WO2021057709A1 - 一种培养雨生红球藻生产虾青素的方法 - Google Patents

一种培养雨生红球藻生产虾青素的方法 Download PDF

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WO2021057709A1
WO2021057709A1 PCT/CN2020/116775 CN2020116775W WO2021057709A1 WO 2021057709 A1 WO2021057709 A1 WO 2021057709A1 CN 2020116775 W CN2020116775 W CN 2020116775W WO 2021057709 A1 WO2021057709 A1 WO 2021057709A1
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cells
culture
source
nitrogen
haematococcus pluvialis
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PCT/CN2020/116775
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English (en)
French (fr)
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郑玉彬
曹培鑫
刘帅帅
王�华
陈方见
谢诗懿
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山东拜昂生物技术有限公司
山东金晶生物技术有限公司
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Priority to JP2022516380A priority Critical patent/JP7535103B2/ja
Priority to CN202080060094.5A priority patent/CN114286856B/zh
Priority to EP20869712.8A priority patent/EP4036216A4/en
Priority to US17/763,171 priority patent/US20220340950A1/en
Priority to CA3153214A priority patent/CA3153214A1/en
Publication of WO2021057709A1 publication Critical patent/WO2021057709A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention relates to the field of astaxanthin production, in particular to a method for producing astaxanthin by using Haematococcus pluvialis, and a medium used in the method.
  • Astaxanthin (Astaxanthin), chemical name 3,3'-dihydroxy-4,4'-diketo ⁇ -carotene, molecular formula C 40 H 52 O 4 , molecular weight 596.86, is a keto carotenoid , Has a strong antioxidant function and coloring effect, and is widely used in functional foods, cosmetics, feed additives and other fields.
  • astaxanthin can be produced by chemical synthesis, its antioxidant activity and biological safety are not as good as natural astaxanthin.
  • the astaxanthin content of Haematococcus pluvialis can reach 1.5-3% of the dry cell weight. Its biological safety has been recognized by major countries in the world. It has been approved as a food raw material by the European Union FSA, the US FDA, and the Chinese Ministry of Health. It is the best organism to produce natural astaxanthin in nature.
  • Haematococcus pluvialis exists as green vegetative cells in a nutrient-rich environment with suitable light and temperature. Under unfavorable conditions, such as high light, high temperature, high salt, and nutrient deficiency, it is thick The immobile cells of the wall exist, and at the same time a large amount of astaxanthin is accumulated to combat adverse conditions. According to its physiological characteristics, the two-step method is currently used to cultivate Haematococcus pluvialis to produce astaxanthin, the first step is the expansion of green vegetative cells, and the second step is astaxanthin induction.
  • Cyanotech a Hawaiian company in the United States, uses an autotrophic method to cultivate Haematococcus pluvialis vegetative cells in a closed photobioreactor, and then uses sunlight in an open track pond to induce redness of the cells, and the astaxanthin content can reach the dry cell weight 1.5% of it.
  • Israel Algatechologies uses a pipeline photobioreactor for vegetative cell culture and astaxanthin induction, and the astaxanthin content can reach 3%.
  • the invention patent (PCT/CN2013/084262) discloses a method for producing astaxanthin by using Haematococcus pluvialis. The first step is to use heterotrophic culture to obtain green vegetative cells, and then add culture medium for dilution, and then use light culture to achieve The accumulation of astaxanthin, the astaxanthin content reached 2.3%.
  • the present invention provides a method for culturing Haematococcus pluvialis to produce astaxanthin under non-light conditions.
  • the present invention provides a method for producing astaxanthin, comprising:
  • Haematococcus pluvialis is a single-celled green algae belonging to the Chlorophyta, Chlorophyceae, Volvox, Haematococaceae, and Haematococcus genus. Under suitable environmental and nutrient-rich conditions, Haematococcus pluvialis rapidly grows and divides and reproduces, producing a large number of vegetative cells with flagella; when the environmental conditions become unsuitable, the swimming cells lose their flagella and become unsuitable.
  • the medium used to produce astaxanthin Haematococcus pluvialis can be any medium that can be used to cultivate Haematococcus pluvialis for its growth and reproduction, and it usually contains a nitrogen source, a phosphorus source, a sulfur source, and magnesium. Source, calcium source and/or trace element, and those skilled in the art can determine its content according to the knowledge and practice requirements in the field. Suitable media for specific algae are known in the art. For this, see media such as BG-11, BBM, C medium, and MCM.
  • the "nitrogen source” that can be used in the culture medium of the present invention refers to an inorganic or organic nitrogen source that can be utilized by cultivated algae, such as but not limited to nitric acid, nitrate, nitrite, ammonia, ammonium, and urea , Amino acids, peptone, yeast extract, protein powder, corn steep liquor and any combination thereof.
  • the "phosphorus source” that can be used in the culture medium of the present invention refers to a phosphorus source that can be used by the cultivated algae, such as but not limited to phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and dihydrogen phosphate. Potassium and any combination thereof.
  • the "sulfur source” that can be used in the culture medium of the present invention refers to a sulfur source that can be utilized by the cultivated algae, and includes, but is not limited to, sulfuric acid, magnesium sulfate, sodium sulfate, and any combination thereof, for example.
  • the "magnesium source” that can be used in the medium of the present invention refers to a magnesium source that can be utilized by the cultivated algae, and includes, but is not limited to, magnesium sulfate, magnesium chloride, and combinations thereof, for example.
  • the "calcium source” that can be used in the culture medium of the present invention refers to a calcium source that can be used by the cultivated algae, and includes, but is not limited to, calcium chloride, calcium sulfate, calcium nitrate, and any combination thereof, for example.
  • the "trace element” that can be used in the culture medium of the present invention refers to the trace element that can be utilized by the cultivated algae, for example, including but not limited to Mn (for example, manganese chloride), Zn (for example, zinc sulfate), B (for example, boric acid) One or more of, I, Mo (such as sodium molybdate), Cu (such as copper sulfate), Co (such as cobalt chloride), Fe (such as ferric chloride).
  • Mn for example, manganese chloride
  • Zn for example, zinc sulfate
  • B for example, boric acid
  • I, Mo such as sodium molybdate
  • Cu such as copper sulfate
  • Co such as cobalt chloride
  • Fe such as ferric chloride
  • the "organic carbon source” that can be used in the culture medium of the present invention refers to the organic carbon source that can be utilized by the target microorganisms to be cultured. Those skilled in the art can determine which organic carbon sources can be used in the culture medium of the present invention based on the technical knowledge in the field.
  • Examples include but are not limited to acetic acid, acetate, propionic acid, propionate, butyric acid, butyrate, lactic acid, lactate, fatty acid, fatty acid salt, amino acid, methanol, ethanol, glycerol, citric acid, citric acid Salt, pyruvate, pyruvate, glucose, fructose, arabinose, lactose, mannose, rhamnose, ribose, or wastewater containing these organic carbon sources, hydrolysate, fermentation broth, and any combination thereof.
  • the addition amount of the organic carbon source can be determined according to the conventional knowledge in the field and the actual growth conditions of the algae cells, which are all within the technical ability of those skilled in the art.
  • the inoculation density of the astaxanthin-producing Haematococcus pluvialis cells can be any density suitable for the growth and reproduction of the astaxanthin-producing Haematococcus pluvialis cells, and those skilled in the art can use their technical knowledge in the art. And empirically determine the appropriate inoculation density.
  • the present invention shrimp producing astaxanthin pluvialis seeding density of algal cells may be at least 104 cells / ml culture medium, e.g. 1-20 ⁇ 10 4 cells / ml culture medium, such as 5, 8 or 10 ⁇ 10 4 cells/ml medium.
  • the inoculation density of astaxanthin-producing Haematococcus pluvialis cells may be at least 0.5-2.0 g cells/L medium, for example at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 , 1.5, 1.6, 1.7, 1.8, 1.9, 2.0g cells/L medium.
  • nutrient-deficient medium refers to a medium lacking nutrient elements, such as one or more, or even all, of nitrogen, phosphorus, sulfur, magnesium, calcium, and trace elements.
  • the nutrient-deficient medium lacks a source of nitrogen, phosphorus, sulfur, calcium, magnesium, and/or trace elements.
  • the nutrient-deficient medium lacks a nitrogen source.
  • the nutrient-deficient medium lacks a source of nitrogen and phosphorus.
  • the nutrient-deficient medium lacks nitrogen sources and trace elements.
  • the nutrient-deficient medium is a medium lacking all the nutrients.
  • the nutrient-deficient medium is acetic acid or an acetate solution, such as a sodium acetate solution.
  • the nutrient-deficient medium is, for example, a medium having a concentration of 60-1050 g/L, such as about 120, 180, 240, 300, 400, 500, 600, 700, 800, 900, 1000 g/L Acetic acid solution.
  • no light conditions refers to no light or insufficient light to autotrophically cultivate the Haematococcus pluvialis.
  • autotrophic is a cultivation method that uses inorganic carbon sources such as carbon dioxide, carbonate, bicarbonate, etc., to grow and reproduce under light conditions through photosynthesis.
  • polyculture is a cultivation method that uses organic carbon sources for growth and reproduction under light conditions.
  • heterotrophic is a cultivation method that uses organic carbon sources to grow and reproduce in the absence of light.
  • the vegetative cells of step (a) are obtained by culturing astaxanthin-producing Haematococcus pluvialis cells.
  • a variety of method steps for culturing algae cell growth and reproduction are known in the art, including, for example, autotrophic, polytrophic or heterotrophic culture methods.
  • step (a) after culturing the Haematococcus pluvialis cells may include the steps of removing the culture medium and/or collecting vegetative cells, and optionally concentrating the vegetative cells. Any suitable method known in the art can be used to remove the culture medium, collect and/or concentrate the nutrient cells, such as by precipitation (natural sedimentation or centrifugation) or filtration (using filters or membranes).
  • the culture temperature and pH of step (a) can be any temperature or pH suitable for the growth and reproduction of Haematococcus pluvialis cells.
  • the culture pH of step (a) is 6.0-9.0, such as 6.0-8.0, 7.0-8.0, 7.0-8.5, 7.5-8.0, 7.5-8.5, 8.0-9.0 or 8.5-9.0, such as about 6.5 , 7.0, 7.5, 8.0, 8.5 or 9.0.
  • the culture pH range of step (a) is controlled to be 7.0-8.0.
  • the culture in step (a) is an autotrophic culture, and mixed air containing carbon dioxide (for example, 0.5%-5% (v/v)) can be passed to control the pH.
  • the culture in step (a) is polyculture and heterotrophic culture, and acid (for example, 0.1-10 mol/L hydrochloric acid, sulfuric acid, and acetic acid) can be used to control pH.
  • the culture temperature of step (a) is 15-25°C, preferably 20-25°C, for example about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C , 23°C, 24°C or 25°C.
  • step (a) obtained contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 3 million or more cells (nutrition Cells)/ml of culture medium.
  • the vegetative cells of step (a) are obtained by autotrophically culturing Haematococcus pluvialis cells.
  • Haematococcus pluvialis cells can be autotrophically cultured under any suitable growth and reproduction and light conditions.
  • Haematococcus pluvialis cells can be inoculated and cultured in a medium containing a nitrogen source (for example, nitrate such as sodium nitrate), and the light intensity can be, for example, but not limited to 10-100, 10-90, 10 -80, 10-70, 20-90, 20-80, 20-70, 30-90, 30-80, 30-70, 40-60 ⁇ E/m 2 /s, for example about 20, 30, 40, 50, 60, 70, 80 or 90 ⁇ E/m 2 /s.
  • a nitrogen source for example, nitrate such as sodium nitrate
  • carbon dioxide or a mixed gas containing carbon dioxide can be introduced to provide an inorganic carbon source, such as mixed air containing 0.5-1.5% (v/v) carbon dioxide;
  • the aeration volume can be, for example, 0.05-0.5vvm, such as 0.1- 0.5 or 0.2-0.5 vvm, for example about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 vvm.
  • the pH of the culture solution can be adjusted by adjusting the carbon dioxide content and the aeration rate.
  • step (a) adopts polyculture or heterotrophic mode to culture Haematococcus pluvialis cells.
  • the medium used in step (a) for polyculture or heteroculture contains 80-700, 90-600, 90-500, 90-400, 100-600, 100-500, 100-400, 100 -350 or 120-350mg/L carbon element organic carbon source, and containing 40-800, 40-700, 40-600, 50-800, 50-700, 50-600, 60-800, 60-700, 60 -600, 70-800, 70-700, 70-600, 80-600mg/L nitrogen source of nitrogen element, preferably the mass ratio of carbon to nitrogen element is 0.1-10:1, 0.2-10:1, 0.1-5:1 , 0.2-5:1 or 0.3-4.5:1, for example, about 0.3:1, 0.5:1, 1:1, 1.4:1, 1.5, 1.8:1, 2:1, 2.4:1, 3:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1 or 9:
  • the vegetative cells of step (a) are obtained by polyculture of Haematococcus pluvialis cells.
  • Polyculture is the growth and reproduction of Haematococcus pluvialis cells using the organic carbon source contained in the culture medium under light conditions.
  • the medium used for polyculture contains an organic carbon source (for example, acetic acid or acetate, such as sodium acetate), optionally a nitrogen source (for example, nitric acid or nitrate, such as sodium nitrate), preferably carbon nitrogen
  • an organic carbon source for example, acetic acid or acetate, such as sodium acetate
  • a nitrogen source for example, nitric acid or nitrate, such as sodium nitrate
  • the element mass ratio is 0.1-10:1, 0.2-10:1, 0.1-5:1, 0.2-5:1 or 0.3-4.5:1, for example, about 0.3:1, 0.5:1, 1:1, 1.4 :1, 1.5, 1.8:1, 2:1, 2.4:1, 3:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1 or 9:1.
  • the light intensity can be, for example, but not limited to 10-100, 10-90, 10-80, 10-70, 20-90, 20-80, 20-70, 30-90, 30-80, 30-70, 40- 60 ⁇ E/m 2 /s, for example about 20, 30, 40, 50, 60, 70, 80 or 90 ⁇ E/m 2 /s.
