JP3181237B2 - Microalgae chlorella and method for immobilizing CO2 using microalgae chlorella - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a high-temperature and high-concentration C
The present invention relates to a novel algae of the genus Chlorella having a CO ₂ fixation ability in an O ₂ atmosphere, a culture method thereof, a CO ₂ fixation method using the Chlorella, and the like.

[0002]

2. Description of the Related Art As one method for fixing CO ₂ generated by burning fossil fuels, etc., CO ₂ generated by photosynthesis of microalgae is used.
₂ There is a method that utilizes the fixing ability. This method has an advantage that sunlight can be directly used as an energy source of the reaction and the reaction is performed at normal pressure. Therefore, development of a bioreactor immobilized with CO _{2 is} currently urgent.

[0003] An important issue in the development of the bioreactor is the selection of the microalgae to be used. Microalgae have a temperature range of 15-30 ° C. and 0.03% CO
₂ adapted to the concentration of the environment, affected metabolism by anesthetic action of CO ₂ in the CO ₂ atmosphere necrosis or or high concentrations causing denaturation of enzymes or the like in the cell at higher elevated temperatures, the growth inhibition It is known to occur. Several species of cyanobacteria and red algae are known to be able to grow at 55 ° C. In green algae, Chlorellasorokin
that iana Shihira et Krarus can grow at 40 ° C
Alogological Studies (1980) 26: 80-86. Microalgae that can grow under high CO ₂ supply conditions include Chlorococcum litto, a green alga Chlorococcum sp.
rale Chihara, Nakayama et Inouye by J Mar Biotechnol
(1993) 1: 21-25.

Japanese Patent Application Laid-Open No. 5-304945 discloses a microalga of the genus Chlorella which fixes high concentration of CO ₂ . Japanese Patent Application Laid-Open No. 5-252930 describes a chlorella culture apparatus.

[0005]

An effective way to keep CO ₂ from diffusing into the atmosphere is to treat the exhaust gas of a thermal power plant. It is said that exhaust gas from thermal power plants has a CO ₂ concentration of about 15% and a temperature of about 100 ° C. As the exhaust gas is cooled and then treated, the temperature in the reaction tank is 30 ° C. or higher,
It is expected to be around 40 ° C. On the other hand, efficient CO
_In order to fix ₂ , it is better to treat CO ₂ in the exhaust gas without diluting it.

[0006] Therefore, to save cooling energy from the or concentrated without diluting the treated gas in CO ₂ CO ₂
It has been desired to provide a novel algae that immobilizes CO ₂ by photosynthesis in an environment of a high-temperature, high-concentration CO ₂ atmosphere in order to immobilize. On the other hand, in order to reduce the size of the CO ₂ -immobilized bioreactor, it is necessary that the bioreactor has a high proliferative ability and can be cultured at high density.

[0007] Furthermore, many microalgae have the property of adhesion or colony formation, but when applied to the bioreactor, especially in order to make the light irradiation uniform, there is no adhesion and the particles are uniformly dispersed. Nature is required. Further, culture of microalgae requires water containing a large amount of nutrients (N, P), and there has been a problem in a method of supplying the water.

[0008] Therefore, the object of the present invention is to provide at least
It has the ability to fix CO ₂ under high temperature and high concentration CO ₂ atmosphere, has high growth ability, enables high-density cultivation, searches for non-adherent microalgae from nature and uses it for CO ₂ fixation. ,
It consists in using eutrophic water as a source of the necessary nutrients.

[0009]

Means for Solving the Problems The present inventors consider that algae having a high temperature tolerance inhabit, and a source temperature of 40 ° C.
An unspecified number of microalgae were collected in the above hot springs.
Next, the microalgae were shake-cultured in an incubator in an air atmosphere at 40 ° C so that strains resistant to the temperature of 40 ° C were selected. Next, by maintaining the temperature at 35 ° C and gradually increasing the CO ₂ concentration in the supply air from 5% → 10% → 30% → 40%, a strain resistant to 40% CO ₂ concentration was selected. I was doing it. Further, regarding the adhesiveness, a strain that grows while dispersing uniformly was selected by visual observation. Furthermore, the growth ability was compared by measuring the amount of algal bodies during the culture, and a strain having a high growth ability was selected. For the strain selected by the above means, the strain was isolated and sterilized by the agar medium method.

