METHOD FOR CULTURING HAEMATOCOCCUS PLUVIALIS TO PRODUCE ASTAXANTHIN

Abstract

A method for producing astaxanthin, comprising: (a) acquiring vegetative cells of astaxanthin-producing Haematococcus pluvialis; (b) heterotrophically culturing the vegetative cells of astaxanthin-producing Haematococcus pluvialis in a nutrient-poor culture medium containing an organic carbon source and under a no-light condition, to obtain spore cells; and (c) harvesting the spore cells and/or astaxanthin, and optionally purifying the astaxanthin. Also provided is a culture medium used in the described method.

Claims

1. A method of producing astaxanthin, comprising: (a) obtaining vegetative cells of an astaxanthin-producing Haematococcus pluvialis; (b) heterotrophically culturing the vegetative cells of the astaxanthin-producing Haematococcus pluvialis in a nutrient-deficient culture medium containing an organic carbon source under a dark condition to obtain spore cells; and (c) harvesting the spore cells and/or astaxanthin, optionally purifying astaxanthin.

2. The method of claim 1, wherein the vegetative cells in step (a) are obtained by autotrophic, mixotrophic or heterotrophic culture of the Haematococcus pluvialis cells at a condition: (i) culture temperature is controlled to be 15-25° C., and/or (ii) the pH is controlled to be 6.0-9.0.

3. The method of claim 1, wherein the vegetative cells in step (a) are obtained by autotrophic or mixotrophic culture of the Haematococcus pluvialis cells.

4. The method of claim 1, wherein the vegetative cells in step (a) are obtained by mixotrophic or heterotrophic culture of the Haematococcus pluvialis cells, wherein dissolved oxygen is controlled to be 1-50%.

5. The method of claim 1, wherein immotile vegetative cells in the vegetative cells obtained in step (a) account for at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the total number of cells.

6. The method of claim 1, wherein the vegetative cells in step (a) are obtained by autotrophic culture of the Haematococcus pluvialis cells.

7. The method of claim 1, wherein the vegetative cells in step (a) are obtained by mixotrophic or heterotrophic culture of the Haematococcus pluvialis cells in a culture medium containing an organic carbon source and a nitrogen source.

8. The method of claim 1, wherein the vegetative cells in step (a) are obtained by mixotrophic or heterotrophic culture of the Haematococcus pluvialis cells in a manner selected from a batch, fed-batch, semi-continuous and continuous culture, wherein, when culturing Haematococcus pluvialis cells by a feeding culture, feeding solution contains 15-1050 g/L of acetic acid or an acetate, a nitrogen source containing 0.3-120 g/L of nitrogen and a 1-50 times concentrated culture medium.

9. The method of claim 7, wherein the nitrogen source contains an inorganic nitrogen source and/or an organic nitrogen source selected from the group consisting of nitric acid, nitrates, nitrites, aqueous ammonium, ammonium salts, urea, amino acids, peptone, yeast extract, protein powder, corn steep liquor, and any combination thereof.

10. The method of claim 1, wherein the organic carbon source is selected from the group consisting of acetic acid, acetates, propionic acid, propionates, butyric acid, butyrates, lactic acid, lactates, fatty acids, fatty acid salts, amino acids, methanol, ethanol, glycerin, citric acid, citrates, pyruvic acid, pyruvates, glucose, fructose, arabinose, lactose, mannose, rhamnose, ribose and waste water, hydrolysate, zymotic fluid containing said organic carbon source(s), and any combination thereof.

11. The method of claim 1, wherein the nutrient-deficient culture medium lacks one or more nutrient elements selected from the group consisting of a nitrogen source, a phosphorus source, a sulfur source, a magnesium source, a calcium source and trace elements, wherein the trace element is one or more selected from the group consisting of Mn, Zn, B, I, Mo, Cu, Co and Fe.

12. The method of claim 1, wherein the step (b) comprises one or more of the following: (i) the culture medium contains 1-15 g/L acetic acid or an acetate, (ii) the culture temperature is controlled to be 15-35° C., (iii) the pH is controlled to be 6.0-11.0, and (iv) the dissolved oxygen is controlled to be 20-90%.

