JPH09252764A - Cultivation of monodus algae producing eicosapentaenoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To culture Monodus alga capable of increased producing of eicosapentaenoic acid by multi-step culturing a monodus alga in specific culturing conditions culturing in an inorganic liquid culture medium in a transparent vertical type oblate tank, aerating CO2 containing air under photo irradiation, suppressing the increase of PCV concentration. SOLUTION: In the cultivation of a single cell fresh water alga in an inorganic liquid culture medium in the transparent vertical type oblate tank under photo irradiation and aeration of carbon dioxide containing air, an alga belonging to Monodus algae (e.g.; monodus subterraneus ATCC30593, etc.) is inoculated to the culture medium in PCV concentration of 0.9-1.7ml/l, cultured under irradiation from the front or back face of the either side of the tank in the brightness of 6,000-12,500lx until the 1.8-4.9ml/l of PCV concentration in the first step, the second culturing step where the culturing liquid having this concentration is cultured under photo irradiation from both side of the tank at the brightness of 11,000-21, 000lx to the PCV concentration of 5.0-5.5ml/l and the third step culturing this liquid until to the PCV concentration of 10.9-11.0ml/l under photo irradiation of the brightness of 10,000-35,000lx capable of producing eicosapentaenoic acid in large amount.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing a freshwater unicellular alga, Monodus algal cells, which produces eicosapentaenoic acid (hereinafter referred to as EPA).

[0002]

2. Description of the Related Art Conventionally, in an inorganic liquid medium in a glass flat flask to which a nitrogen source (sodium nitrate) has been added, Monodas Sazterraneus ATCC30593.CTCC848
After irradiating with 4 to 10 kilolux of light and aeration of air containing 5% of carbon dioxide gas, primary culture was carried out for about 6 to 10 days, and then algae were collected from the obtained culture. The production method of eicosapentaenoic acid, which is a secondary culture of about 10 to 15 days in a medium substantially containing no nitrogen source (sodium nitrate) to accumulate a large amount in the alga body, is disclosed in JP-B-4-383.
It is disclosed in Japanese Patent Publication No. 97 and is publicly known.

[0003]

The present inventors have accomplished the present invention as a result of researching a culture method for culturing a large amount of algae belonging to the genus Monodus, which is a freshwater single-cell microgenus that produces EPA.

[0004]

According to the present invention, an inorganic liquid medium in an acrylic resin or glass flat vertical aquarium is inoculated with algae belonging to the genus Monodus, and the temperature is 10 to 30 ° C.
At the temperature of, the air containing 0.3 to 3.0% of carbon dioxide gas,
In the culture method of culturing while aerating at an aeration rate of 5 to 35 l / min, the culture start concentration is 0.9 to 1.7 ml.
/ L, and irradiate light with an illuminance of 6000 to 21000 lx to either one of the front and rear surfaces of the water tank to obtain a PCV concentration of 1.
First step of culturing up to 8 to 4.9 ml / l and the PCV
A culture solution having a concentration of 1.8 to 4.9 ml / l was irradiated with illuminance of 11000 to 21000 lx on both surfaces before and after the water tank,
The second step of culturing to a PCV concentration of 5.0 to 5.5 ml / l, and using a culture solution having a PCV concentration of 5.0 to 5.5 ml / l, illuminance of 10000 to 35000 l on both sides before and after the water tank.
and a third step of culturing to a PCV concentration of 10.0 to 11.0 by irradiating x light, and a method for culturing an EPA-producing Monodaceous alga.

The vertical flat water tank having an open upper end used in the present invention is made of glass or acrylic having a thickness of about 3 cm, has a height of 1.5 to 2.5 m, and has front and rear surfaces. The width of the surface is about 0.5 to 2.0, and the width of each of the left and right side surfaces is 7 to
A 17 cm vertical, flat water tank with a rectangular opening formed at the upper end and a suitable number of ventilation holes in the peripheral wall, and a ventilation pipe with a closed tip inserted from the tip. Further, it means a vertical flat water tank in which a culture solution discharge pipe is detachably provided at a bottom portion formed in a ship bottom shape. If the width of the side surface of the water tank exceeds 17 cm, light irradiation is not sufficient, which is not preferable. And, the light receiving area of the front and rear surfaces of the aquarium is wide and preferable, and when the carbon dioxide-containing air is fed from the inserted ventilation pipe, the medium in the aquarium is agitated and the absorption efficiency of carbon dioxide is high, Moreover, since the upper end opening is long and narrow, it is suitable for large-scale culturing of microalgae with less contamination of various bacteria. Further, the light irradiation in the present invention means irradiation of a white fluorescent lamp, a daylight fluorescent lamp or an incandescent lamp.

