JPH09234055A - New fine alga belonging to genus botryococcus and its culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new strain of fine alga belonging to the genus Botryococcus braunii A race capable of producing a 33C hydrocarbon and to provide a method for efficiently culturing the strain. SOLUTION: This strain of Botryococcus braunii A race is a new fine alga belonging to the genus Botryococcus. A hydrocarbon produced by the alga of this strain contains a 33C hydrocarbon. The new strain of the fine alga belonging to the genus Botryococcus braunii A race is cultured under intermittently irradiating the strain with an artificial light preferably once daily for 5-15 hours.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microalgae belonging to the genus Botryococcus and a method for culturing the same, and more specifically, a novel Botryococcus brownie A race having an ability to produce a hydrocarbon having 33 carbon atoms. The present invention relates to a strain and an efficient culture method thereof.

[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, Botryococcus brownie A
The microalgae belonging to the race have the characteristic that they contain a large amount of straight-chain alkadienes and alkatrienes in the hydrocarbons produced by the algal bodies, but the strains of the microalgae belonging to the Botulinum coccus brownie A race also differ. The type of hydrocarbons produced and the content of individual hydrocarbons in the total hydrocarbons produced vary. It is known that all of the microalgae belonging to the currently known Botryococcus brownie A race, regardless of the strains, are all hydrocarbons produced by the algal cells having 32 or less carbon atoms. .

On the other hand, hydrocarbons having 33 carbon atoms, particularly straight chain alkadienes and alkatrienes having 33 carbon atoms, are effectively used as raw materials for agricultural chemicals, pharmaceuticals, food additives, physiologically active substances, etc., in addition to being used as fuels. It is attracting attention as a usable hydrocarbon, and it is expected that a hydrocarbon having 33 carbon atoms will be produced by using microalgae belonging to the botulinum Coccus brownie A race that produces a large amount of straight chain alkadienes and alkatrienes. As mentioned above, a strain of microalgae belonging to the Botulinum coccus brownie A race producing a hydrocarbon having 33 carbon atoms has not been known so far.

[0005]

The present invention has been made from the above point of view, and is a novel strain of microalgae belonging to the Botulinum coccus brownie A race having the ability to produce a hydrocarbon having 33 carbon atoms, and It is an object to provide an efficient culture method therefor.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that the hydrocarbons produced by algae are botulinum Coccus brownie containing 33-carbon hydrocarbons. A new strain of microalgae belonging to A race was found, and the culture efficiency was improved by culturing such a new strain of microalgae belonging to A race A while intermittently irradiating with artificial light. Then, the present invention has been completed.

[0007] That is, the present invention provides a novel strain of microalgae belonging to the Botryococcus brownie A race in which the hydrocarbon produced by the alga contains a hydrocarbon having 33 carbon atoms.

Further, the present invention relates to a novel strain of microalgae belonging to the Botryococcus brownie A race of the present invention, wherein the hydrocarbon having 33 carbon atoms is a straight-chain hydrocarbon, and is a Botryococcus brownie A race strain, And
Of these new strains, there is further provided a Botryococcus brownie A race strain in which the hydrocarbon having 33 carbon atoms contains an alkadiene and / or an alkatriene.

Further, the present invention provides a specific strain of a new strain of microalgae belonging to such Botryococcus brownie A race, which is Botryococcus brownie S.
Provide I-30 strain.

The present invention also relates to the above botulinum coccus, which comprises culturing while intermittently irradiating artificial light.
A method for culturing a new strain of microalgae belonging to Brownie A race is provided.

In the culturing method of the present invention, as a specific method of intermittently irradiating with artificial light, light irradiation once a day is preferably performed for about 5 to 15 hours, more preferably about 15 to 15 hours. Can be carried out for about 7 to 13 hours. Other conditions relating to artificial light to be irradiated, or culture conditions other than light irradiation, such as culture medium, aeration and temperature, may be substantially the same as those of the conventional microalgae belonging to the Botryococcus brownie A race. When the culture is carried out while aerating CO ₂ -added air, the growth of microalgae belonging to the known Botryococcus brownie A race is C
In contrast to being easily affected by the O ₂ concentration, in the culture of the novel microalgae belonging to the genus Botryococcus of the present invention, the aeration control of the culture is relatively easy because the CO ₂ concentration is not significantly affected. is there.

