METHOD FOR PRODUCING ASTAXANTHIN BY HETEROTROPHIC CULTURE OF HAEMATOCOCCUS PLUVIALIS

Abstract

Provided is a method for producing astaxanthin, comprising: (a) performing heterotrophic cultivation of astaxanthin-producing Haematococcus pluvialis; (b) performing heterotrophic cultivation of the Haematococcus pluvialis obtained in step (a) under conditions of high acetate concentration and high dissolved oxygen concentration, where the acetate concentration is at least 1800 mg/L and the dissolved oxygen concentration is at least 2.0 mg/L; and (c) collecting algae cells from step (b) and/or harvesting astaxanthin. Also provided is a base medium for culturing Haematococcus pluvialis, a propagation feed medium, and an induction feed medium.

Claims

1. A method of producing astaxanthin, comprising: (a) heterotrophically culturing an astaxanthin-producing Haematococcus pluvialis, optionally until at least about 50% of algal cells are immotile vegetative cells, and/or optionally the number of algal cells after culture is at least about 10.sup.6 cells/mL, and/or optionally the density of cells is at least about 1.0 g/L; (b) heterotrophically culturing the Haematococcus pluvialis cells obtained in step (a) under a condition of increased concentrations of acetate and dissolved oxygen, optionally until the yield of astaxanthin no longer increases, wherein the concentration of acetate is at least about 1800 mg/L, and the concentration of dissolved oxygen is at least about 2 mg/L; and (c) collecting the Haematococcus pluvialis cells and/or harvesting astaxanthin in step (b), optionally purifying astaxanthin.

2. The method of claim 1, wherein in step (a): the pH of the culture solution is about 6.0-9.0; and/or the culture temperature is about 15-25° C.; and/or the concentration of dissolved oxygen in the culture solution is about 0.1-4.0 mg/L, and is lower than the concentration of dissolved oxygen in step (b); and/or the concentration of acetate is about 60-1200 mg/L; and/or the concentration of nitrate is about 100-2000 mg/L; and/or the concentration of phosphate is about 50-500 mg/L; and/or the initial density of the Haematococcus pluvialis cells is at least about 10.sup.4 cells/mL.

3. The method of claim 1, wherein in step (b): the pH of the culture solution is about 6.0-11.0; and/or the culture temperature is about 20-35° C.; and/or the concentration of acetate is about 1800-6000 mg/L; and/or the concentration of dissolved oxygen is about 2-10 mg/L.

4. The method of claim 1, wherein in step (a), the Haematococcus pluvialis is cultured in a basal culture medium, and an expansion feeding medium is fed during the culture process, wherein the basal culture medium comprises: 0.1-2.0 g/L sodium acetate, 0.1-3.0 g/L sodium nitrate, 50-500 mg/L potassium dihydrogen phosphate, 20-200 mg/L dipotassium hydrogen phosphate, 50-500 mg/L magnesium sulfate, 1-50 mg/L calcium chloride, 0.5-5 mg/L disodium edetate, 0.1-5 mg/L boric acid, 100-500 μg/L ferric chloride, 1-100 μg/L manganese chloride, 1-100 μg/L zinc sulfate, 1-100 μg/L sodium molybdate, 0.1-10 μg/L cobalt chloride, 1-100 μg/L copper sulfate, 0-1 μg/L vitamin B12, 0-1 μg/L biotin, and 0-20 μg/L vitamin B1, wherein the expansion feeding medium comprises: 30-600 g/L acetic acid, 2-70 g/L sodium nitrate, 0.2-5.0 g/L potassium dihydrogen phosphate, 0.05-10 g/L magnesium sulfate, 1-40 mg/L disodium edetate, 1-35 mg/L boric acid, 150-4000 μg/L ferric chloride, 30-1000 μg/L manganese chloride, 15-600 μg/L zinc sulfate, 15-500 μg/L sodium molybdate, 3-100 μg/L cobalt chloride, 20-700 μg/L copper sulfate, 0-10 μg/L vitamin B12, 0-10 μg/L biotin, and 0-1000 μg/L vitamin B1.

5. The method of claim 1, wherein in step (b), a high concentration of acetate is maintained by adding acetic acid or an acetate such as sodium acetate and/or by feeding an induction feeding medium, wherein the induction feeding medium comprises: 30-600 g/L acetic acid, 0-70 g/L sodium nitrate, 0-5.0 g/L potassium dihydrogen phosphate, 0-1.0 g/L magnesium sulfate, 1-40 mg/L disodium edetate, 1-35 mg/L boric acid, 150-4000 μg/L ferric chloride, 30-1000 μg/L manganese chloride, 15-600 μg/L zinc sulfate, 15-500 μg/L sodium molybdate, 3-100 μg/L cobalt chloride and 20-700 μg/L copper sulfate.

