JPH099953A - New fine algae belonging to genus botryococcus - Google Patents

Abstract

PURPOSE: To obtain a new Botryococcus braunii B race strain producing hydrocarbons mainly consisting of 30-33C hydrocarbons and containing above a specific weight ratio of a 30C hydrocarbon to the total of hydrocarbons. CONSTITUTION: Planktons on the surface of Ippeki lake (Shizuoka prefecture) are collected by using a Kitahara type plankton net with 25μm mesh. The prepared specimen is observed by a microscope and a colony of an alge of the genus Botryococcus is collected. The colony is cultured in a test tube, a single colony of the alga of the genus Botryococcus is raked by a platinum wire while observing characteristic properties of the alga of the genus Botryococcus in a colony state, etc., by using a stereoscopic microscope and cultured in a float plate medium to give a new Botryococcus braunii B race strain which produces hydrocarbons consisting mainly of 30C to 33C hydrocarbons and containing >=5wt.% 30C hydrocarbon based on the total of hydrocarbons and is Botryococcus braunii SI-1 strain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microalgae belonging to the genus Botryococcus, more specifically, a novel microalga that efficiently contains a hydrocarbon having 30 carbon atoms and has 30 to 33 carbon atoms. Botulio Coccus brownie
Regarding B race stock.

[0002]

2. Description of the Related Art In recent years, with global warming caused by CO ₂ becoming a problem, expectations are increasing for the use of microalgae having the ability to fix CO ₂ by light energy and convert it into hydrocarbons. Of the microalgae that fix CO ₂ by using light energy to produce hydrocarbons in this manner, the microalgae belonging to the genus Botryococcus are particularly excellent in the ability to produce hydrocarbons and are hydrocarbons based on the total amount of algal cells. The content thereof is remarkably high at 20 to 40% of the dry weight, and various studies have already been conducted to extract hydrocarbons from cultured alga bodies of microalgae belonging to the genus Botryococcus and utilize them effectively.

Among the above-mentioned microalgae belonging to the genus Botryococcus, the hydrocarbons produced by the algal bodies of the microalgae belonging to the B race of the genus Botryococcus are characteristic hydrocarbons collectively called Botryococcenes. There are various types of hydrocarbons produced and the content of individual hydrocarbons in the total produced hydrocarbons also varies depending on the strain of the microalgae belonging to the B race of the genus Botryococcus.

Here, in the currently known microalgae belonging to the Botryococcus brownie B race, most of the hydrocarbons produced are strains having carbon atoms of 34 or more, and most of them are carbonaceous strains. Strains containing no more than 34 hydrocarbons in the produced hydrocarbons and strains containing very few hydrocarbons are known, but these strains contain 3 hydrocarbons.
3 or 32 hydrocarbons are the main components in the produced hydrocarbons. Such a Botryococcus brownie
The hydrocarbons produced by the microalgae belonging to B race are purified and then used as fuel as they are, or the obtained hydrocarbons are used as a saturated hydrocarbon by a reduction reaction such as hydrogenation to be used as a cosmetic base. Although it can be used, a large amount of energy is required for distillation and the like required for purification because the hydrocarbon produced as described above has a large number of carbon atoms.

[0005] Therefore, in the microalgae belonging to the Botryococcus brownie B race excellent in the ability to produce hydrocarbons, the number of carbon atoms of the produced hydrocarbons is 30 for the purpose of saving the energy required for purification and the like.
A novel strain of microalgae belonging to the genus Botryococcus, which is composed of about 33 to about 33 hydrocarbons and more efficiently produces hydrocarbons having a small carbon number among these hydrocarbons, has been desired.

[0006]

SUMMARY OF THE INVENTION The present invention has been made from the above viewpoint, and is composed of hydrocarbons having about 30 to 33 carbon atoms. Among these hydrocarbons, hydrocarbons having smaller carbon number are more efficient. It is an object of the present invention to provide a novel strain of microalgae belonging to the genus Botryococcus which is produced in Japan.

[0007]

As a result of intensive studies to solve the above problems, the present inventor has found that the hydrocarbons produced by algae are mainly hydrocarbons having 30 to 33 carbon atoms. The present invention was completed by discovering a new strain of microalgae belonging to the genus Botuliococcus having a ratio of hydrocarbons having 30 carbon atoms to the total amount of hydrogen of 5% by weight or more.

That is, according to the present invention, the hydrocarbons produced by the alga are mainly hydrocarbons having 30, 31, 32 and 33 carbon atoms, and the ratio of the hydrocarbon having 30 carbon atoms to the total amount of the hydrocarbons is 5% by weight. We will provide new strains of Botulio Coccus Brownie B races as described above.