  • the dissolved oxygen is controlled at 1-50%, such as 5-30% or 5-10%, and the dissolved oxygen can be controlled by, for example, adjusting the aeration rate (for example, air) and stirring speed
  • the ventilation rate is 0.05-0.5 or 0.05-0.1 vvm, such as about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 vvm
  • the stirring speed is 50-150 or 50-80 revolutions per minute, such as about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 revolutions per minute.
  • step (a) performs polyculture culture in a fed-batch manner, wherein a feed liquid is added to the culture broth.
  • the feed solution comprises a medium containing an organic carbon source (e.g., acetic acid or acetate, such as sodium acetate) and optionally a nitrogen source (e.g., nitric acid or nitrate, such as sodium nitrate).
  • the feed liquid contains an organic carbon source of 6-420 g/L carbon element and a nitrogen source of 0.3-120 g/L nitrogen element.
  • the mass ratio of carbon to nitrogen is 1-50:1, for example, about 1.
  • the feed solution is a concentrated medium containing an organic carbon source and optionally a nitrogen source, such as a 5-50 times concentrated medium.
  • the vegetative cells of step (a) are obtained by heterotrophically culturing Haematococcus pluvialis cells.
  • Heterotrophic culture is a culture method in which Haematococcus pluvialis uses organic carbon sources in the culture medium to grow and reproduce under conditions of no light or insufficient light for autotrophic culture.
  • Haematococcus pluvialis is different in a medium containing an organic carbon source (such as acetic acid or acetate, such as sodium acetate) and optionally a nitrogen source (such as nitric acid or nitrate, such as sodium nitrate).
  • an organic carbon source such as acetic acid or acetate, such as sodium acetate
  • a nitrogen source such as nitric acid or nitrate, such as sodium nitrate
  • the mass ratio of carbon to nitrogen is preferably 0.1-10:1, 0.2-10:1, 0.1-5:1, 0.2-5:1 or 0.3-4.5:1, for example, about 0.3:1, 0.5: 1, 1:1, 1.4:1, 1.5:1, 1.8:1, 2:1, 2.4:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1 or 9:1.
  • the dissolved oxygen is controlled at 1-50%, preferably 5-30%, such as about 15%, 20%, 25%, 30%, 35%, 40% , 45% or 50%
  • the dissolved oxygen can be controlled by, for example, adjusting the ventilation (such as air) and stirring speed, for example, the ventilation is 0.05-0.5vvm, such as about 0.1, 0.2, 0.3, 0.4, 0.5vvm, and/or stirring
  • the speed is 50-150 revolutions per minute, for example about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 revolutions per minute.
  • the heterotrophic culture of step (a) is performed in a fed-batch manner, wherein a feed liquid is added to the culture broth.
  • the feed solution comprises a medium containing an organic carbon source (such as acetic acid or acetate, such as acetic acid) and optionally a nitrogen source (such as nitric acid or nitrate, such as sodium nitrate).
  • the feed liquid contains an organic carbon source of 6-420g/L carbon element and a nitrogen source of 0.3-120g/L nitrogen element, preferably the mass ratio of carbon to nitrogen is 1-50:1, 1-40: 1, 1-35:1, 5-50:1, 5-40:1, or 5-35:1, for example, about 1:1, 2:1, 3:1, 4:1, 5:1, 10 :1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1.
  • the feed solution is a concentrated medium containing an organic carbon source and optionally a nitrogen source, such as a 5-50 times concentrated medium.
  • the vegetative cells of step (a) are obtained by inoculating Haematococcus pluvialis cells in a polyculture or heterotrophic culture in a medium containing an organic carbon source and a nitrogen source, preferably the medium contains 80-700 mg/ The organic carbon source of L carbon element, the nitrogen source of 40-800 mg/L nitrogen element, preferably the mass ratio of carbon to nitrogen is 0.1-10:1.
  • the vegetative cells of step (a) are obtained by culturing Haematococcus pluvialis cells in a manner selected from batch, fed-batch, semi-continuous and continuous culture, or heterotrophically cultured Haematococcus pluvialis; when fed-batch culture
  • the feed solution preferably contains 15-1050g/L, more preferably 15-600 or 60-300g/L acetic acid or acetate, and 0.3-120g/L nitrogen source And a 1-50 times concentrated medium, preferably the carbon-nitrogen mass ratio is about 1-50:1, 1-40:1, 1-35:1, 5-50:1, 5-40:1, or 5-35 :1.
  • Step (b) is to heterotrophically culture the Haematococcus pluvialis vegetative cells of step (a) in a nutrient-deficient medium containing organic carbon sources under no light conditions to stimulate the production of astaxanthin by the algae cells.
  • the inoculation density, culture temperature and pH of step (b) can be any density, temperature and pH value suitable for heterotrophic culture of Haematococcus pluvialis vegetative cells.
  • the seeding density of Haematococcus pluvialis vegetative cells in step (b) may be at least 0.5-2.0 g cells/L medium, such as 0.5-1.7 g cells/L medium, such as at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0g cells/L medium.
  • the culture temperature of step (b) is 15-35°C, 20-30°C, or 25-30°C, for example about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C. °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C or 34°C.
  • the pH of step (b) is 6.0-11.0, 7.0-10.0, 7.0-9.0 or 7.5-9.0, for example about 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or 10.5.
  • the dissolved oxygen is controlled at 20-90%, preferably 30-70%, for example, the dissolved oxygen is controlled by adjusting the ventilation volume and the stirring speed, and the ventilation volume is preferably 0.2-3.0 vvm, such as 0.5 , 1.0, 1.5, 2.0, 2.5 vvm, and/or preferably the stirring speed is 100-300 revolutions per minute, such as 150, 200, 250 revolutions per minute.
  • the carbon element content of the organic carbon source is at least about 200 mg/L, such as at least about 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 4000, 5000mg/L.
  • the organic carbon source contained in the nutrient-deficient medium is acetic acid or acetate, such as 1-15, 1-14, 1-13, 1-12, 1- 11, 1-10, 1-9, 1-8, 2-10, 2-9, 2-8, 3-8, 3.5-8, 4-8g/L, such as about 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14g/L.
  • the organic carbon source contained in the nutrient-deficient medium in step (b) is sodium acetate, such as 1-12, 1-11, 1-10, 1-9, 1-8, 2-10, 2-9, 2-8, 3-8, 3.5-8, 4-8 g/L, such as about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9 or 10 g/L.
  • step (b) is cultured in a feeding manner
  • the preferred feeding solution contains acetic acid or acetate (for example, 30-1050g/L, especially 30-600 or 100-600g/L, such as about 50, 100, 200, 300, 400, 500, 550, 600, 700, 800, 900, 1000g/L) nutrient-deficient medium (for example, lack of nitrogen, phosphorus and/or trace elements, or lack all nutrients)
  • the feed solution is an acetic acid solution, for example, a concentration of 60-600 g/L, such as about 60, 120, 180, 240, 300, 400, 550, 560, 565, 570 g/L.
  • the pH range of step (b) is controlled to be 7.0-8.5, which can be controlled by adding, for example, 0.1-10 mol/L hydrochloric acid, sulfuric acid, and acetic acid.
  • steps (a) and/or (b) can be carried out in a bioreactor.
  • the bioreactor includes various photobioreactors, such as a flat plate, a column type, a hanging bag type, and a tube type. , Runway ponds and fermentation tanks.
  • step (c) The harvesting of algae spore cells and/or astaxanthin described in step (c) can be used to harvest the spore cells (such as sedimentation or centrifugation, etc.) and/or destroy the cell wall (mechanical, chemical or enzymatic methods). Method) and harvesting astaxanthin, optionally using any suitable method to separate and/or purify astaxanthin.
  • the present invention provides a method for culturing Haematococcus pluvialis to produce astaxanthin under non-light conditions, including:
  • Haematococcus pluvialis cells preferably to obtain concentrated Haematococcus pluvialis cells, for example by natural sedimentation, centrifugation or filtration;
  • step (b) Haematococcus pluvialis cells obtained in step (a) of heterotrophic culture by adding an organic carbon source in a nutrient-deficient medium under non-light conditions, for example, by batch, fed-batch, semi-continuous or Perform heterotrophic culture in a continuous culture mode, preferably wherein the culture temperature is controlled at 15-35°C, the pH is controlled at 6.0-11.0 and/or the dissolved oxygen is controlled at 20-90%; and
  • the medium for the autotrophic culture of Haematococcus pluvialis that can be used in step (a) of the present invention contains or consists of the following components:
  • Magnesium sulfate 50-500mg/L
  • Disodium ethylenediaminetetraacetic acid 0.5-6mg/L
  • Zinc sulfate 10-100 ⁇ g/L
  • Cobalt chloride 5-50 ⁇ g/L
  • nitrogen sources such as nitric acid or nitrate such as sodium nitrate, where the nitrogen content is about 40-800mg/L,
  • the pH is 7.0-8.0 (e.g. 7.5).
  • the medium for polyculture or heterotrophic culture of Haematococcus pluvialis that can be used in step (a) of the present invention contains or consists of the following components:
  • Organic carbon source 80-700mg/L (carbon element content)
  • Nitrogen source 40-800mg/L (nitrogen content), wherein the mass ratio of carbon to nitrogen: 0.1-10:1, such as 0.2-10:1, 0.1-5:1, 0.2-5:1, preferably 0.3-4.5 :1,
  • Magnesium sulfate 50-500mg/L
  • Disodium ethylenediaminetetraacetic acid 0.5-6mg/L
  • Zinc sulfate 10-100 ⁇ g/L
  • Cobalt chloride 5-50 ⁇ g/L
  • the pH is 7.0-8.0 (e.g. 7.5).
  • the culture medium contains 90-600, 90-500, 90-400, 100-600, 100-500, 100-400, 100-350 or 120-350 mg/L of organic carbon source,
  • organic carbon source for example, about 90mg/L, 100mg/L, 110mg/L, 120mg/L, 130mg/L, 140mg/L, 150mg/L, 200mg/L, 210mg/L, 220mg/L, 230mg/L, 240mg/L, Organic carbon source of 250mg/L, 260mg/L, 270mg/L, 280mg/L, 290mg/L, 300mg/L or 350mg/L.
  • the organic carbon source contained in the culture medium includes, but is not limited to, acetic acid or acetate such as sodium acetate, glucose, ribose, and any combination thereof.
  • the culture medium comprises 40-800, 40-700, 40-600, 50-800, 50-700, 50-600, 60-800, 60-700, 60-600, 70-800 , 70-700, 70-600, 80-600mg/L nitrogen source, for example about 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L, 100mg/L, 150mg/L, 200mg/ L, 250mg/L, 300mg/L, 350mg/L, 400mg/L, 450mg/L, 500mg/L, 550mg/L or 600mg/L nitrogen source.
  • the nitrogen source contained in the culture medium includes, but is not limited to, nitric acid or nitrates such as sodium nitrate, ammonium sulfate, urea, and any combination thereof.
  • the mass ratio of carbon and nitrogen elements in the medium is about 0.2:1; 0.3:1; 0.4:1; 0.5:1; 1:1; 1.5:1; 2:1; 2.5:1; 3:1; 3.5:1; 4:1; 4.5:1; 5:1; 6:1; 7:1; 8:1 or 9:1, such as 1.4:1; 1.8:1; 2.4:1; 4.4 :1.
  • the medium for heterotrophic culture of Haematococcus pluvialis that can be used in step (b) of the present invention contains or consists of the following components:
  • Magnesium sulfate 50-500mg/L
  • Disodium ethylenediaminetetraacetic acid 0.5-6mg/L
  • Zinc sulfate 10-100 ⁇ g/L
  • Cobalt chloride 5-50 ⁇ g/L
  • the pH is 7.0-8.0 (e.g. 7.5).
  • nutrient-deficient medium means that the medium does not contain the nutrient element, or the nutrient element is lower than the target microorganism needs to grow, resulting in the target microorganism being starved for the nutrient element. In one embodiment, “nutrient-deficient medium” means that the nutrient element is not contained in the medium.
  • the nutrient-deficient medium described in step (b) of the present invention is the medium described in the present invention, which does not contain corresponding nutrient elements.
  • a nitrogen-deficient medium may be a medium having the composition described in the present invention, but in which a nitrogen-containing compound (disodium ethylenediaminetetraacetic acid) is not present.
  • a phosphorus-deficient medium is a medium having the composition described in the present invention, but the phosphorus-containing compound (potassium dihydrogen phosphate) is not present.
  • the medium of the present invention contains about 0.05g/L, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7 g/L, 0.8g/L, 0.9g/L or 1.0g/L potassium dihydrogen phosphate.
  • the medium of the present invention contains about 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, 300 mg/L, 350 mg/L, 400 mg/L, 450 mg/L or 500mg/L of magnesium sulfate.
  • the medium of the present invention contains about 5 mg/L, 10 mg/L, 11 mg/L, 12 mg/L, 13 mg/L, 14 mg/L, 15 mg/L, 20 mg/L, 25 mg/L, 27mg/L, 30mg/L, 35mg/L, 36mg/L, 40mg/L, 45mg/L or 50mg/L calcium chloride.
  • the culture medium of the present invention contains 0.5-5.5 mg/L of disodium edetate, such as about 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 2.5mg/L, 3.0mg/L, 3.5mg/L, 4.0mg/L, 4.5mg/L, 5.0mg/L or 5.5mg/L disodium edetate.
  • the medium of the present invention contains about 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 1.6 mg/L, 1.7 mg/L, 1.8 mg/L, 1.9 mg/L, 2.0 mg/L, 2.5mg/L, 3.0mg/L, 3.5mg/L, 4.0mg/L, 4.5mg/L or 5.0mg/L boric acid.
  • the culture medium of the present invention contains 100-950, 100-900 or 120-900 ⁇ g/L ferric chloride, for example about 110 ⁇ g/L, 120 ⁇ g/L, 130 ⁇ g/L, 140 ⁇ g/L, 150 ⁇ g /L, 200 ⁇ g/L, 250 ⁇ g/L, 300 ⁇ g/L, 400 ⁇ g/L, 500 ⁇ g/L, 600 ⁇ g/L, 700 ⁇ g/L, 800 ⁇ g/L or 900 ⁇ g/L ferric chloride.
