WO2014015841A2 - 一种利用微藻高效生产虾青素的新方法 - Google Patents

一种利用微藻高效生产虾青素的新方法 Download PDF

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WO2014015841A2
WO2014015841A2 PCT/CN2013/084262 CN2013084262W WO2014015841A2 WO 2014015841 A2 WO2014015841 A2 WO 2014015841A2 CN 2013084262 W CN2013084262 W CN 2013084262W WO 2014015841 A2 WO2014015841 A2 WO 2014015841A2
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culture
light
astaxanthin
microalgae
heterotrophic
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PCT/CN2013/084262
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English (en)
French (fr)
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WO2014015841A3 (zh
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李元广
章真
范建华
万民熙
侯冬梅
张京奎
黄建科
梁松涛
王俊
陈杰
王伟良
王军
李淑兰
沈国敏
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华东理工大学
嘉兴泽元生物制品有限责任公司
上海泽元海洋生物技术有限公司
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Priority to EP13823422.4A priority Critical patent/EP2878676A4/en
Priority to CN201380039500.XA priority patent/CN104662162A/zh
Priority to US14/417,166 priority patent/US20150252391A1/en
Priority to AU2013295436A priority patent/AU2013295436A1/en
Priority to BR112015001637A priority patent/BR112015001637A2/pt
Priority to IN1606DEN2015 priority patent/IN2015DN01606A/en
Publication of WO2014015841A2 publication Critical patent/WO2014015841A2/zh
Publication of WO2014015841A3 publication Critical patent/WO2014015841A3/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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • 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
    • 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

Definitions

  • the invention belongs to the field of microalgae biotechnology, and relates to a method for cultivating microalgae to produce astaxanthin. Background technique
  • Astaxanthin chemical name 3,3'-dihydroxy-4,4'-diketo- ⁇ , ⁇ '-carotene, has a molecular formula of C 40 H 52 O 4 and a relative molecular mass of 596.86.
  • the chemical structure of astaxanthin is composed of four isoprene units linked by a conjugated double bond type, and two isoprene units at both ends form a six-membered ring structure. The chemical structure is shown in the following figure. Because of the chemical structure of astaxanthin, it contains a long conjugated unsaturated double bond system.
  • Astaxanthin is one of the carotenoids and the highest grade of carotenoids. Beta-carotene, lutein, canthaxanthin, and lycopene are intermediates in the synthesis of carotenoids. Therefore, astaxanthin has the strongest antioxidant activity in nature. Natural astaxanthin is by far the most powerful antioxidant found in nature, and its antioxidant activity far exceeds that of existing antioxidants. It is known as a “super oxidant”. Astaxanthin has a wide range of applications and can be used not only as a feed additive for aquaculture but also as a human food additive, and has great potential for application in the fields of medicines, cosmetics and nutraceuticals.
  • Haematococcus pluvialis contains astaxanthin, which accounts for 1-5% of the dry weight of cells, and is the natural species with the highest astaxanthin content in nature.
  • astaxanthin is mainly in the form of monoesters in microalgae, its structure is trans-structure, and its bioavailability is higher than that of chemically synthesized cis-structure.
  • the growth cycle of microalgae is short, and the production equipment occupies land. The area is small, the product quality and yield are relatively stable.
  • microalgae itself is a high-value product containing a large amount of active ingredients such as protein, oil, polysaccharide, etc., which can be separated and extracted. Realize the comprehensive utilization of microalgae cells.
  • the cultivation modes of astaxanthin produced by microalgae mainly include photoautotrophic and heterotrophic.
  • microalgae photoautotrophic culture slow growth of microalgae cells, low cell density and low astaxanthin yield.
  • the maximum cell dry weight of photoautotrophic cultured microalgae is 6.8 g/L achieved by Ranjbar et al. in a 16 liter bubble column reactor (cell production). Rate of 0.2 g/L/d) (Ranjbar R, Inoue R, Shiraishi H, Katsuda T, Katoh S: High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor. Biochemical Engineering Journal 2008, 39(3) ): 575-580. ).
