CN109722407B - Method for compensating growth of microalgae by starting at ice temperature - Google Patents
Method for compensating growth of microalgae by starting at ice temperature Download PDFInfo
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Abstract
The invention relates to a method for microalgae ice temperature starting compensation growth, which comprises the following steps: and (3) under the dark condition, uniformly reducing the culture temperature of the microalgae in the logarithmic growth phase to 0-minus 10 ℃, and maintaining the temperature for 1-15 h. The invention induces the internal absorption and metabolism mechanism of the cell organism to change by external ice temperature stimulation to the microalgae, starts the ice temperature compensation growth mechanism and promotes the organism activity. The method is simple and effective, has low treatment cost, can be directly used for the industrial production of the microalgae, greatly improves the industrial yield of the microalgae, increases the growth rate of the microalgae treated by the method by more than 2 times compared with the growth rate of the untreated microalgae, and has great popularization and application values for improving the culture efficiency of the microalgae, prolonging the culture period and culturing the microalgae in large scale.
Description
Technical Field
The invention relates to a method for improving the growth rate of microalgae, in particular to a method for starting the microalgae to grow in a compensation manner at an ice temperature.
Background
The spirulina, the dunaliella salina and the chlorella belong to economic microalgae which can be industrialized in a large scale, contain rich protein, fat, vitamin, biological polysaccharide, unsaturated fatty acid, chlorophyll, carotenoid and various mineral substances needed by human bodies, and have good nutrition and health care functions. At present, besides leading a product, one of the microalgae food processing tablets also sequentially appears in that algae powder nutrient extracts are added into common food as food to prepare food with microalgae nutrition and flavor. On the other hand, biofuels are becoming more and more important over several days of fossil fuel mass consumption, and microalgae biofuels, as third generation biofuels, have greater advantages than previous first generation biofuels produced using crops such as starch and corn, and second generation biofuels produced using cellulose and crop straw, and the like.
The development of plants having a total dry weight greater than that of the control plants in the convalescent period after being subjected to stress by certain environmental factors, including temperature, light, pH and salt stress, is called compensatory growth, and is now widely studied in connection with land, aquatic animals and higher plants. At present, most of researches on the compensatory growth of algae with more success are concentrated on two aspects of illumination and temperature, and the previous researches find that the compensatory growth effect of the algae is better due to light saturation and low light stress than high light stress; however, the high temperature stress and the low temperature stress have the effect of compensating growth of algae, but the current literature reports show that although the high temperature stress can compensate growth of algae to a certain extent, the algae filament is damaged to a certain extent, the damage needs to be recovered for a long time, the low temperature stress obviously greatly reduces the damage, but the efficiency of compensating growth is not obvious, the temperature range of the current research on compensating growth of the low temperature stress is only 2-15 ℃, and the zero-temperature ice temperature stress promotes compensating growth, which is not reported.
In China, the production of microalgae is generally limited by local environmental factors, the culture production period of microalgae is greatly limited due to environmental influence, and the whole-year culture cannot be realized, taking spirulina as an example, the culture production period is only about 6 months per year, the breeding period before entering the culture production period every year is long due to the low environmental temperature of local winter and spring, and the yield in the initial stage of the culture production period is very low, so that the yield is the highest in only 6 months, 7 months and 8 months in the whole year. On the other hand, microalgae are cultivated in the world by adopting a traditional laggard cultivation mode of greenhouse runway cultivation, so that the cultivation efficiency of the whole area is low, and the production cost is high.
In order to solve the problems of low culture production efficiency, low biomass during culture, short culture period and difficult breeding, the method is developed by combining local practice and developing culture technical researches in autumn and winter, and a method for starting hyper-compensation growth of microalgae at ice temperature is found so as to improve the total yield, reduce the production cost and obtain the maximum economic benefit.
Disclosure of Invention
In order to improve the industrial yield of microalgae and reduce the production cost, according to local conditions and in combination with local actual environmental conditions, the invention carries out a great deal of research on how to start the ice temperature compensation growth of microalgae, and the inventor carries out external ice temperature stimulation on the microalgae to induce the internal absorption and metabolism mechanism of the cell organism to change, starts the ice temperature compensation growth mechanism and promotes the organism activity of the microalgae, thereby providing a method for starting the ice temperature compensation growth of the microalgae.
The method specifically comprises the following steps: and under the dark condition, uniformly reducing the culture temperature of the microalgae in the logarithmic growth phase to 0-minus 10 ℃, and maintaining the culture temperature for 1-15 h.
