Method for improving astaxanthin produced by haematococcus pluvialis

Abstract

The invention belongs to the field of industrial application of microalgae, and provides a method for improving astaxanthin produced by haematococcus pluvialis. Comprising the following steps: 1, culturing haematococcus pluvialis to a logarithmic phase; 2, the haematococcus pluvialis is diluted to a proper concentration, and a seed solution for producing astaxanthin from the haematococcus pluvialis is prepared; 3, respectively adding six pre-prepared inducers including creatine, glycine, 6-aminocaproic acid, glutamic acid, L-hydroxyproline and L-cysteine into the prepared haematococcus pluvialis liquid for induction; and 4, continuously filling sterile air into the haematococcus pluvialis liquid added with the inducer in a photobioreactor, culturing, and inducing haematococcus pluvialis cells to accumulate astaxanthin. The invention discloses a method for improving astaxanthin produced by haematococcus pluvialis. The haematococcus pluvialis can be promoted to accumulate a large amount of astaxanthin, and the production cycle of the astaxanthin is shortened. The method is easy to operate, high in efficiency, low in cost and completely suitable for industrial culture of haematococcus pluvialis.

Description

Technical Field

[0001] The present invention relates to a method for producing natural astaxanthin from Haematococcus pluvialis, and belongs to the field of microalgae biotechnology. Background Art

[0002] Astaxanthin belongs to terpene unsaturated compounds and is well-known for its excellent antioxidant properties and outstanding coloring ability. It is reported that its antioxidant capacity is 10 times that of β-carotene and 6000 times that of vitamin C. In addition, astaxanthin also has activities such as anti-inflammatory, anti-tumor, anti-diabetic, and immunomodulatory effects. Therefore, astaxanthin is widely used in various industries such as aquaculture, food, health products, cosmetics, and pharmaceuticals. Therefore, the market demand for astaxanthin is increasing.

[0003] Astaxanthin exists in three different configurations, including left-handed (3S, 3'S), right-handed (3R, 3'R), and meso (3S, 3'R). Among them, the left-handed isomer has the highest antioxidant activity, followed by the right-handed, and the meso configuration has a lower antioxidant activity.

[0004] Currently, the methods for producing astaxanthin mainly include chemical synthesis method, natural extraction method, and microbial fermentation method. Among them, the market price of chemically synthesized astaxanthin is 2000 US dollars per kilogram, while that of natural astaxanthin is 7000 US dollars per kilogram. Among them, (1) the chemical synthesis method is the main production method of astaxanthin at present. However, the production process of the chemical synthesis method is complex, and the synthesized product is a mixture of astaxanthin with various configurations (generally left-handed: racemic: right-handed = 1:2:1). Along with the chemical synthesis, a variety of by-products are accumulated, and there are safety risks. Therefore, chemically synthesized astaxanthin is prohibited for human use. Moreover, the absorption and utilization rate of chemically synthesized astaxanthin in the body is much lower than that of natural astaxanthin. (2) The natural extraction method is to extract astaxanthin from crustacean wastes such as lobsters and crabs, but the yield is low and the process is complex. Moreover, it is restricted by the source of raw materials and large-scale production is limited. (3) The microbial fermentation method is a way to synthesize astaxanthin using microorganisms. Microbial production of astaxanthin has the advantages of clear structure, few by-products, and low production cost, and is one of the most effective ways to realize industrial production of astaxanthin, and can better meet the needs of consumers for natural compounds.

[0005] Haematococcus pluvialis is a freshwater single-celled green alga. Haematococcus pluvialis is currently the species known to have the highest astaxanthin content. Under stress conditions, it can accumulate a large amount of astaxanthin, with the content reaching 1-5% of the cell dry weight. At the same time, the astaxanthin produced by Haematococcus pluvialis is the astaxanthin ester with the strongest antioxidant activity in the left-handed configuration. Among them, the monoester accounts for about 80%, and the diester accounts for about 15%. It is considered to be one of the most commercially valuable sources of astaxanthin. Trans-astaxanthin has higher absorption utilization rate, coloring ability and antioxidant activity for farmed animals. The astaxanthin derived from Haematococcus pluvialis has been widely used in various fields, and the current market supply falls short of demand.