Hereinafter, the present invention will be described in detail. The hydrocarbons produced by the algal cells of the present invention are mainly those having 30 or 3 carbon atoms.
A new strain of Botryococcus braunii belonging to B-race consisting of 1, 32 and 33 hydrocarbons, wherein the proportion of hydrocarbons having 30 carbon atoms to the total amount of said hydrocarbons is 5% by weight or more, Microalgae belonging to the genus Botryococcus collected from lake water (hereinafter,
It is possible to separate from a sample containing "Botryococcus algae") using the following method.

The sample containing the algae of the genus Botryococcus is, for example, a plankton net (mesh: 10 μm) from the surface of fresh water (including ponds, swamps, etc.) and brackish water.
m-200 μm) or the like, but the sample is not particularly limited as long as it is a sample containing Botryococcus algae.

In order to isolate the novel strain of Botryococcus brownie B race of the present invention from the sample thus collected, first, the above-mentioned sample is treated with available chlorine so that microorganisms other than the alga of the genus Botryococcus To sterilize. At this time, the sample to be treated may be previously cultured in an appropriate medium to increase the amount of algal cells in order to facilitate the effective chlorine treatment and the subsequent treatment. Examples of the medium include CHU medium, JM medium, MDM medium and other inorganic media, but are not particularly limited as long as they are suitable for culturing Botryococcus algae. Further, the sample or the culture solution itself may be treated with available chlorine, or the algal cells or the concentrated solution obtained from the sample or the culture solution by centrifugation, filtration or the like.

In order to make available chlorine act on a sample containing the above-mentioned Botryococcus alga, for example, an aqueous solution of hypochlorous acid or an aqueous solution of hypochlorite such as sodium hypochlorite or calcium hypochlorite. A method of suspending the sample in (hereinafter, simply referred to as “hypochlorous acid”) can be mentioned. The concentration of hypochlorous acid used to process the sample is
Usually, the effective chlorine concentration is 0.01 to 1000 pp
m, preferably 0.1 to 100 ppm, and the treatment time is 0.1 to 1000 minutes, preferably 1 to 100 minutes. If this concentration is lower than 0.01 ppm or if the treatment time is shorter than 0.1 minutes, various bacteria may not be sterilized sufficiently. Further, if the concentration exceeds 1000 ppm or if the treatment time is longer than 1000 minutes, the Botryococcus alga itself may be killed.

When the culture solution of the above sample is treated with hypochlorous acid, hypochlorous acid may be added so as to have the above concentration, and when algal cells are treated with hypochlorous acid. For this purpose, the algal cells may be suspended in the hypochlorous acid aqueous solution diluted to the above concentration or in the hypochlorous acid diluted with the culture solution.

After treating the sample with hypochlorous acid as described above, the sample may be used as it is, but an operation of separating algal cells by filtration or centrifugation and suspending them in a culture solution or a buffer solution. It is preferable to remove the available chlorine by washing by repeating the above, or by adding sodium thiosulfate or the like. The amount of sodium thiosulfate added is preferably the amount required to remove all of the available chlorine resulting from the added hypochlorous acid.

Subsequently, the hypochlorous acid-treated sample is treated with a plate medium suitable for culturing Botryococcus algae, for example, an inorganic medium such as CHU medium, JM medium, MDM modified medium, or the like. The culture is performed by applying the medium to a medium in which an organic carbon source such as glucose, citric acid, and acetic acid is added. An antibiotic or the like that does not affect the growth of Botryococcus alga may be added to the medium used here. When the amount of application is too large when applying to the plate medium, colonies formed on the plate medium come close to each other and it becomes difficult to separate the colonies. Therefore, it is advisable to apply the diluted solution in several steps.

The culture is preferably performed under light irradiation using a fluorescent lamp or the like. However, if the irradiation illuminance is too high, the growth may be hindered. Therefore, the culture is usually 0 to 300 μE / m.
Irradiation is performed at ² · s, preferably 5 to 100 μE / m ² · s. The culture temperature is usually 0 to 80 ° C,
It is preferably 15 to 35 ° C.

The culture is usually carried out for 1 to 60 days, preferably 1 to
Do 30 days. A certain culture period is required to determine whether the colonies generated during the culture are colonies of remaining bacteria or a single colony of Botryococcus algae, and if the culture period is too long Since various bacteria grow, it is preferable to culture within the above period, but since it varies depending on the medium, the culture conditions, the degree of remaining of various bacteria, etc., colonies may be observed during the culture and the culture may be appropriately stopped.