The invention induces the internal absorption and metabolism mechanism of the cell organism to change by external ice temperature stimulation to the microalgae, starts the ice temperature compensation growth mechanism and promotes the organism activity.
The invention further provides that the microalgae is selected from one or more of spirulina, dunaliella salina and chlorella;
wherein the Spirulina includes Spirulina maxima, Spirulina platensis, and Spirulina subsalsa;
the Dunaliella salina comprises Dunaliella salina; (ii) a
The invention has the best effect of promoting the growth of the spirulina platensis, the dunaliella salina and the chlorella.
The method of the present invention is applied to microalgae of the above type and is not limited to the types listed in the examples.
The invention further provides that the microalgae enter the logarithmic growth phase by adopting the following pre-culture mode: inoculating the microalgae into a culture medium, and culturing at the temperature of 26-38 ℃ and under the illumination of 5000-30000 Lux;
preferably, the spirulina is inoculated into a Z culture medium with the pH value of 8.25-9.5;
inoculating the dunaliella salina into a DM culture medium with the pH value of 7.2-7.8;
the chlorella is inoculated into BG-11 culture medium with the pH value of 7.0-7.4.
Further preferably, the spirulina is inoculated into a Z culture medium with the pH value of 8.5-9;
inoculating the dunaliella salina into a DM culture medium with the pH value of 7.5;
the chlorella is inoculated into BG-11 culture medium with pH value of 7.1.
Wherein the formula of the Z culture medium is as follows: 16.8g/L of sodium bicarbonate, 0.5g/L of dipotassium phosphate, 2.4g/L of sodium nitrate, 1.0g/L of potassium sulfate, 1.0g/L of sodium chloride, 0.2g/L of magnesium sulfate heptahydrate, 0.04g/L of dihydrate and calcium chloride, 0.2g/L of ferrous sulfate heptahydrate and 0.08g/L of EDTA disodium, and the components are uniformly mixed.
The formula of the DM culture medium is as follows: 87.69g/L of sodium chloride, 0.42g/L of sodium nitrate, 0.0156g/L of sodium dihydrogen phosphate dihydrate, 0.044g/L of calcium chloride dihydrate, 0.074g/L of potassium chloride, 1.23g/L of magnesium sulfate heptahydrate, 0.84g/L of sodium bicarbonate and 0.5mL of ferric citrate (1%).
The formula of BG-11 is as follows: 10mL of sodium nitrate (15g/100 mLdH)2O), dipotassium hydrogen phosphate 10mL (2g/500 mLdH)2O), magnesium sulfate heptahydrate 10mL (3.75g/500 mLdH)2O), 10mL of calcium chloride dihydrate (1.8g/500 mLdH)2O), citric acid 10mL (0.3g/500 mLdH)2O), 10mL of ferric ammonium citrate (0.3g/500 mLdH)2O), 10mL of EDTA disodium salt (0.05g/500 mLdH)2O), calcium carbonate 10mL (1.0g/500 mLdH)2O)。
The invention further provides that the pre-culture is carried out under the conditions that the temperature is 31-33 ℃ and the illumination is 5000-7000 Lux.
The invention further provides that the pH value can be adjusted by adopting the conventional means in the field; adjusting the pH value by adopting a regulating solution which is more beneficial to the growth of algae, more preferably adopting a phosphoric acid solution rich in a phosphorus source;
specifically, a phosphoric acid solution with the concentration of 0.1-0.5 mol/L is adopted, and 0.1mol/L is preferable.
The inoculation concentration of the microalgae in the culture medium is 8-12 ml/L, and preferably 10 ml/L. The microalgae is in the state of algae mud, and the OD value of each 10ml/L of algae mud is about 0.3.
The number of days of pre-culture is 1-3 days, preferably 2 days.
The invention further provides that the culture temperature is reduced at a constant speed at a rate of 1 ℃ per 20-60 minutes, preferably at a rate of 1 ℃ per 30-42 minutes, and more preferably at a rate of 1 ℃ per 36 minutes.
The invention further provides that the culture temperature is reduced to 0 to-7 ℃ at a constant speed, and preferably 0 to-5 ℃; especially when the temperature is reduced to-5 ℃, the efficiency of compensating growth is highest.
The invention further provides that the time for maintaining the culture temperature is 6-15 hours, preferably 9-12 hours.