[0006] Most of the process methods for culturing Haematococcus pluvialis to produce astaxanthin are divided into two stages according to its biological characteristics; the first stage is the green cell stage, providing suitable production conditions for Haematococcus pluvialis to allow the rapid division and growth of Haematococcus pluvialis, accumulating algal cells, and providing microalgal seed liquid; the second stage is the red cell stage, changing the culture environment of Haematococcus pluvialis to stress Haematococcus pluvialis to stop dividing and start accumulating astaxanthin.

[0007] The accumulation of astaxanthin in Haematococcus pluvialis is regulated by a variety of signaling factors and at multiple levels. High light, nitrogen deficiency and the addition of inducers can effectively promote astaxanthin accumulation. Under high light conditions, the accumulation of astaxanthin increases significantly, but high light has the disadvantages of high energy consumption and significant inhibition of growth. Nitrogen deficiency can also significantly induce Haematococcus pluvialis to accumulate astaxanthin, but there are disadvantages such as long culture period and easy contamination. Adding inducers such as α-ketoglutaric acid, indoleacetic acid, melatonin, naphthylacetic acid, fulvic acid, brassinolide, jasmonic acid, gibberellin, inositol, etc. in the late logarithmic growth phase of Haematococcus pluvialis culture can also effectively induce astaxanthin accumulation.

[0008] In addition, the long culture period of culturing Haematococcus pluvialis leads to too high production costs, becoming a bottleneck in the development of this technology. Shortening the culture period is an important means to reduce production costs. However, in the natural culture process, Haematococcus pluvialis accumulates a small amount of astaxanthin, and the period of producing astaxanthin is relatively long. How to shorten the culture period of Haematococcus pluvialis accumulating astaxanthin has become an urgent need in the industry of producing astaxanthin from Haematococcus pluvialis. Summary of the Invention

[0009] In view of the above problems, the purpose of the present invention is to provide a method for improving the production of astaxanthin by Haematococcus pluvialis, which is applied to the culture method for the cultivation of Haematococcus pluvialis and the efficient accumulation of astaxanthin.

[0010] In one embodiment, the present invention provides a method for producing astaxanthin, including:

[0011] Step 1, the green cell culture stage.

[0012] Step 2: dilute the algae cell seed solution cultured in step 1 to an appropriate concentration, add 0.5 g / L of carrot powder, and prepare a seed solution for producing astaxanthin by Haematococcus pluvialis.

[0013] Step 3: adding creatine, glycine, 6-aminocaproic acid, glutamic acid, L-hydroxyproline, and L-cysteine as inducers to the algae solution of Haematococcus pluvialis prepared in step 2 for induction.

[0014] Step 4: Add six different inducers, namely creatine, glycine, 6-aminocaproic acid, glutamic acid, L-hydroxyproline, and L-cysteine, to the algae solution in step 3 and continuously fill it with sterile carbon dioxide for cultivation to induce the Haematococcus pluvialis cells to accumulate astaxanthin.

[0015] Furthermore, the step 1 is carried out by culturing Haematococcus pluvialis in an indoor photobioreactor to produce a green cell seed solution of Haematococcus pluvialis;

[0016] Furthermore, in step 1, the following ingredients per liter of culture medium were added: MgSO4·7H2O 100 mg, CaCl2·H2O 24.99 mg, FeCl3·6H2O 0.2 mg, H3BO3 0.6 mg, CuSO4·5H2O 0.1563 mg, (NH4)6Mo7O4 0.4 mg, K2HPO4·3H2O 26.2 mg, Na2ENTA 0.2 mg, KNO3 200 mg, CoCl2 0.1 mg, 0.5 g / L carrot powder, and 1000 mL of distilled water.