By collecting a single colony of Botryococcus algae formed on the plate medium as described above, various purified strains of Botryococcus algae can be obtained. When separating colonies from the plate medium, use a microscope,
It is preferable to carry out the observation under a stereoscopic microscope, since it can be collected while confirming that it is a colony of Botryococcus alga. The method of collecting colonies is not particularly limited,
For example, it may be scraped with a sterilized platinum wire or sucked up with a Pasteur pipette. At this time, in order to prevent contamination by airborne bacteria, it is advisable to carry out the operation in a sterile room or cleave bench.

It is possible to confirm that these are purified strains by culturing again the strains isolated as described above and confirming that the germs do not grow. Furthermore, it is advisable to confirm that the obtained purified strain is an alga of the genus Botryococcus by microscopic observation or the like.

Purified strains of Botryococcus alga obtained in this manner are separated, and each purified strain is
The hydrocarbons produced by the algal bodies of these purified strains are analyzed by the following method, and the hydrocarbons produced by the algal bodies consist mainly of hydrocarbons having 30, 31, 32, and 33 carbon atoms, and A new strain of the genus Alga of the genus Botryococcus of the present invention can be obtained by screening a strain having a hydrocarbon ratio of 30 carbon atoms of 5% by weight or more. Here, in the present invention, "hydrocarbons mainly have 30 carbon atoms,
"Comprising 31, 32, and 33 hydrocarbons" means that the proportion of the total amount of hydrocarbons having 30, 31, 32, and 33 carbon atoms in the total amount of hydrocarbons produced by the alga is approximately 90% by weight.
The above is shown.

As a method for analyzing the hydrocarbons produced by the algal bodies of Botryococcus algae, conventionally used methods may be used. For example, the algae are grown by culturing Botryococcus algae. Then, the wet algal cells taken out from the obtained culture solution by filtration and the like are freeze-dried or heated and dried, and then a fixed amount of n-pentacosane or the like is added as an internal standard to the dried algal cells, and n-hexane is added. , Methanol-chloroform (1: 1) soaks in an extraction solvent to extract hydrocarbons (Phytochemistry, vol.19,1081-1085,1980),
Then, the algal cells and the elution solvent were removed, and the extracted hydrocarbons were analyzed by NMR, IR, gas chromatography and GC.
-A method such as MS may be used for analysis.

The hydrocarbons produced by the alga in this manner are mainly composed of hydrocarbons having 30, 31, 32 and 33 carbon atoms, and the ratio of the hydrocarbon having 30 carbon atoms to the total amount of hydrocarbons is 5% by weight or more. The strains of the genus Alga of the genus Botryococcus of the present invention can be obtained by screening certain strains. These strains are identified by examining their taxonomic properties. Regarding the hydrocarbon production capacity, the above characteristics (hydrocarbons produced by algae are mainly hydrocarbons having 30, 31, 32 and 33 carbon atoms, and the ratio of hydrocarbons having 30 carbon atoms to the total amount of hydrocarbons is 5% by weight or more. The algae of the genus Botryococcus of the present invention having the following) was confirmed to be a strain belonging to Botryococcus brownie B race by the investigation of such taxonomic properties. Further, the above-mentioned characteristics of the hydrocarbon produced by the alga are not found in other strains of Botryococcus brownie B race found so far, as will be described later. Botulio Coccus
It can be said that the Brownie B race strain is new.

The hydrocarbons produced by such algal cells are mainly hydrocarbons having 30, 31, 32 and 33 carbon atoms, and the ratio of the hydrocarbons having 30 carbon atoms to the total amount of hydrocarbons is 5% by weight or more. Among the Botryococcus brownie B race strains of the present invention, the ratio of hydrocarbons having 30 carbon atoms to the total amount of hydrocarbons produced by algal cells is 1
Botryococcus brownie B with 0% by weight or more
Race strains are more preferred in the present invention.

As an example of such a Botryococcus brownie B race strain of the present invention, there can be mentioned the Botryococcus brownie SI-1 strain isolated in the below-mentioned Example. 1
The strain is a novel strain obtained by the present invention for the first time, and the hydrocarbon produced by the alga of this strain has 30 carbon atoms,
The ratio of the total amount of hydrocarbons of 31, 32, and 33 to the total amount of hydrocarbons is generally 95% by weight or more, and the ratio of the hydrocarbon having 30 carbon atoms to the total amount of hydrocarbons is generally 10 to 10.
It is 30% by weight and can be said to be more preferable in the present invention. Although the deposit of this strain to the Institute of Biotechnology, Institute of Biotechnology, AIST was refused, the distribution of the strain to confirm the establishment of the present invention is guaranteed here.