  • the culture medium of the present invention contains 15-100 ⁇ g/L of manganese chloride, such as about 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 40 ⁇ g/L, 50 ⁇ g /L, 60 ⁇ g/L, 70 ⁇ g/L, 72 ⁇ g/L, 80 ⁇ g/L, 90 ⁇ g/L or 100 ⁇ g/L manganese chloride.
  • 15-100 ⁇ g/L of manganese chloride such as about 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 40 ⁇ g/L, 50 ⁇ g /L, 60 ⁇ g/L, 70 ⁇ g/L, 72 ⁇ g/L, 80 ⁇ g/L, 90 ⁇ g/L or 100 ⁇ g/L manganese chloride.
  • the culture medium of the present invention contains 10-100, 10-90, or 14-90 ⁇ g/L zinc sulfate, such as about 10 ⁇ g/L, 11 ⁇ g/L, 12 ⁇ g/L, 13 ⁇ g/L, 14 ⁇ g/L L, 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 36 ⁇ g/L, 40 ⁇ g/L, 50 ⁇ g/L, 60 ⁇ g/L, 66 ⁇ g/L, 70 ⁇ g/L, 80 ⁇ g/L, 88 ⁇ g/L, 90 ⁇ g/L or 95 ⁇ g/L zinc sulfate.
  • 10-100, 10-90, or 14-90 ⁇ g/L zinc sulfate such as about 10 ⁇ g/L, 11 ⁇ g/L, 12 ⁇ g/L, 13 ⁇ g/L, 14 ⁇ g/L L, 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35
  • the medium of the present invention contains about 10 ⁇ g/L, 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 40 ⁇ g/L, 45 ⁇ g/L, 50 ⁇ g/L, 60 ⁇ g/L, 70 ⁇ g/L, 80 ⁇ g/L, 87 ⁇ g/L, 90 ⁇ g/L or 100 ⁇ g/L sodium molybdate.
  • the culture medium of the present invention contains about 5 ⁇ g/L, 10 ⁇ g/L, 15 ⁇ g/L, 20 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 33 ⁇ g/L, 35 ⁇ g/L, 36 ⁇ g/L, 40 ⁇ g/L, 45 ⁇ g/L or 50 ⁇ g/L cobalt chloride.
  • the culture medium of the present invention contains 20-100 ⁇ g/L copper sulfate, such as about 21 ⁇ g/L, 22 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 40 ⁇ g/L, 45 ⁇ g/L L, 50 ⁇ g/L, 55 ⁇ g/L, 60 ⁇ g/L, 65 ⁇ g/L, 70 ⁇ g/L, 75 ⁇ g/L, 79 ⁇ g/L, 80 ⁇ g/L, 85 ⁇ g/L, 90 ⁇ g/L, 95 ⁇ g/L or 100 ⁇ g/L Copper sulfate.
  • 20-100 ⁇ g/L copper sulfate such as about 21 ⁇ g/L, 22 ⁇ g/L, 25 ⁇ g/L, 30 ⁇ g/L, 35 ⁇ g/L, 40 ⁇ g/L, 45 ⁇ g/L L, 50 ⁇ g/L, 55 ⁇ g/L, 60 ⁇ g/L, 65 ⁇ g/L, 70 ⁇ g/L,
  • the present invention provides a method for culturing Haematococcus pluvialis to produce astaxanthin under non-light conditions, the specific steps are as follows:
  • (I) Vegetative cell culture Connect Haematococcus pluvialis species into a bioreactor equipped with a culture medium for autotrophic, polyculture or heterotrophic culture, where the culture temperature is controlled at 15-25°C, and the pH is controlled at 6.0-9.0.
  • the cultivation is stopped when the algae cells no longer divide and multiply, and change from green swimming cells to green vegetative cells.
  • step (II) Cell preparation: removing the culture medium, for example, removing the culture medium from the algae liquid in step (I) through methods such as natural sedimentation, centrifugation, filtration, etc., to obtain concentrated algal cells.
  • step (III) Heterotrophic induction The concentrated algae cells of step (II) are connected to a bioreactor equipped with nutrient-deficient medium, and are cultured heterotrophically by adding organic carbon sources, such as batch and fed-batch , Semi-continuous or continuous culture and other methods for heterotrophic culture, in which the culture temperature is controlled at 15-35°C, the pH is controlled at 6.0-11.0, and the dissolved oxygen is controlled at 20-90%.
  • the culture is terminated when the algae cells change from green vegetative cells to red spore cells and the astaxanthin content no longer increases.
  • the present invention provides a method for culturing Haematococcus pluvialis to produce astaxanthin under non-light conditions, including:
  • the organic carbon source is selected from acetic acid or acetate such as sodium acetate;
  • the nitrogen source is selected from nitric acid or nitrates such as sodium nitrate, urea, tryptone and yeast extract;
  • the carbon element content of the organic carbon source is 150-300mg/L, for example 175-300mg/L;
  • the mass ratio of carbon and nitrogen elements in the medium is 0.3-3:1, for example 0.3-2.5:1;
  • the feed liquid contains an organic carbon source and a nitrogen source, wherein the mass ratio of carbon to nitrogen is 5-35:1, such as 5-33:1;
  • the immobile vegetative cells in the obtained vegetative cells account for at least 80%, preferably 100% of the total number of cells;
  • step (b) Heterotrophically culture the nutrient cells obtained in step (a) in a nutrient-deficient medium containing an organic carbon source in a fed-batch manner under no light conditions to obtain spore cells, including one or more of the following, preferably All:
  • the organic carbon source is selected from acetic acid or acetate such as sodium acetate, for example 4.0-5.5 g/L;
  • the medium lacks (i) nitrogen source, (ii) nitrogen source and phosphorus source, or (iii) all nutrient elements,
  • the culture temperature is 25-30°C, preferably about 30°C;
  • -pH is controlled at 7.5-8.0, preferably about 8.0;
  • the feed solution is (i) a medium containing an organic carbon source lacking a nitrogen source and a phosphorus source or (ii) a medium containing an organic carbon source lacking all nutrients, preferably an acetic acid solution, for example, a concentration of 60-300g/L Or 180-300g/L;
  • step (b) When at least 90%, 95% or 100% of the vegetative cells are transformed into spore cells, step (b) is terminated;
  • the culture medium of the present invention contains or consists of the following components:
  • Magnesium sulfate 150-400mg/L
  • Disodium ethylenediaminetetraacetic acid 1.0-3.5mg/L
  • Manganese chloride 25-75 ⁇ g/L, such as 25-72 ⁇ g/L,
  • Zinc sulfate 20-50 ⁇ g/L
  • Cobalt chloride 10-35 ⁇ g/L, for example 10-33 ⁇ g/L,
  • the pH is 7.5-8.0.
  • the astaxanthin content determination method is as follows:
  • step (3) Repeat step (3) 2-3 times until the color of the algae turns white, and measure the absorbance value A 492 at a wavelength of 492 nm.
  • the Haematococcus pluvialis of the present invention can be any species of Haematococcus pluvialis, for example including but not limited to Haematococcus pluvialis CCTCC M2018809, Haematococcus pluvialis AC136, AC143, AC587, AC588 (Algobank-Caen Microalgal Culture Collection of University of Caen Basse-Normandie, France), Haematococcus pluvialis ATCC 30453, ATCC 30402 (American Type Culture Collection, USA), Haematococcus pluvialis CS-321 (Australian National Algae Culture Collection, Australia) , Haematococcus pluvialis G 1002 (Culture Collection of Algae of Charles University, Czech Republic), Haematococcus pluvialis ETTL 1958/3, TAKACOVAL 1983/1, PRIBYL 2005/4, PRIBYL 2008/3 (Culture Collection of Autotrophic Organisms,
  • Haematococcus pluvialis AQHP0 was deposited at the China Center for Type Culture Collection (CCTCC) (Wuhan University, Wuhan, China, 430072) under the deposit number CCTCC M 2018809 on November 21, 2018.
  • CTCC China Center for Type Culture Collection
  • step refers to the presence or absence of the step.
  • the term “about” refers to a range of values that includes a specific value, and those skilled in the art can reasonably consider it to be similar to the specific value. In certain embodiments, the term “about” means within the standard error of using a measurement commonly accepted in the art. For example, in certain embodiments, about refers to +/- 10% or 5% of the specified value.
  • the present invention has the following advantages and effects:
  • the present invention provides a method for cultivating Haematococcus pluvialis to produce astaxanthin, which overcomes the high requirements for light in the traditional scheme, and can realize the accumulation of high content of astaxanthin under completely dark conditions.
  • the astaxanthin content of Haematococcus pluvialis can reach 2.5% or even 3.21% at the end of astaxanthin induction.
  • the reactor design does not need to consider factors such as specific surface area and light path.
  • Large-volume bioreactors such as fermenters can be used to reduce the number of reactors and floor space, thereby reducing production cost.
  • the present invention gets rid of the dependence of traditional large-scale cultivation of Haematococcus pluvialis on climate, season and geography, and will promote the transformation of traditional agricultural cultivation mode to industrialized large-scale production.
  • Figure 1 shows the production of astaxanthin in algal cells obtained by induction of autotrophic culture using a batch heterotrophic method under non-light conditions.
  • Figure 2 shows the production of astaxanthin in algae cells obtained by polyculture inducing heterotrophic batches under non-light conditions.
  • Figure 3 shows the induction of astaxanthin production by algal cells heterotrophically cultured with sodium acetate and sodium nitrate using a batch-feeding heterotrophic method under non-light conditions.
  • Figure 4 shows the induction of astaxanthin production by algal cells heterotrophically cultured with sodium acetate and ammonium sulfate using a batch fed heterotrophic method under non-light conditions.
  • Figure 5 shows the astaxanthin production of algal cells cultured heterotrophically with sodium acetate and urea using a batch fed heterotrophic method under non-light conditions.
  • Figure 6 shows the astaxanthin production of algal cells cultured heterotrophically with glucose and sodium nitrate using a batch fed heterotrophic method under non-light conditions.
  • Figure 7 shows the induction of astaxanthin production by algal cells heterotrophically cultured with sodium acetate, ribose and sodium nitrate in a fed-batch heterotrophic manner under non-light conditions.
  • Figure 8 shows the induction of astaxanthin production by algal cells heterotrophically cultured with sodium acetate, yeast extract and peptone using a fed-batch heterotrophic method under non-light conditions.
  • the basic medium formula is: potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 500mg/L, calcium chloride 36mg/L, disodium edetate 5mg/L, boric acid 4.5mg/L, ferric chloride 900 ⁇ g/ L, manganese chloride 100 ⁇ g/L, zinc sulfate 88 ⁇ g/L, sodium molybdate 90 ⁇ g/L, cobalt chloride 50 ⁇ g/L, copper sulfate 79 ⁇ g/L.
  • CCTCC M2018809 Preserved in the China Type Culture Collection (CCTCC)) in a sterile basal medium containing 1.5g/L sodium nitrate, and place it in a hanging bag film photobioreactor ,
  • the initial cell number is 50,000 cells/mL
  • the light path of the reactor is 6cm
  • the volume is 5L
  • the filling volume is 70%
  • the culture temperature is 22°C
  • the white fluorescent lamp is continuously illuminated on one side for 24 hours
  • the light intensity is 60 ⁇ E/m 2 /s
  • the mixed air containing 0.5-1.5% (v/v) carbon dioxide is introduced for stirring
  • the aeration rate is 0.2-0.5vvm
  • the pH of the algae liquid is controlled at 7.5 by adjusting the carbon dioxide content and the aeration rate.
  • the algae cells no longer divide and multiply.
  • the number of immobile vegetative cells accounts for 90% of the total number of cells, and the total number of cells
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.05g/L, magnesium sulfate 50mg/L, calcium chloride 5mg/L, disodium edetate 0.5mg/L, boric acid 1.9mg/L, ferric chloride 120 ⁇ g /L, manganese chloride 15 ⁇ g/L, zinc sulfate 14 ⁇ g/L, sodium molybdate 10 ⁇ g/L, cobalt chloride 5 ⁇ g/L, copper sulfate 22 ⁇ g/L.
  • Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal medium containing 0.5g/L sodium acetate (carbon content of 146mg/L) and 0.5g/L sodium nitrate (nitrogen content of 82mg/L), the carbon-nitrogen ratio It is 1.8/1, the initial cell number is 80,000 cells/mL, and it is placed in a 5L glass fermentor, the filling amount is 70%, the culture temperature is 20°C, the white fluorescent lamp is continuously illuminated on one side for 24 hours, and the light intensity is 40 ⁇ E /m 2 /s, the dissolved oxygen is controlled at 5-10% by adjusting the air ventilation rate of 0.05-0.1vvm and the stirring speed of 50-80 rpm.
  • the pH of the fermentation broth was maintained at 7.0 by adding a 50-fold concentrated basal medium containing 600g/L acetic acid and 70g/L sodium nitrate, and the carbon to nitrogen ratio of the feed medium was 20.8/1. After 240 hours of polyculture, the number of immobile vegetative cells accounted for 85% of the total number of cells, and the total number of cells reached 3 million cells/ml.
  • the charging amount is 70%
  • the culture temperature is 25°C
  • the dissolved oxygen is controlled at 30-40% by adjusting the air ventilation 0.2-1.0vvm and the stirring speed 100-150 revolutions/min, and by adding 565g/L acetic acid.
  • the nitrogen basal medium maintains the pH of the algae solution at 9.0.
  • Astaxanthin content determination method refers to the above.
  • the medium formula is: potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 200mg/L, calcium chloride 12mg/L, disodium edetate 3mg/L, boric acid 3mg/L, ferric chloride 500 ⁇ g/L, Manganese chloride 72 ⁇ g/L, zinc sulfate 50 ⁇ g/L, sodium molybdate 45 ⁇ g/L, cobalt chloride 33 ⁇ g/L, copper sulfate 65 ⁇ g/L. After the preparation is complete, adjust the pH to 8.0 with dilute sulfuric acid or sodium hydroxide solution.
  • the ratio is 2.4/1
  • the initial cell number is 100,000 cells/ml, and it is placed in a 5L fermenter
  • the filling volume is 70%
  • the culture temperature is 20°C
  • the air ventilation volume is adjusted to 0.1-0.4vvm and the number of stirring revolutions.