[0017] Furthermore, the pH value of the culture medium in step 1 is adjusted to 7.0-8.5;

[0018] Furthermore, the photobioreactor in step 1 is equipped with an LED as a light source, whose characteristic spectrum is white or red and whose light quantum intensity is 80-120 μmol / m 2 ·s, photoperiod was 12L:12D;

[0019] Furthermore, the ventilation in step 1 is a sterile gas, which is carbon dioxide gas, and the gas is filtered through a 0.22 μm filter membrane to remove bacteria;

[0020] Furthermore, in step 1, the culture temperature of the Haematococcus pluvialis is 20-26°C;

[0021] Further, in step 1, the concentration of the Haematococcus pluvialis culture reaches 10 5 ~10 7 When the number of cells / mL is reached, proceed to step 2;

[0022] Further, the dilution solution used in step two is as follows per liter of culture medium: 100 mg of MgSO4·7H2O, 24.99 mg of CaCl2·H2O, 0.2 mg of FeCl3·6H2O, 0.6 mg of H3BO3, 0.1563 mg of CuSO4·5H2O, 0.4 mg of (NH4)6Mo7O4, and

[0023] 26.2 mg of K2HPO4·3H2O, 0.2 mg of Na2ENTA, 40 mg of KNO3, 0.1 mg of CoCl2, 0.5 g / L of carrot powder, and 1000 mL of distilled water.

[0024] Further, in step two, the Haematococcus pluvialis is diluted to a density of 1×10 4 cells / mL to 9×10 4 cells / mL;

[0025] Further, in step three, the concentrations of the six different inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, are 0.1 - 80 mg / L respectively;

[0026] Further, in step four, the culture temperature of the Haematococcus pluvialis is 25 - 30 °C;

[0027] Further, in step four, the pH value of the Haematococcus pluvialis culture solution is 7.5 - 9.0.

[0028] Further, in step four, the photobioreactor uses LEDs as the light source, its characteristic spectrum is white, light with a wavelength of 380 - 500 nm is added, the light intensity is adjusted to 160 - 200 μmol / m 2 ·s, and the light cycle is changed to continuous light.

[0029] Furthermore, the present invention provides the application of the six inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, in the culture or cultivation of Haematococcus pluvialis during the induction stage.

[0030] Furthermore, the present invention provides the application of adding the six inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, during the induction stage to induce Haematococcus pluvialis to produce astaxanthin.

[0031] The present invention further provides the culture conditions of the above - mentioned bacteria - algae co - culture system. The specific culture conditions are adjusted according to the proliferation of the cell number of Haematococcus pluvialis, and are successively divided into green cell culture conditions and induction culture conditions;

[0032] Furthermore, the application of the above - mentioned Haematococcus pluvialis culture method in the production of astaxanthin.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1) By regulating the amount of carbon dioxide introduced, the present invention adjusts the pH value of the Haematococcus pluvialis culture system, solving the problem of unstable pH during the large-scale cultivation of Haematococcus pluvialis.

[0035] 2) The six inducing agents of creatine, glycine, 6-aminohexanoic acid, glutamic acid, L-hydroxyproline, and L-cysteine used in the present invention are very cheap compared with other chemical inducing agents. While saving costs, the content of natural astaxanthin in Haematococcus pluvialis is significantly increased.

[0036] 3) By adding six inducing agents of creatine, glycine, 6-aminohexanoic acid, glutamic acid, L-hydroxyproline, and L-cysteine to induce Haematococcus pluvialis to accumulate astaxanthin, the induction period is shortened, and the astaxanthin yield of Haematococcus pluvialis is rapidly increased in a short period.

[0037] 4) The operation of the present invention is simple and feasible, and is completely applicable to the large-scale cultivation of Haematococcus pluvialis. Description of the Drawings

[0038] Figure 1 Flowchart of the application method of the inducing agent provided in the embodiment of the present invention in promoting the production of astaxanthin by Haematococcus pluvialis.

[0039] Figure 2 Effect of different inducing agents on the astaxanthin accumulation amount of Haematococcus pluvialis. Detailed Embodiments

[0040] The technical solution of the present invention will be further described below in conjunction with specific embodiments. The reagents and raw materials used in the present invention are all conventional commercially available products without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0041] Example 1

[0042] Application method of adding creatine to induce Haematococcus pluvialis to accumulate astaxanthin, the specific steps are as follows:

[0043] 1) Cultivation of green cells of Haematococcus pluvialis: Cultivate Haematococcus pluvialis at 24 °C, light intensity of 80 μmol / m 2 ·s, and a light cycle of 12L:12D. The Haematococcus pluvialis used is the Haematococcus pluvialis strain, and it is cultivated until the late logarithmic growth phase, and the cell concentration is 5×10 6 cells / mL.