[0010] As a result, four isolated and sterile microalgae which are resistant to high temperatures of 40 ° C. or higher and high CO ₂ concentrations of up to 40%, are non-adherent and have high growth ability, namely, HAK-2
Strains, HFK-6 strain, YSK strain, and HHK-6 strain were found. The morphology and physiological properties of the HAK-2 strain collected from the drainage pool of Asahidake Onsen in Hokkaido were examined by light microscopic observation, electron microscopic observation, culture experiment, and photosynthetic activity measurement experiment using the selected HAK-2 strain as a representative strain. The results are as follows. FIG. 3 shows an electron micrograph image. 1. Morphological properties (1) Cells are spherical single cells having a diameter of 3 to 6 μm and do not form a colony. (2) The cell is surrounded by a cell wall, with one nucleus 2 inside.
One chloroplast 23 and one pyrenoid 24 are found. (3) One chloroplast 23 is cup-shaped and exhibits a green color. (4) The lamella is composed of two to several thylakoids. (5) Single cell wall. (6) The pyrenoid substrate is covered by two watchglass-shaped starch sheaths, and thylakoid 22 penetrates. (7) Only asexual reproduction is performed, and the number of autologous spores formed in the mother cell is often two, but sometimes four. The cell wall of the mother cell that has released the spores divides into petals. 2. Physiological properties (1) Photosynthetic ability It is an inorganic nutrient by photosynthesis. At an irradiation light intensity of 400 μE / m ² / s or more, the photosynthesis rate is 300 μmo
a l over O ₂ / mg over chl / h. (2) Pigment system chlorophyll a, chlorophyll b (3) Growth temperature range 25-42 ° C (optimum growth temperature: 35-40 ° C) (4) Growth CO ₂ concentration range 0.03-40% (optimal growth CO ₂ Concentration: 20-40%) The above traits are Chlorella sorokini (Chlorella sorokini)
ana), which indicates that this algae is Chlorella sorokinian (Chlorella sorokinian).
a) was concluded.

Chlorella sorkiniana (Chlorella sor) is the only genus Chlorella that grows under high temperature conditions of 40 ° C.
okiniana) is known. Thus, the algal strain was ordered from the University of Texas, USA, and compared with the HAK-2 strain in terms of morphology and physiological properties. The results are as follows. 1. Morphological Comparison As shown in FIG. 3, in the HAK-2 strain, one thylakoid penetrates the pyrenoid in a straight line, whereas as shown in FIG. 9, the known chlorella solokiniana bends at several places and penetrates. are doing. 2. Physiological comparison As shown in FIG.
_{2 The} optimal growth CO ₂ concentration of Chlorella solokiniana is 10%, while the concentration is in the range of 20-40%.

From the above comparative studies, both algae are of the same species, but the HAK-2 strain is different from the strain because it is resistant to high concentrations of CO ₂ , and the HAK-2 strain is a new strain. I concluded that there was. The properties of the other three types, that is, the resistance to CO ₂ , the temperature resistance, and the adhesion, are HA
All have the same properties as the K-2 strain,
It was determined to be a new strain similar to -2 strains. Note that, at present, no algae have been deposited at the National Institute of Bioscience and Biotechnology, and no new strain according to the present invention has been deposited.

Table 1 shows the collection locations and characteristics of each algae strain. Hereinafter, the four new chlorella strains are referred to as the present algae.

[0014]

[Table 1]

[0015] Accordingly, the first invention is directed to Chlorella solokiniana having a growth ability at a temperature of 25 to 40 ° C and in a mixed gas containing 0.03 to 40% CO _2.
sorokiniana) HAK strain, Chlorella solokiniana HF
K strain, Chlorella Solokiniana YSK strain and Chlorella
It is a microalga Chlorella consisting of any of the Solokiniana HHK strains.

By the way, the present inventors have found that this algae is 0.03%
When a substance that had been in a CO ₂ concentration atmosphere was directly exposed to a 40% CO ₂ concentration atmosphere, a phenomenon that the growth was temporarily stopped might be observed. However, the CO ₂ concentration was increased stepwise or gradually. 40% C while keeping maximum growth ability by going
It has been found that continuous growth is possible even in an O ₂ concentration atmosphere.