According to such a culturing method of the present invention, it is possible to efficiently cultivate the microalgae belonging to the novel Botryococcus genus of the present invention.

[0013]

Embodiments of the present invention will be described below. (1) Botryococcus brownie A novel strain of microalgae belonging to A race Botryococcus brownie of which the hydrocarbon produced by the alga contains a hydrocarbon having 33 carbon atoms (Botryo)
coccus braunii) A new strain of microalgae belonging to A race is a microalgae belonging to the genus Botryococcus collected from lake water etc. (hereinafter referred to as "Botryococcus algae").
It is possible to separate from the sample containing the following using the following method.

The sample containing the algae of the genus Botryococcus is, for example, a plankton net (mesh: 1 μm) from the surface of fresh water (including ponds, swamps, etc.) and brackish water.
To about 1000 μm, preferably 10 μm to 200 μm
It can be collected by pulling the plankton net so that it does not sink, but it is not particularly limited as long as it is a sample containing Botryococcus algae.

In order to isolate the novel strain of Botryococcus brownie A race of the present invention from the sample thus collected, first, effective chlorine is allowed to act on the above-mentioned sample, and 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 was 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 carried out by applying to a medium in which sugars such as glucose, mannose and fructose and organic acids such as citric acid and acetic acid are added to the medium as an organic carbon source. 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 culturing is preferably carried out under light irradiation using a fluorescent lamp or the like. However, if the irradiation illuminance is too high, the growth may be hindered, so that it 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. In addition, Botryococcus algae form colonies in which pear-shaped cells containing chloroplasts in the cells are aggregated. Further, oil droplets may be generated when the colony is observed under a microscope.

The method for collecting the colonies is not particularly limited, and 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 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.

The purified strains of Botryococcus alga obtained in this way are separated, and each purified strain is
Analysis of the hydrocarbons produced by the algal bodies of these purified strains was carried out by the following method, and the number of carbon atoms contained in the hydrocarbons produced by the algal bodies was 33.
Having the following hydrocarbons, and the produced hydrocarbons have the taxonomic characteristics of strains belonging to the Botryococcus brownie A race (the produced hydrocarbons are mainly linear olefins having 27, 29, and 31 carbon atoms). By screening the strains, the novel strain of Botryococcus alga of the present invention can be obtained.

As a method for analyzing the hydrocarbons produced by the algal bodies of Botryococcus alga, conventionally used methods may be used. For example, the alga bodies are grown by culturing Botryococcus algae. Then, the wet algal cells taken out from the resulting culture solution by filtration or the like are freeze-dried or heated and dried, and then the dried algal cells are extracted with an extraction solvent such as n-hexane or methanol-chloroform (1: 1). Hydrocarbons are extracted by immersion (Phytochemistry, vol.19,1081-1085,198
0) After that, the method of removing the algal cells and the eluting solvent and analyzing the extracted hydrocarbon by NMR, IR, gas chromatography, GC-MS or the like may be used.

Thus, a strain containing a hydrocarbon having 33 carbon atoms in the hydrocarbon produced by the alga and having a taxonomic characteristic of the strain in which the produced hydrocarbon belongs to the Botulinum coccus brownie A race is selected. By screening, the strain of the genus Botryococcus of the present invention can be obtained.

The Botryococcus alga of the present invention has the taxonomic characteristics of the strain belonging to the Botryococcus brownie A race with respect to the hydrocarbon-producing ability as described above,
That is, the Botryococcus alga of the present invention is a strain belonging to the Botryococcus brownie A race,
In addition, it is characterized in that the hydrocarbon produced by the alga contains a hydrocarbon having 33 carbon atoms. The above-mentioned characteristics regarding the hydrocarbon-producing ability of the Botryococcus brownie A race strain of the present invention (the hydrocarbon produced by the alga contains a hydrocarbon having a carbon number of 33) can be observed as described below. It is not found in any other Botryococcus brownie A race strains that have been issued, and the Botryococcus
It can be said that the Brownie A race stock is new.

