JP2000050861A - Marine microalga with high proliferation rate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject marine microalgae for the purpose of attaining the improved productivity thereof through mass culture thereof. SOLUTION: The marine microalgae, Chlamydomonas perigranulata as a kind of marine green algae, has a peculiar characteristic in respect of that numerous granules exist on the surface of the cells, and has the following parameters: chlorophyll content: 2.0×10-15 mol/cell, chlorophyll composition ratio (chlorophyll(a)/chlorophyll(b)): 1.9 mol/mol, and proliferation rate: 32 g/m2 a day on a dry basis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to microalgae useful for producing fuels, chemical raw materials and food raw materials (proteins) and suitable for mass culture.

[0002]

2. Description of the Related Art In recent years, a technique of culturing microalgae in large quantities and using it for industrial raw materials, feed and fine chemical raw materials has attracted attention. In addition, this technology has attracted attention as one of the biological means for fixing CO ₂ . But,
Few of these technologies have been put to practical use, and one of the causes is that productivity is low due to insufficient growth ability of microalgae. Therefore, when microalgae are mass-cultured and used effectively, improving the productivity has been an issue.

On the other hand, as means for improving productivity, improvement of a reactor for culture and selection and screening of microalgae suitable for culture production have been conventionally performed. But,
As shown in Table 1 below, the maximum production rate reported so far is only 25 g / m ² day, and further improvement of the production rate is an important issue in microalgal mass culture.

[0004]

[Table 1]

In addition, the present inventors have found that when cultivating in an outdoor culture pond, it is possible to improve productivity by a method using microalgae with reduced light-collecting pigment, and a patent application for this method has already been filed. (Japanese Patent Application Hei 7-32296)
No. 6).

[0006] Despite such improved techniques, techniques for mass cultivation of microalgae are not yet sufficient, and there is a demand for techniques that can achieve higher productivity.

[0007]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a novel seawater microalgae having a high growth rate in order to achieve an improvement in productivity in mass culture of microalgae. is there.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have isolated (screened) a novel green alga belonging to the genus Chlamydomonas, namely, Chlamydomonas perigranulata ATRC-1 from the sea.
However, it was confirmed that the growth rate was higher than that of the conventional reports.

[0009] Furthermore, the above-mentioned isolates are subjected to a mutation treatment to obtain a light-reducing pigment-reducing mutant, Chlamydomonas.
When perigranulata ATRC-1 LHC-1 was prepared, it was confirmed that this mutant had a higher production rate.

That is, in order to achieve the above object, the present invention relates to a green alga belonging to the genus Chlamydomonas, which has the following characteristics: Chlamydomonas perigranulata ATRC-
Provide one share. -Photosynthetic pigment: chlorophyll a, chlorophyll b, and carotenoid are detected by thin-layer chromatography. -Chlorophyll content: 2.0 ( ^10-15 mol / c)
ell) ・ Chlorophyll composition ratio (chlorophyll a / chlorophyll b): 1.9 (mol / mol)

This Chlamydomonas perigranulata ATR
If the C-1 strain is used, 32 (dry weight g / m ² day)
Production speed can be obtained.

The present invention also relates to the above Chlamydomonas perigr
A mutant strain of the anulata ATRC-1 strain, which has the following characteristics: Chlamydomonas perigranulata ATRC-
1LHC-1 is provided. -Chlorophyll content: 1.5 ( ^10-15 mol / c)
ell) ・ Chlorophyll composition ratio (chlorophyll a / chlorophyll b): 2.7 (mol / mol)

With this mutant, 39 (dry weight g
/ M ² day).

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a novel green alga according to the present invention will be described in detail.

<I>: Chlamydomonas perigranulata A
TRC-1 The entitled microalgae was isolated from the sea area as follows.

First, seawater obtained from various places in Japan and overseas (about 5
After L) was centrifuged (3000 g, 10 min), most of the supernatant was discarded, and the remaining precipitate was suspended in a small amount of seawater. 1 mL of the suspension was applied to A medium (see Table 3) using agar as a support (1.1%). Next, this A medium was added to a 10 μm
The cells were placed under culture conditions of a light intensity of Em ^- 2sec- ¹ and a temperature of 25 [deg.] C., followed by standing culture for about one month. As a result, it was possible to obtain an expanded green-yellow colony-like microalga. However, the colony-like microalga obtained here was determined to contain a plurality of microalgae by microscopic observation.
Therefore, from the mixed microalgae, one microalgae was pulled up by a capillary pipette under microscopic observation, and A
The cells were transferred to a microtiter plate containing 100 μL of the medium. About 600 strains of these microalgae were added to 10 μEm ^−2.
After static culture for about 2 months under the conditions of light intensity of sec- ¹ and a temperature of 25 ° C., 250 strains grew to tens of thousands as a single kind of microalga.