Next, the present algae are cultured by gradually supplying high concentration of CO ₂ to a chlorella collected in a place having an acidic and high temperature state, for example, a hot spring or the like, to isolate a viable strain. It can be created or sorted out. That is, the second invention is to gradually supply CO ₂ to chlorella collected in a place exhibiting an acidity and in a high temperature state, to culture the chlorella, and to isolate a surviving strain,
Any of the chlorella solokiniana HAK strain, the chlorella solokiniana HFK strain, the chlorella solokiniana YSK strain, and the chlorella solokiniana HHK strain, which have a growth ability at a temperature of 25 to 40 ° C. and in a mixed gas containing CO ₂ at a concentration of 0.03 to 40%. It is a method of creating or selecting microalga Chlorella.

The medium for culturing the algae is an ordinary medium containing carbon, nitrogen and phosphorus. As a carbon source, C
It is a gas obtained by bringing a gas containing O ₂ into contact with a medium and dissolved, or a carbonate ion obtained by dissolving a carbonate such as Na ₂ CO ₃ . As the nitrogen source, NaNO _3, KNO _3, etc. Furthermore ammonium nitrogen, such as nitrate or of NH ₄ Cl is an organic nitrogen such as urea. Other minor components include Mg, Fe, M
n, B, Zn, Mo, etc. are appropriately used. The synthetic medium for maintaining and preserving the algae is, for example, Fitzgerald as shown in Table 2.
(Fitzgerald) modified medium is effective.

[0019]

[Table 2]

The algae are cultured at a high temperature of 40 ° C. and an air atmosphere containing 0.03 to 40% CO _{2 at} a pH of 5.5 to 7.0, as shown in Examples described later. Since the cultivation of the present algae can be performed under such conditions, for example, when the present algae is used to fix CO ₂ in exhaust gas from a thermal power plant or the like, it is necessary to adjust the concentration of CO _{2 in} the exhaust gas. There is no. In addition, by using the present algae, it is sufficient that the temperature is controlled to 40 ° C. or less in the temperature control at the time of cooling the exhaust gas, and there is no need to cool the reaction tank when irradiating light. In terms of growth ability, the algae show a doubling time of 3.9 hours.

Accordingly, a third invention is a chlorella solokiniana HAK strain, which has a growth ability at a temperature of 25 to 40 ° C. and in a mixed gas containing 0.03 to 40% CO ₂ .
Chlorella Sorokiniana HFK strain, Chlorella Sorokiniana YSK Inc. and Chlorella solo Kinnear Na HHK any more made microalga chlorella cultivated in a medium containing a CO ₂ method immobilization CO _2, characterized in that to fix the CO ₂ strains It is in.

The culture for immobilizing CO _{2 is performed} by subjecting the algae to a culture medium containing CO ₂ under the above culture conditions. In particular, the algae are, for example, CO _{2 in} exhaust gas from thermal power plants.
When fixing the, show advantageous properties not generally of microalgae, as the CO ₂ to be treated, incineration, engine, furnace, heating or the like, or exhaust gas resulting by burning a carbon-containing material, Exhaust gas generated by fermentation and decay or dissolved CO _{2 in} waste liquid, or even air, at least CO ₂
Or a liquid in which CO ₂ or carbonate ions are dissolved.

In the culture for immobilizing CO ₂ , a large amount of medium may need to be supplied. As the water containing the carbon source, the nitrogen source and the phosphorus, so-called eutrophic water such as polluted river water, lake water, domestic wastewater, industrial wastewater, and human waste is suitable. Therefore, in the CO ₂ immobilization method of the present invention, for example, eutrophic water can be used as a medium. Here, the eutrophic water is water containing at least nitrogen or phosphorus, such as wastewater generated by fermentation or decay, deep sea water, or dam lake water, in addition to the above.

The characteristics of the present algae, which enable high growth ability and high-density cultivation, are not limited to CO ₂ immobilization, for example, when microalgae are cultured to recover substances synthesized by the microalgae. In addition, by using the algae, the time required for culturing can be reduced and the volume of the culture tank can be reduced. In addition, by culturing at a high temperature and a high CO ₂ concentration, propagation of mixed bacteria and the like can be suppressed without performing a sterilization treatment. Furthermore, the cost can be reduced by using eutrophic water as the medium.