Among the Botryococcus brownie A race strains in which the hydrocarbons produced by such algal cells contain hydrocarbons having 33 carbon atoms, the hydrocarbons having 33 carbon atoms are linear hydrocarbons. Strains and, among these strains, strains in which the hydrocarbon having 33 carbon atoms contains alkadiene and / or alkatriene are more preferable in the present invention from the viewpoint of usefulness of the produced hydrocarbon.

As an example of such a Botryococcus brownie A race strain of the present invention, there can be mentioned the Botryococcus brownie SI-30 strain isolated in the following embodiment. This Botulio Coccus Brownie S
The I-30 strain is a novel strain obtained for the first time by the present invention, and the hydrocarbons produced by the algal cells of this strain contain approximately 2 to 45% of hydrocarbons having 33 carbon atoms. The hydrocarbons are all straight chain alkadienes or straight chain alkatrienes and can be said to be preferred strains in the present invention.

Although the deposit of this strain to the Institute of Biotechnology, Institute of Biotechnology, AIST was refused, the distribution of this strain for confirmation of the establishment of the present invention is guaranteed here. is there.

(2) Method for culturing a new strain of microalgae belonging to Botryococcus brownie A race of the present invention The method of culturing a new strain of microalgae belonging to Botulinum coccus brownie A race of the present invention is It is characterized by culturing while intermittently irradiating with artificial light in order to efficiently perform growth.

In the culture method of the present invention, artificial light is intermittent during the culture, specifically once a day, preferably for about 5 to 15 hours, more preferably for about 7 to 13 hours. Irradiated at a rate. As a light source of artificial light, a light source normally used for culturing Botryococcus algae, for example, an incandescent lamp or a fluorescent lamp may be used. The irradiation illuminance may be generally the same as when culturing Botryococcus algae, specifically, 20 to 800 μE / m.
The irradiation illuminance is about ² · s, preferably about 40 to 500 μE / m ² · s.

The culturing method of the present invention is generally carried out in the same manner as described above except that the artificial light is intermittently irradiated.
A method similar to that for culturing Botryococcus alga can be used. For example, in the culture method of the present invention, as a medium, an inorganic medium such as CHU medium, JM medium, or MDM modified medium, or a sugar such as glucose, mannose, fructose or the like as an organic carbon source, citric acid, acetic acid or the like is added to these inorganic mediums. Usually, the same medium as that used for culturing Botryococcus alga can be used, such as a medium containing an organic acid. Similarly, the culture temperature is usually 0 to 80 ° C, preferably about 15 to 35 ° C. Examples of the culturing method include ordinary methods such as static culturing and shaking culturing.

In the process of culturing the present invention, when performing the aeration media, CO ₂ concentration of about 0.5 to 20% CO ₂
It is preferable to pour the added air into a ventilation amount of about 0.01 to 1 vvm. Further, in the culture method of the present invention, during the time when the artificial light irradiation is not performed, it may be aerated,
It is preferred not to vent.

In the culture of microalgae belonging to the previously known Botryococcus brownie A race, the CO ₂ concentration during aeration is set within the above range of 0.5 to 2
When it is 0%, the growth amount of the alga body tends to decrease in the high concentration range, whereas in the culture of the microalgae belonging to the novel Botryococcus genus of the present invention, the growth amount of the alga body is the CO ₂ concentration. It was confirmed in the examples described later that it was not significantly affected by. This proves that the Botryococcus brownie A race strain of the present invention is novel,
Because of this feature, it can be said that the control of aeration during the cultivation of the novel microalgae belonging to the genus Botryococcus of the present invention is easier than that of conventional strains.

[0037]

[Examples] Examples of the present invention will be described below using Botryococcus brownie SI-30 strain as an example.