Inorganic liquid medium in the present invention means medium 100
Sodium nitrate is 150 to 180 per liter
g, potassium phosphate 20 to 30 g, magnesium sulfate 20 to 30 g, calcium chloride 10 to 20 g, ferric citrate 0.45 to 2.45 g, and boric acid 0.11 to
It means an inorganic liquid medium containing 0.55 g, molybdic acid 30 to 150 mg, cobalt chloride 10 to 50 mg, copper sulfate 10 to 50 mg, and zinc sulfate 10 to 5 mg.

The monodus algae used in the present invention include:
Monodas subterraneus (mon) belonging to true eye spot algae
odus ・ subterraneus) and Phaeodactylums belonging to diatoms
p. ).

In the present invention, monodus algae are inoculated into an inorganic liquid medium in a vertical flat water tank at a PCV concentration of 0.9 to 1.7 ml / l, and carbon dioxide gas of 0.
Illuminance of 8000 to 180 in the first step while aerating 5 to 5.0% contained air at a rate of 5 to 35 l / min.
By irradiating either side of the front or rear of the water tank with 001x of light for culturing, it is possible to cultivate smoothly without agglutinating algal cells, and to cultivate at a PCV concentration of 1.8 to 4.9 ml / l. Is stopped when the PCV concentration exceeds 4.9 ml / l,
This is because illuminance becomes insufficient and the EPA production rate decreases, and the alga bodies cannot be aggregated to smoothly transfer the culture to the second step.

The concentration of PCV cultured in the first step is 1.8-
In the second step, the culture solution in a water tank of 4.9 ml / l was applied to both front and rear surfaces of the water tank with an illuminance of 11000 to 21000 l.
When culturing by irradiating x-ray, culturing is performed smoothly and EPA is performed.
Because it will be possible to produce
Stopping the culture at a V concentration of 5.0 to 5.5 ml / l is due to P
Stopping the culture at a CV concentration of 5.5 ml / l is because if the culture is continued until the PCV concentration exceeds 5.5 ml / l, the production rate of EPA is decreased due to insufficient illuminance and algae are aggregated, This is because it is not possible to smoothly shift to the third step.

The culture solution having a PCV concentration of 5.0 to 5.5 ml / l in the water tank cultivated in the second step was irradiated with light having an illuminance of 10,000 to 35000 lx in both the front and rear sides of the water tank in the third step. The culturing was carried out in order to allow the EPA production to proceed smoothly, and the culturing was performed at a PCV concentration of 10.
The reason for stopping at 0 to 11.0 is that the increase in PCV concentration is scarcely recognized, and therefore the production of EPA is also stopped, so that the culture is stopped.

[0011]

Example 1 150 g of sodium nitrate, 20 g of potassium phosphate, 20 g of magnesium sulfate, 10 g of calcium chloride, 0.49 g of ferric citrate, 0.11 g of boric acid and 100 g of molybdic acid per 100 liters of a medium. Is 3
0 mg, molybdenum chloride 10 mg, cobalt chloride 1
100 liters of an inorganic liquid medium (pH 7.5) containing 0 mg, 10 mg of copper sulfate and 10 mg of zinc sulfate, and a height of 2
m, the width of each of the front and rear surfaces is 1 m, and the width of each of the left and right sides is 13 cm. The container is placed in a vertical flat water tank made of acrylic, the culture temperature is 25 ° C., the irradiation time is 18 hours / day, and the air containing 1% carbon dioxide gas is 20 l. As the first step, while ventilating at a rate of / min, use Monodus subterraneus with a PCV concentration of 1.2 m.
Inoculate at a ratio of 1 / l, irradiate 12,000 lx of white fluorescent lamp on either one of the front and back surfaces of the water tank, and culture
After the PCV concentration reached 2.2 ml / l, in the second step, both front and rear surfaces of the water tank were irradiated with 15000 lx white fluorescent lamps and cultured to reach a PCV concentration of 5.5 ml / l. , As the third step, on both front and rear surfaces of the aquarium,
Irradiate with 20000 lx white fluorescent lamp, incubate, and
After the V concentration reached 10.6 ml / l, the culture treatment was stopped, and the obtained culture solution was subjected to a Sharpless centrifuge (150
The algal cells obtained by fractionation at 00 rpm were washed with distilled water and freeze-dried to obtain 203 g of dried algal cells.

The obtained dried algal cells were placed in a methanol solvent containing 5% hydrochloric acid and refluxed at 95 ° C. for 3 hours, and then fatty acid methyl ester was extracted with a hexane solvent, and trichosanoic acid was analyzed by gas chromatography using an internal standard. As a result of quantification by the method, 18.3 g of EPA was obtained.