Accordingly, a fourth invention is a strain of Chlorella solokiniana HAK, which has a growth ability at a temperature of 25 to 40 ° C. and in a mixed gas containing 0.03 to 40% CO ₂ .
A useful substance characterized by culturing a microalga Chlorella consisting of any of Chlorella solokiniana HFK strain, Chlorella solokiniana YSK strain and Chlorella solokiniana HHK strain, accumulating the useful substance in a medium, and collecting the useful substance. It is a manufacturing method.

Here, useful substances synthesized by microalgae include not only organic substances such as proteins, peptides, fats, sugars, and organic acids, but also, for example, fluorescence emitted by the algae, gases such as oxygen and CO ₂ , and inorganic substances. Etc. Further, as described above, the algae have advantageous characteristics in large-scale culture as compared with general algae, so that alga bodies obtained by culturing and growing microalgae are used as fuels, foods, fertilizers, soil conditioners, building materials. , Civil engineering materials,
Use of the algae when used as a material such as paper can save time, cost, and site.

Therefore, the fifth invention provides a chlorella solokiniana HAK strain, which has a growth ability at a temperature of 25 to 40 ° C. and in a mixed gas containing 0.03 to 40% CO ₂ .
A fuel comprising the microalgae grown by culturing and growing a microalga, chlorella, comprising any of the chlorella solokiniana HFK strain, the chlorella solokiniana YSK strain, and the chlorella solokiniana HHK strain.

[0028]

[Action] As described above, this algae is hot (at least 40 ° C)
And in order to have CO ₂ fixing ability under 40% CO ₂ atmosphere,
Saving cooling energy is possible to fix the CO ₂ from Without or concentrated diluting the treated gas CO _2, further high proliferative capacity, so capable of high-density culture, CO ₂ immobilized bio Since the reactor can be reduced in size and has a property of being uniformly dispersed without adhesion, it is possible to fix CO _{2 with high} light efficiency.

Next, in the present invention, since CO ₂ is gradually supplied to chlorella at a high concentration, the chlorella can continuously grow even in a 40% CO ₂ concentration atmosphere while maintaining the maximum growth ability. . Next, in the present invention, eutrophic water is used as a source of nutrients necessary for the growth of the algae, so that eutrophic water can be purified simultaneously with CO ₂ fixation.

Next, the present algae is cultured to recover the substance synthesized by the present algae, so that the growth is fast, that is, the synthesis of the substance is fast, and high-density cultivation is possible, so that the size of the culture tank can be reduced. Further, the cost can be reduced by using eutrophic water as a medium.

[0031]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described specifically with reference to examples. However, the present invention is not limited to these examples. [Example 1] Hot springs with a source temperature of 40 ° C or higher at which high temperature resistant microalgae are likely to grow (Asahidake Onsen, Horoka Onsen, Sounkyo Onsen, Daisetsu Kogen Onsen, Tokachidake Onsen, Fukiage Onsen, Aizankei Onsen , Shirokane Onsen, Tenninkyo Onsen, etc.), lakes (West Lake), ponds, etc., to collect green hot water or suspended matter deposits. 0.03 l of the modified Fitzgerald medium shown in Table 2
The collected water or the collected material was added to a 0.05-l Erlenmeyer flask, and 100 (μE /
m ² / s) and cultivated by shaking with irradiation of a fluorescent lamp. As a result, 4
Microalgae capable of growing at 0 ° C were selected. Next, the microalgae were inoculated into a 0.1-l Erlenmeyer flask containing 0.08 l of the modified Fitzgerald medium and irradiated with a 100 (μE / m ² / s) fluorescent lamp from the bottom in a water bath maintained at 35 ° C. did. Cultivation was performed while gradually increasing the supplied CO ₂ concentration to 5%, 10%, 30%, and 40%, and high CO ₂ -resistant algae were selected.