[0038]

[Example 1] Botryococcus brownie SI-
Acquisition of 30 strains (1) Sampling of lake water Planktons on the lake surface of Mannoike (Kagawa Prefecture) were collected using a Kitahara type plankton net with a mesh of 25 µm. Observation of the obtained sample with a microscope revealed that colonies with a characteristic morphology of Botryococcus algae (oil droplets secreted from the colonies after 10 to 30 minutes under the microscope)
Was observed.

(2) Botryococcus brownie S
Isolation of strain I-30 The above lake water sample was divided into four test tubes, and the effective chlorine concentration of each was 1 ppm, 5 ppm, 10 ppm, 20 p.
Sodium hypochlorite was added to reach pm, and the mixture was allowed to stand at room temperature for 10 minutes. After standing, sodium thiosulfate was added to remove effective chlorine by 7.16 per 1 mg of effective chlorine.
It was added to be mg.

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] 1 mL each of stock solutions 1 to 9 in Table 1 below is mixed, and the volume is adjusted to 1 L with distilled water or deionized water, and the pH is adjusted to around 7.

[0042]

[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 colony shape using a stereoscopic microscope (magnification: 20) 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 Botryococcus algae obtained above was scraped off with a platinum wire, transferred to a JM medium liquid medium, and further cultured under light irradiation for 2 weeks. Take a portion of the culture solution with a platinum loop and use a regular agar medium (1 L of meat extract:
5 g, peptone: 10 g, sodium chloride: 5 g, agar: 15 g, and applied to a plate medium of pH 7.0),
After culturing at 25 ° C. for 7 days, it was confirmed that various bacteria did not grow. In addition, microscopic observation of the purified strain thus obtained showed that only Botryococcus brownie had the unique characteristics of the algae such as colony shape (the pear-shaped cells containing chloroplasts in the cells gathered). And form colonies.
Furthermore, when the colony is observed under a microscope, oil drops are produced. ).

The purified strain thus obtained was designated as Botryococcus brownie SI-30 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.

(3) Botryococcus brownie S
Identification of strain I-30 Next, the Botryococcus brownie obtained above was obtained.
The SI-30 strain was inoculated into a JM medium containing glucose and cultured for 5 days while repeating subculture. The algal cells obtained by this plating are CHU-13 × 2 whose composition is shown in Table 2.
A flat flask containing 0.3 L of the medium (pH 7) was inoculated, and liquid culture was carried out for 8 days (culture conditions; illuminance: 350 μE /
s ・ m ² , aeration rate: 0.5 vvm (5% CO ₂ ), temperature 2
5 ° C.).

[0047]

[Table 2]

[0048]

[Table 3]

Alga bodies were collected from the culture solution obtained by the above liquid culture using filter paper, and the alga bodies were dried at 105 ° C. With respect to this dried alga, hydrocarbons were extracted three 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 this was analyzed using NMR, IR, and GC-MS.

In the NMR analysis, a peak derived from unsaturated proton was observed at 4.8 to 6.2 ppm, and a peak derived from saturated proton was observed at 0.7 to 2.4 ppm. The ratio of saturated protons to unsaturated protons was approximately 10: 1. IR
In, 3080cm ^-1, absorption of from 1650 cm ^-1 double bond, 2950 cm ^-1, the absorption derived from CH bonds to 2850 cm ^-1 were observed respectively. In addition, CH bond,
Since no absorption other than CC bond was observed, it was confirmed to be hydrocarbon.

From GC-MS analysis, 404, 43
Parent peaks of 2, 458, and 460 were observed, and these peaks were each alkadiene (C2
₉ H ₅₆ ), 31 alkadienes (C ₃₁ H ₆₀ ), 33 alkatrienes (C ₃₃ H ₆₂ ), 33 alkadienes (C ₃₃
The peak was determined to represent H ₆₄ ).