[0044] 2) Dilution of algal cells. Dilute the algal cells with sterile nitrogen-reduced MCM medium (KNO₃ 40 mg / L) until the cell concentration reaches 5×10 4 cells / mL.

[0045] 3) Add creatine to the diluted culture medium to a final concentration of 40 mg / L.

[0046] 4) Culture the algal cell culture medium with creatine added in step 3) in a photobioreactor. Culture conditions: Culture Haematococcus pluvialis under continuous light at 28 °C and a light intensity of 200 μmol / m 2 ·s. After 14 days of culture, collect the algal cells to measure the astaxanthin content. Additionally, set up a control group: Inoculate only Haematococcus pluvialis with the same initial cell density into a tubular photobioreactor and perform algal cell culture. The culture conditions are exactly the same as those of the above experimental group.

[0047] For the algal liquid cultured in this example, the biomass was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific steps are as follows: (1) Measurement of biomass: Take 10 mL of the algal liquid, centrifuge it and discard the supernatant, collect it in a pre-weighed 1.5 mL centrifuge tube, and perform freeze-drying until a constant weight is obtained. Calculate according to the following formula: Biomass (g / L) = dry weight (g) / volume (L). (2) Measurement of astaxanthin content: Take 5 mL of the algal liquid, centrifuge it (3500×g) for 10 min, add 2 mL of a mixed solution of 30% methanol (containing 5% KOH), mix well, and heat in a water bath at 65 °C for 15 min. Centrifuge the mixed solution (3500×g) for 5 min, discard the supernatant, and wash it twice with deionized water. Add 5 mL of dimethyl sulfoxide (DMSO), and use a 200 W ultrasonic wave to break the algal bodies until they are colorless. After centrifugation, take the supernatant and measure its absorbance value at 490 nm. Calculate according to the following formula: Astaxanthin concentration (mg / L) = 4.5×A×Va / Vb. Where, A represents the absorbance value at 490 nm; Va represents the volume of DMSO added; Vb represents the volume of the algal liquid. (3) Calculation of astaxanthin content: Astaxanthin content (mg / g) = astaxanthin concentration (mg / L) / cell biomass (g / L).

[0048] The test results showed that when the creatine concentration was 40 mg / L, the highest astaxanthin content in microalgae was 36.13 mg / g. And the culture medium after culturing the algal cells was non-polluted and could be directly discharged.

[0049] Example 2

[0050] Application method of adding glycine in inducing Haematococcus pluvialis to accumulate astaxanthin, the specific steps are as follows:

[0051] 1) Culture of green cells of Haematococcus pluvialis: At 24 °C and a light intensity of 80 μmol / m2 ·s, Haematococcus pluvialis was cultured under the condition of 12L:12D light cycle. Haematococcus pluvialis strain was used, and when cultured to the late logarithmic growth phase, the cell concentration was 5×10 6 cells / mL.

[0052] 2) Dilution of algal cells. The algal cells were diluted with sterile nitrogen-reduced MCM medium (KNO3 40 mg / L) until the cell concentration reached 5×10 4 cells / mL.

[0053] 3) Glycine was added to the diluted culture medium at a final concentration of 10 mg / L.

[0054] 4) The algal cell culture medium added with glycine in step 3) was cultured in a photobioreactor. Culture conditions: Haematococcus pluvialis was cultured under continuous light at 28 °C and light intensity of 200 μmol / m 2 ·s to induce the accumulation of astaxanthin in Haematococcus pluvialis cells. After 14 days of culture, the algal cells were collected to measure the astaxanthin content. In addition, a control group was set up: only Haematococcus pluvialis with the same initial cell density was inoculated into the tubular photobioreactor for algal cell culture, and the culture conditions were exactly the same as those of the above experimental group.

[0055] For the algal liquid cultured in this example, the biomass was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific measurement steps were as in Example 1.

[0056] The test results showed that when the glycine concentration was 10 mg / L, the highest astaxanthin content in microalgae was 34.37 mg / g. And the culture medium after culturing the algal cells was non-polluted and could be directly discharged.