Among these strains, 2 ml of the medium was withdrawn over time during the cultivation, and the proliferation ability was determined by measuring the turbidity. By performing the selective cultivation in this way, microalgae having high temperature, high CO ₂ concentration and high proliferation ability were obtained. The effect of temperature on growth was examined for the HAK-2 and HFK-6 strains. 120m inside diameter
m, a modified Fitzgerald medium was placed in a 110 mm-high transparent cylindrical culture tank, and irradiation was performed from the outer periphery using a fluorescent lamp. The culture temperature was kept constant by circulating constant temperature water through a water jacket around the periphery of the flask. A mixed gas of CO ₂ and air is immersed in a porous glass diffuser tube in the medium, and 0.5 l / min
Supplied with The CO ₂ concentration in the mixed gas was 30%. Under the same conditions described above, four types of cultures were performed at temperatures of 30, 35, 40, and 45 ° C. As shown in FIGS. 1 and 8,
The best growth was observed at 35-40 ° C.

By culturing the algae, it is possible to fix CO ₂ efficiently without requiring cooling when irradiating light. For the HAK-2 strain, the relationship between the CO ₂ concentration and the growth ability was examined. Using the same culture tank as above, C in ventilated air
The O ₂ concentration and cultured at 5,10,20,30,40% conditions. As shown in FIG. 2, the best growth was observed when a mixed gas having a CO ₂ concentration of 20 to 40% was supplied.

By culturing the algae, CO in the exhaust gas is reduced.
₂ can be immobilized without dilution. HAK
-2 strains were cultured at high density. Width 110mm, thickness 2
The Fitzgerald modified medium and the HAK-2 strain were placed in a 6 mm, 280 mm high transparent flat culture vessel, and irradiated with a fluorescent lamp from the outer periphery. The mixture was agitated from the bottom at a temperature of 40 ° C. and a flow rate of 500 l / min. The CO ₂ concentration in the supply air was 10%. During the culture, 2 ml of the medium was withdrawn over time, and the turbidity was measured to determine the proliferation ability. As shown in FIG. 4, high-density culture up to 4.0 g / l in dry weight was possible.

By culturing the present algae, the size of the CO ₂ immobilized bioreactor can be reduced. [Example 2] Secondary treatment water of sewage was supplied with an inner diameter of 120 mm and a height of 110 mm.
Was inoculated with the HAK-2 strain and irradiated with a fluorescent lamp. As shown in FIG. 5, good growth was exhibited.
By culturing the algae while supplying the secondary treatment water, C
O ₂ fixation and secondary treatment water purification can be performed simultaneously. Example 3 An example of a CO ₂ fixation in the present invention will be described. In the CO ₂ fixing device shown in FIG.
Reference numeral 1 denotes a photosynthesis reaction tank, and combustion exhaust gas from a thermal power plant 2 is supplied to the reaction tank by a blower-4. In the embodiment of the present invention, the generation source of the combustion exhaust gas is the thermal power plant 2. For example, the combustion power of a carbon-containing substance such as a garbage incineration plant, or the fermentation or decay of a garbage fermentation apparatus or the like is used. It is not particularly limited.
The blower 4 is not particularly limited as long as it is a means for sending the exhaust gas to the photosynthesis reaction tank 1.

Chlorella H is contained in the photosynthesis reaction tank 1 described above.
The AK-2 strain, HFK-6 strain, YSK strain, HHK-6 strain alone or as a mixture were used as shown in FIG.
In suspension. Reference numeral 3 denotes a sedimentation tank, which is a pretreatment tank for the water to be treated. Reference numeral 5 denotes a pump for supplying wastewater to the photosynthesis reaction tank 1. In this embodiment, water from which domestic wastewater is removed by the sedimentation tank 3 is treated. Although the water 19 is supplied to the photosynthesis reaction tank 1, the water 19 to be treated may be, for example, industrial wastewater such as industrial wastewater or agricultural wastewater, or wastewater from a garbage fermentation apparatus. , Biological treatment etc. may be used, and furthermore, rivers, lakes,
The water may be pond water or the like, and is not particularly limited as long as it contains nitrogen and phosphorus as water-soluble substances. In addition, instead of using the pump 5, the photosynthetic reaction occurs in river water, lake water and sea water. Tank 1 is immersed, and river flow, convection,
The water 19 to be treated may flow into the photosynthesis reaction tank 1 using a sea current or the like. Numeral 7 denotes algae collecting means for moving the collected alga that has been collected in the photosynthesis reaction tank 1, and the collected alga is dried as biomass 8 and used as fuel or as fertilizer.