Further, in the NMR analysis, the peak derived from the methyl group was 0.78 to 1 ppm, the peak derived from the methylene group was 1.05 to 1.6 ppm, and the peak derived from the proton of the carbon adjacent to the double bond was 1. 0.82 to 2.2 ppm,
3. The peak derived from the proton bonded to the unsaturated carbon is 4.
4 kinds of peaks, namely, a peak derived from a methyl group, a peak derived from a methylene group, a peak derived from a proton of a carbon adjacent to a double bond, and a peak derived from a proton bonded to an unsaturated carbon, which are observed at 7 to 6.1 ppm, respectively. The area ratio is 3:
It was 5: 44: 6. In addition, it was confirmed from the molecular weight measurement results obtained by GC-MS analysis that there was only one methyl group. Therefore, these hydrocarbons were identified as straight-chain hydrocarbons having a double bond at the end.

As a result of such an analysis, it was found that Botryococcus
Regarding the above-mentioned hydrocarbons produced by the Brownie SI-30 strain, the characteristic properties of the hydrocarbons produced by the microalgae belonging to the Botryococcus brownie A race were confirmed, and from this, it was confirmed that Botryococcus brownie S
The strain I-30 was identified as Botryococcus brownie A race.

[0054]

Example 2 The Botryococcus brownie strain SI-30 obtained in Example 1 above was inoculated into a JM medium containing glucose and cultured for 5 days while repeating subculture. The algal cells obtained by this plate culture were inoculated into 0.3 L of CHU-13 × 2 medium (pH 7) placed in a flat flask, and liquid culture was carried out for 8 days (culture conditions; illuminance: 350 μE /
s ・ m ² , aeration rate: 0.5 vvm (5% CO ₂ ), temperature 2
5 ° C.). 5 ml of the obtained culture broth was taken out and the algal cells were collected using a filter paper, which was dried at 105 ° C. Then, a solution 1 in which n-pentacosane was dissolved in hexane at a rate of 1 g / L as an internal standard in this dried alga
After adding ml, the mixture was extracted 3 times with n-hexane. The obtained n-hexane phase was concentrated and then analyzed by gas chromatography. A phenylmethylsilicon-based capillary column (25 m) was used as a gas chromatography column. The composition of the hydrocarbon produced by the Botryococcus brownie strain SI-30 in the above liquid culture was confirmed by gas chromatography analysis.

For comparison, instead of the algal cells obtained by plating the Botryococcus brownie SI-30 strain used in Example 2 above, instead of the Botryococcus brownie CCAP807 / 2 purified strain (comparison Example 1)
Liquid culture was performed in exactly the same manner as in Example 1 except that the above was used, or in the same manner as in Example 1 except that the purified Botryococcus brownie Austin strain (Comparative Example 2) was used. The hydrocarbons produced by the respective strains were extracted from the obtained algal cells, and the composition of the hydrocarbons was confirmed by the same method (gas chromatography analysis) as in Example 2 above.

The results are shown in Table 4 below together with the results of other examples and the composition of hydrocarbons produced by the conventionally known Botryococcus brownie A race strain.

[0057]

[Example 3] 0.3 L of CHU-13 x 2 placed in a flat flask was prepared by substituting the algal cells of the Botryococcus brownie strain SI-30 subcultured in the same manner as in Example 2 above.
Inoculation into a medium (pH 7), liquid culture for 5 days (culture conditions; illuminance: 350 μE / s · m ² , aeration: 0.5 vv
m (5% CO ₂ ), temperature 25 ° C.). The alga bodies obtained by this culture were treated in the same manner as in Example 2 above, and in this liquid culture, Botryococcus brownie SI-3 was used.
The hydrocarbon produced by 0 was extracted, and the composition of the obtained hydrocarbon was confirmed by the same method as in Example 2 (gas chromatography analysis). The results are shown in Table 4.