As a preferred scheme of the invention, the method for starting the microalgae to compensate the growth at the ice temperature comprises the following steps:
1) inoculating spirulina, dunaliella salina or chlorella into a culture medium at an inoculation concentration of 8-12 ml/L, and culturing at 31-33 ℃ under the illumination of 5000-7000 Lux to enable the spirulina, dunaliella salina or chlorella to enter a logarithmic phase;
wherein the content of the first and second substances,
the spirulina is inoculated into a Z culture medium with the pH value of 8.25-9.5;
inoculating the dunaliella salina into a DM culture medium with the pH value of 7.2-7.8;
inoculating the chlorella into a BG-11 culture medium with a pH value of 7.0-7.4;
2) and (2) under a dark condition, uniformly reducing the culture temperature of the spirulina, the dunaliella salina or the chlorella in the logarithmic growth phase in the step 1) to 0-5 ℃ at the rate of reducing the temperature by 1 ℃ every 30-40 minutes, and maintaining the culture temperature for 9-12 hours.
Further provides a preferable scheme, a microalgae ice temperature starting and growth compensation method, which comprises the following steps:
1) inoculating spirulina, dunaliella salina or chlorella into a culture medium at an inoculation concentration of 10ml/L, and culturing at 31-33 ℃ under the illumination of 5000-7000 Lux to enable the spirulina, dunaliella salina or chlorella to enter a logarithmic phase;
wherein the content of the first and second substances,
the spirulina is inoculated into a Z culture medium with the pH value of 8.5-9;
inoculating the dunaliella salina into a DM culture medium with the pH value of 7.2-7.8;
inoculating the chlorella into a BG-11 culture medium with a pH value of 7.0-7.4;
2) and (2) under the dark condition, uniformly reducing the culture temperature of the spirulina, the dunaliella salina or the chlorella in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes, and maintaining the culture temperature for 12 hours.
The method for starting the ice temperature compensation growth of the microalgae is simple and effective, has low treatment cost, can be directly used for the industrial production of the microalgae, greatly improves the industrial yield of the microalgae, increases the growth rate of the microalgae treated by the method by more than 2 times compared with the common microalgae, and has immeasurable industrial application value.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The formulation of the Z's medium used in the following examples was as follows:
16.8g/L of sodium bicarbonate, 0.5g/L of dipotassium phosphate, 2.4g/L of sodium nitrate, 1.0g/L of potassium sulfate, 1.0g/L of sodium chloride, 0.2g/L of magnesium sulfate heptahydrate, 0.04g/L of dihydrate and calcium chloride, 0.2g/L of ferrous sulfate heptahydrate and 0.08g/L of EDTA disodium, and the components are uniformly mixed.
The formula of the DM culture medium is as follows: 87.69g/L of sodium chloride, 0.42g/L of sodium nitrate, 0.0156g/L of sodium dihydrogen phosphate dihydrate, 0.044g/L of calcium chloride dihydrate, 0.074g/L of potassium chloride, 1.23g/L of magnesium sulfate heptahydrate, 0.84g/L of sodium bicarbonate and 0.5mL of ferric citrate (1%).
The formula of BG-11 is as follows: 10mL of sodium nitrate (15g/100 mLdH)2O), dipotassium hydrogen phosphate 10mL (2g/500 mLdH)2O), magnesium sulfate heptahydrate 10mL (3.75g/500 mLdH)2O), 10mL of calcium chloride dihydrate (1.8g/500 mLdH)2O), citric acid 10mL (0.3g/500 mLdH)2O), 10mL of ferric ammonium citrate (0.3g/500 mLdH)2O), 10mL of EDTA disodium salt (0.05g/500 mLdH)2O), calcium carbonate 10mL (1.0g/500 mLdH)2O)。
The formula of BG-11 is as follows: 10mL of sodium nitrate (15g/100 mLdH)2O), dipotassium hydrogen phosphate 10mL (2g/500 mLdH)2O), magnesium sulfate heptahydrate 10mL (3.75g/500 mLdH)2O), 10mL of calcium chloride dihydrate (1.8g/500 mLdH)2O), citric acid 10mL (0.3g/500 mLdH)2O), 10mL of ferric ammonium citrate (0.3g/500 mLdH)2O), 10mL of EDTA disodium salt (0.05g/500 mLdH)2O), calcium carbonate 10mL (1.0g/500 mLdH)2O)。
The spirulina used in the examples is Spirulina platensis obtained from the alga species preserved in Spirulina research institute of Beijing university of forestry;
dunaliella salina (FACHB-435) and Chlorella vulgaris (FACHB-8) were purchased from the stock of wild organisms belonging to Chinese academy of sciences, freshwater algae.