  • the dissolved oxygen is controlled at 15-20% at 50-100 revolutions per minute.
  • the pH of the fermentation broth is maintained at 8.0 by adding a 5-fold concentrated basal medium containing 60g/L acetic acid and 5g/L sodium nitrate, and the feed medium carbon and nitrogen The ratio is 29.1/1.
  • the algae cells no longer divide and reproduce, the number of immobile vegetative cells accounted for 100% of the total number of cells, and the total number of cells reached 2.9 million cells/ml.
  • the charging amount is 70%
  • the culture temperature is 30°C
  • the dissolved oxygen is controlled at 50-70% by adjusting the air ventilation rate of 1.0-3.0vvm and the stirring speed of 100-200 rpm, and by adding nutrients containing 180g/L acetic acid All missing medium maintains the pH of the algae solution at 8.0.
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.3g/L, magnesium sulfate 300mg/L, calcium chloride 27mg/L, disodium edetate 4mg/L, boric acid 3.5mg/L, ferric chloride 700 ⁇ g/ L, manganese chloride 80 ⁇ g/L, zinc sulfate 90 ⁇ g/L, sodium molybdate 87 ⁇ g/L, cobalt chloride 40 ⁇ g/L, copper sulfate 100 ⁇ g/L.
  • the Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal medium containing 1.2g/L sodium acetate (carbon content of 351mg/L) and 0.38g/L ammonium sulfate (nitrogen content of 81mg/L).
  • the ratio is 4.4/1, the initial cell number is 100,000 cells/mL, and it is placed in a 5L fermentor, the filling volume is 70%, the culture temperature is 25°C, and the air ventilation volume is adjusted to 0.1-0.3vvm and the number of stirring revolutions.
  • the dissolved oxygen is controlled at 10-15% at 50-80 revolutions per minute.
  • the pH of the fermentation broth is maintained at 7.5 by adding a 20-fold concentrated basal medium containing 180 g/L acetic acid and 11.5 g/L ammonium sulfate to maintain the pH of the fermentation broth at 7.5.
  • the nitrogen ratio is 29.5/1.
  • Stop aeration and stirring remove the supernatant after natural sedimentation, and inoculate the concentrated algae cells into a medium containing 6.8g/L sodium acetate and all the nutrients lacking, and the inoculation concentration is 1.68g/L, and placed in a 5L fermentor
  • the filling volume is 70%
  • the culture temperature is 30°C
  • the dissolved oxygen is controlled at 35-50% by adjusting the air ventilation volume of 0.5-1.5vvm and the stirring speed of 100-150 revolutions/min
  • the dissolved oxygen is controlled at 35-50% by adding 120g/L acetic acid.
  • the nitrogen-deficient basal medium maintains the pH of the algal solution at 8.0.
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.2g/L, magnesium sulfate 400mg/L, calcium chloride 50mg/L, disodium edetate 3.5mg/L, boric acid 4mg/L, ferric chloride 200 ⁇ g/ L, manganese chloride 35 ⁇ g/L, zinc sulfate 25 ⁇ g/L, sodium molybdate 35 ⁇ g/L, cobalt chloride 20 ⁇ g/L, copper sulfate 45 ⁇ g/L.
  • adjust the pH to 8.0 with dilute sulfuric acid or sodium hydroxide solution.
  • Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal medium containing 1.0g/L sodium acetate (carbon content of 293mg/L) and 0.31g/L urea (nitrogen content of 145mg/L), the ratio of carbon to nitrogen It is 2.0/1, the initial cell number is 80,000 cells/mL, and it is placed in a 5L fermenter, the filling volume is 70%, the culture temperature is 23°C, and the air ventilation volume is adjusted to 0.1-0.4vvm and the stirring speed is 50 -100 revolutions/min to control the dissolved oxygen at 20-25%.
  • the pH of the fermentation broth is maintained at 8.0 by adding a 10-fold concentrated basal medium containing 120g/L acetic acid and 3.1g/L urea, and the carbon to nitrogen ratio of the feed medium It is 33.2/1. After 240 hours of heterotrophic culture, the algae cells no longer divide and reproduce, the number of immobile vegetative cells accounted for 100% of the total number of cells, and the total number of cells reached 2.7 million cells/mL.
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.6g/L, magnesium sulfate 100mg/L, calcium chloride 10mg/L, disodium edetate 2mg/L, boric acid 0.5mg/L, ferric chloride 600 ⁇ g/ L, manganese chloride 20 ⁇ g/L, zinc sulfate 36 ⁇ g/L, sodium molybdate 25 ⁇ g/L, cobalt chloride 45 ⁇ g/L, copper sulfate 80 ⁇ g/L.
  • Haematococcus pluvialis CCTCC M2018809 was inoculated into a sterile basal medium containing 0.3g/L glucose (carbon content of 120mg/L) and 1.5g/L sodium nitrate (nitrogen content of 247mg/L), the carbon-nitrogen ratio It is 0.5/1, the initial cell number is 40,000 cells/mL, and placed in a 250mL Erlenmeyer flask, the filling volume is 100ml, the culture is shaken at 100 revolutions/min, the culture temperature is 20°C, and after 120 hours of heterotrophic culture, no The number of dynamic vegetative cells accounted for 70% of the total number of cells, and the total number of cells reached 250,000 cells/mL.
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.8g/L, magnesium sulfate 250mg/L, calcium chloride 40mg/L, disodium edetate 5.5mg/L, boric acid 5mg/L, ferric chloride 400 ⁇ g/ L, manganese chloride 90 ⁇ g/L, zinc sulfate 66 ⁇ g/L, sodium molybdate 100 ⁇ g/L, cobalt chloride 36 ⁇ g/L, copper sulfate 90 ⁇ g/L. After the preparation is complete, adjust the pH to 7.5 with dilute sulfuric acid or sodium hydroxide solution.
  • Haematococcus pluvialis CCTCC M2018809 was inoculated with 0.4g/L sodium acetate (carbon content of 117mg/L), 0.3g/L ribose (carbon content of 120mg/L) and 1.0g/L sodium nitrate (nitrogen content of 165mg/L) sterile basal medium, the carbon to nitrogen ratio is 1.4/1, the initial cell number is 40,000 cells/mL, and placed in a 250mL Erlenmeyer flask, the filling volume is 100ml, 100 revolutions/min. Bed culture, culture temperature 20°C, after 120 hours of heteroculture, the number of immobile vegetative cells accounted for 60% of the total number of cells, and the total number of cells reached 300,000 cells/mL.
  • the inoculation concentration is 1.24g/L and placed in 5L
  • the filling volume is 70%
  • the culture temperature is 25°C
  • the dissolved oxygen is controlled at 30-40% by adjusting the air ventilation volume 0.5-1.0vvm and the stirring speed 100-150 revolutions/min
  • the dissolved oxygen is controlled at 30-40% by adding 240g/min.
  • the basal medium lacking acetic acid and nitrogen and trace elements maintains the pH of the algae solution at 7.0.
  • Astaxanthin content determination method refers to the above.
  • the basic medium formula is: potassium dihydrogen phosphate 0.1g/L, magnesium sulfate 150mg/L, calcium chloride 15mg/L, disodium edetate 1mg/L, boric acid 2mg/L, ferric chloride 150 ⁇ g/L , Manganese chloride 25 ⁇ g/L, Zinc sulfate 20 ⁇ g/L, Sodium molybdate 20 ⁇ g/L, Cobalt chloride 10 ⁇ g/L, Copper sulfate 30 ⁇ g/L. After the preparation is complete, adjust the pH to 7.5 with dilute sulfuric acid or sodium hydroxide solution.
  • the Haematococcus pluvialis CCTCC M2018809 was inoculated with 0.6g/L sodium acetate (carbon content of 176mg/L), 4.0g/L tryptone (nitrogen content of 400mg/L) and 2.0g/L yeast extract (nitrogen content).
  • the carbon-nitrogen ratio is 0.3/1