6. The method of claim 1, comprising an activation step before the step (a), which comprises: obtaining a Haematococcus pluvialis seed solution, optionally the proportion of green vegetative cells in the seed solution is at least about 90%, and/or optionally the density of cells is at least about 50×10.sup.4 cells/mL.

7. The method of claim 6, wherein, in the activation step: the pH is about 6.0-9.0; and/or the culture temperature is about 15-25° C.

8. The method of claim 6, wherein the step (a) comprises heterotrophically culturing the seed solution to obtain a culture solution, wherein at least about 50% of motile vegetative cells in the obtained culture solution are converted to immotile vegetative cells; optionally the initial inoculation amount of the seed solution is about 1-50×10.sup.4 cells/mL; optionally the number of algal cells in the obtained culture solution is at least about 1, 2, 3, 4, 5 or 6×10.sup.6 cells/mL, and/or optionally the density of algal cells is at least about 2.0 g/L.

9. The method of claim 6, wherein the activation step comprises the step of culturing the Haematococcus pluvialis in a culture medium comprising Tris (tris(hydroxymethylaminomethane)) to obtain the seed solution.

10. The method of claim 6, wherein in the activation step, the Haematococcus pluvialis is cultured in a basal culture medium, wherein the basal culture medium comprises: 0.1-2.0 g/L sodium acetate, 0.1-3.0 g/L sodium nitrate, 50-500 mg/L potassium dihydrogen phosphate, 20-200 mg/L dipotassium hydrogen phosphate, 50-500 mg/L magnesium sulfate, 1-50 mg/L calcium chloride, 0.5-5 mg/L disodium edetate, 0.1-5 mg/L boric acid, 100-500 μg/L ferric chloride, 1-100 μg/L manganese chloride, 1-100 μg/L zinc sulfate, 1-100 μg/L sodium molybdate, 0.1-10 μg/L cobalt chloride, 1-100 μg/L copper sulfate, 0-1 μg/L vitamin B12, 0-1 μg/L biotin, and 0-20 μg/L vitamin B1.

11. 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.

12. The method of claim 1, comprising: (1) culturing the Haematococcus pluvialis in a basal culture medium containing 0.5-0.6 g/L Tris (trishydroxymethylaminomethane) to obtain a seed solution, wherein the pH of the culture medium in the beginning of culture is 7.5-8.0 and the culture temperature is about 20-25° C., and the proportion of green vegetative cells in the obtained seed solution is at least about 90%; (2) heterotrophically culturing the seed solution in step (1) in a basal culture medium to obtain a culture solution, wherein during the culturing, the pH is maintained at about 7.5-8.0, the culture temperature is maintained at about 20-25° C., the concentration of dissolved oxygen in the culture solution is maintained at about 1.0-4.0 mg/L, the concentration of acetate is maintained at about 300-900 mg/L, the concentration of nitrate is maintained at about 100-900 mg/L, and the concentration of phosphate is maintained at about 50-250 mg/L, and an expansion feeding medium was fed during the culturing; optionally performing step (3) when at least about 90% of motile vegetative cells are converted to immotile vegetative cells and/or optionally the density of cells in the obtained culture solution is at least 4×10.sup.6 cells/mL; (3) adding acetic acid or an acetate to the culture solution in step (2) to provide the concentration of acetate of at least about 3000 mg/L and adding an induction feeding medium during the culturing, wherein during the culturing, the pH is maintained at about 7.5-8.0, the concentration of acetate is maintained at least about 3000-5000 mg/L, the culture temperature is maintained at about 20-35° C. and the concentration of dissolved oxygen in the culture solution is maintained at about at least 4.0 mg/L; and (4) collecting algae cells and/or harvesting astaxanthin obtained in step (3), optionally purifying astaxanthin; wherein the basal culture medium comprises: 0.8-1.0 g/L sodium acetate, 0.5-1.0 g/L sodium nitrate, 60-200 mg/L potassium dihydrogen phosphate, 20-100 mg/L dipotassium hydrogen phosphate, 50-200 mg/L magnesium sulfate, 1-25 mg/L calcium chloride, 1.5-3 mg/L disodium edetate, 0.1-3 mg/L boric acid, 200-350 μg/L ferric chloride, 4-70 μg/L manganese chloride, 10-35 or 12-35 μg/L zinc sulfate, 1-30 μg/L sodium molybdate, 3-7 μg/L cobalt chloride, 1-50 μg/L copper sulfate, 0-0.1 μg/L vitamin B12, 0-0.1 μg/L biotin, and 0-10 μg/L vitamin B1; wherein the expansion feeding medium comprises: 30-60 g/L acetic acid, 5.0-7.0 g/L sodium nitrate, 0.5-1.0 g/L potassium dihydrogen phosphate, 0.1-1.0 g/L magnesium sulfate, 3-24 mg/L disodium edetate, 1-6 mg/L boric acid, 350-4000 μg/L ferric chloride, 50-300 or 70-280 μg/L manganese chloride, 35-600 μg/L zinc sulfate, 30-60 μg/L sodium molybdate, 5-100 or 7-100 μg/L cobalt chloride and 50-100 μg/L copper sulfate; wherein the induction feeding medium comprises: 60-600 g/L acetic acid, 0-50 g/L sodium nitrate, 0.5-5.0 g/L potassium dihydrogen phosphate, 0.05-1.0 g/L magnesium sulfate, 1-30 mg/L disodium edetate, 1-30 mg/L boric acid, 150-3500 μg/L ferric chloride, 30-700 μg/L manganese chloride, 15-350 μg/L zinc sulfate, 15-300 μg/L sodium molybdate, 3-70 μg/L cobalt chloride and 20-500 μg/L copper sulfate.