13. The method of claim 1, wherein in step (b), heterotrophic culture is carried out in a batch or fed-batch manner, wherein, for fed-batch culture, feeding solution is a nutrient-deficient culture medium containing 15-1050 g/L acetic acid or an acetate or is acetic acid.

14. The method of claim 1, wherein, when at least 60% of vegetative cells changed into spore cells and/or the content of astaxanthin no longer increases, the step (b) is stopped.

15. The method of claim 1, wherein the culture medium comprises: TABLE-US-00001 an organic carbon source 80-700 mg/L (content of carbon element) a source of nitrogen 40-800 mg/L (content of nitrogen element) the mass ratio of 0.1-10:1 carbon to nitrogen potassium dihydrogen 0.05-1 g/L, phosphate magnesium sulfate 50-500 mg/L, calcium chloride 5-50 mg/L, disodium edetate 0.5-6 mg/L, boric acid 0.5-5 mg/L, ferric chloride 100-1000 μg/L, manganese chloride 10-100 μg/L, zinc sulfate 10-100 μg/L, sodium molybdate 10-100 μg/L, cobalt chloride 5-50 μg/L, copper sulfate 10-100 μg/L, optionally a pH of 7.0-8.0.

16. The method of claim 1, wherein the Haematococcus pluvialis is selected from the group consisting of Haematococcus pluvialis CCTCC M2018809, AC136, AC143, AC587, AC588, ATCC 30453, ATCC 30402, CS-321, G 1002, ETTL 1958/3, TAKACOVAL 1983/1, PRIBYL 2005/4, PRIBYL 2008/3, CCCryo 188-04, CCCryo 189-04, CCCryo 190-04, SCCAP K-0084, IPPAS H-239, NIVA-CHL 9, FWAC 7072, FWAC 7039, CPCC 93, ACOI 816, ACOI 815, ACOI 276, ACOI 255, ACOI 133, ACOI 51, CCAP 34/1D, CCAP 34/1F, CCAP 34/6, CCAP 34/7, CCAP34/8, CCAP 34/12, CCAP 34/13, CCAP 34/14, NIES-144, NIES-2263, NIES-2264, NIES-2265, SAG 192.80, SAG 44.96, SAG 34-1a, SAG 34-1b, SAG 34-1c, CCAC 0055, CCAC 0125, CCAC 0129, CCAC 2072B, UTEX 2505, UTEX 16, UTEX B 294, CWU-MACC20, TISTR 8647, FACHB-712, FACHB-827, FACHB-797, FACHB-955, FACHB-1164 and CCMP 3127.

17. The method of claim 1, comprising: (a) heterotrophically culturing an astaxanthin-producing Haematococcus pluvialis in a culture medium containing an organic carbon source and a nitrogen source in a fed-batch manner to obtain vegetative cells, comprising one or more of the following: the organic carbon source is selected from acetic acid or an acetate; the nitrogen source is selected from nitric acid or a nitrate, urea, tryptone or a yeast extract; the content of carbon element in the organic carbon source is 150-300 mg/L; the content of nitrogen element is 100-600 mg/L; the mass ratio of carbon to nitrogen in the culture medium is 0.3-3:1; culture temperature is 20-25° C.; dissolved oxygen is controlled to be 15-30%; pH is controlled to be 7.5-8.0; feeding solution contains an organic carbon source and a nitrogen source, wherein the mass ratio of carbon to nitrogen is 5-35:1; immotile vegetative cells in the obtained vegetative cells account for at least 80% of the total number of cells; (b) heterotrophically culturing the vegetative cells obtained in step (a) in a nutrient-deficient culture medium containing an organic carbon source in a fed-batch manner under a dark condition to obtain spore cells, comprising one or more of the following: the organic carbon source is selected from acetic acid or an acetate, the culture medium lacks (i) a nitrogen source, (ii) a nitrogen source and a phosphorus source, or (iii) all nutrient elements, culture temperature is 25-30° C.; dissolved oxygen is controlled to be 45-70%; pH is controlled to be 7.5-8.0; and feeding solution is (i) a culture medium containing an organic carbon source and lacking a nitrogen source and a phosphorus source or (ii) a culture medium containing an organic carbon source and lacking all nutrients; and when at least 90%, 95% or 100% of the vegetative cells change to spore cells, stopping the step (b); and (c) harvesting the spore cells and/or astaxanthin, optionally purifying astaxanthin.