  • the highest volumetric yield of echinococcin is 23.04 mg/L/d obtained by Ranjbar et al. in a 1 L airlift photobioreactor for culturing Haematococcus pluvialis (Ranjbar R, Inoue R, Katsuda T, Yamaji H, Katoh S: High efficiency production of astaxanthin in an airlift photobioreactor. Journal of Bioscience and Bioengineering 2008, 106(2): 204-207. ) , the highest area yield of astaxanthin is Olaizola et al.
  • microalgae culture mainly carried out the cultivation of Haematococcus pluvialis to make it grow rapidly.
  • continuous culture refers to the cultivation of microalgae under stable culture conditions, so that Haematococcus pluvialis continuously produces under the condition of maintaining constant growth rate and stable physiological characteristics; semi-continuous culture means that the cultured algae cells reach a certain level.
  • the second stage is a series of stresses such as high light, high temperature, high salt, nutrient salt hunger, etc., which promotes the transformation of Haematococcus pluvialis into thick-walled spores in a harsh living environment to achieve accumulation.
  • the purpose of astaxanthin In these two stages, the nutrient and environmental conditions required for microalgae are different. At present, domestic and foreign research mainly focuses on the selection and control of conditions in these two stages and the influence of environmental factors.
  • the first stage of photoautotrophic culture does not accumulate astaxanthin
  • the purpose is to increase the number and weight of cells, when reaching the end of the exponential growth phase (at this time, the cell density is 0.5 ⁇ 1.5g/L, the number of cells is 20 ⁇ 50 10,000/ml), due to the consumption of nutrients such as nitrogen and phosphorus, the photoautotrophic cells are directly transferred to the second stage without dilution, etc., supplemented by strong light, high temperature, high salt and other stress conditions and added with nitrogen and phosphorus. The lack of medium promotes the accumulation of astaxanthin. At this stage, the number of cells no longer increases.
  • the number of cells decreases with the severity of stress conditions, but the cell weight increases slowly due to cell sporulation and expansion, and the stress culture ends.
  • the cell weight in the unit volume of the culture solution is increased by 2 to 4 times compared to the beginning, to about 2 to 3 g/L.
  • Astaxanthin accumulation stage medium and photoautotrophic culture medium are not completely the same, the latter N, P is rich and requires reasonable ratio between elements (required carbon, nitrogen, phosphorus, sulfur, sodium, calcium, potassium, magnesium and other elements) , the former only needs to add calcium salt
  • the physical environmental factors and nutrients affecting the photoautotrophic culture of Haematococcus pluvialis mainly include parameters such as temperature, light intensity, pH value, dissolved oxygen and nutrient content. There have been quite a lot of reports in domestic and foreign literature, as shown in Table 1.
  • the optimum light intensity is 30 ⁇ 50 ⁇ 1 ⁇ - 2 s—the optimum pH is neutral to slightly alkaline, and NaAc can be used for mixed vegetative growth.
  • High ambient light, high temperature, nutrient salt (nitrogen, phosphorus) starvation, salt stress (NaCl, NaAc, etc.) and oxidative stress (active oxygen, oxygen free radicals and dissolved oxygen) and many other environmental conditions can induce intracellular astaxanthin Accumulation, which are collectively referred to as induction conditions or stress conditions, are invariably inhibited by cell growth and division, and have synergistic effects.
  • the traditional photoautotrophic two-stage culture system cannot overcome problems such as low yield, easy pollution, large seasonal changes, large area, and high cost.
  • the cell density of photoautotrophic culture is not high, because Haematococcus pluvialis has strict requirements on the physical and chemical conditions of culture, and it is impossible to keep it in a state of vegetative cells for a long time. Although the cell weight in the spore state can be slowly increased, it is no longer vegetatively reproduced, and the number of cell populations cannot be rapidly increased. This limits the maximum amount of cells that can be achieved when photoautotrophic.