13-15. (canceled)

16. A product, which is one of I) to III): I) a basal culture medium for culturing Haematococcus pluvialis, comprising: 0.1-2.0 g/L sodium acetate, 0.1-3.0 g/L sodium nitrate, 50-500 mg/L potassium dihydrogen phosphate, 20-200 mg/L dipotassium hydrogen phosphate, 50-500 mg/L magnesium sulfate, 1-50 mg/L calcium chloride, 0.5-5 mg/L disodium edetate, 0.1-5 mg/L boric acid, 100-500 μg/L ferric chloride, 1-100 μg/L manganese chloride, 1-100 μg/L zinc sulfate, 1-100 μg/L sodium molybdate, 0.1-10 μg/L cobalt chloride, 1-100 μg/L copper sulfate, 0-1 μg/L vitamin B12, 0-1 μg/L biotin, and 0-20 μg/L vitamin B1; II) an expansion feeding medium for culturing Haematococcus pluvialis, comprising: 30-600 g/L acetic acid, 2-70 g/L sodium nitrate, 0.2-5.0 g/L potassium dihydrogen phosphate, 0.05-10 g/L magnesium sulfate, 1-40 mg/L disodium edetate, 1-35 mg/L boric acid, 150-4000 μg/L ferric chloride, 30-1000 μg/L manganese chloride, 15-600 μg/L zinc sulfate, 15-500 μg/L sodium molybdate, 3-100 μg/L cobalt chloride and 20-700 μg/L copper sulfate, 0-10 μg/L vitamin B12, 0-10 μg/L biotin, and 0-1000 μg/L vitamin B1; III) an induction feeding medium for inducing Haematococcus pluvialis to produce astaxanthin, comprising: 30-600 g/L acetic acid, 0-70 g/L sodium nitrate, 0-5.0 g/L potassium dihydrogen phosphate, 0-1.0 g/L magnesium sulfate, 1-40 mg/L disodium edetate, 1-35 mg/L boric acid, 150-4000 μg/L ferric chloride, 30-1000 μg/L manganese chloride, 15-600 μg/L zinc sulfate, 15-500 μg/L sodium molybdate, 3-100 μg/L cobalt chloride and 20-700 μg/L copper sulfate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG. 1 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 1.

[0128] FIG. 2 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 2.

[0129] FIG. 3 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 3.

[0130] FIG. 4 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 4.

[0131] FIG. 5 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 5.

[0132] FIG. 6 shows a variation curve of the content vs. yield of astaxanthin during heterotrophic induction of Haematococcus pluvialis in Example 6.

[0133] The techniques and methods in 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 present invention will be further described in combination with the examples. However, it should be understood that these examples are for illustrative purposes only, and not for limiting the present invention.

EXAMPLES

[0134] The present invention is further illustrated by the following examples. However, any example or combinations 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 field, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modification or change to the technical solutions of the present invention, and such modifications and changes are also fallen within the scope of the present invention.

Example 1

[0135] A preferred basal culture medium of this example has a formula of: 0.82 g/L sodium acetate, 1.0 g/L sodium nitrate, 152 mg/L potassium dihydrogen phosphate, 64 mg/L dipotassium hydrogen phosphate, 100 mg/L magnesium sulfate, 15 mg/L calcium chloride, 3 mg/L disodium edetate, 3 mg/L boric acid, 350 μg/L ferric chloride, 70 μg/L manganese chloride, 35 μg/L zinc sulfate, 30 μg/L sodium molybdate, 7 μg/L cobalt chloride, and 50 μg/L copper sulfate.