Example 1
The embodiment is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) inoculating spirulina platensis into a fresh Z culture medium at the inoculation concentration of 10ml/L, and culturing for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9 to ensure that the spirulina platensis adapts to a new growth environment and enters a logarithmic growth phase;
2) under the dark condition, uniformly reducing the culture temperature of the spirulina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes; the incubation temperature was maintained and the treatment was carried out for 12 h.
The spirulina cultured in the above steps is cultured for 4 days at 32 ℃ under 6000 Lux. The daily relative growth rate was measured, and the daily relative growth rate (OD) was measured to be 0.210.
Example 2
The embodiment is a method for starting compensation growth of dunaliella salina at ice temperature, which comprises the following steps:
1) the dunaliella salina is inoculated into a fresh DM culture medium at the inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 7.5, so that the dunaliella salina is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the dark condition, uniformly reducing the culture temperature of the dunaliella salina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes; the culture temperature was maintained for 9 h.
Culturing the dunaliella salina cultured in the above steps at 32 deg.C and 6000Lux for 4 days. The daily relative growth rate was measured, and the daily relative growth rate (OD) was measured to be 0.168.
Example 3
The embodiment is a method for starting up chlorella to compensate growth by using ice temperature, which comprises the following steps:
1) chlorella was inoculated into fresh BG-11 medium at an inoculation concentration of 10ml/L, and cultured under culture conditions of 32 deg.C, 6000Lux, pH 7.1 for 2 days to adapt to a new growth environment, and then, it entered a logarithmic growth phase.
2) Under the dark condition, uniformly reducing the culture temperature of the chlorella in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes; the culture temperature was maintained for 3 h.
Culturing the chlorella cultured in the above steps at 32 deg.C and 6000Lux for 4 days. The daily relative growth rate was measured, and the daily relative growth rate (OD) was measured to be 0.089.
Example 4
The embodiment is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) the spirulina platensis is inoculated into a fresh Z culture medium at the inoculation concentration of 10ml/L, and is cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the spirulina platensis is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the dark condition, uniformly reducing the culture temperature of the spirulina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 30 minutes, and maintaining the culture temperature for 12 hours;
3) the spirulina was cultured at 32 ℃ under 6000Lux for 4 days. The daily relative growth rate (OD) was 0.179.
Example 5
The embodiment is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) the spirulina platensis is inoculated into a fresh Z culture medium at the inoculation concentration of 10ml/L, and is cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the spirulina platensis is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the dark condition, uniformly reducing the culture temperature of the spirulina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 42 minutes, and maintaining the culture temperature for 12 hours;
3) the spirulina was cultured at 32 ℃ under 6000Lux for 4 days. The daily relative growth rate (OD) was 0.147.
Example 6
The embodiment is a method for starting compensation growth of dunaliella salina at ice temperature, which comprises the following steps:
1) the Dunaliella salina is inoculated into a fresh Z culture medium at the inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the Dunaliella salina is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the dark condition, uniformly reducing the culture temperature of the dunaliella salina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 30 minutes, and maintaining the culture temperature for 9 hours;
3) the above dunaliella salina was cultured at 32 ℃ and 6000Lux for 4 days. The daily relative growth rate (OD) was 0.129.
Comparative example 1
The comparative example is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) the spirulina is inoculated into a fresh Z culture medium at an inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the spirulina is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the condition of illumination, uniformly reducing the culture temperature of the spirulina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes; the incubation temperature was maintained for 12 h.
The spirulina cultured in the above steps is cultured for 4 days at 32 ℃ under 6000 Lux. The daily relative growth rate was measured, and the daily relative growth rate (OD) was 0.011.
Comparative example 2
The comparative example is a method for the ice temperature starting compensation growth of dunaliella salina, which comprises the following steps:
1) the dunaliella salina is inoculated into a fresh DM culture medium at the inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 7.5, so that the dunaliella salina is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the dark condition, carrying out low-temperature treatment on the dunaliella salina in the logarithmic growth phase in the step 1), adjusting the temperature to-5 ℃, and continuing the whole low-temperature process for 12 h;
the spirulina cultured in the above steps is cultured for 4 days at 32 ℃ under 6000 Lux. Detecting the daily relative growth rate of the plants to obtain
The daily relative growth rate (OD) was 0.013.
Comparative example 3
The comparative example is a method for the cold starting and the compensatory growth of chlorella, which comprises the following steps:
1) chlorella was inoculated into fresh BG-11 medium at an inoculation concentration of 10ml/L, and cultured under culture conditions of 32 deg.C, 6000Lux, pH 7.1 for 2 days to adapt to a new growth environment, and then, it entered a logarithmic growth phase.