FIG. 7 shows the details of the photosynthesis reaction tank 1 shown in FIG. The exhaust gas is supplied to the photosynthesis reaction tank 1 from the exhaust gas supply pipe 11 and the wastewater is supplied from the wastewater supply pipe 13 to the photosynthesis reaction tank 1.
These supply pipes are connected below the liquid surface 16 of the water 19 to be treated in the photosynthesis reaction tank 1 as shown in the figure. A part of the photosynthesis reaction tank 1 has a filtration membrane 15 having holes or gaps smaller than the size of the algae held in the photosynthesis reaction tank 1.
To open the treated water discharge pipe 6. A discharge pump 14 is installed in the treated water discharge pipe 6. The luminescent carrier 17 is immersed in the water 19 to be treated in the photosynthesis reaction tank 1. An optical fiber 18 is connected to the light emitting carrier 17.

Next, the operation of such an apparatus will be described. First, the domestic wastewater from which particles have been removed by the sedimentation tank 3 is guided to the drainage supply pipe 12 by the pump 5 and is supplied to the photosynthesis reaction tank 1.
Is discharged to the treated water discharge pipe 6 through the filtration membrane 15 by the discharge pump 14. Chlorella is inoculated into this photosynthesis reaction tank 1. The exhaust gas from the thermal power plant 2 is cooled by the exhaust gas cooling device 2 by the blower-4.
After being drawn into 0 and cooled, it is supplied to the photosynthesis reaction tank 1 via an exhaust gas supply pipe 11. The optical fiber 18 introduces external light, for example, sunlight, into the light-emitting carrier 17, and the light-emitting carrier 17 uniformly irradiates the introduced light from the entire surface. The chlorella in the photosynthesis reaction tank 1 receives light irradiation from the luminous carrier 17 and performs photosynthesis to obtain CO ₂ in the exhaust gas and the water 1 to be treated.
Proliferates by absorbing nitrogen and phosphorus in 9. Drainage supply pipe 13
Is supplied to the photosynthesis reaction tank 1 sequentially, the liquid level 16 rises, and dissolved nitrogen,
The treated water from which phosphorus has been removed is filtered by the filtration membrane 15 and discharged from the treated water discharge pipe 6. The processing gas containing oxygen from which CO ₂ has been removed and released by the chlorella is discharged into the atmosphere from the liquid surface 16 or the discharge gas discharge port 9.

As described above, the exhaust gas and the eutrophic water are simultaneously purified. However, as described above, in order to culture a large amount of algae for treating the exhaust gas, a large amount of nitrogen and phosphorus are supplied. There is a need to. By the way, when supplying nitrogen and phosphorus, in the present invention, eutrophic water is continuously supplied into the photosynthesis reaction tank 1. Therefore, the exhaust gas and the eutrophic water can be simultaneously purified without a shortage of nitrogen and phosphorus. In particular, since nitrogen and phosphorus in eutrophic water are removed, it is possible to prevent the occurrence of blue water in the hydrosphere. [Embodiment 4] An embodiment of CO ₂ fixation in the present invention will be described. First, the amount of carbon immobilized in the algal cells of the Chlorella solokiniana HAK strain was examined. After the culture, the alga bodies were collected by centrifugation, and in order to remove inorganic salts and the like in the medium adhered to the alga bodies, a washing operation of suspending in distilled water and centrifuging again was performed twice or more. The collected alga bodies were frozen at -20 ° C, degassed and dried at 60 ° C for 5 hours, and pulverized in a mortar. The combustion gas obtained by burning the sample under oxygen was analyzed using a CHN analyzer.

Next, the Chlorella solokiniana HAK strain was cultured, and the algal body increase rate was determined. The amount of the nitrate in the MDM medium was previously increased to about 10 mM. diameter
4 L of medium was placed in a 16 cm cylindrical glass container, a stirring rod installed inside the container was rotated at 100 rpm, and an air mixed gas having a CO ₂ concentration of 5% was supplied into the medium at 0.3 L / min through a glass aeration tube. . The culture temperature was 40 ° C. Light irradiation was performed by using four 15 W fluorescent lamps arranged on the outer periphery of the culture tank, and the average light intensity at the center of the culture tank was set to 101 μE / m ² / s. During the culture, 2 ml of the medium was withdrawn over time, and the wavelength was 750 nm using a spectrophotometer.
Was measured for absorbance. To convert the absorbance into dry weight, the algal suspension was diluted stepwise, and the absorbance and dry weight were measured to prepare a calibration curve of absorbance and algal dry weight concentration. From the graph of the algal cell concentration with respect to the culture time, the algal cell increasing rate was determined by the following equation.