[0136] A preferred expansion feeding medium of this example has a formula of: 7.0 g/L sodium nitrate, 1.0 g/L potassium dihydrogen phosphate, 0.3 g/L magnesium sulfate, 6 mg/L disodium edetate, 6 mg/L boric acid, 700 μg/L ferric chloride, 140 μg/L manganese chloride, 70 μg/L zinc sulfate, 60 μg/L sodium molybdate, 14 μg/L cobalt chloride and 100 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after cooling to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 60 g/L.

[0137] A preferred induction feeding medium of this example has a formula of: 50 g/L sodium nitrate, 5.0 g/L potassium dihydrogen phosphate, 1.0 g/L magnesium sulfate, 30 mg/L disodium edetate, 30 mg/L boric acid, 3500 μg/L ferric chloride, 700 g/L manganese chloride, 350 μg/L zinc sulfate, 300 μg/L sodium molybdate, 70 μg/L cobalt chloride, and 500 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after cooling to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 600 g/L.

[0138] The steps were as follows:

[0139] (1) Seed activation: Haematococcus pluvialis CCTCC M2018809 (deposited at the China Center for Type Culture Collection (CCTCC)) preserved in a slant medium was inoculated into the sterile basal culture medium with 0.5 g/L Tris (Tris(hydroxymethylaminomethane)) at an initial pH of 7.5, and placed in a 100 mL Erlenmeyer flask at a loading volume of 50 ml for static culture (hand shaking 2 times every day) at a culture temperature of 20° C. for 20 days, where 90% of algae cells were green vegetative cells, and the number of algae cells reached 570,000 cells/mL.

[0140] (2) Heterotrophic expansion culture: the prepared basal culture medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated into a fermenter at an initial inoculation amount of 180,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 300-900 mg/L, the concentration of nitrate at 600-900 mg/L, the concentration of phosphate ion at 100-150 mg/L, and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 1.0 mg/L-2.0 mg/L by adjusting aeration volume at 0.1-0.3 vvm and stirring speed at 50-100 rpm. Sampling was conducted every day for observation. After 14 days of cultivation, the density of algal cells reached 7.82 g/L, the number of cells reached 4.5 million cells/mL, and 90% of motile vegetative cells were converted into immotile vegetative cells.

[0141] (3) Heterotrophic induction: a concentrated sodium acetate mother solution was added to the fermenter in step (2) through an aseptic operation, to increase the concentration of acetate in the broth to 3000 mg/L, before starting heterotrophic culture. During the culture, the induction feeding medium was fed to maintain the pH of the broth to 8.0, the concentration of acetate at 3000-4000 mg/L, and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 4.0 mg/L-8.0 mg/L by adjusting aeration volume at 1.0-1.5 vvm and stirring speed at 100-200 rpm. Sampling was conducted every day for observation. After culturing for 20 days, all vegetative cells were converted into red spore cells, and the density of algal cells reached 37.25 g/L. At this time, the content of astaxanthin reached 2.89%, and the yield of astaxanthin reached 1076.53 mg/L (as shown in FIG. 1).

Example 2

[0142] A preferred basal culture medium of this example has a formula of: 0.95 g/L sodium acetate, 0.4 g/L sodium nitrate, 200 mg/L potassium dihydrogen phosphate, 100 mg/L dipotassium hydrogen phosphate, 200 mg/L magnesium sulfate, 25 mg/L calcium chloride, 1.5 mg/L disodium edetate, 1.5 mg/L boric acid, 200 μg/L ferric chloride, 40 μg/L manganese chloride, 20 μg/L zinc sulfate, 20 μg/L sodium molybdate, 3 μg/L cobalt chloride, and 20 μg/L copper sulfate.

[0143] A preferred expansion feeding medium of this example has a formula of: 5.0 g/L sodium nitrate, 0.5 g/L potassium dihydrogen phosphate, 0.1 g/L magnesium sulfate, 3 mg/L disodium edetate, 3 mg/L boric acid, 350 μg/L ferric chloride, 70 μg/L manganese chloride, 35 μg/L zinc sulfate, 30 μg/L sodium molybdate, 7 μg/L cobalt chloride and 50 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 60 g/L.

[0144] A preferred induction feeding medium of this example has a formula of: 5.0 g/L sodium nitrate, 0.5 g/L potassium dihydrogen phosphate, 0.1 g/L magnesium sulfate, 3 mg/L disodium edetate, 3 mg/L boric acid, 350 μg/L ferric chloride, 70 μg/L manganese chloride, 35 μg/L zinc sulfate, 30 μg/L sodium molybdate, 7 μg/L cobalt chloride, and 50 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 300 g/L.