  • the initial cell number is 80,000 cells/mL
  • it is placed in a 5L fermentor with a filling volume of 70% and a culture temperature 25°C, control the dissolved oxygen to 25-30% by adjusting the air ventilation 0.2-0.5vvm and the stirring speed 80-150 rpm, and by adding 15g/L acetic acid, 8.0g/L tryptone and 4.0g/
  • the 30-fold concentrated basal medium of L yeast extract maintains the pH of the fermentation broth at 7.5, and the carbon to nitrogen ratio of the feed medium is 5.0/1.
  • the number of immobile vegetative cells accounted for 80% of the total number of cells, and the total number of cells reached 2.8 million cells/mL.
  • the inoculation concentration is 1.55g/L and placed in 5L fermentation.
  • the filling volume is 70%
  • the culture temperature is 30°C
  • the dissolved oxygen is controlled at 50-70% by adjusting the air ventilation rate of 1.0-2.0vvm and the stirring speed of 100-250 revolutions/min
  • the content of 60g/L is added by adding
  • the basal medium lacking acetic acid and nitrogen and phosphorus keeps the pH of the algae liquid at 8.0.
  • Astaxanthin content determination method refers to the above.

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Abstract

提供了一种生产虾青素的方法，包括：(a)获得产虾青素雨生红球藻的营养细胞；(b)在含有有机碳源的营养缺失的培养基中在无光照条件下异养培养所述产虾青素雨生红球藻的营养细胞，以获得孢子细胞；和(c)收获所述孢子细胞和/或虾青素，任选纯化虾青素。还提供了用于所述方法的培养基。

Description

一种培养雨生红球藻生产虾青素的方法 技术领域
本发明涉及生产虾青素的领域，具体涉及通过使用雨生红球藻生产虾青素的方法，以及用于所述方法的培养基。
背景技术
虾青素(Astaxanthin)，化学名称3,3′-二羟基-4,4′-二酮基β-胡萝卜素，分子式C 40H 52O 4，分子量为596.86，是一种酮式类胡萝卜素，具有很强的抗氧化功能以及着色效果，广泛应用于功能食品、化妆品、饲料添加剂等领域。
虾青素虽然可以通过化学合成的方式进行生产，但其抗氧化活性和生物安全性均不如天然虾青素。雨生红球藻虾青素含量可达细胞干重的1.5-3％，其生物安全性获得了世界主要国家的认可，先后被欧盟FSA、美国FDA、中国卫生部批准为食品原料，被公认为自然界中生产天然虾青素的最好生物。
雨生红球藻(Haematococcus pluvialis)在营养丰富、光照和温度适宜的环境中以游动的绿色营养细胞存在，在不利的条件下，如高光照、高温、高盐、营养缺失等，以厚壁的不动细胞存在，同时大量积累虾青素以对抗不利的条件。根据其生理特征，目前主要采用两步法培养雨生红球藻生产虾青素，第一步绿色营养细胞扩培，第二步虾青素诱导。美国夏威夷Cyanotech公司采用自养的方式，在封闭式光生物反应器培养雨生红球藻营养细胞，而后在开放式跑道池中利用太阳光诱导细胞变红，虾青素含量可以达到细胞干重的1.5％。以色列Algatechologies采用管道式光生物反应器进行营养细胞培养和虾青素诱导，虾青素含量可以达到3％。发明专利(PCT/CN2013/084262)公开了一种利用雨生红球藻生产虾青素的方法，第一步采用异养培养获得绿色营养细胞，而后加入培养基进行稀释，再通过光照培养实现虾青素的积累，虾青素含量达到2.3％。
当前普遍认为，高光照强度是获得高含量虾青素的核心因素，高光照强度条件下藻细胞通过光合作用会产生过量的活性氧(ROS)，雨生红球藻通过合成和积累虾青素可以抵御活性氧对藻细胞的氧化损伤(Li et al.2008，Consumption of oxygen by astaxanthin biosynthesis:a protective mechanism against oxidative stress in Haematococcus pluvialis(Chlorophyceae).J.Plant Physiol.165:1783-1797；and Li et al.2010，Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis(Chlorophyceae).J.Appl.Phycol.22:253-263)。因此，雨生红球藻绿色营养细胞的获得无论是采用自养或异养的方式培养，虾青素的诱导必须在有光照的条件下进行，而且藻细胞受到的光照强度越高，虾青素的含量越高。然而，光的穿透性与细胞密度是反比关系，因此降低细胞密度、增加反应器的比表面积、减小反应器的光径、补充人工光照是提高虾青素含量的有效手段，但会导致反应器体积小、数量多、占地面积大、成本高、规模放大困难等问题。另外，为了降低生产成本，规模化雨生红球藻的培养都是采用太阳光，然而太阳光照的时间和强度是随季节和天气情况不断变化的，导致虾青素的含量不稳定。
有文献报道通过高盐度刺激能够在无光的条件下诱导雨生红球藻绿色营养细胞转变为红色孢子细胞，但虾青素的含量只有0.5％(30pg/cell)，没有实际应用的价值(Kobayashi et al.1997，Light-independent,astaxanthin production by the green microalga Haematococcus pluvialis under salt stress.Biotechnol Lett 19(6):507–509)。因此，如果能够开发一种在无光照条件下诱导雨生红球藻积累高含量虾青素的方法，将有助于改变目前靠天吃饭的农业化生产状态，实现工业化的生产。
发明内容
除非另有定义，本文所用的技术和科学术语具有本领域技术人员通常理解的含义。参见例如，Singleton et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed.,J.Wiley&Sons(New York,NY 1994)；Sambrook et al.,MOLECULAR CLONING,A LABORATORY MANUAL,Cold Springs Harbor Press(Cold Springs Harbor,NY 1989)。
本发明提供了在无光条件下培养雨生红球藻生产虾青素的方法。
在一个实施方案中，本发明提供了一种生产虾青素的方法，包括：
(a)获得产虾青素雨生红球藻的营养细胞；
(b)在含有有机碳源的营养缺失的培养基中在无光照条件下异养培养所述营养细胞，获得孢子细胞；和
(c)收获所述孢子细胞和/或虾青素，任选纯化虾青素。
如本文所用，雨生红球藻是一种单细胞绿藻，属于绿藻门、绿藻纲、团藻目、红球藻科、红球藻属。在环境适宜、营养丰富的条件下，雨生红球藻快速生长并分裂繁殖，产生大量带鞭毛游动的营养细胞；当环境条件变得不适宜的时候，游动细胞失去鞭毛，变为不动的营养细胞；在持续的不利环境条件刺激下，如高光照、高盐、营养饥饿等，营养细胞将不再继续分裂繁殖，并通过在细胞内积累大量的虾青素以对抗不利的环境条件，形成红色的厚壁孢子。
如本文所用，用于产虾青素雨生红球藻的培养基可以是任何可用于培养雨生红球藻使其生长繁殖的培养基，其通常含有氮源、磷源、硫源、镁源、钙源和/或微量元素，并且本领域技术人员可以根据本领域知识和实践要求确定其含量。本领域已知合适的用于特定藻类的培养基，对此可参见BG-11、BBM、C培养基、MCM等培养基。
可用于本发明所述培养基的“氮源”是指可以被培养的藻类利用的无机氮源或有机氮源，例如包括但不限于硝酸、硝酸盐、亚硝酸盐、氨水、铵盐、尿素、氨基酸、蛋白胨、酵母提取物、蛋白粉、玉米浆及其任意组合。
可用于本发明所述培养基的“磷源”是指可以被培养的藻类利用的磷源，例如包括但不限于磷酸、磷酸二氢钠、磷酸氢二钠、磷酸氢二钾、磷酸二氢钾及其任意组合。
可用于本发明所述培养基的“硫源”是指可以被培养的藻类利用的硫源，例如包括但不限于硫酸、硫酸镁、硫酸钠及其任意组合。
可用于本发明所述培养基的“镁源”是指可以被培养的藻类利用的镁源，例如包括但不限于硫酸镁、氯化镁及其组合。
可用于本发明所述培养基的“钙源”是指可以被培养的藻类利用的钙源，例如包括但不限于氯化钙、硫酸钙、硝酸钙及其任意组合。
可用于本发明所述培养基的“微量元素”是指可以被培养的藻类利用的微量元素，例如包括但不限于Mn(例如氯化锰)、Zn(例如硫酸锌)、B(例如硼酸)、I、Mo(例如钼酸钠)、Cu(例如硫酸铜)、Co(例如氯化钴)、Fe(例如氯化铁)的一或多种。培养基中微量元素的添加量可以根据本领域常规知识确定。
可用于本发明所述培养基的“有机碳源”是指可以被培养的靶微生物利用的有机碳源，本领域技术人员根据本领域技术知识可以确定哪些有机碳源可用于本发明培养基，例如包括但不限于乙酸、乙酸盐、丙酸、丙酸盐、丁酸、丁酸盐、乳酸、乳酸盐、脂肪酸、脂肪酸盐、氨基酸、甲醇、乙醇、甘油、柠檬酸、柠檬酸盐、丙酮酸、丙酮酸盐、葡萄糖、果糖、阿拉伯糖、乳糖、甘露糖、鼠李糖、核糖或者含有这些有机碳源的废水、水解液、发酵液、及其任意组合。有机碳源的添加量可以根据本领域常规知识以及藻类细胞的实际生长状况确定，这均在本领域技术人员的技术能力范围内。
如本文所用，所述产虾青素雨生红球藻细胞的接种密度可以是任何适合所述产虾青素雨生红球藻细胞生长繁殖的密度，本领域技术人员可以根据本领域技术知识以及经验 确定合适的接种密度。例如，本发明产虾青素雨生红球藻细胞的接种密度可以是至少10 4个细胞/ml培养基，例如1-20×10 4个细胞/ml培养基，如5、8或10×10 4个细胞/ml培养基。或者，产虾青素雨生红球藻细胞的接种密度可以是至少0.5-2.0g细胞/L培养基，例如至少约0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0g细胞/L培养基。
如本文所用，“营养缺失的培养基”是指缺少营养元素的培养基，例如缺少氮源、磷源、硫源、镁源、钙源以及微量元素的一种或者多种、或者甚至全部。
在一个实施方案中，所述营养缺失的培养基缺少氮源、磷源、硫源、钙源、镁源和/或微量元素。
在一个实施方案中，所述营养缺失的培养基缺少氮源。
在一个实施方案中，所述营养缺失的培养基缺少氮源和磷源。
在一个实施方案中，所述营养缺失的培养基缺少氮源和微量元素。
在一个实施方案中，所述营养缺失的培养基是缺失所述全部营养的培养基。在一个实施方案中，营养全部缺失的培养基是乙酸或乙酸盐溶液，例如乙酸钠溶液。在一个进一步的实施方案中，营养全部缺失的培养基是例如浓度为60-1050g/L，例如约120、180、240、300、400、500、600、700、800、900、1000g/L的乙酸溶液。
如本文所用，“无光照条件”是指没有光照或者光照不足以自养培养所述雨生红球藻。
如本文所用，“自养”是在光照条件下，通过光合作用利用无机碳源如二氧化碳、碳酸盐、碳酸氢盐等生长繁殖的培养方式。
如本文所用，“混养”是在光照条件下，利用有机碳源生长繁殖的培养方式。
如本文所用，“异养”是在无光照条件下，利用有机碳源生长繁殖的培养方式。
在一个实施方案中，步骤(a)的营养细胞通过培养产虾青素雨生红球藻细胞而获得。本领域已知多种培养藻类细胞生长繁殖的方法步骤，例如包括自养、混养或异养培养方法。
任选地，步骤(a)培养雨生红球藻细胞后，可以包括去除培养基和/或收集营养细胞、任选浓缩所述营养细胞的步骤。去除培养基、收集和/或浓缩营养细胞可以采用本领域已知的任何合适方法，例如通过沉淀(自然沉降或离心)或过滤(使用滤器或滤膜)等。
步骤(a)的培养温度和pH可以是适合雨生红球藻细胞生长繁殖的任何温度或pH值。
在一个实施方案中，步骤(a)的培养pH值为6.0-9.0，例如6.0-8.0、7.0-8.0、7.0-8.5、7.5-8.0、7.5-8.5、8.0-9.0或8.5-9.0，如约6.5、7.0、7.5、8.0、8.5或9.0。在一个实施方案中，步骤(a)的培养pH范围控制为7.0-8.0。在一个实施方案中，步骤(a)的培养是自养培养，可以通入含有二氧化碳(例如0.5％-5％(v/v))的混合空气控制pH。在一个实施方案中，步骤(a)的培养是混养和异养培养，可以采用酸(例如0.1-10mol/L盐酸、硫酸和乙酸)控制pH。
在一个实施方案中，步骤(a)的培养温度为15-25℃，优选20-25℃，例如约15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃或25℃。
在一个实施方案中，步骤(a)获得了含有至少约10、20、30、40、50、60、70、80、90、100、150、200、250、300万或更多个细胞(营养细胞)/ml的培养液。
在一个实施方案中，步骤(a)的营养细胞通过自养培养雨生红球藻细胞而获得。雨生红球藻细胞可以在任何适合生长繁殖以及光照条件下进行自养培养。在一个实施方案中，可以将雨生红球藻细胞接种在含有氮源(例如硝酸盐如硝酸钠)的培养基中培养，光照强度可以是例如但不限于10-100、10-90、10-80、10-70、20-90、20-80、20-70、30-90、30-80、30-70、40-60μE/m 2/s，例如约20、30、40、50、60、70、80或90μE/m 2/s。自 养培养中，可以通入二氧化碳或含有二氧化碳的混合气体以提供无机碳源，例如含有0.5-1.5％(v/v)二氧化碳的混合空气；通气量可以例如为0.05-0.5vvm，例如0.1-0.5或0.2-0.5vvm，例如约0.05、0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5vvm。通过调整二氧化碳含量和通气量可以调节培养液的pH。
在一个实施方案中，步骤(a)采用混养或异养方式培养雨生红球藻细胞。在一个实施方案中，步骤(a)用于混养或异养的培养基含有80-700、90-600、90-500、90-400、100-600、100-500、100-400、100-350或120-350mg/L碳元素的有机碳源，以及含有40-800、40-700、40-600、50-800、50-700、50-600、60-800、60-700、60-600、70-800、70-700、70-600、80-600mg/L氮元素的氮源，优选碳氮元素质量比为0.1-10:1、0.2-10:1、0.1-5:1、0.2-5:1或0.3-4.5:1，例如为约0.3:1、0.5:1、1:1、1.4:1、1.5、1.8:1、2:1、2.4:1、3:1、4:1、4.5:1、5:1、6:1、7:1、8:1或9:1。