[0013]

[Embodiment 2] As in the case of Embodiment 1, 10
0 liters of an inorganic liquid medium was added, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and the air containing 1% carbon dioxide was aerated at a rate of 20 l / min, and the first step was Monodus subterraneus. CTCC848 was inoculated at a culture starting concentration of 5 ml / l, and 12500 lx of white fluorescent lamp was irradiated from either one of the front and rear surfaces of the water tank,
When the culture reached a PCV concentration of 2.1 ml / l,
As the second step, 16000l from both front and back of the water tank
x white fluorescent lamp, cultivated and PCV concentration 5.4m
When it becomes 1 / l, as a third step, irradiate a white fluorescent lamp of 20,000 lx from both front and rear surfaces of the water tank,
When the culture reached a PCV concentration of 10.3 ml / l,
After stopping the culturing process, 70 liters of the culture broth separated from the obtained 100 liters of culture broth were subjected to Sharpless centrifugation (15,000 rpm) to wash the alga bodies with distilled water and freeze-drying. 128 g of dried algal cells was obtained. Then, as a result of quantifying the EPA in the obtained dried algal cells in the same manner as described in Example 1, 12.3 g of EPA was obtained.

The amount of EPA in the obtained dried algal cells was quantified by the gas chromatography analysis method as in Example 1, and as a result, the amount of EPA in the dried algal cells was 14.3 g.

70 liters of the inorganic liquid medium of Example 1 was added to the remaining 30 liters of the PCV concentration of 10.3 ml / l, which was obtained by separating the above 70 liters, and a 20000 lx white fluorescent lamp was irradiated. , The culture solution obtained by repeatedly culturing 6 times was washed by distilled water with a Sharpless centrifuge (15000 rpm), and the freeze-dried dried alga bodies As a result of quantifying EPA, 78 g of EPA was obtained.

[0016]

Example 3 For 200 liters of medium, 300 g of sodium nitrate, 40 g of potassium phosphate, 40 g of magnesium sulfate, 20 g of calcium chloride, 0.98 g of ferric citrate, 0.22 g of boric acid and molybdic acid were used. 60
Inorganic liquid medium (pH 7.5) containing 20 mg, 20 mg of cobalt chloride, 20 mg of copper sulfate and 20 mg of zinc sulfate.
0 liters were placed in a vertical flat water tank made of acrylic with a height of 2 m, a front / rear surface width of 2 m, and left / right side surfaces of 13 cm width, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and carbon dioxide gas was added. As a first step, 1.3 ml / l of pheoductilam was inoculated into the above-mentioned inorganic liquid medium while aerating 1% -containing air at a rate of 35 l / ml, and a 12,000 lx white fluorescent lamp was provided on either side of the front and rear of the water tank. Irradiate, incubate,
After the PCV concentration was 2.3, in the second step, both front and rear surfaces of the water tank were irradiated with a white fluorescent lamp with an illuminance of 15000 lx and cultured to give a PCV concentration of 5.0 ml / l. After that, as the third step, the illuminance of 2 on both the front and rear surfaces of the water tank.
Irradiate with 0000 lx white fluorescent lamp, incubate, and
After the concentration reached 10.1 ml / l, the culturing process was stopped, and the obtained culture broth was transferred to a shear press centrifuge (150
(00 revolutions / minute), the algal cells obtained by separation are washed with distilled water,
It was freeze-dried to obtain 395 g of dried algal cells. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1 and the result was 36.3 g.

[0017]

Example 4 75 g of sodium nitrate, 10 g of potassium phosphate, 10 g of magnesium sulfate, 5 g of calcium chloride, 0.25 g of ferric citrate, 0.055 g of boric acid and 50% of molybdic acid per 50 liters of medium. Is 15
Inorganic liquid medium (pH 7.5) containing 5 mg of cobalt chloride, 5 mg of cobalt chloride, 5 mg of copper sulfate and 5 mg of zinc sulfate 5
0 liter, height 2m, front / rear width 0.5m, left
It was placed in an acrylic vertical flat water tank with a width of 13 cm on the right side, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and air containing 1% carbon dioxide was aerated at a rate of 20 l / min. Meanwhile, in the first step, the culture starting concentration of Monodus subterraneus CTCC848 was 1.
Inoculate 1 ml / l, irradiate one side of the front surface of the water tank with a white fluorescent lamp with an illuminance of 12000 lux, and culture it until the PCV concentration became 2.0 ml / l.
After irradiating 000 lx white fluorescent lamps on both sides of the front and rear of the water tank, and culturing to reach an algal concentration of 5.4 ml / l, as a third step, illuminance 20000 lx white fluorescent lamps on both sides of the front and rear of the water tank. To a PCV concentration of 10.8
When the amount reached to ml / l, the culture was stopped, and the obtained culture solution was fractionated with a Sharpless centrifuge (15,000 revolutions / minute) to obtain algal cells, which were washed with distilled water and freeze-dried to give 97 g.
The dried alga bodies of The amount of EPA in the obtained dried algal cells was quantified by the Cass chromatography analysis method as in Example 1, and as a result, the amount of EPA was 8.9 g.