  • Haematococcus pluvialis is very sensitive to environmental changes, with short exponential growth period, poor resistance to bacterial and protozoan contamination during vegetative growth, and loss of reproductive capacity in extreme environments, making it difficult to establish stable and efficient culture. Technology System. Therefore, the cultivation of astaxanthin from Haematococcus pluvialis has considerable difficulty in the design of algae species, photobioreactors, high-density culture conditions, and ecological regulation techniques for astaxanthin accumulation.
  • the internationally successful production mode adopts a two-stage mode, that is, the closed photobioreactor culture system is used to achieve high-density vegetative growth of cells, and the pollution problem can be overcome, and the conventional open pool system is used in the stress condition.
  • the cells are allowed to accumulate astaxanthin.
  • Cyanotech and Aquasearch in the United States can realize the large-scale cultivation of Haematococcus pluvialis.
  • Haematococcus pluvialis The growth conditions of Haematococcus pluvialis are relatively mild. Many kinds of predatory organisms such as rotifers, protozoa and other microalgae can grow and multiply in the Haematococcus pluvialis culture medium. The prevention and control of biological pollution becomes very important when the algae is grown in large scale. Hard to overcome problems. Early experiments showed that in the open pond culture process, rotifers swallowing Haematococcus appeared in the culture medium for about 4 to 5 days, and then the whole culture failed. If the algae cells are completely transformed into immobile spores, their ability to resist enemy organisms is greatly enhanced.
  • the microalgae heterotrophic culture has disadvantages such as low intracellular astaxanthin and chlorophyll, the microalgae can be cultured in a fermenter at a high density, and the cell growth rate is fast. Heterotrophic culture can achieve high cell density and high cell growth rate.
  • the highest reported algal cell dry weight reported in the literature is 7g/L ( Hata N, Ogbonna JC, Hasegawa Y, Taroda H, Tanaka H: Production of astaxanthin by Haematococcus pluvialis in a Sequential heterotrophic-photoautotrophic culture.
  • cell yield is 0.3g/L/d, but its astaxanthin yield is low (only 4.4mg/L/ d)
  • the astaxanthin content was 1.85% after 8 days of light stress.
  • Heterotrophic culture was carried out in a 2.3 L fermenter.
  • the photoautotrophic culture is carried out in an indoor glass container (16 cm in diameter, 900 ml in liquid, 5.5 cm in liquid level), and artificial light is irradiated from the top to the bottom, and the light intensity at the liquid surface is 950 ⁇ 1 ⁇ - 2 s.
  • the temperature is 30 ° C.
  • the mixing is achieved by magnetic stirring (100 rpm), and the gas to the algae solution is 5% CO 2 air, and the aeration is 0.22 wm. Although it adopts two sections of heterotrophic-photoautotrophic Mode, but there are mainly four problems:
  • the selected medium is ordinary basal medium, no growth-promoting plant growth hormone substances are added, and the culture process is controlled by intermittent flow plus unoptimized feed medium to control the pH to 7.5-8.0.
  • This method does not consider the difference in nutrient requirements between Haematococcus pluvialis heterotrophic and ordinary photoautotrophic, resulting in poor culture of the culture medium and slow cell growth.
  • intermittent feeding causes pH fluctuation and culture.
  • the algae solution is directly added to the photoautotrophic culture without adding the medium, and there are two problems: 1) First, the cells will die in large numbers: Since the algae liquid was not diluted to a lower density and the cell density at the end of the heterotrophic culture was maintained at 5.5 g/L, high-density photoautotrophic due to the self-shading effect of the algae cells, a large number of cells could not be sufficiently illuminated, so that A large number of algae cells died, from 650,000 cells/ml at initial light autotrophy to 210,000 cells/ml at the end of photoautotrophication, and the loss of algae cells was about 70%; 2) the second is intracellular shrimp
  • the increase in the content of chlorophyll is effective: since the original solution is used for photoautotrophic culture without dilution, the heterotrophic and autotrophic of Haematococcus pluvialis has different nutritional requirements, resulting in an increase in the content of astaxanthin in algae cells. Large,
  • microalgae In addition to heterotrophic culture and photoautotrophic culture mode, microalgae also has an unusual culture mode, namely, mixed nutrient culture.