2) Under the illumination condition, uniformly reducing the culture temperature of the pellets in the logarithmic growth phase in the step 1) to 0 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes, and maintaining the culture temperature for 3 hours;
the spirulina cultured in the above steps is cultured for 4 days at 32 ℃ under 6000 Lux. Detecting the daily relative growth rate of the plants to obtain
The daily relative growth rate (OD) was 0.039.
Comparative example 4
The comparative example is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) the spirulina is inoculated into a fresh Z culture medium at an inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the spirulina is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the condition of illumination, the spirulina is continuously cultured for 4 days at 32 ℃ and 6000 Lux. The daily relative growth rate (OD) was 0.077.
Comparative example 5
The embodiment is a method for the ice temperature starting compensation growth of spirulina, which comprises the following steps:
1) the spirulina platensis is inoculated into a fresh Z culture medium at the inoculation concentration of 10ml/L, and is cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the spirulina platensis is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the illumination condition, uniformly reducing the culture temperature of the spirulina in the logarithmic growth phase in the step 1) to 0 ℃ at the rate of reducing the temperature by 1 ℃ every 36 minutes, and maintaining the culture temperature for 12 hours;
3) the spirulina was cultured at 32 ℃ under 6000Lux for 4 days. The daily relative growth rate (OD) was 0.019.
Comparative example 6
The embodiment is a method for starting up chlorella to compensate growth by using ice temperature, which comprises the following steps:
1) the chlorella is inoculated into a fresh Z culture medium at an inoculation concentration of 10ml/L, and cultured for 2 days under the culture conditions of 32 ℃, 6000Lux and pH 9, so that the chlorella is adapted to a new growth environment and enters a logarithmic growth phase.
2) Under the condition of illumination, uniformly reducing the culture temperature of the chlorella in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 42 minutes, and maintaining the culture temperature for 12 hours;
3) culturing the above Chlorella at 32 deg.C and 6000Lux for 4 days. The daily relative growth rate (OD) was 0.032.
The treatment parameters and daily relative growth rates of the examples and comparative examples are shown in Table 1.
TABLE 1
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (7)
1. A method for starting compensation growth of microalgae at ice temperature is characterized in that under a dark condition, the culture temperature of microalgae in a logarithmic growth phase is reduced to-5 ℃ at a constant speed, and the culture temperature is maintained for 9-12 h; the culture temperature is reduced at a constant speed of 1 ℃ every 30-42 minutes; the microalgae is selected from one or more of spirulina and dunaliella salina; the spirulina is one of Spirulina maxima, Spirulina platensis, and Spirulina subsalsa; the Dunaliella salina is Dunaliella salina; the microalgae enter logarithmic growth phase by adopting the following pre-culture mode: inoculating the microalgae into a culture medium, and culturing at the temperature of 26-38 ℃ and under the illumination of 5000-30000 Lux; the spirulina is inoculated into a Z culture medium with the pH value of 8.5-9; the dunaliella salina is inoculated into DM culture medium with the pH value of 7.5.
2. The method according to claim 1, wherein the pre-culture is performed under the conditions of a temperature of 31 to 33 ℃ and an illuminance of 5000 to 7000 Lux.
3. The method of claim 1, wherein the agent that adjusts the pH is a phosphoric acid solution.
4. The method according to claim 3, wherein the phosphoric acid solution is used at a concentration of 0.1 to 0.5 mol/L.
5. The method according to any one of claims 1 to 4, wherein the microalgae is inoculated in the culture medium at a concentration of 8 to 12 ml/L.
6. The method of claim 5, wherein the microalgae is inoculated into the culture medium at a concentration of 10 ml/L.
7. Method according to claim 1, characterized in that it comprises the following steps:
1) inoculating spirulina and dunaliella salina into a culture medium at an inoculation concentration of 8-12 ml/L, and culturing under the conditions that the temperature is 31-33 ℃ and the illumination is 5000-7000 Lux to enable the spirulina and the dunaliella salina to enter a logarithmic phase;
wherein the content of the first and second substances,
the spirulina is inoculated into a Z culture medium with the pH value of 8.25-9.5;
inoculating the dunaliella salina into a DM culture medium with the pH value of 7.2-7.8;
2) and (3) under a dark condition, uniformly reducing the culture temperature of the spirulina and the dunaliella salina in the logarithmic growth phase in the step 1) to-5 ℃ at the rate of reducing the temperature by 1 ℃ every 30-40 minutes, and maintaining the culture temperature for 9-12 hours.
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