18. A culture medium, comprising: TABLE-US-00002 an organic carbon source 80-700 mg/L (content of carbon element) a nitrogen source 40-800 mg/L (content of nitrogen element) the mass ratio of 0.1-10:1 carbon to nitrogen potassium dihydrogen 0.05-1 g/L, phosphate magnesium sulfate 50-500 mg/L, calcium chloride 5-50 mg/L, disodium edetate 0.5-6 mg/L, boric acid 0.5-5 mg/L, ferric chloride 100-1000 μg/L, manganese chloride 10-100 μg/L, zinc sulfate 10-100 μg/L, sodium molybdate 10-100 μg/L, cobalt chloride 5-50 μg/L, copper sulfate 10-100 μg/L, optionally pH 7.0-8.0.

19. The method of claim 7, wherein the mass ratio of carbon to nitrogen in the culture medium is 0.1-10:1.

20. The method of claim 17, wherein the feeding solution in step (b) is an acetic acid solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0178] FIG. 1 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by autotrophic culture in a batch manner under a dark condition.

[0179] FIG. 2 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by mixotrophic in a fed-batch manner under a dark condition.

[0180] FIG. 3 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with sodium acetate and sodium nitrate in a fed-batch manner under a dark condition.

[0181] FIG. 4 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with sodium acetate and ammonium sulfate in a fed-batch manner under a dark condition.

[0182] FIG. 5 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with sodium acetate and urea in a fed-batch manner under a dark condition.

[0183] FIG. 6 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with glucose and sodium nitrate in a fed-batch manner under a dark condition.

[0184] FIG. 7 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with sodium acetate, ribose and sodium nitrate in a fed-batch manner under a dark condition.

[0185] FIG. 8 shows the production of astaxanthin by heterotrophically inducing algal cells obtained by heterotrophic culture with sodium acetate, a yeast extract and peptone in a fed-batch manner under a dark condition.

EXAMPLES

[0186] The techniques and methods of the present invention are generally carried out according to conventional methods well known in the art and described in the references cited in this specification. The invention is further illustrated by the following examples. However, it should be understood that such example are for illustrative purpose, and any example or combination thereof should not be construed as limiting the scope or embodiments of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art may clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modification or change to the technical solutions of the present invention, and such modifications and changes are also included in the scope of the present invention.

Example 1

[0187] A basal culture medium has a formula of: 1.0 g/L potassium dihydrogen phosphate, 500 mg/L magnesium sulfate, 36 mg/L calcium chloride, 5 mg/L disodium edetate, 4.5 mg/L boric acid, 900 μg/L ferric chloride, 100 RA manganese chloride, 88 RA zinc sulfate, 90 RA sodium molybdate, 50 μg/L cobalt chloride, and 79 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.5 with a diluted sulfuric acid or sodium hydroxide solution.

[0188] Haematococcus pluvialis CCTCC M2018809 (deposited at the China Center for Type Culture Collection (CCTCC)) was inoculated in the sterile basal culture medium containing 1.5 g/L sodium nitrate, and placed in a hanging bag film photobioreactor at an initial cell number of 50,000 cells/mL, wherein the light path of the reactor was 6 cm, the volume was 5 L, the loading volume was 70%, and the culture temperature was 22° C. It was subjected to continuous illumination on one side under a white fluorescent lamp for 24 hours, at a light intensity of 60 μE/m.sup.2/s, and mixed air containing 0.5-1.5% (v/v) carbon dioxide was aerated with an aeration volume of 0.2-0.5 vvm and agitated. The pH of the broth was controlled to be 7.5 by adjusting the content of carbon dioxide and the aeration volume. After 240 hours of autotrophic culture, the algae cells no longer divided and reproduced. The number of immotile vegetative cells accounted for 90% of the total cell number, and the total cell number reached 1.1 million cells/mL.