[0025]

EXAMPLES Examples of the present invention will be described below.

[0026]

Example 1 Planktons on the surface of lake water of Lake Ippiki (Shizuoka Prefecture) were collected using a Kitahara type plankton net having a mesh of 25 μm. When the obtained sample was observed under a microscope, colonies having a morphology characteristic of Botryococcus algae (oil droplets were secreted from the colonies after 10 to 30 minutes under the microscope) were observed.

The lake water sample was divided into four test tubes,
Effective chlorine concentration for each is 1ppm, 5ppm, 10p
Sodium hypochlorite was added to pm of 20 ppm, and the mixture was allowed to stand at room temperature for 10 minutes. After standing, sodium thiosulfate was added to remove active chlorine so that the amount of 7.16 mg was 1 mg of active chlorine.

The above treatment solution was applied to a plate medium of JM medium containing glucose 0.1% and agar 1.5%, and irradiated with a fluorescent lamp as a light source at about 15 μE / m ² · s,
The cells were cultured at 25 ° C for one week. The composition of the JM medium is as follows. [Composition of JM medium] Stock solutions 1 to 9 in Table 1 below
Mix each mL and use distilled or deionized water to 1 L
And adjust the pH to around 7.

[0029]

[Table 1]

After culturing, a single colony of Botryococcus algae was scratched with a platinum wire while observing the characteristics peculiar to Botryococcus algae such as the colony shape using a stereoscopic microscope (20 times magnification) in a clean bench. It was taken and transferred to a JM plate medium containing glucose 0.1% and agar 1.5%. This was cultured at 25 ° C. for 4 weeks under irradiation with light of about 15 μE / m ² · s by a fluorescent lamp.

The algae of Botryococcus algae obtained above were scraped using a platinum wire and transferred to a JM liquid medium,
The cells were further cultured under light irradiation for 2 weeks. Take a part of the culture solution with a platinum loop and use a normal agar medium (meat extract: 5 g in 1 L,
Peptone: 10 g, sodium chloride: 5 g, agar: 15
g; pH 7.0), and cultured at 25 ° C. for 7 days to confirm that no bacteria grow. In addition, microscopic observation of the purified strain obtained in this way revealed that only Botryococcus brownies showed the unique properties of the algae, such as colony shape (a pear-shaped cell containing chloroplasts in the cells was collected. An approximately spherical colony was formed, and the colony was further observed under a microscope to produce oil droplets.)

The purified strain thus obtained was designated as Botulinum coccus brownie SI-1 strain. The lake water sample was directly applied to a plate (JM medium and JM medium containing glucose) and cultured, but no colonies of Botryococcus alone were obtained.

Next, the Botryococcus
The Brownie SI-1 strain was inoculated into a JM medium containing glucose and cultured for 5 months while repeating subculture.
CH 2 whose composition is shown in Table 2 is obtained from the algal cells obtained by this solid culture.
A flat flask containing 0.3 L of U-13 × 2 medium (pH 7) was inoculated, and liquid culture was carried out for 8 days (culture conditions; illuminance: 2
80 μE / s · m ² , ventilation rate: 0.5 vvm (5% C
O ₂ ), temperature 25 ° C.).

[0034]

[Table 2]

[0035]

[Table 3]

The obtained algal cells were dried at 105 ° C., and hydrocarbons were extracted 3 times with 50 times the amount of n-hexane. After concentrating the n-hexane phase, the hydrocarbon was purified by silica gel column chromatography. The hydrocarbon fraction eluted with n-hexane was concentrated, and it was collected by NMR, IR, G
It analyzed using C-MS. In the NMR analysis, a peak derived from an unsaturated proton was observed at 4.4 to 6 ppm, and a peak derived from a saturated proton was observed at 0.6 to 2.7 ppm.
The ratio of saturated to unsaturated protons was approximately 4: 1. In IR, absorption derived from the double bond was observed at 3080 cm ^-1 and 1640 cm ^-1 . Others, CH bond, CC
Since no bond other than the bond was observed, it was confirmed to be hydrocarbon.