Next, each of these 250 single strains was
After transferring to an L-shaped tube containing 10 mL of A medium, 30 μEm
^The cells were cultured with shaking at ^-2 sec ^-1 at 25 ° C. and 60 rpm for about 3 weeks. As a result, 100 out of 250 shares
The turbidity (7
(Wavelength of 50 nm) was 3 or more. This one
0 mL was further transferred to a 100 mL Erlenmeyer flask containing 60 mL of the A medium, and subjected to shaking culture under the conditions of 25 ° C. and 60 rpm for 2 weeks, whereby a seed used for mass culture of microalgae was obtained.

Next, about 100 strains were transformed into 1.3L scale.
200μEm in a flat large culture bottle ^-2sec^-1,
0.5% CO₂And grow them at a rate
evaluated. Based on the results, the number of shares increased
One strain having the best breeding speed was selected.

The taxonomic characteristics of this strain were as follows: 1) Shape: oval Size: Vegetative cells: 7 to 12 μm (preferred growth environment, cells in vigorous growth under abundant nutrient conditions) Immobilized cells: 10 to 16 μm (deterioration of growth environment, nutrient depletion conditions) 2) Flagella: Has two vegetative nutrients. 3) Motility: Yes (only vegetative cell state) 4) Photosynthetic pigment: Has chlorophyll a, chlorophyll b, and carotenoid. (Detected by thin-layer chromatography) Chlorophyll content 2.0 (10 ⁻¹⁵ mol / cell) Chlorophyll composition ratio (Chlorophyll a / Chlorophyll b) 1.9 (mol / mol) 5) Cell structure: Many cells on the cell surface Has small granules. The cell has a cell wall on the outer periphery. Has eye spots. It has a cup-shaped chloroplast. It has a round pyrenoid at the base of the chloroplast. It has a large number of starch granules inside the chloroplast. 6) Salt tolerance: The salt concentration suitable for growth in culture under photoautotrophic conditions is 2 to 5% NaCl, and the optimum around seawater salinity (about 3.5%) is considered.

These characteristics are characteristic of the genus Chlamydomonas, that is, that the vegetative cells are unicellular, have two flagella, a cell wall, and a pyrenoid, and basically have a swimming but often immobile stage. Therefore, the isolated strain was identified as a member of the genus Chlamydomonas.

Many types of Chlamydomonas are described, and they are classified according to cell size and morphology, chloroplasts, morphology and arrangement of pyrenoids, morphology of papillae, and the like. The isolate of the present application is considered to belong to the Euchlamydomonas group because the chloroplast is cup-shaped and the pyrenoid is located at the bottom. There are few reports on marine Chlamydomonas, and among them, small species (10 μm or less) belonging to the Euchlamydomonas group were compared with the isolates of the present invention (Table 2). This isolate is most similar to C. coccoides Butcher in terms of cell size and papillary morphology. However, the isolated strain of the present invention has a unique feature that a large number of granules are present on the cell surface, and this feature is not a known species. Is done.

[0022]

[Table 2]

Therefore, a new species of green algae of the present application is Chlamydomonas
perigranulata and the isolate was Chlamydomonas perig
ranulata ATRC-1. By culturing this isolate, as shown in the Examples below,
A production rate of (dry weight g / m ² day) could be achieved.

Since this strain is a green algae, its acceptance was refused by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Heisei 1).
March 18, 0). Therefore, this strain is stored under the applicant's control at the applicant's responsibility.

<II>: Chlamydomonas perigranulata A
TRC-1 LHC-1 Chlamydomonas perigranulata ATR isolated above
C-1 is irradiated with ultraviolet rays (1 mW / cm ² , 30 s)
After ec), colonies were formed on an agar medium. Chlorophyll (Chl) was extracted from the colonies on the agar medium, and chlorophyll a (Chla) and chlorophyll b were extracted.
The ratio (Chla / Chlb, hereinafter referred to as Chla / b) with (Chlb) was determined, and screening was performed based on the ratio. That is, by selecting a colony having the highest Chla / b value, the mutant strain of the present invention, namely, Chlamydomonas perigranulata ATRC-1 is selected.
LHC-1 strain was obtained.

The value of Chla / b is an indicator of the magnitude of the concentration of the light-collecting dye (Fujita et al., 1989, Plant an).
d Cell Physiology, 30: 1029-1037), the larger the value, the less the light-collecting dye can be considered.
See that reference for details.