The algal increasing rate _{= (X 2 -X 1) /} (t 2 -t 1) ........................ (1) X 1: algae body dry weight concentration in the incubation time t _{1 (1} = 1,2),
t ₁ : cultivation time (h) In addition, from the algal increase rate and the carbon content in the algal body, CO
_{(2) The} immobilization speed was calculated by the following equation. CO ₂ immobilization rate (g-CO ₂ / m ² / h) = algal increase rate × algal carbon content × 44 (CO ₂ molecular weight) / 12 (carbon atomic weight) (gd.w./m ² / h) (wt.% × 1/100) ……………………… (3) For comparison, Chlorella vulgaris (Chlorella v.
ulgaris) (NIES-227) (hereinafter referred to as known chlorella strains)
Was cultured in the same manner as the Chlorella solokiniana HAK strain except that it was cultured at 25 ° C. in a medium in which the nitrate of the C medium was increased to 7.6 mM, and CO _{2 was} fixed based on the algal growth rate and the carbon content in the alga. The conversion rate was calculated.

Chlorella Solokiniana HAK strain and the known Chlorella strain both had a carbon content in the alga of about 47%, which was almost the same. As shown in FIG. 11, the algal cells increased faster in the Chlorella solokiniana HAK strain. The CO ₂ immobilization rate obtained from this graph was 16 (g-CO ₂ / m ² / h) for the Chlorella solokiniana HAK strain and 15 for the known Chlorella strain.
(g-CO ₂ / m ² / h). Thus, the Chlorella solokiniana HAK strain of the present invention exhibited a higher CO ₂ immobilization rate than known Chlorella.

By culturing the algae, fast CO ₂ fixation becomes possible. [Example 5] As an example of a method of culturing the present algae and recovering a substance synthesized by the microalgae, a method of recovering β-carotene from the present algae will be described. For example, the algae grown in a large amount and at a high density by a device as in Example 3 are allowed to stand for one day, or by centrifugation at 5000 rpm for 15 minutes, and the supernatant is discarded to collect the precipitate. I do. 60% potassium hydroxide and then ethanol are added thereto and heated. The supernatant is separated by centrifugation or spontaneous sedimentation, and ethanol is added, and the extraction is repeated. Transfer the ethanol solution to a separatory funnel, add an equal volume of ether, and then add water until it separates into two phases. The ether solution is further washed with water and then dehydrated to remove the solvent. By the above operation, a crude lipid is obtained.

The crude lipid is dissolved in ethanol, and 1/10 amount of 60% potassium hydroxide is added. After mixing well, the gas phase is replaced with nitrogen gas, and the mixture is allowed to stand at room temperature in a dark place. 2 to this
Add 3 volumes of water, add an equal volume of ether and extract into ether phase. The ether extraction is repeated three to four times, transferring all carotenoids to ether. 1/2 ether solution
Wash with an amount of water to remove lipids, ethanol, and alkali, and add anhydrous sodium sulfate to dehydrate. It is concentrated under reduced pressure. Thereby, a crude carotenoid is obtained.

This crude carotenoid is dissolved in petroleum ether, and 90% methanol is added and mixed well. The petroleum ether layer is washed several times with water to remove methanol sufficiently, dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure. An equal amount of ether is added and mixed well, and water is added little by little to separate two layers. The ether layer is washed with water to remove methanol, dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure. Thus, a crude carotene standard is obtained.

Next, it is applied to a column chromatograph. A crude carotene sample is adsorbed on an activated alumina column, and a mixed solution of petroleum ether and acetone is used as a developing solution. At first, use only petroleum ether, then add acetone little by little, and gradually increase its concentration. The yellow dye is eluted first, followed by the yellow dye. The former is α-carotene and the latter is β-carotene. The alga provides 0.33 g / kg-dw of β-carotene.