  • this culture mode can only be carried out in a steam-sterilizable closed photobioreactor, and the culture process must ensure absolute sterility, and at the same time requires a reasonable configuration of the light source, which cannot be achieved in actual production. Therefore, the use of a mixed nutrition model to culture microalgae to produce astaxanthin has no industrial value.
  • the present invention designs a "heterotrophic-dilution-light-induced" tandem culture mode for the production of astaxanthin microalgae, the process is as follows: 1) Firstly, the astaxanthin-producing microalgae can be cultured in a bioreactor to obtain high-density cells; (2) the organic carbon source and nitrogen source in the culture solution are almost the same. After the consumption is completed, the algae solution is diluted with the medium without the organic carbon source; (3) The astaxanthin in the algae cells is rapidly accumulated in a large amount by light induction.
  • the heterotrophic phase in this mode is carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, bubbling, etc., in order to obtain higher density algae cells in a short time;
  • the light-inducing phase can be carried out in any system that can be used for photoautotrophic culture of microalgae, in order to increase the content of astaxanthin in algal cells by light induction; the heterotrophic phase and the light-inducing phase are separately carried out, and the heterotrophic phase is released separately.
  • the algae liquid depends on its cell density and the level of nutrients in it, and the intensity of outdoor light intensity. It is considered whether it is diluted with light-inducing medium and then transferred to the light-induced culture stage.
  • the invention divides the process of producing astaxanthin by the microalgae culture method into a heterotrophic growth culture for the purpose of rapidly obtaining high-density cells, diluting for the purpose of reducing algal cell density and causing nutritional stress, and improving microalgae cells.
  • the astaxanthin content simultaneously increases the amount of microalgae cells for the purpose of three stages of light-induced culture, namely heterotrophic culture, dilution and light-induced culture.
  • heterotrophic culture a large number of microalgae cells that can accumulate astaxanthin can be obtained in a short time.
  • the algae solution is diluted and transferred to light-induced culture, and the astaxanthin content in the algae is rapidly increased to several times or more.
  • Heterotrophic culture can make Haematococcus pluvialis long-term in the stage of vegetative reproduction, and the cell yield in heterotrophic stage is high.
  • the average cell yield is 1.53g/L/d, the highest 5.74g / L / d, the final cell density in the heterotrophic stage can be as high as 26.01g / L, so the light-induced algal cell density is very high (2 ⁇ 10g / L), is the conventional photoautotrophic culture algae cell density (about 0.2 ⁇ 5 to 10 times 2g/L);
  • the heterotrophic released algae cells can be directly induced by stress to accumulate astaxanthin, which does not require adaptation or transition period, so the light induction time can be shorter (about 5c! ⁇ 7d); However, the traditional autotrophic culture of Haematococcus pluvialis is very long (about 14c! ⁇ 30d).
  • the same as the accumulation phase of astaxanthin in the traditional photoautotrophic culture process is the cell at the end of induction.
  • the dry weight can be increased, so the volume yield of astaxanthin in the unit algae liquid can be increased several times or more than the traditional photoautotrophic at this stage;
  • Heterotrophic culture is almost independent of weather and weather.
  • Light-induced culture can be carried out in a glass room.
  • the light source can be used with natural light or artificial light, and the temperature range of light induction is wide (15 ⁇ 35 °C). It can promote the accumulation of astaxanthin.
  • low temperature conditions do not promote the accumulation of astaxanthin, due to the small area of induced culture, it is still possible to achieve shrimp green by artificial warming, high salt, high carbon to nitrogen ratio, strong light and other synergistic stress conditions.
  • the rapid accumulation of hormones Therefore, large-scale continuous production of astaxanthin can be achieved by the method of the present invention.