[0145] The steps were as follows:

[0146] (1) Seed activation: Haematococcus pluvialis CCTCC M2018809 preserved in a slant medium was inoculated into a sterile basal medium with 0.6 g/L Tris at an initial pH of 8.0, and placed in a 100 mL Erlenmeyer flask at a loading volume of 50 ml for static culture (hand shaking 2 times every day) at a culture temperature of 20° C. for 20 days, where 95% of algae cells were green vegetative cells, and the number of cells reached 570,000 cells/mL.

[0147] (2) Heterotrophic expansion culture: the prepared basal medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated in a fermenter at an initial inoculation amount of 50,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 300-700 mg/L, the concentration of nitrate at 100-400 mg/L, the concentration of phosphate at 150-250 mg/L and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 1.5 mg/L-2.5 mg/L by adjusting aeration volume at 0.1-0.4 vvm and stirring speed at 60-100 rpm. Sampling was conducted every day for observation. After 15 days of culture, the density of algal cells reached 3.38 g/L, the number of cells reached 4.08 million cells/mL, and 90% of motile vegetative cells were converted to immotile vegetative cells.

[0148] (3) Heterotrophic induction: a concentrated sodium acetate mother solution was added to the fermenter in step (2) through an aseptic operation, to increase the concentration of acetate in the broth to 3500 mg/L, before starting heterotrophic culture. During the culture, the induction feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 3500-4800 mg/L, and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 5.0 mg/L-8.0 mg/L by adjusting aeration volume at 1.2-1.8 vvm and stirring speed at 150-200 rpm. Sampling was conducted every day for observation. After 20 days of culture, all vegetative cells were converted to red spore cells, and the density of algal cells reached 27.63 g/L. At this time, the content of astaxanthin reached 2.55%, and the yield of astaxanthin reached 704.57 mg/L (as shown in FIG. 2).

Example 3

[0149] A preferred basal culture medium of this example has a formula of: 0.82 g/L sodium acetate, 0.5 g/L sodium nitrate, 60 mg/L potassium dihydrogen phosphate, 20 mg/L dipotassium hydrogen phosphate, 50 mg/L magnesium sulfate, 1 mg/L calcium chloride, 3 mg/L disodium edetate, 0.1 mg/L boric acid, 350 μg/L ferric chloride, 4 μg/L manganese chloride, 12 g/L zinc sulfate, 1 μg/L sodium molybdate, 7 μg/L cobalt chloride, 1 μg/L copper sulfate, 0.1 μg/L Vitamin B12, 0.1 μg/L biotin, and 10 μg/L Vitamin B1.

[0150] A preferred expansion feeding medium of this example has a formula of: 6.0 g/L sodium nitrate, 0.7 g/L potassium dihydrogen phosphate, 1.0 g/L magnesium sulfate, 24 mg/L disodium edetate, 1 mg/L boric acid, 4000 μg/L ferric chloride, 280 μg/L manganese chloride, 600 μg/L zinc sulfate, 30 μg/L sodium molybdate, 100 μg/L cobalt chloride and 50 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with filter-sterilized Vitamin B12 of 5 μg/L, biotin of 5 μg/L, Vitamin B1 of 500 μg/L, and glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 30 g/L.

[0151] A preferred induction feeding medium of this example has a formula of: 0.5 g/L potassium dihydrogen phosphate, 0.05 g/L magnesium sulfate, 1 mg/L disodium edetate, 1 mg/L boric acid, 150 μg/L ferric chloride, 30 μg/L manganese chloride, 15 μg/L zinc sulfate, 15 μg/L sodium molybdate, 3 μg/L cobalt chloride, and 20 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 60 g/L.

[0152] The steps were as follows:

[0153] (1) Seed activation: Haematococcus pluvialis NIES-144 (Microbial Culture Collection of National Institute for Environmental Studies, Japan) preserved in a slant medium was inoculated in a sterile basal medium with 0.5 g/L Tris at an initial pH of 7.5, and placed in a 250 mL Erlenmeyer flask at a loading volume of 50 ml for shake cultivation at 30 rpm and a culture temperature of 25° C. for 12 days, where 90% of algae cells were green vegetative cells, and the number of algae cells reached 800,000 cells/mL.

[0154] (2) Heterotrophic expansion culture: the prepared basal medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated into a fermenter at an initial inoculation amount of 50,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 7.5, the concentration of acetate at 300-900 mg/L, the concentration of nitrate at 300-900 mg/L and the concentration of phosphate at 50-100 mg/L, and the culture temperature was controlled at 25° C. During the fermentation, the concentration of dissolved oxygen was controlled at 2.0 mg/L-4.0 mg/L by adjusting aeration volume at 0.1-0.3 vvm and stirring speed at 50-100 rpm. Sampling was conducted every day for observation. After 12 days of culture, the density of algal cells reached 8.15 g/L, the number of cells reached 6.16 million cells/mL, and 95% of motile vegetative cells were converted to immotile vegetative cells.