[0058]

[Example 4] 10 L of CHU-1 was prepared by placing the alga of Botryococcus brownie SI-30 strain subcultured in the same manner as in Example 2 in an acrylic culture device.
3 × 2 medium (pH 7) was inoculated, and liquid culture was carried out for 10 days (culture conditions; illuminance: 350 μE / s · m ² , aeration amount:
0.5 vvm (5% CO ₂ ), temperature 25 ° C.).
The alga body obtained by this culture is treated in the same manner as in Example 2 above, and the hydrocarbon produced by Botryococcus brownie SI-30 is extracted in this liquid culture, and the composition of the obtained hydrocarbon is as described above. It was confirmed by the same method (gas chromatography analysis) as in Example 2. The results are shown in Table 4.

[0059]

Example 5 The algal cells of the Botryococcus brownie strain SI-30 subcultured in the same manner as in Example 2 above were inoculated into a JM medium containing glucose, and static culture was carried out for 49 days (culture conditions). Illuminance: 30 μE / s · m ² , temperature 2
5 ° C.). The alga obtained by this culturing was scraped off with a platinum loop and diluted with 5 ml of sterile water, and this was treated in the same manner as the 5 ml taken out from the culture solution in Example 2 above, and this static culturing was carried out. Rio Coccus Brownie S
The hydrocarbon produced by I-30 was extracted, and the composition of the obtained hydrocarbon was confirmed by the same method as in Example 2 (gas chromatography analysis). The results are shown in Table 4.

Table 4 shows the results of analysis of the composition of hydrocarbons produced by the Botryococcus brownie strain SI-30 of the present invention in the cultures of Examples 2 to 5 and the cultures of Comparative Examples 1 and 2 above. The analysis results of the composition of the hydrocarbons produced by the conventionally known Botryococcus brownie A race strain and the analysis value of the hydrocarbon produced by the conventionally known Botryococcus brownie A race strain ( It is a table showing (reference value).

[0061]

[Table 4]

From these results, unlike the other strains of Botryococcus brownie A race found so far, which produce no hydrocarbon having 33 carbon atoms as shown in the table, it was obtained by the present invention. The hydrocarbon produced by the alga body of the above-mentioned Botryococcus brownie SI-30 strain contains 4.7 to 33.8% by weight of a hydrocarbon having 33 carbon atoms.
You can see that it contains. In addition, it is known from the above analysis results that the C 33 hydrocarbons produced by the alga of the Botryococcus brownie SI-30 strain are all linear alkadienes or linear alkatrienes.

Botryococcus brownie like this
The characteristics of the hydrocarbons produced by the SI-30 strain are as follows: The conventionally known Botryococcus brownie A
It is not found in race strains, and it can be said that the Botryococcus brownie SI-30 strain is a novel strain obtained for the first time by the present invention.

[0064]

[Example 6] 3.0 in an agitated culture tank of external / internal reflection type
L of CHU-13 × 2 medium (pH 7) was added for sterilization. This medium was inoculated with the algal cells of the Botryococcus brownie SI-30 strain subcultured in the same manner as in Example 2, and the liquid culture was performed for 7 days (culture conditions; illuminance: external illuminance was 140 μE / s. m ² , internal illuminance of 360 μE
/ S · m ² , aeration rate: 0.5 vvm (5% CO ₂ ), temperature 25 ° C., stirring speed: 100 rpm). The concentration of the algal cells in the culture solution of the algal cells obtained by this culture was measured by the dry weight method (3 ml of the culture solution was collected, and the algal cells were collected by filtration, and the temperature was adjusted to
After 1 hour of drying, the weight of the algal cells is measured and the concentration of the algal cells is determined), and the concentration is measured every day during the culturing period, and from the obtained algal cell concentration, Botryococcus brownie SI-30 is obtained.
The amount of growth of the strain algal cells per day was calculated. Further, in the above-mentioned culture, the same culture was performed except that the CO ₂ concentration was changed to 10% or 15%, and in the same manner as above, the Botryococcus brownie SI-30 strain alga body per day in each culture was obtained. Was calculated. Also, for comparison, Botryococcus brownie CCAP80
The same culture test was performed using the 7/2 purified strain. Table 5 shows the results.