[0057] Example 3

[0058] Application method of adding 6-aminocaproic acid in inducing the accumulation of astaxanthin in Haematococcus pluvialis, the specific steps are as follows:

[0059] 1) Culture of green cells of Haematococcus pluvialis: Haematococcus pluvialis was cultured at 24 °C and light intensity of 80 μmol / m 2 ·s under the condition of 12L:12D light cycle. Haematococcus pluvialis strain was used, and when cultured to the late logarithmic growth phase, the cell concentration was 5×10 6 cells / mL.

[0060] 2) Dilution of algal cells. The algal cells were diluted with sterile nitrogen-reduced MCM medium (KNO3 40 mg / L) until the cell concentration reached 5×10 4 cells / mL.

[0061] 3) Add 6 - aminocaproic acid to the diluted culture medium to a final concentration of 0.5 mg / L.

[0062] 4) Culture the algal cell culture medium added with 6 - aminocaproic acid in step 3) in a photobioreactor. Culture conditions: Culture Haematococcus pluvialis under continuous light at 28 °C and a light intensity of 200 μmol / m 2 ·s. After culturing for 14 days, collect the algal cells to measure the astaxanthin content. Additionally, set up a control group: Only inoculate Haematococcus pluvialis with the same initial cell density into a tubular photobioreactor for algal cell culture, and the culture conditions are exactly the same as those of the above experimental group.

[0063] For the algal liquid cultured in this example, the biomass was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific measurement steps are as in Example 1.

[0064] The test results showed that when the concentration of 6 - aminocaproic acid was 0.5 mg / L, the highest astaxanthin content in microalgae was 22.54 mg / g. And the culture medium after culturing the algal cells was pollution - free and could be directly discharged.

[0065] Example 4

[0066] Application method of adding glutamic acid in inducing Haematococcus pluvialis to accumulate astaxanthin, the specific steps are as follows:

[0067] 1) Culture of green cells of Haematococcus pluvialis: Culture Haematococcus pluvialis at 24 °C and a light intensity of 80 μmol / m 2 ·s, with a light cycle of 12L:12D. The Haematococcus pluvialis used is the algal strain Haematococcus pluvialis. Culture until the late logarithmic growth phase, and the cell concentration is 5×10 6 cells / mL.

[0068] 2) Dilution of algal cells. Dilute the algal cells with sterile nitrogen - reduced MCM medium (KNO3 40 mg / L) until the cell concentration is 5×10 4 cells / mL.

[0069] 3) Add glutamic acid to the diluted culture medium to a final concentration of 5 mg / L.

[0070] 4) Culture the algal cell culture medium added with glutamic acid in step 3) in a photobioreactor. Culture conditions: At 28 °C and a light intensity of 200 μmol / m 2·s, cultivate Haematococcus pluvialis under continuous light conditions to induce the accumulation of astaxanthin in Haematococcus pluvialis cells. After 14 days of cultivation, collect the algal cells and measure the astaxanthin content. Additionally, set up a control group: only inoculate Haematococcus pluvialis with the same initial cell density into a tubular photobioreactor for algal cell cultivation, and the cultivation conditions are exactly the same as those of the above experimental group.

[0071] For the algal liquid cultured in this example, the biomass was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific measurement steps were as in Example 1.

[0072] The test results showed that when the glutamic acid concentration was 5 mg / L, the highest astaxanthin content in the microalgae was 26.81 mg / g. Moreover, the culture medium of the cultured algal cells was pollution-free and could be directly discharged.

[0073] Example 5

[0074] The application method of adding L-hydroxyproline in inducing the accumulation of astaxanthin in Haematococcus pluvialis is as follows:

[0075] 1) Cultivation of green cells of Haematococcus pluvialis: Cultivate Haematococcus pluvialis at 24 °C, light intensity of 80 μmol / m 2 ·s, and a light cycle of 12L:12D. Haematococcus pluvialis uses the algal strain Haematococcus pluvialis and is cultured until the late logarithmic growth phase, with a cell concentration of 5×10 6 cells / mL.

[0076] 2) Dilution of algal cells. Dilute the algal cells with sterile nitrogen-reduced MCM medium (KNO3 40 mg / L) to a cell concentration of 5×10 4 cells / mL.

[0077] 3) Add L-hydroxyproline to the diluted culture solution to a final concentration of 1 mg / L.