[0155] (3) Heterotrophic induction: a concentrated sodium acetate mother solution was added to the fermenter in step (2) through an aseptic operation, to increase the concentration of acetate in the broth to 3000 mg/L, before starting the heterotrophic culture. During the culture, the induction feeding medium was fed to maintain the pH of the broth at 7.5, the concentration of acetate at 3000-4000 mg/L, and the culture temperature was controlled at 25° C. The temperature was increased to 30° C. on Day 10 and to 35° C. on Day 12. During the fermentation, the dissolved oxygen was controlled at 4.0 mg/L-8.0 mg/L by adjusting aeration volume at 1.0-1.5 vvm and stirring speed at 100-200 revolutions per minute. Sampling was conducted every day for observation. After culturing for 15 days, all vegetative cells were converted to red spore cells, and the density of algal cells reached 18.35 g/L. At this time, the content of astaxanthin reached 1.33%, and the yield of astaxanthin reached 244.06 mg/L (as shown in FIG. 3).

Example 4

[0156] A preferred basal culture medium of this example has a formula of: 0.50 g/L sodium acetate, 1.5 g/L sodium nitrate, 300 mg/L potassium dihydrogen phosphate, 100 mg/L dipotassium hydrogen phosphate, 300 mg/L magnesium sulfate, 30 mg/L calcium chloride, 4 mg/L disodium edetate, 4 mg/L boric acid, 500 μg/L ferric chloride, 80 μg/L manganese chloride, 50 μg/L zinc sulfate, 40 μg/L sodium molybdate, 10 μg/L cobalt chloride, and 70 μg/L copper sulfate.

[0157] A preferred expansion feeding medium of this example has a formula of: 10.0 g/L sodium nitrate, 2.0 g/L potassium dihydrogen phosphate, 0.6 g/L magnesium sulfate, 12 mg/L disodium edetate, 12 mg/L boric acid, 1400 μg/L ferric chloride, 280 μg/L manganese chloride, 140 μg/L zinc sulfate, 120 μg/L sodium molybdate, 30 μg/L cobalt chloride and 200 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 120 g/L.

[0158] A preferred induction feeding medium of this example has a formula of: 0.4 g/L magnesium sulfate, 12 mg/L disodium edetate, 12 mg/L boric acid, 1400 μg/L ferric chloride, 280 μg/L manganese chloride, 140 μg/L zinc sulfate, 120 μg/L sodium molybdate, 30 μg/L cobalt chloride, and 200 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 240 g/L.

[0159] The steps were as follows:

[0160] (1) Seed activation: Haematococcus pluvialis CCTCC M2018809 preserved in a slant medium was inoculated in a sterile basal medium with 1.0 g/L Tris at an initial pH of 7.0, and placed in a 100 mL Erlenmeyer flask at a loading volume of 50 ml for static culture (hand shaking for 2 times every day) at a culture temperature of 20° C. for 20 days, where 92% of algae cells were green vegetative cells, and the number of cells reached 550,000 cells/mL.

[0161] (2) Heterotrophic expansion culture: the prepared basal medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated into a fermenter at an initial inoculation amount of 100,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 100-400 mg/L, the concentration of nitrate at 1000-1500 mg/L, and the concentration of phosphate at 200-300 mg/L, and the culture temperature was controlled at 25° C. During the fermentation, the concentration of dissolved oxygen was controlled at 0.1 mg/L-1.0 mg/L by adjusting aeration volume at 0.05-0.15 vvm and stirring speed at 40-60 rpm. Sampling was conducted every day for observation. After 12 days of culture, the density of algal cells reached 1.87 g/L, the number of cells reached was 1.9 million cells/mL, and 70% of motile vegetative cells were converted to immotile vegetative cells.

[0162] (3) Heterotrophic induction: a concentrated sodium acetate mother solution was added to the fermenter in step (2) through an aseptic operation, to increase the concentration of acetate in the broth to 2700 mg/L, before starting the heterotrophic culture. During the culture, the induction feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 2700-4000 mg/L, and the culture temperature was controlled at 30° C. During the fermentation, the concentration of dissolved oxygen was controlled at 2.0 mg/L-3.0 mg/L by adjusting aeration volume at 0.1-0.5 vvm and stirring speed at 60-100 rpm. Sampling was conducted every day for observation. After culturing for 10 days, all vegetative cells were converted to red spore cells, and the density of algal cells reached 7.74 g/L. At this time, the content of astaxanthin reached 1.32%, and the yield of astaxanthin reached 102.17 mg/L (as shown in FIG. 4).