Next, according to GC-MS, the parent ions of the peaks observed in GC were 410, 424, 434, and 446. Therefore, it was confirmed that the botulinum Coccus brownie B races having 30, 31, 32, and 33 carbon atoms were obtained. It was confirmed to be a characteristic hydrocarbon, and from this, the Botryococcus brownie SI-1 strain was identified as a botulococcus brownie B race. Further, the composition of the hydrocarbon produced by the Botryococcus brownie SI-1 strain by the liquid culture was 26% by weight of hydrocarbons having 30, 31, 32, and 33 carbon atoms by the GC-MS analysis, respectively. %, 44% by weight, 15% by weight, and it was found that a hydrocarbon having a carbon number of 34 was also contained in a small amount.

[0038]

Example 2 Botryococcus brownie SI-1 strain obtained in the same manner as in Example 1 was inoculated into a JM medium containing glucose and cultured for 4 weeks. After scraping off the algal cells obtained by this solid culture and drying this at 105 ° C.,
The dried algal cells were extracted 3 times with n-hexane. After n-hexane was concentrated and removed from the extracted solution, it was analyzed by gas chromatography. A phenylmethylsilicon-based capillary column (25 m) was used as the gas chromatography column. As a result, the composition of the hydrocarbons produced in this culture was 11% by weight, 6% by weight, 61% by weight of hydrocarbons having 30, 31, 32 and 33 carbon atoms, respectively.
It was found to be 22% by weight.

[0039]

[Example 3] In the same manner as in Example 1 above, the botulinum Coccus brownie SI-1 strain was mixed with JM containing glucose.
After solid-cultured in the medium, the obtained algal cells were liquid-cultured in CHU-13 × 2 medium (pH 7) having the composition shown in Table 2 above. The obtained algal cells were mixed with CHU-13 × 2 medium (pH 7) 3
The mixture was inoculated into a stirred culture tank containing L and further liquid-cultured for 8 days (culture conditions; illuminance: 180 μE / s · m ² , aeration amount:
0.5 vvm (5% CO ₂ ), temperature 25 ° C.).
10 mL of this culture broth was taken out, the algal cells were collected using a filter paper, and then dried at 105 ° C. N- was added to the obtained dried algal cells.
Hexane was added and extraction treatment was performed 3 times. The extract was concentrated and then analyzed by gas chromatography. As a result, the composition of the hydrocarbons produced in this culture has 30 and 3 carbon atoms.
Hydrocarbons of 1, 32 and 33 are 16% by weight and 15% respectively
It was found to be wt%, 52 wt% and 17 wt%.

The results of analysis on the composition of hydrocarbons produced by the Botryococcus brownie SI-1 strain of the present invention in the culture of Examples 1 to 3 above were analyzed and the results of other Botryococcus brownie B race strains were produced. It is shown in Table 4 together with the hydrocarbon analysis values (reference values).

[0041]

[Table 4]

From the above, in the hydrocarbon produced by the alga of the above-mentioned Botryococcus brownie SI-1 strain obtained by the present invention, the number of carbon atoms is 30, 31, 32.
And the proportion of the total amount of hydrocarbons 33 and 33 in the total amount of hydrocarbons is 97% by weight or more, and the proportion of the hydrocarbons having 30 carbon atoms in the total amount of hydrocarbons is 11 to 26% by weight. all right. Such a feature in the produced hydrocarbon of the Botryococcus brownie SI-1 strain of the present invention is not found in other strains of the Botryococcus brownie B race found so far, as can be seen from the table. Not possible, Botulio Coccus Brownie SI
It can be said that the -1 strain is a novel strain obtained for the first time by the present invention.

[0043]

INDUSTRIAL APPLICABILITY In the novel Botryococcus brownie B race strain of the present invention, the hydrocarbons produced by the alga of this strain are mainly hydrocarbons having 30, 31, 32 and 33 carbon atoms, The ratio of hydrocarbons having 30 carbon atoms is 5% by weight or more, which is a characteristic not found in the hitherto known Botryococcus brownie B race strains. Therefore, the Botryococcus of the present invention
By using the Brownie B race strain, it becomes possible to efficiently produce a hydrocarbon having 30 to 33 carbon atoms, which contains a large amount of hydrocarbon having 30 carbon atoms.

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Claims (2)

[Claims] 1. The hydrocarbon produced by the alga is composed mainly of hydrocarbons having 30, 31, 32 and 33 carbon atoms, and the ratio of the hydrocarbon having 30 carbon atoms to the total amount of said hydrocarbons is 5% by weight or more. Botulio Coccus Brownie B
Race stock. 2. A Botryococcus brownie SI-
The Botryococcus brownie B race strain according to claim 1, which is one strain.