  • heterotrophic-dilution-light-induced tandem culture mode of the present invention reasonably combines the respective advantages of heterotrophic and light-induced culture, and has higher production efficiency and culture system than other modes.
  • the combination of flexibility and low production cost can fully exploit the advantages of heterotrophic culture mode to obtain rapid accumulation of astaxanthin in high-density algae liquid and light-induced phase, and provide a solution to the large-scale industrialization of microalgae astaxanthin.
  • the present invention provides a novel method for "heterotrophic-dilution-light-induced" tandem culture of rapid culture of microalgae accumulation of astaxanthin, which comprises a heterotrophic culture step of microalgae, and the obtained microalgae heterotrophic The step of diluting the culture solution, and the step of light-induced culture.
  • Another aspect of the present invention provides a method for rapidly increasing the content of astaxanthin in microalgae, which comprises the step of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae and performing light-induced culture.
  • the invention also provides a method for producing astaxanthin, which comprises the steps of heterotrophic microalgae, the step of diluting the heterotrophic culture solution of the microalgae for light-induced culture, and the harvesting of algae cells and the separation of astaxanthin. The steps of extraction.
  • the microalgal cells obtained in the heterotrophic culture can also be directly subjected to light-induced culture.
  • the method of the present invention enables rapid accumulation of intracellular astaxanthin, significantly improves production efficiency, reduces production costs, and provides high quality astaxanthin.
  • the pH is controlled by feeding and the elements such as carbon, nitrogen and/or phosphorus are stabilized within a certain concentration range, and carbon, nitrogen and/or at the end of heterotrophic culture.
  • concentration of nutrients such as phosphorus is low or even zero.
  • the pH of the algal fluid is controlled by feeding to a constant value in the range of 4.0-10.0, such as pH 7.5.
  • the pH of the algal solution is usually controlled to a constant value in the range of 5.0 to 9.0, more preferably in the range of 7.0 to 8.0.
  • the pH of the algal fluid is controlled to be ⁇ ⁇ ⁇ by feeding, wherein the gentry 9.0.
  • the pH of the algal solution was controlled to be within the range of 7.5 ⁇ 0.3 by feeding.
  • the elements such as carbon, nitrogen and/or phosphorus are stabilized in a certain concentration range by feeding.
  • the content of carbon in the algae liquid can be controlled to be in the range of 0.5 to 50 mM by feeding, the content of nitrogen is controlled in the range of 0.5 to 10 mM, and the content of phosphorus is controlled in the range of 0.01 to 0.5 mM.
  • the three elements of carbon, nitrogen and phosphorus are stabilized within a certain concentration range by feeding.
  • the content of carbon in the algae solution can be controlled to be in the range of 0.5 to 50 mM by feeding, the content of nitrogen is controlled in the range of 0.5 to 10 mM, and the content of phosphorus is controlled in the range of 0.01 to 0.5 mM.
  • the content of magnesium in the algae solution is controlled to be in the range of 0.00001 - 0.00 I mM by feeding.
  • the microalgae is selected from the group consisting of Haematococcus pluvialis, Chlorella zofingiensis, and the like.
  • the step of heterogeneous culture of the microalgae comprises: adding a medium having a pH of 4.0 to 10.0 in the bioreactor, and accessing the microalgae species according to a working volume of 0.1 to 50% for batching Culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture, culture temperature is 10 ⁇ 40 °C, control pH is less than 10.0, and controlled dissolved oxygen is above 0.1%.
  • the microalgae heterotrophic culture solution is diluted with a light-inducing medium to dilute the algal liquid obtained by heterotrophy to a cell density of 0.1 to 20 g/liter and a pH of 4.0 to 9.0.
  • the light-induced culture comprises transferring the diluted algal liquid into a light-inducing device for light induction, continuous illumination or intermittent illumination, the culture temperature is 5 to 50 ° C, and the illumination intensity is 0.1 to 150 klx.
  • the light-induced culture period is from 1 to 480 hours.