[0065]

[Table 5]

From these results, in the culture of the microalgae belonging to the Botryococcus brownie A race known so far, the CO ₂ concentration during aeration was 10%, 15%.
%, The growth amount of algal cells decreases, but in the culture of microalgae belonging to the novel Botryococcus genus of the present invention, the growth amount of algal cells is in the range of 5 to 15% CO ₂ concentration. , CO ₂ concentration was not so affected. This is the Botryococcus brownie of the present invention
This is because the A race strain is novel, and due to this characteristic, it can be said that the control of aeration during the cultivation of the novel algae belonging to the genus Botryococcus of the present invention is easier than that of the conventional strain. .

[0067]

Example 7 Next, an example of the culture method of the present invention relating to the above-mentioned novel microalgae belonging to the genus Botryococcus of the present invention will be described.

0.3 L in a flat flask (A / V = 41)
CHU-13 × 2 medium (pH 7) was added for sterilization.
This medium was inoculated with the algal cells of the Botryococcus brownie SI-30 strain subcultured in the same manner as in Example 2 above, and liquid culture was carried out for 8 days (culture conditions; aeration rate: 0.
5 vvm (5% CO ₂ ), temperature 25 ° C.). The light irradiation during the culture was performed by intermittent irradiation in which light of a fluorescent lamp having an illuminance of 350 μE / s · m ² was irradiated once a day for 10 hours, and non-irradiated for the remaining 14 hours. Aeration was stopped only during non-irradiation. For comparison, light irradiation in the above culture was performed using artificial light similar to that described above, that is, illuminance of 35.
The alga body of Botulinum coccus brownie SI-30 strain was cultured in exactly the same manner as above except that continuous irradiation with 0 μE / s · m ² of fluorescent light was performed for 24 hours / day.

The concentration of algal cells in the culture solution of the algal cells obtained by culturing under each of the above culturing conditions was measured daily during the culturing period by the same dry weight method as in Example 6 above, and the obtained algae were obtained. From the body concentration, the amount of growth of the Botryococcus brownie SI-30 strain under each culture condition per day was calculated.

For further reference, Botryococcus
The same culture test as above was carried out using the Brownie CCAP807 / 2 purified strain. Table 6 shows the results.

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[Table 6]

As is clear from these results, the novel Botryococcus brownie of the present invention was obtained by the culture method of the present invention.
By culturing the algal cells of the A race strain, it is possible to efficiently grow the algal cells.

[0073]

INDUSTRIAL APPLICABILITY The novel Botryococcus brownie A race strain of the present invention is a hitherto known Botryococcus that the hydrocarbon produced by the alga of this strain contains a hydrocarbon having 33 carbon atoms.・ Brownie It has characteristics not found in the A race. Therefore, by using the Botryococcus brownie A race strain of the present invention,
In addition to its use as a fuel, it has 3 carbon atoms that can be effectively used as a raw material for agricultural chemicals, pharmaceuticals, food additives, bioactive substances, etc.
It is possible to produce the hydrocarbon of 3 and the more useful linear alkadiene and alkatriene having 33 carbon atoms.

Further, according to the culture method of the present invention, it is possible to efficiently grow the alga bodies of the novel Botryococcus brownie A race strain of the present invention.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 5/02 C12R 1:89)

Claims (6)

[Claims] 1. A Botryococcus brownie A in which a hydrocarbon produced by an algal cell contains a hydrocarbon having 33 carbon atoms.
Race stock. 2. A botulococcus brownie A according to claim 1, wherein the hydrocarbon having 33 carbon atoms is a straight chain hydrocarbon.
Race stock. 3. The Botulinum coccus brownie A race strain according to claim 1 or 2, wherein the hydrocarbon having 33 carbon atoms contains an alkadiene and / or an alkatriene. 4. A Botryococcus brownie SI-
The Botryococcus brownie A race strain according to any one of claims 1 to 3, which is 30 strains. 5. The method for culturing a Botryococcus brownie A race strain according to any one of claims 1 to 4, which comprises culturing while intermittently irradiating with artificial light. 6. The culture method according to claim 5, wherein the irradiation with artificial light is performed for 5 to 15 hours each.