Example 5

[0163] A preferred basal culture medium of this example has a formula of: 1.10 g/L sodium acetate, 0.7 g/L sodium nitrate, 152 mg/L potassium dihydrogen phosphate, 64 mg/L dipotassium hydrogen phosphate, 100 mg/L magnesium sulfate, 15 mg/L calcium chloride, 3 mg/L disodium edetate, 3 mg/L boric acid, 350 μg/L ferric chloride, 70 μg/L manganese chloride, 35 μg/L zinc sulfate, 30 μg/L sodium molybdate, 7 μg/L cobalt chloride, 50 μg/L copper sulfate, 0.05 μg/L Vitamin B12, 0.05 μg/L biotin, and 5 μg/L Vitamin B1.

[0164] A preferred expansion feeding medium of this example has a formula of: 25 g/L sodium nitrate, 2.5 g/L potassium dihydrogen phosphate, 0.5 g/L magnesium sulfate, 15 mg/L disodium edetate, 15 mg/L boric acid, 1750 μg/L ferric chloride, 350 μg/L manganese chloride, 175 μg/L zinc sulfate, 150 μg/L sodium molybdate, 35 μg/L cobalt chloride and 250 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with filter-sterilized Vitamin B12 of 2 μg/L, biotin of 2 μg/L, Vitamin B1 of 200 μg/L, and glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 300 g/L.

[0165] A preferred induction feeding medium of this example has a formula of: 2.5 g/L potassium dihydrogen phosphate, 0.5 g/L magnesium sulfate, 15 mg/L disodium edetate, 15 mg/L boric acid, 1750 μg/L ferric chloride, 350 μg/L manganese chloride, 175 μg/L zinc sulfate, 150 μg/L sodium molybdate, 35 μg/L cobalt chloride, and 250 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 300 g/L.

[0166] The steps were as follows:

[0167] (1) Seed activation: Haematococcus pluvialis CCTCC M2018809 preserved in a slant medium was inoculated in a sterile basal medium with 0.5 g/L Tris (Tris(hydroxymethylaminomethane)) at an initial pH of 7.5, and placed in a 100 mL Erlenmeyer flask at a loading volume of 50 ml for static culture (hand shaking for 2 times every day) at a culture temperature of 20° C. for 20 days, where 98% of algae cells were green vegetative cells, and the number of algae cells reached 600,000 cells/mL.

[0168] (2) Heterotrophic expansion culture: the prepared basal medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated into a fermenter at an initial inoculation amount of 20,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 7.0, the concentration of acetate at 500-900 mg/L, the concentration of nitrate at 400-700 mg/L, and the concentration of phosphate at 100-150 mg/L, and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 0.5 mg/L-1.5 mg/L by adjusting aeration volume at 0.1-0.2 vvm and stirring speed at 50-70 rpm. Sampling was conducted every day for observation. After 10 days of culture, the density of algal cells reached 1.26 g/L, the number of cells reached was 670,000 cells/mL, and 60% of motile vegetative cells were converted to immotile vegetative cells.

[0169] (3) Heterotrophic induction: a concentrated sodium acetate mother solution was added to the fermenter in step (2) through an aseptic operation, to increase the concentration of acetate in the broth to 4800 mg/L, before starting the heterotrophic culture. During the culture, the induction feeding medium was fed to maintain the pH of the broth at 9.0, the concentration of acetate at 4000-6000 mg/L, and the culture temperature was controlled at 30° C. During the fermentation, the concentration of dissolved oxygen was controlled at 4.0 mg/L-8.0 mg/L by adjusting aeration volume at 1.0-1.5 vvm and stirring speed at 100-200 rpm. Sampling was conducted every day for observation. After culturing for 14 days, all vegetative cells were converted to red spore cells, and the density of algal cells reached 6.80 g/L. At this time, the content of astaxanthin reached 2.05%, and the yield of astaxanthin reached 139.40 mg/L (as shown in FIG. 5).

Example 6

[0170] A preferred basal culture medium of this example has a formula of: 0.66 g/L sodium acetate, 0.8 g/L sodium nitrate, 100 mg/L potassium dihydrogen phosphate, 40 mg/L dipotassium hydrogen phosphate, 150 mg/L magnesium sulfate, 20 mg/L calcium chloride, 2 mg/L disodium edetate, 2 mg/L boric acid, 300 μg/L ferric chloride, 50 μg/L manganese chloride, 30 μg/L zinc sulfate, 25 μg/L sodium molybdate, 5 g/L cobalt chloride, and 30 g/L copper sulfate.

[0171] A preferred expansion feeding medium of this example has a formula of: 50 g/L sodium nitrate, 5 g/L potassium dihydrogen phosphate, 1 g/L magnesium sulfate, 30 mg/L disodium edetate, 30 mg/L boric acid, 3500 μg/L ferric chloride, 700 μg/L manganese chloride, 350 μg/L zinc sulfate, 300 g/L sodium molybdate, 70 μg/L cobalt chloride and 500 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 600 g/L.