This mutant strain Chlamydomonas perigranulata
ATRC-1 LHC-1 is derived from Chla and Chlb
Chlamydomonas perigranulat, which is a parent cell that contains two types of Chl and has motility (two flagella)
a It had the same characteristics as ATRC-1. Chlamydomo
Only the Chl dye (content and composition ratio) and the growth rate could be detected as differences from nas perigranulata ATRC-1, as described in detail in the Examples below. That is, the chlorophyll content, chlorophyll composition ratio and growth rate of this mutant strain were as follows. -Chlorophyll content: 1.5 ( ^10-15 mol / c)
ell) ・ Chlorophyll composition ratio (chlorophyll a / chlorophyll b): 2.7 (mol / mol) ・ Growth rate: 39 (dry weight g / m ² day)

Since this mutant strain is a green algae like the parent strain, it was rejected by the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology (March 18, 1998).
It is stored under the applicant's control at the applicant's responsibility.

The microalga Chlamydomo isolated from the above seawater
nas perigranulata ATRC-1 and its light-harvesting pigment mutant Chlamydomonas perigranulata ATRC-1
Since LHC-1 has a remarkably higher growth rate than conventional microalgae, it can be used as a fuel, a chemical raw material, and a food raw material (protein), and can be cultured and produced in large quantities.

As shown in Table 1, most of the high productivity microalgae reported so far are fresh water. On the other hand, since the microalgae according to the present invention is seawater, water required for culture can be obtained from inexhaustible seawater. Therefore, the fermentation does not require fertile land, and has the advantage of using degraded land such as a desert and culturing and producing using seawater.

[0031]

EXAMPLES Hereinafter, examples of culture of the above two strains will be described as examples.

<Experimental Materials and Experimental Methods> Example 1 1) Chlamydomonas perigranulata ATR as microalgae
C-1 was used.

2) Chlamydomonas perigr used for culture
About anulata ATRC-1, the measurement of the light absorption spectrum and quantification of chlorophyll were performed.

3) For the culture experiment, a culture apparatus shown in FIG. 2 was used. In FIG. 2, reference numeral 1 denotes a flat culture bottle. The culture bottle 1 is filled with 1 liter of the culture solution 2. Culture bottle 1
The cotton filter tube 3 is connected to the upper opening of the. Air containing 1% CO ₂ is introduced into the inlet of the cotton filter tube 3. The introduced air is sterilized in the cotton filter tube 3 and then bubbled into the culture solution 2 filled in the culture bottle 1 through the aeration unit 4 of the cotton filter tube. Further, the flat culture bottle 1 was kept at 25 ° C.
In a constant temperature water bath 5. Further, an incandescent lamp 6 is arranged on the outer side of the constant temperature water tank 5 so as to irradiate the culture solution 2 in the flat culture bottle 1 with light. The intensity of light irradiation is adjusted by changing the distance between the incandescent lamp 6 and the culture bottle 1. In the figure, reference numeral 7 denotes a cotton plug.

4) A culture medium shown in Table 3 below was used as a culture solution, and 1% carbon dioxide gas + air was aerated (300 mL).
/ Min). All experiments were performed at 25 ° C. In order to simulate outdoor culture, culture was performed while irradiating light with an incandescent lamp 6. The intensity of light irradiation was adjusted so that the irradiation amount per day was 6000 kcal / m ² d in fine weather.

[0036]

[Table 3]

5) During the culture, as shown in FIG.
Semi-continuous culture was performed with turbidity = 0.4 set as the initial concentration. That is, after culturing was started by inoculating the seeds so that turbidity = 0.4, the cells were grown until the end point concentration of turbidity = 1.
Next, the culture solution was extracted and replaced with a fresh culture solution so as to obtain the initial turbidity. Thereafter, this culture cycle was repeated. In this case, the microalgae grow linearly in one culture cycle (the algal mass increases in proportion to the time).
Is shown.

6) The production rate of the microalgae was measured based on the dry weight. That is, the dry weight of the alga bodies was measured both at the initial start and at the end point marked with a circle in the figure, and the production rate was calculated according to the following equation. Production rate = (end weight-initial weight) / culture time

Example 2 1) As microalgae, mutant strain Chlamydomonas perigranulat
a ATRC-1 LHC-1 was used.

2) The mutant strain Chlamydomonas perigr used
Anulata ATRC-1 LHC-1 was measured for its light absorption spectrum and quantified for chlorophyll.

3) A culture experiment was performed in the same manner as in Example 1.

<Experimental results> 1) Parent strain Chlamydomona used in Examples 1 and 2 above
s perigranulata ATRC-1 and mutant Chlamydomo
The absorption spectra of nas perigranulata ATRC-1 LHC-1 are as shown in FIG.

