix obtained in the cell-cell matrix recovery step, an organic compound acquisition step of obtaining an organic compound derived from the microalgae, and an organic compound acquisition step.
A method for producing an organic compound containing. The method for producing an organic compound according to claim 1.
A method in which the organic compound comprises hydrocarbons and arginane. The method for producing an organic compound according to claim 1.
A method in which the microalgae are microalgaes belonging to the genus Botryococcus. The method for producing an organic compound according to claim 1.
The method, wherein the microalgae is a Botryococcus brownie or a Botryococcus brownie strain derived from Botryococcus brownie. The method for producing an organic compound according to claim 1.
The microalgae slurry has a water content of 75 to 99.9% by weight.
The release step comprises a step of freezing the microalgae slurry. The method for producing an organic compound according to claim 1.
The microalgae slurry has a water content of 75 to 99.9% by weight.
The release step comprises a step of heating the microalgae slurry at 50 ° C. or higher and 70 ° C. or lower for 5 minutes or longer and 5 days or shorter. The method for producing an organic compound according to claim 1.
The microalgae slurry has a water content of 75 to 99.9% by weight.
The method comprising the step of adding sodium chloride and / or both of sodium chloride and glycerol to the microalgae slurry.

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