在一个实施方案中，步骤(a)的营养细胞通过混养培养雨生红球藻细胞而获得。混养是雨生红球藻细胞在光照条件下利用培养基中含有的有机碳源生长繁殖。在一个实施方案中，用于混养的培养基含有有机碳源(例如乙酸或乙酸盐，例如乙酸钠)，任选含有氮源(例如硝酸或硝酸盐，例如硝酸钠)，优选碳氮元素质量比为0.1-10:1、0.2-10:1、0.1-5:1、0.2-5:1或0.3-4.5:1，例如为约0.3:1、0.5:1、1:1、1.4:1、1.5、1.8:1、2:1、2.4:1、3:1、4:1、4.5:1、5:1、6:1、7:1、8:1或9:1。光照强度可以是例如但不限于10-100、10-90、10-80、10-70、20-90、20-80、20-70、30-90、30-80、30-70、40-60μE/m 2/s，例如约20、30、40、50、60、70、80或90μE/m 2/s。在步骤(a)混养培养的一个实施方案中，溶氧控制在1-50％，如5-30％或5-10％，可以通过例如调节通气量(例如空气)和搅拌速度控制溶氧，例如通气量为0.05-0.5或0.05-0.1vvm，例如约0.05、0.1、0.2、0.3、0.4、0.5vvm，和/或搅拌速度为50-150或50-80转/分钟，例如约50、60、70、80、90、100、110、120、130、140、150转/分钟。
在一个实施方案中，步骤(a)以补料分批方式进行混养培养，其中向培养液中添加补料液。在一个实施方案中，补料液包含含有有机碳源(例如乙酸或乙酸盐，例如乙酸钠)和任选包含氮源(例如硝酸或硝酸盐，例如硝酸钠)的培养基。在一个实施方案中，补料液含有6-420g/L碳元素的有机碳源和0.3-120g/L氮元素的氮源，优选碳氮元素质量比为1-50:1，例如为约1:1、2:1、3:1、4:1、5:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1或50:1。在一个实施方案中，补料液是含有有机碳源和任选包含氮源的浓缩培养基，例如5-50倍浓缩的培养基。
在一个实施方案中，步骤(a)的营养细胞通过异养培养雨生红球藻细胞而获得。异养培养是在无光照或光照不足以进行自养培养的条件下，雨生红球藻利用培养基中的有机碳源生长繁殖的培养方式。在一个实施方案中，雨生红球藻在包含有机碳源(例如乙酸或乙酸盐，例如乙酸钠)和任选包含氮源(例如硝酸或硝酸盐，例如硝酸钠)的培养基中异养培养，优选碳氮元素的质量比为0.1-10:1、0.2-10:1、0.1-5:1、0.2-5:1或0.3-4.5:1，例如为约0.3:1、0.5:1、1:1、1.4:1、1.5:1、1.8:1、2:1、2.4:1、2.5:1、3:1、3.5:1、4:1、4.5:1、5:1、5.5:1、6:1、6.5:1、7:1、7.5:1、8:1、8.5:1或9:1。在一个实施方案中，在步骤(a)异养培养过程中，溶氧控制在1-50％、优选5-30％，如约15％、20％、25％、30％、35％、40％、45％或50％，可以通过例如调节通气量(例如空气)和搅拌速度控制溶氧，例如通气量为0.05-0.5vvm，例如约0.1、0.2、0.3、0.4、0.5vvm，和/或搅拌速度为50-150转/分钟，例如约50、60、70、80、90、100、110、120、130、140、150转/分钟。
在一个实施方案中，以补料分批方式进行步骤(a)的异养培养，其中向培养液中添加补料液。在一个实施方案中，补料液包含含有有机碳源(例如乙酸或乙酸盐，例如乙酸)和任选包含氮源(例如硝酸或硝酸盐，例如硝酸钠)的培养基。在一个实施方案中，补料液含有6-420g/L碳元素的有机碳源和0.3-120g/L氮元素的氮源，优选碳氮元素质量比为1-50:1、1-40:1、1-35:1、5-50:1、5-40:1或5-35:1，例如为约1:1、2:1、3:1、 4:1、5:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1或50:1。在一个实施方案中，补料液是含有有机碳源和任选包含氮源的浓缩培养基，例如5-50倍浓缩的培养基。
在一个实施方案中，步骤(a)的营养细胞通过接种在含有有机碳源和氮源的培养基中通过混养或异养培养雨生红球藻细胞获得，优选培养基含有80-700mg/L碳元素的有机碳源，40-800mg/L氮元素的氮源，优选碳氮质量比为0.1-10：1。
在一个实施方案中，步骤(a)的营养细胞以选自分批、补料分批、半连续和连续培养的方式混养或异养培养雨生红球藻细胞而获得；当以补料培养的方式培养雨生红球藻细胞时，优选补料液含有15-1050g/L、更优选15-600或60-300g/L的乙酸或乙酸盐，0.3-120g/L氮元素的氮源以及1-50倍浓缩的培养基，优选碳氮质量比为约1-50:1、1-40:1、1-35:1、5-50:1、5-40:1或5-35:1。
步骤(b)是在无光照条件下在含有有机碳源的营养缺失的培养基中异养培养步骤(a)的雨生红球藻营养细胞，以刺激藻类细胞生产虾青素。步骤(b)的接种密度、培养温度和pH可以是适合异养培养雨生红球藻营养细胞的任何密度、温度和pH值。
在一个实施方案中，步骤(b)中雨生红球藻营养细胞的接种密度可以是至少0.5-2.0g细胞/L培养基，例如0.5-1.7g细胞/L培养基，例如至少约0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0g细胞/L培养基。
在一个实施方案中，步骤(b)的培养温度为15-35℃、20-30℃或25-30℃，例如约15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33℃或34℃。
在一个实施方案中，步骤(b)的pH值为6.0-11.0、7.0-10.0、7.0-9.0或7.5-9.0，例如约6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0或10.5。
在一个实施方案中，在步骤(b)中控制溶氧在20-90％，优选30-70％，例如通过调节通气量和搅拌转速控制溶氧，优选通气量为0.2-3.0vvm，例如0.5、1.0、1.5、2.0、2.5vvm，和/或优选搅拌转速为100-300转/分钟，例如150、200、250转/分钟。
在一个实施方案中，在步骤(b)中，含有有机碳源的营养缺失的培养基中，有机碳源的碳元素含量为至少约200mg/L，例如至少约250、300、400、500、600、700、800、900、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、3000、4000、5000mg/L。在一个实施方案中，在步骤(b)中，营养缺失的培养基中含有的有机碳源为乙酸或乙酸盐，例如1-15、1-14、1-13、1-12、1-11、1-10、1-9、1-8、2-10、2-9、2-8、3-8、3.5-8、4-8g/L，如约1、2、3、4、5、6、7、8、9、10、11、12、13、14g/L。在一个实施方案中，步骤(b)中营养缺失的培养基含有的有机碳源为乙酸钠，例如1-12、1-11、1-10、1-9、1-8、2-10、2-9、2-8、3-8、3.5-8、4-8g/L，如约2、3、3.5、4、4.5、5、5.5、6、7、8、9或10g/L。
在一个实施方案中，步骤(b)采用补料的方式培养，优选的补料液为含有乙酸或乙酸盐(例如30-1050g/L、特别是30-600或100-600g/L，如约50、100、200、300、400、500、550、600、700、800、900、1000g/L)的营养缺失的培养基(例如缺氮、磷和/或微量元素，或者缺失全部营养元素)。在一个实施方案中，补料液为乙酸溶液，例如浓度为60-600g/L，例如约60、120、180、240、300、400、550、560、565、570g/L。
在一个实施方案中，步骤(b)的pH范围控制为7.0-8.5，可以采用添加例如0.1-10mol/L盐酸、硫酸和乙酸进行控制。
在一个实施方案中，步骤(a)和/或(b)可以在生物反应器中进行，所述生物反应器包括各类光生物反应器，如平板式、立柱式、吊袋式、管式、跑道池以及发酵罐。
在一个实施方案中，当至少60％(例如至少70％、80％、90％、95％、99％，甚至100％)的营养细胞转变为孢子细胞和/或虾青素含量不再增加时结束步骤(b)。
步骤(c)所述的收获藻类孢子细胞和/或虾青素可以采用任何已知的方法收获所述孢子细胞(例如沉降或离心等)和/或破坏细胞壁(机械方式、化学方式或酶学方式)及收获虾青素，任选采用任何合适方法分离和/或纯化虾青素。
在一个实施方案中，本发明提供了一种在无光照条件下培养雨生红球藻生产虾青素的方法，包括：
(a)在培养基中培养雨生红球藻细胞，如通过自养、混养或异养进行培养，优选其中培养温度控制在15-25℃，pH控制在6.0-9.0；
任选收获雨生红球藻细胞，优选获得浓缩的雨生红球藻细胞，例如通过自然沉降、离心或过滤；
(b)在无光照条件下，在营养缺失的培养基中通过添加有机碳源异养培养步骤(a)获得的雨生红球藻细胞，例如通过分批、补料分批、半连续或连续培养的方式进行异养培养，优选其中培养温度控制在15-35℃，pH控制在6.0-11.0和/或溶氧控制在20-90％；和
(c)任选收集藻类细胞和/或收获虾青素，任选纯化虾青素。
在一个实施方案中，可用于本发明步骤(a)的所述雨生红球藻自养培养的培养基包含或由如下组分组成：
磷酸二氢钾：0.05-1g/L，
硫酸镁：50-500mg/L，
氯化钙：5-50mg/L，
乙二胺四乙酸二钠：0.5-6mg/L，
硼酸：0.5-5mg/L，
氯化铁：100-1000μg/L，
氯化锰：10-100μg/L，
硫酸锌：10-100μg/L，
钼酸钠：10-100μg/L，
氯化钴：5-50μg/L，
硫酸铜：10-100μg/L，
任选含有其它氮源，例如硝酸或硝酸盐如硝酸钠，其中氮元素含量为约40-800mg/L，
pH为7.0-8.0(例如7.5)。
在一个实施方案中，可用于本发明步骤(a)的所述雨生红球藻混养或异养培养的培养基包含或由如下组分组成：
有机碳源：80-700mg/L(碳元素含量)
氮源：40-800mg/L(氮元素含量)，其中碳氮元素质量比：0.1-10:1，例如0.2-10:1、0.1-5:1、0.2-5:1，优选0.3-4.5:1，
磷酸二氢钾：0.05-1g/L，
硫酸镁：50-500mg/L，
氯化钙：5-50mg/L，
乙二胺四乙酸二钠：0.5-6mg/L，
硼酸：0.5-5mg/L，
氯化铁：100-1000μg/L，
氯化锰：10-100μg/L，
硫酸锌：10-100μg/L，
钼酸钠：10-100μg/L，
氯化钴：5-50μg/L，
硫酸铜：10-100μg/L，
pH为7.0-8.0(例如7.5)。
在一个实施方案中，所述培养基包含90-600、90-500、90-400、100-600、100-500、100-400、100-350或120-350mg/L的的有机碳源，例如约90mg/L、100mg/L、110mg/L、120mg/L、130mg/L、140mg/L、150mg/L、200mg/L、210mg/L、220mg/L、230mg/L、240mg/L、250mg/L、260mg/L、270mg/L、280mg/L、290mg/L、300mg/L或350mg/L的有机碳源。
在一个实施方案中，所述培养基中含有的有机碳源包括但不限于乙酸或乙酸盐例如乙酸钠、葡糖糖、核糖及其任意组合。
在一个实施方案中，所述培养基包含40-800、40-700、40-600、50-800、50-700、50-600、60-800、60-700、60-600、70-800、70-700、70-600、80-600mg/L的氮源，例如约50mg/L、60mg/L、70mg/L、80mg/L、90mg/L、100mg/L、150mg/L、200mg/L、250mg/L、300mg/L、350mg/L、400mg/L、450mg/L、500mg/L、550mg/L或600mg/L的氮源。
在一个实施方案中，所述培养基中含有的氮源包括但不限于硝酸或硝酸盐例如硝酸钠、硫酸铵、尿素及其任意组合。
在一个实施方案中，所述培养基中碳氮元素质量比为约0.2:1；0.3:1；0.4:1；0.5:1；1:1；1.5:1；2:1；2.5:1；3:1；3.5:1；4:1；4.5:1；5:1；6:1；7:1；8:1或9:1，例如1.4:1；1.8:1；2.4:1；4.4:1。
在一个实施方案中，可用于本发明步骤(b)的所述雨生红球藻异养培养的培养基包含或由如下组分组成：
磷酸二氢钾：0.05-1g/L，
硫酸镁：50-500mg/L，
氯化钙：5-50mg/L，
乙二胺四乙酸二钠：0.5-6mg/L，
硼酸：0.5-5mg/L，
氯化铁：100-1000μg/L，
氯化锰：10-100μg/L，
硫酸锌：10-100μg/L，
钼酸钠：10-100μg/L，
氯化钴：5-50μg/L，
硫酸铜：10-100μg/L，
pH为7.0-8.0(例如7.5)。
如本文所用，“营养缺失的培养基”是指所述培养基中不含所述营养元素，或者所述营养元素低于靶微生物生长所需，导致靶微生物对于该营养元素处于饥饿状态。在一个实施方案中，“营养缺失的培养基”是指所述培养基中不含所述营养元素。
在一个实施方案中，本发明步骤(b)所述的营养缺失的培养基是本发明所述的培养基，其中不含相应营养元素。例如，缺氮的培养基可以是具有本发明所述组成的培养基，但是其中含氮化合物(乙二胺四乙酸二钠)不存在。作为另一个例子，缺磷的培养基是具有本发明所述组成的培养基，但是其中含磷化合物(磷酸二氢钾)不存在。
在一个实施方案中，本发明所述培养基包含约0.05g/L、0.1g/L、0.2g/L、0.3g/L、0.4g/L、0.5g/L、0.6g/L、0.7g/L、0.8g/L、0.9g/L或1.0g/L的磷酸二氢钾。
在一个实施方案中，本发明所述培养基包含约50mg/L、100mg/L、150mg/L、200mg/L、250mg/L、300mg/L、350mg/L、400mg/L、450mg/L或500mg/L的硫酸镁。
在一个实施方案中，本发明所述培养基包含约5mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、20mg/L、25mg/L、27mg/L、30mg/L、35mg/L、36mg/L、40mg/L、45mg/L或50mg/L的氯化钙。
在一个实施方案中，本发明所述培养基包含0.5-5.5mg/L的乙二胺四乙酸二钠，例如约0.5mg/L、1.0mg/L、1.5mg/L、2.0mg/L、2.5mg/L、3.0mg/L、3.5mg/L、4.0mg/L、4.5mg/L、5.0mg/L或5.5mg/L的乙二胺四乙酸二钠。
在一个实施方案中，本发明所述培养基包含约0.5mg/L、1.0mg/L、1.5mg/L、1.6mg/L、1.7mg/L、1.8mg/L、1.9mg/L、2.0mg/L、2.5mg/L、3.0mg/L、3.5mg/L、4.0mg/L、4.5mg/L或5.0mg/L的硼酸。
在一个实施方案中，本发明所述培养基包含100-950、100-900或120-900μg/L的氯化铁，例如约110μg/L、120μg/L、130μg/L、140μg/L、150μg/L、200μg/L、250μg/L、300μg/L、400μg/L、500μg/L、600μg/L、700μg/L、800μg/L或900μg/L的氯化铁。
在一个实施方案中，本发明所述培养基包含15-100μg/L的氯化锰，例如约15μg/L、20μg/L、25μg/L、30μg/L、35μg/L、40μg/L、50μg/L、60μg/L、70μg/L、72μg/L、80μg/L、90μg/L或100μg/L的氯化锰。
在一个实施方案中，本发明所述培养基包含10-100、10-90或14-90μg/L的硫酸锌，例如约10μg/L、11μg/L、12μg/L、13μg/L、14μg/L、15μg/L、20μg/L、25μg/L、30μg/L、35μg/L、36μg/L、40μg/L、50μg/L、60μg/L、66μg/L、70μg/L、80μg/L、88μg/L、90μg/L或95μg/L的硫酸锌。
在一个实施方案中，本发明所述培养基包含约10μg/L、15μg/L、20μg/L、25μg/L、30μg/L、35μg/L、40μg/L、45μg/L、50μg/L、60μg/L、70μg/L、80μg/L、87μg/L、90μg/L或100μg/L的钼酸钠。
在一个实施方案中，本发明所述培养基包含约5μg/L、10μg/L、15μg/L、20μg/L、25μg/L、30μg/L、33μg/L、35μg/L、36μg/L、40μg/L、45μg/L或50μg/L的氯化钴。
在一个实施方案中，本发明所述培养基包含20-100μg/L的硫酸铜，例如约21μg/L、22μg/L、25μg/L、30μg/L、35μg/L、40μg/L、45μg/L、50μg/L、55μg/L、60μg/L、65μg/L、70μg/L、75μg/L、79μg/L、80μg/L、85μg/L、90μg/L、95μg/L或100μg/L的硫酸铜。