[0018]

Example 5 To 50 liters of a medium, 75 g of sodium nitrate, 10 g of potassium phosphate, 10 g of magnesium sulfate, 5 g of calcium chloride, and ferric citrate of 0.
25g, boric acid 0.055g, molybdic acid 15g,
Cobalt chloride 5 mg, copper sulfate 5 mg, zinc sulfate 5
50 liters of inorganic liquid medium (pH 5.5) containing 50 mg in a height of 1.5 m, width of front / rear surface of 0.5 m, width of left / right side of 7 cm in an acrylic vertical flat water tank, and cultured The temperature was set to 10 ° C., the irradiation time was set to 24 hours / day, and 5% carbon dioxide gas-containing air was aerated at a rate of 35 l / min, and the first step was to add 50 mL of an inorganic liquid medium in a water tank to Monodasu. Subterraneus was treated with a PCV concentration of 0.
Inoculate at a rate of 9 ml / 6000l on one side of the front of the aquarium
PCV concentration is 1.
When it reaches 8 ml / l, as the second step, in front of the water tank
When both sides of the rear are irradiated with 11,000 lx of daylight fluorescent light and cultured, and the PCV concentration becomes 5.0 ml / l, the third step is 10,000 lx of daylight fluorescent on both the front and rear of the water tank. Irradiate with light and culture, PCV concentration 10.0m
When it reached 1 / l, the culture was stopped, and the obtained culture solution was separated by a Sharpless centrifuge (15000 rpm), the alga was washed with distilled water, freeze-dried, and dried to 95 g. I got algae. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1, and the result was 8.1 g.

[0019]

Example 6 90 g of sodium nitrate, 15 g of potassium phosphate, 15 g of magnesium sulfate, 10 g of calcium chloride, 1.23 g of ferric citrate, 0.28 g of boric acid and 50 g of molybdic acid per 50 liters of a medium. Is 75m
g, cobalt chloride 23 mg, copper sulfate 25 mg, and zinc sulfate 25 mg in an inorganic liquid medium (pH 8.
0) 50 liters were placed in a vertical glass flat water tank with a height of 2.5 m, a width of front and rear sides of 2 m, and widths of left and right sides of 17 cm, the culture temperature was 30 ° C., and the irradiation time was 24 hours / hour, 35 l / min of air containing 5% carbon dioxide
As a first step, 50 L of an inorganic liquid medium in a water tank was inoculated with Monodas subterraneus at a PCV concentration of 1.7 ml / l as a first step while aeration of 21,000 lx of incandescent water was applied to the front surface of the water tank. When the PCV concentration reached 4.9 ml / l by illuminating with a lamp and culturing, the second step was irradiating with 21000 lx incandescent lamps on both the front and rear sides of the water tank, culturing, and culturing the PCV concentration. Is 5.0 ml / l
Then, as a third step, both the front and the rear of the water tank are irradiated with an incandescent lamp of 35000 lx, cultured, and
When the V concentration reached 11.0 ml / l, the culturing was stopped, and the obtained culture broth was collected using a sherbless centrifuge (150
(00 revolutions / minute), the algal cells obtained by separation are washed with distilled water,
It was freeze-dried to obtain 97 g of dried algal cells. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1, and the result was 8.9 g.

[0020]

EFFECTS OF THE INVENTION According to the present invention, a useful culture method has been obtained in which large-scale culture of Monodus algae can be easily carried out and EPA can be produced with high efficiency.

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Claims (1)

[Claims] 1. A culture method in which freshwater unicellular algae are inoculated into an inorganic liquid medium in a vertical flat water tank and cultured while irradiating with light and aeration containing carbon dioxide, monodus algae are cultured in the inorganic liquid medium. PCV concentration of 0.9 to 1.7 ml / l was inoculated into the water, and illuminance of 6000 to 6000 from either one of the front and back surfaces of the water tank.
By irradiating 12,500 lx of light, the PCV concentration is 1.8-
The first step of culturing to 4.9 ml / l and the P
A culture solution having a CV concentration of 1.8 to 4.9 ml / l is irradiated with light having an illuminance of 11000 to 21000 lx from both front and rear surfaces of a water tank to culture until a PCV concentration of 5.0 to 5.5 ml / l. 2 steps and the PCV concentration 5.0 to 5.5 m
In 1 / l culture solution, illuminance 1000
A PCV concentration of 10.
And a third step of culturing to 9 to 11.0 ml / l, which is a culturing method of monodus algae for producing eicosapentaene.