[0172] A preferred induction feeding medium of this example has a formula of: 0.1 g/L potassium dihydrogen phosphate, 1 mg/L disodium edetate, 1 mg/L boric acid, 150 μg/L ferric chloride, 30 μg/L manganese chloride, 15 μg/L zinc sulfate, 15 μg/L sodium molybdate, 3 μg/L cobalt chloride, and 20 μg/L copper sulfate. Upon preparation, it was autoclaved at 121° C. for 21 minutes, and after being cooled to room temperature, added with glacial acetic acid (with a content of acetic acid of over 98%) through an aseptic operation, to provide a concentration of acetic acid of 60 g/L.

[0173] The steps are as follows:

[0174] (1) Seed activation: Haematococcus pluvialis CCTCC M2018809 preserved in a slant medium was inoculated in a sterile basal medium with 0.7 g/L Tris at an initial pH of 7.8, and placed in a 100 mL Erlenmeyer flask at a loading volume of 50 ml for static culture (hand shaking for 2 times every day) at a culture temperature of 20° C. for 20 days, where 95% of algae cells were green vegetative cells, and the number of cells reached 490,000 cells/mL.

[0175] (2) Heterotrophic expansion culture: the prepared basal medium was added to a 5 L fermenter at a loading coefficient of 0.6. The seed solution obtained in step (1) was inoculated into a fermenter at an initial inoculation amount of 60,000 cells/mL for heterotrophic culture, wherein the expansion feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 200-600 mg/L, the concentration of nitrate at 300-800 mg/L, and the concentration of phosphate at 80-120 mg/L, and the culture temperature was controlled at 20° C. During the fermentation, the concentration of dissolved oxygen was controlled at 0.5 mg/L-1.5 mg/L by adjusting aeration volume at 0.1-0.2 vvm and stirring speed at 50-70 rpm. Sampling was conducted every day for observation. After 8 days of culture, the density of algal cells reached 1.19 g/L, the number of cells reached was 1.5 million cells/mL, and 50% of motile vegetative cells were converted to immotile vegetative cells.

[0176] (3) Heterotrophic induction: During the culture, the induction feeding medium was fed to maintain the pH of the broth at 8.0, the concentration of acetate at 600-900 mg/L, and the culture temperature was controlled at 25° C. During the fermentation, the concentration of dissolved oxygen was controlled at 4.0 mg/L-6.0 mg/L by adjusting aeration volume at 0.5-1.0 vvm and stirring speed at 150-200 rpm. Sampling was conducted every day for observation. After culturing for 15 days, all vegetative cells were converted to red spore cells, and the density of algal cells reached 9.07 g/L. At this time, the content of astaxanthin reached 1.78%, and the yield of astaxanthin reached 161.45 mg/L (as shown in FIG. 6).

REFERENCES

[0177] Li, Y., Sommerfeld, M., Chen, F. & Hu, Q. 2008. Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J. Plant Physiol. 165:1783-1797. [0178] Li, Y., Sommerfeld, M., Chen, F. & Hu, Q. 2010. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae). J. Appl. Phycol. 22:253-263 [0179] Hata N, Ogbonna J C, Hasegawa Y, Taroda H, Tanaka H. 2001. Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture. J Appl Phycol 13(5):395-402. [0180] Wan, M., Zhang, Z., Wang, J., Huang, J., Fan, J., Yu, A., Wang, W., Li, Y. 2015. Sequential Heterotrophy-Dilution-Photoinduction Cultivation of Haematococcus pluvialis for efficient production of astaxanthin. Bioresour Technol 198:557-563. [0181] Zhang, Z., Wang B., Hu Q, Sommerfeld M., Li Y., Han D. 2016. A New Paradigm for Producing Astaxanthin From the Unicellular Green Alga Haematococcus pluvialis. Biotechnology and Bioengineering 113(10): 2088-2099. [0182] Li, Y., Zhang, Z, Fan, J., Wan, M., Hou, D., Zhang, J., Huang, J., Liang, S., Wang, J., Chen, J., Wang, W. 2013. Method Using micro-algae for high-efficiency production of astaxanthin. PCT/CN2013/084,262. [0183] Kobayashi, M., Kakizono, T., Yamaguchi, K., Nishio, N., Nagai, S. 1992. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. J Ferment Bioeng 74(1):17-20. [0184] Kobayashi, M., Kurimura, Y., Tsuji, Y. 1997. Light-independent, astaxanthin production by the green microalga Haematococcus pluvialis under salt stress. Biotechnol Lett 19(6):507-509. [0185] Shi, X., Zhang, X., Chen, F. 2000. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme and Microbial Technology 27: 312-318.