本发明提供了在无光条件下培养雨生红球藻生产虾青素的方法，具体步骤如下：
(I)营养细胞培养：将雨生红球藻藻种接入装有培养基的生物反应器中进行自养、混养或异养培养，其中培养温度控制在15-25℃，pH控制在6.0-9.0。优选地，当藻细胞不再进行分裂繁殖，由绿色游动细胞转变为绿色营养细胞时停止培养。
(II)细胞制备：去除培养基，例如将步骤(I)中的藻液通过自然沉降、离心、过滤等方法将培养基去除，获得浓缩的藻细胞。
(III)异养诱导：将步骤(II)的浓缩藻细胞接接入装有营养缺失的培养基的生物反应器，通过添加有机碳源并异养培养，例如采用分批、补料分批、半连续或连续培养等多种方式进行异养培养，其中培养温度控制在15-35℃，pH控制在6.0-11.0以及溶氧控制在20-90％。优选地，当藻细胞由绿色营养细胞转变为红色孢子细胞，虾青素含量不再增加时结束培养。
在一个优选实施方案中，本发明提供了一种在无光照条件下培养雨生红球藻生产虾青素的方法，包括：
(a)在含有有机碳源和氮源的培养基中以补料方式异养培养产虾青素雨生红球藻获得营养细胞，其包括如下一或多个，优选全部：
-有机碳源选自乙酸或乙酸盐例如乙酸钠；
-氮源选自硝酸或硝酸盐例如硝酸钠、尿素、胰蛋白胨和酵母提取物；
-有机碳源的碳元素含量为150-300mg/L，例如175-300mg/L；
-氮元素的含量为100-600mg/L；
-培养基中碳氮元素质量比为0.3-3:1，例如0.3-2.5:1；
-培养温度为20-25℃；
-溶氧控制在15-30％；
-pH控制在7.5-8.0；
-补料液含有有机碳源和氮源，其中碳氮元素质量比为5-35:1，例如5-33:1；
-获得的营养细胞中不动营养细胞占总细胞数的至少80％，优选100％；
(b)在含有有机碳源的营养缺失的培养基中在无光照条件下以补料分批方式异养培养步骤(a)获得的营养细胞以获得孢子细胞，包括如下一或多个，优选全部：
-有机碳源选自乙酸或乙酸盐例如乙酸钠，例如4.0-5.5g/L；
-培养基缺失(i)氮源，(ii)氮源和磷源，或(iii)全部营养元素，
-培养温度为25-30℃，优选约30℃；
-溶氧控制在45-70％；
-pH控制在7.5-8.0，优选约8.0；
-补料液是(i)含有有机碳源的缺失氮源和磷源的培养基或(ii)含有有机碳源的缺失全部营养的培养基，优选乙酸溶液，例如浓度为60-300g/L或180-300g/L；
-当至少90％、95％或100％的营养细胞转变为孢子细胞时，终止步骤(b)；
以及
(c)收获所述孢子细胞和/或虾青素，任选纯化虾青素。
在一个优选实施方案中，本发明的培养基包含或由如下组分组成：
磷酸二氢钾：0.1-0.5g/L，
硫酸镁：150-400mg/L，
氯化钙：12-50mg/L，
乙二胺四乙酸二钠：1.0-3.5mg/L，
硼酸：2-4mg/L，
氯化铁：150-500μg/L，
氯化锰：25-75μg/L，例如25-72μg/L，
硫酸锌：20-50μg/L，
钼酸钠：20-45μg/L，
氯化钴：10-35μg/L，例如10-33μg/L，
硫酸铜：30-65μg/L，
pH为7.5-8.0。
本发明中，虾青素含量测定方法如下：
(1)取1mL诱导结束藻液，8000rpm离心5min，弃去上清液；
(2)向藻细胞沉淀中加入1mL甲醇-氢氧化钾溶液(5％氢氧化钾和30％甲醇的混合液)，70℃水浴10min，期间进行数次漩涡震荡，之后以8000rpm离心5min，弃去上清液；
(3)向藻细胞沉淀中加入45μL冰醋酸和1mL DMSO后70℃水浴10min，期间进行数次旋涡震荡，8000rpm离心5min，取上清液于洁净的5mL离心管中；
(4)重复步骤(3)2-3次至藻体颜色变白，于492nm波长下测定吸光度值A 492。
雨生红球藻虾青素含量按下列公式计算：(i)雨生红球藻中提取得到的总虾青素的浓度
Figure PCTCN2020116775-appb-000001
其中
Figure PCTCN2020116775-appb-000002
(ii)雨生红球藻中总虾青素的提取得率：ω(％)＝(C 1*V)/M*100％，其中V＝DMSO的体积(mL)(稀释倍数×DMSO抽提次数)，M＝1mL雨生红球藻液所含藻细胞的干重值(mg)。
本发明所述的雨生红球藻可以是任何雨生红球藻藻种，例如包括但不限于雨生红球藻CCTCC M2018809、雨生红球藻AC136、AC143、AC587、AC588(Algobank-Caen Microalgal Culture Collection of University of Caen Basse-Normandie,France)、雨生红球藻ATCC 30453、ATCC 30402(American Type Culture Collection,USA)、雨生红球藻CS-321(Australian National Algae Culture Collection,Australia)、雨生红球藻G 1002 (Culture Collection of Algae of Charles University,Czech Republic)、雨生红球藻ETTL 1958/3、TAKACOVAL 1983/1、PRIBYL 2005/4、PRIBYL 2008/3(Culture Collection of Autotrophic Organisms,Czech Republic)、雨生红球藻CCCryo 188-04、CCCryo 189-04、CCCryo 190-04(Culture Collection of Cryophilic Algae,Germany)、雨生红球藻SCCAP K-0084(Scandinavian Culture Collection of Algae and Protozoa at the University of Copenhagen,Denmark)、雨生红球藻IPPAS H-239(Culture Collection of Microalgae,Institute of Plant Physiology,Russian Academy of Science,Russia)、雨生红球藻NIVA-CHL 9(Norwegian Institute for Water Research Culture Collection of Algae,Norway)、雨生红球藻FWAC 7072、FWAC 7039(Canadian Center for the Culture of Microorganisms,Canada)、雨生红球藻CPCC 93(Canadian Phycological Culture Centre of University of Waterloo,Canada)、雨生红球藻ACOI 816、ACOI 815、ACOI 276、ACOI 255、ACOI 133、ACOI 51(Coimbra Culture Collection of Algae,Portugal)、雨生红球藻CCAP 34/1D、CCAP 34/1F、CCAP 34/6、CCAP 34/7、CCAP34/8、CCAP 34/12、CCAP 34/13、CCAP 34/14(Culture Collection of Algae and Protozoa of the Centre for Hydrology and Ecology,UK)、雨生红球藻NIES-144、NIES-2263、NIES-2264、NIES-2265(Microbial Culture Collection of National Institute for Environmental Studies,Japan)、雨生红球藻SAG 192.80、SAG 44.96、SAG 34-1a、SAG 34-1b、SAG 34-1c(Culture Collection of Algae at University of
Figure PCTCN2020116775-appb-000003
Germany)，雨生红球藻CCAC 0055、CCAC 0125、CCAC 0129、CCAC 2072B(Culture Collection of Algae at the University of Cologne,Germany)、雨生红球藻UTEX 2505、UTEX 16、UTEX B 294(University of Texas Culture Collection of Algae,USA)、雨生红球藻CWU-MACC20(Herbarium of Kharkov University-MicroAlgae Cultures Collection,Ukraine)、雨生红球藻TISTR 8647(Thailand Institute of Scientific and Technological Research Culture Collection,Thailand)、雨生红球藻FACHB-712、FACHB-827、FACHB-797、FACHB-955、FACHB-1164(Freshwater Culture Algae Collection at Institute of Hydrobiology,The Chinese Academy of Sciences,China)和雨生红球藻CCMP 3127(Provasoli-Guillard National Centre for Marine Algae and Microbiota,USA)。
雨生红球藻AQHP0于2018年11月21日以保藏号CCTCC M 2018809保藏在中国典型培养物保藏中心(CCTCC)(中国武汉市武汉大学，邮编430072)。
除非上下文另有指示，词语“或”旨在包括“和”。
如本文所用，“任选”或“任选地”是指随后描述的事件或情况发生或不发生，该描述包括其中所述事件或情况发生及不发生的情况。例如，任选包括的步骤是指该步骤存在或不存在。
如本文所用，术语“约”是指包括具体数值的数值范围，本领域技术人员可以合理认为其类似于具体数值。在某些实施方案中，术语“约”是指在使用本领域通常接受的测量的标准误差内。例如，在某些实施方案中，约是指到具体数值的+/-10％或5％。
如本文所用，当说明书中针对一个特征列出具体数值或比例时，也涵盖了任意其两个数值或比例组成的范围。例如列出数值1、2、3、4时，也涵盖了1-2、1-3、1-4、2-3、2-4和3-4。
本发明相比于现有技术具有如下优点和效果：
(1)本发明提供了一种培养雨生红球藻生产虾青素的方法，克服了传统方案对于光照的高要求，在完全无光条件下也能实现高含量虾青素的积累。在某些实施方案中，雨生红球藻在虾青素诱导结束时，虾青素含量可以达到2.5％，甚至3.21％。
(2)本发明由于不再依赖光照，在反应器设计方面不用考虑比表面积、光径等因素，可以采用大体积的生物反应器如发酵罐，减少反应器数量和占地面积，从而降低生产成本。
(3)本发明摆脱了传统雨生红球藻规模化培养对于气候、季节、地理的依赖，将促进传统农业化培养模式向工业化规模生产的转变。
附图说明
图1示出采用分批异养方式诱导自养培养获得的藻细胞在无光照条件下虾青素的生产。
图2示出采用分批补料异养方式诱导混养培养获得的藻细胞在无光照条件下虾青素的生产。
图3示出采用分批补料异养方式诱导利用乙酸钠和硝酸钠异养培养的藻细胞在无光照条件下的虾青素生产。
图4示出采用分批补料异养方式诱导利用乙酸钠和硫酸铵异养培养的藻细胞在无光照条件下的虾青素生产。
图5示出采用分批补料异养方式诱导利用乙酸钠和尿素异养培养的藻细胞在无光照条件下的虾青素生产。
图6示出采用分批补料异养方式诱导利用葡萄糖和硝酸钠异养培养的藻细胞在无光照条件下的虾青素生产。
图7示出采用分批补料异养方式诱导利用乙酸钠、核糖和硝酸钠异养培养的藻细胞在无光照条件下的虾青素生产。
图8示出采用分批补料异养方式诱导利用乙酸钠、酵母提取物和蛋白胨异养培养的藻细胞在无光照条件下的虾青素生产。
实施例
本发明所述技术和方法通常根据本领域熟知的及在本说明书引用的参考文献所述的常规方法进行。本发明通过下述实施例进一步阐明。然而应当理解，这些实施例是为了说明目的，任何实施例或其组合不应当理解为对本发明的范围或实施方案的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
实施例1
基础培养基配方为：磷酸二氢钾1.0g/L，硫酸镁500mg/L，氯化钙36mg/L，乙二胺四乙酸二钠5mg/L，硼酸4.5mg/L，氯化铁900μg/L，氯化锰100μg/L，硫酸锌88μg/L，钼酸钠90μg/L，氯化钴50μg/L，硫酸铜79μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.5。
将雨生红球藻CCTCC M2018809(保藏在中国典型培养物保藏中心(CCTCC))接种于含有1.5g/L硝酸钠的无菌基础培养基中，并置于吊袋式薄膜光生物反应器中，初始细胞数为5万个细胞/mL，反应器光径为6cm，体积为5L，装料量为70％，培养温度22℃，白色荧光灯24小时连续单侧光照，光照强度为60μE/m 2/s，通入含有0.5-1.5％(v/v)二氧化碳的混合空气进行搅拌，通气量为0.2-0.5vvm，通过调整二氧化碳含量和通气量将藻液pH控制在7.5。自养240小时后，藻细胞不再进行分裂繁殖，不动的营养细胞数占总细胞数90％，细胞总数达到110万个细胞/mL。
收集藻液置于离心机，3000转/分钟离心5分钟，去除上清液，将浓缩的藻细胞接种至含有8.2g/L乙酸钠的缺氮基础培养基中，接种密度为0.51g/L，并置于0.5L气升式立柱反应器中，装料量为50％，空气通气量为1.0vvm，溶氧为30-40％，培养温度25℃， 通过添加0.5mol/L稀硫酸将pH控制在8.5。在无光照的条件下异养培养192个小时后，超过80％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到1.58g/L，虾青素含量达到1.78％(如图1所示)。虾青素含量测定方法参照上文。
实施例2
基础培养基配方为：磷酸二氢钾0.05g/L，硫酸镁50mg/L，氯化钙5mg/L，乙二胺四乙酸二钠0.5mg/L，硼酸1.9mg/L，氯化铁120μg/L，氯化锰15μg/L，硫酸锌14μg/L，钼酸钠10μg/L，氯化钴5μg/L，硫酸铜22μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.0。
将雨生红球藻CCTCC M2018809接种于含有0.5g/L乙酸钠(碳含量为146mg/L)和0.5g/L硝酸钠(氮含量82mg/L)的无菌基础培养基中，碳氮比为1.8/1，初始细胞数为8万个细胞/mL，并置于5L玻璃发酵罐中，装料量为70％，培养温度20℃，白色荧光灯24小时连续单侧光照，光照强度为40μE/m 2/s，通过调整空气通气量0.05-0.1vvm和搅拌转数50-80转/分钟把溶氧控制在5-10％。通过添加含有600g/L乙酸和70g/L硝酸钠的50倍浓缩基础培养基使发酵液pH维持在7.0，补料培养基碳氮比为20.8/1。混养240小时后，不动的营养细胞数占总细胞数85％，细胞总数达到300万个细胞/ml。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有6.1g/L乙酸钠的缺氮基础培养基中，接种浓度为1.21g/L，并置于5L发酵罐中，装料量为70％，培养温度25℃，通过调整空气通气量0.2-1.0vvm和搅拌转数100-150转/分钟把溶氧控制在30-40％，通过添加含有565g/L乙酸的缺氮基础培养基使藻液pH维持在9.0。在无光照的条件下异养培养336个小时后，超过70％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到5.93g/L，虾青素含量达到2.02％(如图2所示)。虾青素含量测定方法参照上文。
实施例3
培养基配方为：磷酸二氢钾0.5g/L，硫酸镁200mg/L，氯化钙12mg/L，乙二胺四乙酸二钠3mg/L，硼酸3mg/L，氯化铁500μg/L，氯化锰72μg/L，硫酸锌50μg/L，钼酸钠45μg/L，氯化钴33μg/L，硫酸铜65μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到8.0。