[0189] The algae broth was collected and centrifuged in a centrifuge at 3000 rpm for 5 minutes. After removing the supernatant, the concentrated algae cells were inoculated into a nitrogen-deficient basal culture medium containing 8.2 g/L sodium acetate in an inoculation density of 0.51 g/L, and placed in a 0.5 L air-lift column reactor, wherein the loading volume was 50%, the air aeration volume was 1.0 vvm, the dissolved oxygen was 30-40% and the culture temperature was 25° C. The pH was controlled to be 8.5 by adding 0.5 mol/L diluted sulfuric acid. After 192 hours of heterotrophic culture in the absence of light, more than 80% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 1.58 g/L, and the content of astaxanthin reached 1.78% (as shown in FIG. 1). The content of astaxanthin was determined through the method as described above.

Example 2

[0190] A basal culture medium has a formula of: 0.05 g/L potassium dihydrogen phosphate, 50 mg/L magnesium sulfate, 5 mg/L calcium chloride, 0.5 mg/L disodium edetate, 1.9 mg/L boric acid, 120 μg/L ferric chloride, 15 μg/L manganese chloride, 14 μg/L zinc sulfate, 10 μg/L sodium molybdate, 5 μg/L cobalt chloride, and 22 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.0 with a diluted sulfuric acid or sodium hydroxide solution.

[0191] Haematococcus pluvialis CCTCC M2018809 was inoculated into sterile basal culture medium containing 0.5 g/L sodium acetate (carbon content of 146 mg/L) and 0.5 g/L sodium nitrate (nitrogen content of 82 mg/L), with a ratio of nitrogen to nitrogen of 1.8/1, at an initial number of cells of 80,000 cells/mL, and placed in a 5 L glass fermenter with a loading volume of 70% and a culture temperature of 20° C. It was subjected to continuous illumination on one side under a white fluorescent lamp for 24 hours at a light intensity of 40 μE/m.sup.2/s. The dissolved oxygen was controlled to be 5-10% by adjusting air aeration rate at 0.05-0.1 vvm and stirring speed at 50-80 rpm. The pH of the broth was maintained at 7.0 by feeding a 50-times concentrated basal culture medium containing 600 g/L acetic acid and 70 g/L sodium nitrate, and the ratio of carbon to nitrogen in the feeding medium was 20.8/1. After 240 hours of mixotrophic culture, the number of immotile vegetative cells accounted for 85% of the total number of cells, and the total number of cells reached 3 million cells/ml.

[0192] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, and inoculating the concentrated algae cells into a nitrogen-deficient basal culture medium containing 6.1 g/L sodium acetate at an inoculation concentration of 1.21 g/L, and placing in a 5 L fermenter with a loading volume of 70% and a culture temperature of 25° C. The dissolved oxygen was controlled to be 30-40% by adjusting air aeration volume at 0.2-1.0 vvm and stirring speed at 100-150 rpm. The pH of the broth was maintained at 9.0 by feeding a nitrogen-deficient basal medium containing 565 g/L acetic acid. After 336 hours of heterotrophic culture in the absence of light, more than 70% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 5.93 g/L, and the content of astaxanthin reached 2.02% (as shown in FIG. 2). The content of astaxanthin was determined through the method as described above.

Example 3

[0193] A culture medium has a formula of: 0.5 g/L potassium dihydrogen phosphate, 200 mg/L magnesium sulfate, 12 mg/L calcium chloride, 3 mg/L disodium edetate, 3 mg/L boric acid, 500 μg/L ferric chloride, 72 μg/L manganese chloride, 50 μg/L zinc sulfate, 45 μg/L sodium molybdate, 33 μg/L cobalt chloride, and 65 μg/L copper sulfate. Upon preparation, pH was adjusted to be 8.0 with a diluted sulfuric acid or sodium hydroxide solution.