As described above, by culturing the present algae, β-carotene, which is valuable as a health food or pharmaceutical, can be produced in large quantities.

[0048]

According to the present invention, high temperature and high CO ₂
The present invention provides a novel algae having a CO ₂ fixation ability under a concentration atmosphere, and makes it possible to prevent global warming by fixing CO ₂ in combustion exhaust gas from a thermal power plant or the like to the algae. In addition, the algae can be used to absorb nitrogen and phosphorus in eutrophic water of lakes and rivers to purify the aquatic environment.

[Brief description of the drawings]

FIG. 1 is a diagram showing the temperature characteristics of the growth of the HAK-2 strain.

FIG. 2 is a graph showing the CO ₂ concentration characteristics of the growth of the HAK-2 strain.

FIG. 3 is an electron micrograph showing the particle structure of the HAK-2 strain.

FIG. 4 is a view showing a growth curve when the HAK-2 strain is cultured at a high density.

FIG. 5 is a view showing a growth curve when the HAK-2 strain is cultured in secondary treatment water.

FIG. 6 is a configuration diagram of a CO ₂ immobilization device according to an embodiment of the present invention.

FIG. 7 is a diagram showing the photosynthesis reaction tank in FIG. 7 in detail.

FIG. 8 is a graph showing the CO ₂ concentration characteristics of the growth of the HFK-6 strain.

FIG. 9 is an electron micrograph showing the particle structure of Chlorella solokiniana.

FIG. 10 is a graph showing the optimal growth CO ₂ concentration characteristics of the HAK-2 strain.

FIG. 11 is a graph showing an increase in algal cell concentration of Chlorella solokiniana HAK strain and known Chlorella strain.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Photosynthetic reaction tank, 2 ... Thermal power plant, 3 ... Sedimentation tank, 4 ...
Blower, 5 ... Pump, 6 ... Treated water discharge pipe, 7 ... Algae recovery means, 8 ... Biomass, 9 ... Discharge gas discharge port, 10 ...
11 ... exhaust gas supply pipe, 12 ... drainage supply pipe, 13 ... drainage supply pipe, 14 ... discharge pump, 15 ... filtration membrane, 16 ... liquid level,
17: luminescent carrier, 18: optical fiber, 19: water to be treated,
20: exhaust gas cooling device, 21: cell nucleus, 22: thylakoid, 23: chloroplast, 24: pyrenoid.

Continued on the front page (51) Int.Cl. ⁷ Identification FI C12P 1/00 B01D 53/34 135Z // (C12N 1/12 C12R 1:89) (C12P 1/00 C12R 1:89) (72) Invention Person Miyuki Kawada 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building Inside the Institute for Global Environmental Technology (72) Inventor Masaru Namba 7-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building Japan Institute for Global Environmental Technology (72) Inventor Yoshiko Shishido 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building Inside the Japan Institute for Global Environmental Technology (72) Inventor Naoto Sakai Tokyo 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo The 7th Oriental Maritime Building Institution for Research on Global Environmental Technology (72) Inventor: Seiou Karube 1-13-16, Higashiarima, Miyamae-ku, Kawasaki-shi, Kanagawa (56) References Special open flat 7-313141 (JP, a) JP flat 4-110395 (JP, a) (58 ) investigated the field (Int.Cl. ^7, DB ) C12N 1/00 - 7/08

Claims (5)

(57) [Claims] 1. A microalga Chlorella solokiniana HAK-2 strain having an optimal CO ₂ concentration of 20 to 40% for growth. 2. The microalga Chlorella solokiniana HAK-2 strain according to claim 1, which is cultured in a medium containing CO ₂ to obtain CO _2.
A method for immobilizing CO ₂ , wherein 3. The microalga Chlorella solokiniana HAK- according to claim 2, wherein the medium is eutrophic water.
Culture method of two strains. 4. A method for producing a useful substance, comprising culturing the microalga Chlorella solorkiniana HAK-2 strain according to claim 1, accumulating the useful substance in a medium, and collecting the useful substance. 5. A fuel comprising the microalgae obtained by culturing and growing the microalga Chlorella solokiniana HAK-2 strain according to claim 1.