将雨生红球藻CCTCC M2018809接种于含有0.8g/L乙酸钠(碳含量为234mg/L)和0.6g/L硝酸钠(氮含量为99mg/L)的无菌基础培养基中，碳氮比为2.4/1，初始细胞数为10万个细胞/ml，并置于5L发酵罐中，装料量为70％，培养温度20℃，通过调整空气通气量0.1-0.4vvm和搅拌转数50-100转/分钟把溶氧控制在15-20％，通过添加含有60g/L乙酸和5g/L硝酸钠的5倍浓缩基础培养基使发酵液pH维持在8.0，补料培养基碳氮比为29.1/1。异养360小时后，藻细胞不再进行分裂繁殖，不动的营养细胞数占总细胞数100％，细胞总数达到290万个细胞/ml。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有4.1g/L乙酸钠的缺氮基础培养基中，接种浓度为1.71g/L，并置于5L发酵罐中，装料量为70％，培养温度30℃，通过调整空气通气量1.0-3.0vvm和搅拌转数100-200转/分钟把溶氧控制在50-70％，通过添加含有180g/L乙酸的营养全部缺失的培养基使藻液pH维持在8.0。在无光照的条件下异养培养384个小时后，100％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到7.87g/L，虾青素含量达到3.21％(如图3所示)。虾青素含量测定方法参照上文。
实施例4
基础培养基配方为：磷酸二氢钾0.3g/L，硫酸镁300mg/L，氯化钙27mg/L，乙二胺四乙酸二钠4mg/L，硼酸3.5mg/L，氯化铁700μg/L，氯化锰80μg/L，硫酸锌90μg/L，钼酸钠87μg/L，氯化钴40μg/L，硫酸铜100μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.5。
将雨生红球藻CCTCC M2018809接种于含有1.2g/L乙酸钠(碳含量为351mg/L)和0.38g/L硫酸铵(氮含量为81mg/L)的无菌基础培养基中，碳氮比为4.4/1，初始细胞数为10万个细胞/mL，并置于5L发酵罐中，装料量为70％，培养温度25℃，通过调整空气通气量0.1-0.3vvm和搅拌转数50-80转/分钟把溶氧控制在10-15％，通过添加含有180g/L乙酸和11.5g/L硫酸铵的20倍浓缩基础培养基使发酵液pH维持在7.5，补料培养基碳氮比为29.5/1。异养312小时后，藻细胞不再进行分裂繁殖，不动的营养细胞数占总细胞数100％，细胞总数达到100万个细胞/mL。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有6.8g/L乙酸钠的营养盐全部缺失的培养基中，接种浓度为1.68g/L，并置于5L发酵罐中，装料量为70％，培养温度30℃，通过调整空气通气量0.5-1.5vvm和搅拌转数100-150转/分钟把溶氧控制在35-50％，通过添加含有120g/L乙酸的缺氮基础培养基使藻液pH维持在8.0。在无光照的条件下异养培养336个小时后，超过90％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到10.7g/L，虾青素含量达到2.29％(如图4所示)。虾青素含量测定方法参照上文。
实施例5
基础培养基配方为：磷酸二氢钾0.2g/L，硫酸镁400mg/L，氯化钙50mg/L，乙二胺四乙酸二钠3.5mg/L，硼酸4mg/L，氯化铁200μg/L，氯化锰35μg/L，硫酸锌25μg/L，钼酸钠35μg/L，氯化钴20μg/L，硫酸铜45μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到8.0。
将雨生红球藻CCTCC M2018809接种于含有1.0g/L乙酸钠(碳含量为293mg/L)和0.31g/L尿素(氮含量为145mg/L)的无菌基础培养基中，碳氮比为2.0/1，初始细胞数为8万个细胞/mL，并置于5L发酵罐中，装料量为70％，培养温度23℃，通过调整空气通气量0.1-0.4vvm和搅拌转数50-100转/分钟把溶氧控制在20-25％，通过添加含有120g/L乙酸和3.1g/L尿素的10倍浓缩基础培养基使发酵液pH维持在8.0，补料培养基碳氮比为33.2/1。异养240小时后，藻细胞不再进行分裂繁殖，不动的营养细胞数占总细胞数100％，细胞总数达到270万个细胞/mL。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有4.5g/L乙酸钠的营养盐全部缺失的培养基中，接种浓度为1.31g/L，并置于5L发酵罐中，装料量为70％，培养温度30℃，通过调整空气通气量1.0-2.0vvm和搅拌转数100-200转/分钟把溶氧控制在45-60％，通过添加含有300g/L乙酸的营养全部缺失的培养基使藻液pH维持在8.0。在无光照的条件下异养培养384个小时后，超过95％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到9.60g/L，虾青素含量达到2.88％(如图5所示)。虾青素含量测定方法参照上文。
实施例6
基础培养基配方为：磷酸二氢钾0.6g/L，硫酸镁100mg/L，氯化钙10mg/L，乙二胺四乙酸二钠2mg/L，硼酸0.5mg/L，氯化铁600μg/L，氯化锰20μg/L，硫酸锌36μg/L，钼酸钠25μg/L，氯化钴45μg/L，硫酸铜80μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.5。
将雨生红球藻CCTCC M2018809接种于含有0.3g/L葡萄糖(碳含量为120mg/L)和1.5g/L硝酸钠(氮含量为247mg/L)的无菌基础培养基中，碳氮比为0.5/1，初始细 胞数为4万个细胞/mL，并置于250mL三角瓶中，装料量为100ml，100转/分摇床培养，培养温度20℃，异养120小时后，不动的营养细胞数占总细胞数70％，细胞总数达到25万个细胞/mL。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有3.7g/L乙酸钠并且氮、磷缺失的基础培养基中，接种浓度为1.37g/L，并置于1L发酵罐中，装料量为70％，培养温度30℃，通过调整空气通气量0.5-1.5vvm和搅拌转数100-200转/分钟把溶氧控制在45-55％，通过添加含有400g/L乙酸的营养全部缺失的培养基使藻液pH维持在7.5。在无光照的条件下异养培养384个小时后，超过85％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到10.54g/L，虾青素含量达到2.39％(如图6所示)。虾青素含量测定方法参照上文。
实施例7
基础培养基配方为：磷酸二氢钾0.8g/L，硫酸镁250mg/L，氯化钙40mg/L，乙二胺四乙酸二钠5.5mg/L，硼酸5mg/L，氯化铁400μg/L，氯化锰90μg/L，硫酸锌66μg/L，钼酸钠100μg/L，氯化钴36μg/L，硫酸铜90μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.5。
将雨生红球藻CCTCC M2018809接种于含有0.4g/L乙酸钠(碳含量为117mg/L)、0.3g/L核糖(碳含量为120mg/L)和1.0g/L硝酸钠(氮含量为165mg/L)的无菌基础培养基中，碳氮比为1.4/1，初始细胞数为4万个细胞/mL，并置于250mL三角瓶中，装料量为100ml，100转/分摇床培养，培养温度20℃，异养120小时后，不动的营养细胞数占总细胞数60％，细胞总数达到30万个细胞/mL。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有5.3g/L乙酸钠并且氮、微量元素缺失的基础培养基中，接种浓度为1.24g/L，并置于5L发酵罐中，装料量为70％，培养温度25℃，通过调整空气通气量0.5-1.0vvm和搅拌转数100-150转/分钟把溶氧控制在30-40％，通过添加含有240g/L乙酸并且氮、微量元素缺失的基础培养基使藻液pH维持在7.0。在无光照的条件下异养培养384个小时后，超过80％藻细胞由绿色营养细胞转变为红色孢子细胞，藻细胞密度达到13.34g/L，虾青素含量达到2.00％(如图7所示)。虾青素含量测定方法参照上文。
实施例8
基础培养基配方为：磷酸二氢钾0.1g/L，硫酸镁150mg/L，氯化钙15mg/L，乙二胺四乙酸二钠1mg/L，硼酸2mg/L，氯化铁150μg/L，氯化锰25μg/L，硫酸锌20μg/L，钼酸钠20μg/L，氯化钴10μg/L，硫酸铜30μg/L。配制完成后，用稀硫酸或者氢氧化钠溶液将pH调整到7.5。
将雨生红球藻CCTCC M2018809接种于含有0.6g/L乙酸钠(碳含量为176mg/L)、4.0g/L胰蛋白胨(氮含量为400mg/L)和2.0g/L酵母提取物(氮含量为200mg/L)的无菌基础培养基中，碳氮比为0.3/1，初始细胞数为8万个细胞/mL，并置于5L发酵罐中，装料量为70％，培养温度25℃，通过调整空气通气量0.2-0.5vvm和搅拌转数80-150转/分钟把溶氧控制在25-30％，通过添加含有15g/L乙酸、8.0g/L胰蛋白胨和4.0g/L酵母提取物的30倍浓缩基础培养基使发酵液pH维持在7.5，补料培养基碳氮比为5.0/1。异养240小时后，不动的营养细胞数占总细胞数80％，细胞总数达到280万个细胞/mL。
停止通气搅拌，自然沉降后去除上清液，将浓缩的藻细胞接种至含有4.9g/L乙酸钠并且氮、磷缺失的基础培养基中，接种浓度为1.55g/L，并置于5L发酵罐中，装料量为70％，培养温度30℃，通过调整空气通气量1.0-2.0vvm和搅拌转数100-250转/分钟把溶氧控制在50-70％，通过添加含有60g/L乙酸并且氮、磷缺失的基础培养基使藻液pH维持在8.0。在无光照的条件下异养培养360个小时后，超过90％藻细胞由绿色 营养细胞转变为红色孢子细胞，藻细胞密度达到12.91g/L，虾青素含量达到2.65％(如图8所示)。虾青素含量测定方法参照上文。

Claims (18)

  1. 一种生产虾青素的方法，包括：
    (a)获得产虾青素雨生红球藻(Haematococcus pluvialis)的营养细胞；
    (b)在含有有机碳源的营养缺失的培养基中在无光照条件下异养培养所述产虾青素雨生红球藻的营养细胞，获得孢子细胞；和
    (c)收获所述孢子细胞和/或虾青素，任选纯化虾青素。
  2. 权利要求1的方法，其中步骤(a)的营养细胞在如下条件下通过自养、混养或异养培养雨生红球藻细胞而获得：
    (i)培养温度控制在15-25℃，例如20-25℃，和/或
    (ii)pH控制在6.0-9.0，优选7.0-8.0。
  3. 权利要求1或2的方法，其中步骤(a)的营养细胞通过自养或混养培养雨生红球藻细胞而获得，优选光照强度为10-100μE/m 2/s。
  4. 权利要求1-3任一项的方法，其中步骤(a)的营养细胞通过混养或异养培养雨生红球藻细胞而获得，其中控制溶氧在1-50％、优选5-30％。
  5. 权利要求1-4任一项的方法，其中步骤(a)获得的营养细胞中不动的营养细胞数至少为总细胞数的50％、60％、70％、80％、85％、90％或95％，优选为100％。
  6. 权利要求1-5任一项的方法，其中步骤(a)的营养细胞通过自养培养雨生红球藻细胞而获得，优选通入含有0.5-1.5％(v/v)二氧化碳的混合空气以提供无机碳源并通过调整二氧化碳含量和通气量控制藻液pH。
  7. 权利要求1-6任一项的方法，其中步骤(a)的营养细胞通过在含有有机碳源和氮源的培养基中混养或异养培养雨生红球藻细胞获得，优选培养基含有80-700mg/L碳元素的有机碳源，40-800mg/L氮元素的氮源，优选碳氮质量比为0.1-10:1。
  8. 权利要求1-7任一项的方法，其中步骤(a)的营养细胞以选自分批、补料分批、半连续和连续培养的方式混养或异养培养雨生红球藻细胞而获得，其中采用补料培养的方式培养雨生红球藻细胞时，优选补料液含有15-1050g/L、更优选60-300g/L的乙酸或乙酸盐，0.3-120g/L氮元素的氮源以及1-50倍浓缩的培养基，优选碳氮质量比为1-50:1。
  9. 权利要求7或8的方法，其中所述氮源包括无机氮源和/或有机氮源，选自硝酸、硝酸盐、亚硝酸盐、氨水、铵盐、尿素、氨基酸、蛋白胨、酵母提取物、蛋白粉、玉米浆和它们的任意组合。
  10. 权利要求1-9任一项的方法，其中所述有机碳源选自：乙酸、乙酸盐、丙酸、丙酸盐、丁酸、丁酸盐、乳酸、乳酸盐、脂肪酸、脂肪酸盐、氨基酸、甲醇、乙醇、甘油、柠檬酸、柠檬酸盐、丙酮酸、丙酮酸盐、葡萄糖、果糖、阿拉伯糖、乳糖、甘露糖、鼠李糖、核糖以及含有上述有机碳源的废水、水解液、发酵液，和它们的任意组合，优选乙酸或乙酸盐，例如1-15.0g/L的乙酸或乙酸盐。
  11. 权利要求1-10任一项的方法，其中所述营养缺失的培养基缺少选自氮源、磷源、硫源、镁源、钙源以及微量元素的一种或者多种营养元素，更优选缺失氮源和/或磷源和/或微量元素，更优选缺失所述全部营养元素，其中所述微量元素选自Mn、Zn、B、I、Mo、Cu、Co和Fe的一或多种。
  12. 权利要求1-11任一项的方法，其中步骤(b)包括如下一或多个：
    (i)培养基含有1-15g/L乙酸或乙酸盐例如乙酸钠，
    (ii)培养温度控制在15-35℃，优选25-30℃，
    (iii)pH控制在6.0-11.0，优选7.0-9.0，和
    (iv)控制溶氧在20-90％，优选30-70％。
  13. 权利要求1-12任一项的方法，其中步骤(b)以分批或补料分批的方式进行异养培养，其中当以补料分批培养时，优选补料液含有15-1050g/L的乙酸或乙酸盐的营养缺失的培养基或乙酸，例如含有100-600g/L乙酸的缺氮培养基或乙酸。
  14. 权利要求1-13任一项的方法，其中当至少60％的营养细胞转变为孢子细胞和/或虾青素含量不再增加时结束步骤(b)。
  15. 权利要求1-14任一项的方法，其中所述培养基包含：
    Figure PCTCN2020116775-appb-100001
  16. 权利要求1-15任一项的方法，其中所述雨生红球藻选自雨生红球藻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和CCMP 3127。
  17. 权利要求1-16任一项的方法，包括：
    (a)在含有有机碳源和氮源的培养基中以补料方式异养培养产虾青素雨生红球藻获得营养细胞，其包括如下一或多个，优选全部：
    -有机碳源选自乙酸或乙酸盐例如乙酸钠；
    -氮源选自硝酸或硝酸盐例如硝酸钠、尿素、胰蛋白胨和酵母提取物；
    -有机碳源的碳元素含量为150-300mg/L，例如175-300mg/L；
    -氮元素的含量为100-600mg/L；
    -培养基中碳氮元素质量比为0.3-3:1，例如0.3-2.5:1；
    -培养温度为20-25℃；
    -溶氧控制在15-30％；
    -pH控制在7.5-8.0；
    -补料液含有有机碳源和氮源，其中碳氮元素质量比为5-35:1，例如5-33:1；
    -获得的营养细胞中不动营养细胞占总细胞数的至少80％，优选100％；
    (b)在含有有机碳源的营养缺失的培养基中在无光照条件下以补料分批方式异养培养步骤(a)获得的营养细胞以获得孢子细胞，包括如下一或多个，优选全部：
    -有机碳源选自乙酸或乙酸盐例如乙酸钠，例如4.0-5.5g/L，
    -培养基缺失(i)氮源，(ii)氮源和磷源或(iii)全部营养元素，
    -培养温度为25-30℃，优选约30℃；
    -溶氧控制在45-70％；
    -pH控制在7.5-8.0，优选约8.0；
    -补料液是(i)含有有机碳源的缺失氮源和磷源的培养基或(ii)含有有机碳源的缺失全部营养的培养基，优选乙酸溶液，例如浓度为60-300g/L或180-300g/L；
    -当至少90％、95％或100％的营养细胞转变为孢子细胞时，终止步骤(b)；
    以及
    (c)收获所述孢子细胞和/或虾青素，任选纯化虾青素。
  18. 一种培养基，其包含：
    Figure PCTCN2020116775-appb-100002
PCT/CN2020/116775 2019-09-23 2020-09-22 一种培养雨生红球藻生产虾青素的方法 WO2021057709A1 (zh)

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