[0078] 4) Cultivate the algal cell culture solution added with L-hydroxyproline in step 3) in a photobioreactor. Cultivation conditions: Cultivate Haematococcus pluvialis at 28 °C, light intensity of 200 μmol / m 2 ·s, under continuous light conditions to induce the accumulation of astaxanthin in Haematococcus pluvialis cells. After 14 days of cultivation, collect the algal cells and measure the astaxanthin content. Additionally, set up a control group: only inoculate Haematococcus pluvialis with the same initial cell density into a tubular photobioreactor for algal cell cultivation, and the cultivation conditions are exactly the same as those of the above experimental group.

[0079] For the algal liquid cultured in this example, the biomass was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific measurement steps were as in Example 1.

[0080] The detection results showed that when the concentration of L-hydroxyproline was 1 mg / L, the highest astaxanthin content in microalgae was 27.01 mg / g. And the culture medium after culturing algal cells was pollution-free and could be directly discharged.

[0081] Example 6

[0082] The application method of adding L-cysteine to induce Haematococcus pluvialis to accumulate astaxanthin is as follows:

[0083] 1) Culture of green cells of Haematococcus pluvialis: Haematococcus pluvialis was cultured under the conditions of 24 °C, light intensity of 80 μmol / m 2 ·s, and a light cycle of 12L:12D. Haematococcus pluvialis strain Haematococcus pluvialis was cultured until the late logarithmic growth phase, and the cell concentration was 5×10 6 cells / mL.

[0084] 2) Dilution of algal cells. The algal cells were diluted with sterile nitrogen-reduced MCM medium (KNO3 40 mg / L) to a cell concentration of 5×10 4 cells / mL.

[0085] 3) Add L-cysteine to the diluted culture solution to a final concentration of 80 mg / L.

[0086] 4) The algal cell culture solution added with L-cysteine in step 3) was cultured in a photobioreactor. Culture conditions: Haematococcus pluvialis was cultured under the conditions of 28 °C, light intensity of 200 μmol / m 2 ·s, and continuous light to induce Haematococcus pluvialis cells to accumulate astaxanthin. After culturing for 14 days, the algal cells were collected to measure the astaxanthin content. In addition, a control group was set up: only Haematococcus pluvialis with the same initial cell density was inoculated into a tubular photobioreactor for algal cell culture, and the culture conditions were exactly the same as those of the above experimental group.

[0087] The biomass of the algal solution cultured in this example was measured by the dry weight method, and the astaxanthin content of the algal cells was measured by spectrophotometry. The specific measurement steps were as in Example 1.

[0088] The detection results showed that when the concentration of L-cysteine was 80 mg / L, the highest astaxanthin content in microalgae was 35.23 mg / g. And the culture medium after culturing algal cells was pollution-free and could be directly discharged.

Claims

1. A method for improving the production of astaxanthin by Haematococcus pluvialis, characterized in that, Cultivate green cells of Haematococcus pluvialis using MCM medium until the cell concentration reaches 10 5 ~10 7 cells / mL; dilute the Haematococcus pluvialis cells to 1×10 4 cells / mL to 9×10 4 cells / mL; then add six kinds of inducers, namely creatine, glycine, 6-aminocaproic acid, glutamic acid, L-hydroxyproline, and L-cysteine, with concentrations ranging from 0.1 to 80 mg / L respectively, to induce Haematococcus pluvialis to accumulate astaxanthin; the specific steps are as follows:

1. Cultivate Haematococcus pluvialis in an indoor photobioreactor to produce a green cell seed solution of Haematococcus pluvialis; In step 1, the MCM medium is as follows: 100 mg of MgSO4·7H2O, 24.99 mg of CaCl2·H2O, 0.2 mg of FeCl3·6H2O, 0.6 mg of H3BO3, 0.1563 mg of CuSO4·5H2O, 0.4 mg of (NH4)6Mo7O4, 26.2 mg of K2HPO4·3H2O, 0.2 mg of Na2ENTA, 200 mg of KNO3, 0.1 mg of CoCl2, and 1000 mL of distilled water; In step 1, the pH value of the medium is 7.0 - 8.5; In Step 1, the photobioreactor uses LEDs as the light source, with a characteristic spectrum of white or red, a photon flux density of 80-120 μmol / m 2 ·s, and a light cycle of 12L:12D; In step 1, the aeration is to introduce sterile carbon dioxide gas, and the gas is passed through a 0.22 μm filter membrane to remove bacteria; In step 1, the cultivation temperature of Haematococcus pluvialis is 20°C - 26°C; In Step 1, when the culture concentration of Haematococcus pluvialis reaches 10 5 to 10 7 cells / mL, Step 2 is entered; 2. Dilute the algal cell seed solution cultivated in step 1 to an appropriate concentration to prepare a seed solution of Haematococcus pluvialis for producing astaxanthin; In step two, the Haematococcus pluvialis is diluted to a density of 1×10 4 cells / mL to 9×10 4 cells / mL; 3. Add six different inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, respectively, to the Haematococcus pluvialis solution prepared in step 2 for induction; In step 3, the concentrations of the six different inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, are 0.1 - 80 mg / L respectively; 4. Place the induced algal solutions added with six different inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, in step 3 into a photobioreactor, continuously charge sterile carbon dioxide, and conduct cultivation to induce Haematococcus pluvialis cells to accumulate astaxanthin. In step 4, the cultivation temperature of Haematococcus pluvialis is 25°C - 30°C; In step 4, the pH value of the Haematococcus pluvialis culture solution is 7.5 - 9.0; In Step 4, the photobioreactor uses LEDs as the light source, with a characteristic spectrum of white light, adding light with a wavelength of 380 - 500 nm, and the light intensity is adjusted to 160 - 200 μmol / m 2 ·s, and the light cycle is changed to continuous light illumination.

2. In the first step and the fourth step according to claim 1, the method for increasing the production of astaxanthin by Haematococcus pluvialis is characterized in that, Control the pH value of the medium by introducing carbon dioxide.

3. The algal-bacterial co-culture system according to claim 1, characterized in that: The cultivation conditions are adjusted according to the proliferation of the number of Haematococcus pluvialis cells, and are divided into green cell cultivation conditions and astaxanthin induction cultivation conditions in sequence; The green cell culture conditions are as follows: MCM medium is used: 100 mg of MgSO4·7H2O, 24.99 mg of CaCl2·H2O, 0.2 mg of FeCl3·6H2O, 0.6 mg of H3BO3, 0.1563 mg of CuSO4·5H2O, 0.4 mg of (NH4)6Mo7O4, 26.2 mg of K2HPO4·3H2O, 0.2 mg of Na2ENTA, 200 mg of KNO3, 0.1 mg of CoCl2, and 1000 mL of distilled water. The culture temperature is 20 - 26 °C, the light intensity is 80 - 120 μmol / m 2 ·s, and the light cycle is 12L:12D; carbon dioxide gas is introduced during the culture process; When the cell count of Haematococcus pluvialis reaches 10 5 ~10 6 cells / mL, adjust the initial culture conditions to the astaxanthin induction culture conditions as follows: add 0.5 g / L of crushed carrot; add creatine, glycine, 6-aminocaproic acid, glutamic acid, L-hydroxyproline, and L-cysteine at concentrations of 0.1 - 80 mg / L respectively; The astaxanthin induction culture conditions are as follows: Use nitrogen-deficient MCM medium: 100 mg of MgSO4·7H2O, 24.99 mg of CaCl2·H2O, 0.2 mg of FeCl3·6H2O, 0.6 mg of H3BO3, 0.1563 mg of CuSO4·5H2O, 0.4 mg of (NH4)6Mo7O4, 26.2 mg of K2HPO4·3H2O, 0.2 mg of Na2ENTA, 40 mg of KNO3, 0.1 mg of CoCl2, and 1000 mL of distilled water. The culture temperature is 25-30 °C, the light intensity is 160-200 μmol / m 2 ·s, and the light cycle is changed to maintain continuous light; carbon dioxide gas is introduced during the culture process.

4. A method for increasing the biomass of Haematococcus pluvialis based on the application of Haematococcus pluvialis in aquaculture according to claims 1 - 3.

5. A method for increasing the astaxanthin production of Haematococcus pluvialis by using six different inducers, namely creatine, glycine, 6 - aminohexanoic acid, glutamic acid, L - hydroxyproline, and L - cysteine, according to claims 1 - 3.

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