In the measurement of the absorption spectrum shown in FIG. 1, the light absorption at 750 to 600 nm was measured for a sample in which the cell concentration was adjusted to 1.6 × 10 ⁷ cells / ml. As is clear from the figure, the mutant Chlamydomo
nas perigranulata ATRC-1 LHC-1 shows that its parent strain Chlamydomonas peri
It was confirmed that the amount of light absorption was smaller than that of granulata ATRC-1.

When the Chl of both strains was quantified,
The results shown in Table 4 below were obtained.

[0045]

[Table 4]

The results in Table 4 also show that the parent strain Chlamydomonas pe
rigranulata ATRC-1 and mutant Chlamydomonas peri
It shows a characteristic difference from granulata ATRC-1 LHC-1. The content of Chl, a major photosynthetic pigment of green algae, was determined for the mutant strain Chlamydomonas perigranulata.
ATRC-1 LHC-1 is derived from the parent strain Chlamydomonas perig
ranulata It is about ／ of ATRC-1. In addition, for Chla / b showing the degree of light-harvesting pigment reduction, mutant Chlamydomonas perigranulata ATRC-1 was also used.
LHC-1 is the parent strain Chlamydomonas perigranulata AT
It exceeds RC-1. From these results, Chlamydo
It was concluded that monas perigranulata ATRC-1 LHC-1 was a mutant having less light-harvesting dye than Chlamydomonas perigranulata ATRC-1.

2) FIG. 3 and Table 4 show the results of measurement of the growth rate when the culture was performed. Chlamydomonas perigranulat
a The growth rate of ATRC-1 is 32 g / m ² day, and is an example of the conventional microalgae production rate shown in Table 1 (up to 25 g
/ M ² day).

3) Mutant strain Chlamydomonas perigranulata
When ATRC-1 LHC-1 was cultured under the same conditions, it showed 39 g / m ² day, indicating that the parent strain Chlamydomonas pe
rigranulata was found to exhibit an even higher production rate than ATRC-1. Mutant Chlamydomonas perigranulat
a The reason why the production rate could be further improved by using ATRC-1 LHC-1 was that, as described in Table 4, the chlorophyll of this mutant strain had the parent strain Chlamydomona.
s perigranulata ATRC-1 (Table 4), and as described in the above-mentioned prior application (Japanese Patent Application No. 7-322966), an effect of improving photosynthesis by reducing the concentration of condensed pigments appeared. It is likely.

[0049]

According to the results of the above embodiment, the present invention
Chlamydomonas perigranulata ATRC-1 and Chlamy
By using domonas perigranulata ATRC-1 LHC-1, the production rate of microalgae can be greatly improved, and this is effective as a means of improving economic efficiency by mass culture.

Further, most of the conventional high-productivity microalgae are freshwater, but the microalgae according to the present invention is seawater, and water required for culture can be obtained from inexhaustible seawater. In addition, since fertile land is not required for culturing microalgae, degraded land such as a desert can be used and cultivated and produced using seawater. Therefore, the practical effect is great in that it can be produced without competing with land and water for food production by plants on land.

[Brief description of the drawings]

FIG. 1 shows the absorption spectrum of each of two types of microalgae according to the present invention.

FIG. 2 is an explanatory view showing a culture apparatus used for culturing microalgae of the present invention.

FIG. 3 is an explanatory view showing a method used for measuring a production rate of a microalga according to the present invention.

[Description of Signs] 1 ... flat culture bottle, 2 ... culture solution, 3 ... cotton filter tube, 4 ... aeration section of cotton filter tube, 5 ... constant temperature water bath, 6 ... incandescent lamp, 7 ... cotton plug

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Ryohei Ueda 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture F-term in Mitsubishi Heavy Industries, Ltd. Basic Research Laboratory 4B065 AA83X AC12 AC16 BA16 BB01 BC01 BC03 BC05 BC07 BC11 BC48 BD50 CA41 CA43

Claims (3)

[Claims] [Claim 1] Chlamydomonas pe, a new marine green algae
rigranulata. 2. Chlamydomonas perigran according to claim 1.
ulata having a chlorophyll content of 2.0 (10
^-15 mol / cell), and the chlorophyll composition ratio (chlorophyll a / chlorophyll b) is 1.9 (mol / m).
ol) Chlamydomonas perigranulata ATRC-
One share. 3. The Chlamydomonas perigran of claim 1.
ulata having a chlorophyll content of 1.5 (10
^-15 mol / cell), and the chlorophyll composition ratio (chlorophyll a / chlorophyll b) is 2.7 (mol / m).
ol) Chlamydomonas perigranulata ATRC-
1 LHC-1 strain.