[0194] Haematococcus pluvialis CCTCC M2018809 was inoculated into sterile basal culture medium containing 0.8 g/L sodium acetate (carbon content of 234 mg/L) and 0.6 g/L sodium nitrate (nitrogen content of 99 mg/L), with a ratio of carbon to nitrogen of 2.4/1, at an initial number of cells of 100,000 cells/ml, and placed in a 5 L fermenter with a loading volume of 70% and a culture temperature of 20° C. The dissolved oxygen was controlled to be 15-20% by adjusting air aeration volume at 0.1-0.4 vvm and stirring speed at 50-100 rpm, and the pH of the broth was maintained at 8.0 by feeding a 5-times concentrated basal culture medium containing 60 g/L acetic acid and 5 g/L sodium nitrate, and the ratio of carbon to nitrogen in the feeding medium was 29.1/1. After 360 hours of heterotrophic culture, algae cells no longer divided and reproduced. The number of immotile vegetative cells accounted for 100% of the total cell number, and the total cell number reached 2.9 million cells/ml.

[0195] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, abd inoculating the concentrated algae cells into a nitrogen-deficient basal culture medium containing 4.1 g/L sodium acetate at an inoculation concentration of 1.71 g/L, and placing in a 5 L fermenter with a loading volume of 70% and a culture temperature of 30° C. The dissolved oxygen was controlled to be 50-70% by adjusting air aeration volume at 1.0-3.0 vvm and stirring speed at 100-200 rpm. The pH of the broth was maintained at 8.0 by feeding a culture medium lacking all nutrients and containing 180 g/L acetic acid. After 384 hours of heterotrophic culture in the absence of light, 100% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 7.87 g/L, and the content of astaxanthin reached 3.21% (as shown in FIG. 3). The content of astaxanthin was determined through the method as described above.

Example 4

[0196] A basal culture medium has a formula of: 0.3 g/L potassium dihydrogen phosphate, 300 mg/L magnesium sulfate, 27 mg/L calcium chloride, 4 mg/L disodium edetate, 3.5 mg/L boric acid, 700 μg/L ferric chloride, 80 μg/L manganese chloride, 90 μg/L zinc sulfate, 87 μg/L sodium molybdate, 40 μg/L cobalt chloride, and 100 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.5 with a diluted sulfuric acid or sodium hydroxide solution.

[0197] Haematococcus pluvialis CCTCC M2018809 was inoculated into sterile basal culture medium containing 1.2 g/L sodium acetate (carbon content of 351 mg/L) and 0.38 g/L ammonium sulfate (nitrogen content of 81 mg/L), with a ratio of carbon to nitrogen of 4.4/1, at an initial cell number of 100,000 cells/mL and placed in a 5 L fermenter with a loading volume of 70% at a culture temperature of 25° C. The dissolved oxygen was controlled to be 10-15% by adjusting air aeration volume at 0.1-0.3 vvm and stirring speed at 50-80 rpm, and the pH of the broth was maintained at 7.5 by feeding a 20-times concentrated basal culture medium containing 180 g/L acetic acid and 11.5 g/L ammonium sulfate, and the ratio of carbon to nitrogen in the feeding medium was 29.5/1. After 312 hours of heterotrophic culture, algae cells no longer divided and reproduced. The number of immotile vegetative cells accounted for 100% of the total cell number, and the total number of cells reached 1 million cells/mL.

[0198] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, and inoculating the concentrated algae cells into a basal culture medium absent of all nutrients and containing 6.8 g/L sodium acetate at an inoculation concentration of 1.68 g/L, and placing in a 5 L fermenter, with a loading volume of 70% and a culture temperature of 30° C. The dissolved oxygen was controlled to be 35-50% by adjusting air aeration volume at 0.5-1.5 vvm and stirring speed at 100-150 rpm. The pH of the broth was maintained at 8.0 by feeding a nitrogen-deficient basal medium containing 120 g/L acetic acid. After 336 hours of heterotrophic culture in the absence of light, more than 90% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 10.7 g/L, and the content of astaxanthin reached 2.29% (as shown in FIG. 4). The content of astaxanthin was determined through the method as described above.

Example 5

[0199] A basal culture medium has a formula of: 0.2 g/L potassium dihydrogen phosphate, 400 mg/L magnesium sulfate, 50 mg/L calcium chloride, 3.5 mg/L disodium edetate, 4 mg/L boric acid, 200 μg/L ferric chloride, 35 μg/L manganese chloride, 25 μg/L zinc sulfate, 35 μg/L sodium molybdate, 20 μg/L cobalt chloride, and 45 μg/L copper sulfate. Upon preparation, pH was adjusted to 8.0 with a diluted sulfuric acid or sodium hydroxide solution.

[0200] Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal culture medium containing 1.0 g/L sodium acetate (carbon content of 293 mg/L) and 0.31 g/L urea (nitrogen content of 145 mg/L), with a ratio of carbon to nitrogen of 2.0/1, at an initial cell number of 80,000 cells/mL, and placed in a 5 L fermenter with a loading volume of 70%, at a culture temperature of 23° C. The dissolved oxygen was controlled to be 20-25% by adjusting air aeration volume at 0.1-0.4 vvm and stirring speed at 50-100 rpm, and the pH of the broth was maintained at 8.0 by feeding a 10-times concentrated basal culture medium containing 120 g/L acetic acid and 3.1 g/L urea, and the ratio of carbon to nitrogen in the feeding medium was 33.2/1. After 240 hours of heterotrophic culture, algae cells no longer divided and reproduced. The number of immotile vegetative cells accounted for 100% of the total cell number which reached 2.7 million cells/mL.

[0201] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, and inoculating the concentrated algae cells into a basal culture medium absent of all nutrients and containing 4.5 g/L sodium acetate at an inoculation concentration of 1.31 g/L, and placing in a 5 L fermenter, with a loading volume of 70% at a culture temperature of 30° C. The dissolved oxygen was controlled to be 45-60% by adjusting air aeration volume at 1.0-2.0 vvm and stirring speed at 100-200 rpm. The pH of the broth was maintained at 8.0 by feeding a basal culture medium absent of all nutrients and containing 300 g/L acetic acid. After 384 hours of heterotrophic culture in the absence of light, more than 95% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 9.60 g/L, and the content of astaxanthin reached 2.88% (as shown in FIG. 5). The content of astaxanthin was determined through the method as described above.

Example 6

[0202] A basal culture medium has a formula of: 0.6 g/L potassium dihydrogen phosphate, 100 mg/L magnesium sulfate, 10 mg/L calcium chloride, 2 mg/L disodium edetate, 0.5 mg/L boric acid, 600 μg/L ferric chloride, 20 μg/L manganese chloride, 36 μg/L zinc sulfate, 25 μg/L sodium molybdate, 45 μg/L cobalt chloride, and 80 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.5 with a diluted sulfuric acid or sodium hydroxide solution.

[0203] Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal culture medium containing 0.3 g/L glucose (carbon content of 120 mg/L) and 1.5 g/L sodium nitrate (nitrogen content of 247 mg/L), with a ratio of carbon to nitrogen of 0.5/1 at an initial cell number of 40,000 cells/mL, and placed in a 250 mL Erlenmeyer flask with a loading volume of 100 ml. It was shaking cultured at 100 rpm at a culture temperature of 20° C. After 120 hours of heterotrophic culture, the number of immotile vegetative cells accounted for 70% of the total number of cells, and the total number of cells reached 250,000 cells/mL.

[0204] Stopping the aeration and stirring, removing the supernatant after natural sedimentation and inoculating the concentrated algae cells into a basal culture medium lacking nitrogen and phosphorus and containing 3.7 g/L sodium acetate at an inoculation concentration of 1.37 g/L, and placing in a 1 L fermenter, with a loading volume of 70% at a culture temperature of 30° C. The dissolved oxygen was controlled to be 45-55% by adjusting air aeration volume at 0.5-1.5 vvm and stirring speed at 100-200 rpm. The pH of the broth was maintained at 7.5 by feeding a basal culture medium absent of all nutrients and containing 400 g/L acetic acid. After 384 hours of heterotrophic culture in the absence of light, more than 85% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 10.54 g/L, and the content of astaxanthin reached 2.39% (as shown in FIG. 6). The content of astaxanthin was determined through the method as described above.

Example 7

[0205] A basal culture medium has a formula of: 0.8 g/L potassium dihydrogen phosphate, 250 mg/L magnesium sulfate, 40 mg/L calcium chloride, 5.5 mg/L disodium edetate, 5 mg/L boric acid, 400 μg/L ferric chloride, 90 μg/L manganese chloride, 66 μg/L zinc sulfate, 100 μg/L sodium molybdate, 36 μg/L cobalt chloride, and 90 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.5 with a diluted sulfuric acid or sodium hydroxide solution.

[0206] Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal culture medium containing 0.4 g/L sodium acetate (carbon content of 117 mg/L), 0.3 g/L ribose (carbon content of 120 mg/L), and 1.0 g/L sodium nitrate (nitrogen content of 165 mg/L), with a ratio of carbon to nitrogen of 1.4/1 at an initial cell number of 40,000 cells/mL, and placed in a 250 mL Erlenmeyer flask with a loading volume of 100 ml. It was shaking cultured at 100 rpm at a culture temperature of 20° C. After 120 hours of heterotrophic culture, the number of immotile vegetative cells accounted for 60% of the total number of cells, and the total number of cells reached 300,000 cells/m L.

[0207] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, and inoculating the concentrated algae cells into a basal culture medium lacking nitrogen and trace elements and containing 5.3 g/L sodium acetate at an inoculation concentration of 1.24 g/L, and placing in a 5 L fermenter, with a loading volume of 70% at a culture temperature of 25° C. The dissolved oxygen was controlled to be 30-40% by adjusting air aeration volume at 0.5-1.0 vvm and stirring speed at 100-150 rpm. The pH of the broth was maintained at 7.0 by feeding a basal culture medium lacking nitrogen and trace elements and containing 240 g/L acetic acid. After 384 hours of heterotrophic culture in the absence of light, more than 80% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 13.34 g/L, and the content of astaxanthin reached 2.00% (as shown in FIG. 7). The content of astaxanthin was determined through the method as described above.

Example 8

[0208] A basal culture medium has a formula of: 0.1 g/L potassium dihydrogen phosphate, 150 mg/L magnesium sulfate, 15 mg/L calcium chloride, 1 mg/L disodium edetate, 2 mg/L boric acid, 150 μg/L ferric chloride, 25 μg/L manganese chloride, 20 μg/L zinc sulfate, 20 μg/L sodium molybdate, 10 μg/L cobalt chloride, and 30 μg/L copper sulfate. Upon preparation, pH was adjusted to 7.5 with a diluted sulfuric acid or sodium hydroxide solution.

[0209] Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal culture medium containing 0.6 g/L sodium acetate (carbon content of 176 mg/L), 4.0 g/L tryptone (nitrogen content of 400 mg/L) and 2.0 g/L yeast extract (nitrogen content of 200 mg/L), with a ratio of carbon to nitrogen of 0.3/1, at an initial cell number of 800,000 cells/mL, and placed in a 5 L fermenter with a loading volume of 70% at a culture temperature of 25° C. The dissolved oxygen was controlled to 25-30% by adjusting air aeration volume at 0.2-0.5 vvm and stirring speed at 80-150 rpm, and the pH of the broth was maintained to 7.5 by feeding a 30-times concentrated basal culture medium containing 15 g/L acetic acid, 8.0 g/L tryptone, and 4.0 g/L yeast extract, and the ratio of carbon to nitrogen in the feeding medium was 5.0/1. After 240 hours of heterotrophic culture, the number of immotile vegetative cells accounted for 80% of the total number of cells, and the total number of cells reached 2.8 million cells/mL.

[0210] Stopping the aeration and stirring, removing the supernatant after natural sedimentation, and inoculating the concentrated algae cells into a basal culture medium lacking nitrogen and phosphorus and containing 4.9 g/L sodium acetate at an inoculation concentration of 1.55 g/L, and placing in a 5 L fermenter, with a loading volume of 70% at a culture temperature of 30° C. The dissolved oxygen was controlled to 50-70% by adjusting air aeration volume at 1.0-2.0 vvm and stirring speed at 100-250 rpm. The pH of the broth was maintained to 8.0 by feeding a basal culture medium lacking nitrogen and phosphorus and containing 60 g/L acetic acid. After 360 hours of heterotrophic culture in the absence of light, more than 90% of algae cells changed from green vegetative cells to red spore cells, the algae cell density reached 12.91 g/L, and the content of astaxanthin reached 2.65% (as shown in FIG. 8). The content of astaxanthin was determined through the method as described above.