  • the heterotrophic medium contains or consists of a nitrogen source, an organic carbon source, a small amount of inorganic salts, plant growth hormone, trace elements and water; the light-inducing medium contains plant growth hormone, nitrogen source, inorganic Salt and water are either composed of these ingredients.
  • the medium used for heterotrophy consists essentially of the following components: sodium acetate 0.1 to 5.0 g/L, NaN0 3 0.05 to 1.5 ⁇ / liter CaCl 2 *7H 2 0 0.05 ⁇ 1.5 g / liter, KH 2 PO 4 0.01 ⁇ 1.5 g / liter, MgS0 4 *7H 2 0 0.01 ⁇ 1.0 g / liter, FeS0 4 *7H 2 0 0.01 ⁇ 0.05 g / liter, auxin 0.001-35 mg / liter, trace elements 0.5 ⁇ 4 ml and water.
  • the heterotrophic step is carried out in a shake flask, mechanically agitated, airlifted or bubbling heterotrophic culture bioreactor in a shake flask or open Runway pool or round pool, closed flat photobioreactor or ducted photobioreactor or column photobioreactor or film pouch and sling photobioreactor can be used for microalgae light
  • the apparatus is cultured, and the light source is natural light or various artificial light.
  • the method of the present invention further comprises: performing solid-liquid separation (i.e., harvesting) on the induced algal cells, and drying the obtained algal cells to obtain an astaxanthin-containing algal flour.
  • solid-liquid separation i.e., harvesting
  • the method of the present invention further comprises: mixing the algae body after extracting astaxanthin with other pigments to prepare algal flour, or separating and extracting other biologically active substances in the algae body.
  • the other pigment comprises chlorophyll.
  • the bioactive material comprises proteins, oils, chlorophyll and polysaccharides.
  • Figure 1 shows the growth process of Haematococcus pluvialis in the heterotrophic culture process of the 5L bioreactor (including the optimal growth process of the heterotrophic culture process of the present invention, only controlling the heterotrophic pH but not optimizing the initial and feed medium strategies, And the strategy of controlling the heterotrophic pH alone and optimizing the initial and fed medium, but comparing the growth process data without adding plant growth hormone).
  • Fig. 2 shows the photoinduced culture process of the Haematococcus pluvialis algae solution in the heterotrophic culture solution in the outdoor 2L column photobioreactor with carbon, nitrogen and phosphorus.
  • Figure 3 shows the light-induced culture process of the non-consumed three-nutrient components of carbon, nitrogen and phosphorus in the heterotrophic culture solution in an outdoor 2L column photobioreactor.
  • microalgae suitable for use in the present application include those which can synthesize astaxanthin and can be heterotrophically cultured, including but not limited to Haematococcus pluvialis, Chlorella zofingiensis and the like.
  • the invention employs Haematococcus pluvialis to produce astaxanthin.
  • Plant growth hormones for use in the media of the invention include, but are not limited to,
  • the medium may contain one or more plant growth hormones.
  • the total amount of plant growth hormone in the medium may be from 0.001 to 35 mg/liter of medium, usually from 0.001 to 20 mg/liter, more usually from 0.001 to 15 mg/liter, from 0.005 to 10 mg/liter, from 0.01 to 10 mg. /L, 0.1-5 mg / liter.
  • each plant growth hormone if present, may be at a concentration such as 2,4-dichlorophenoxyacetic acid
  • each plant growth hormone is preferably, for example, 0.01 to 4 mg/liter, 0.1 to 4 mg/liter, 0.3 to 4 mg/liter, 0.3 to 3 mg/liter, and 0.5 to 2.5 mg/liter.
  • the plant growth hormone can be obtained from a commercially available route and then directly added to a medium known in the art for heterotrophic culture and light-induced culture of microalgae which can synthesize astaxanthin and can be heterotrophically cultured.
  • a medium known in the art for heterotrophic culture and light-induced culture of microalgae which can synthesize astaxanthin and can be heterotrophically cultured. Examples of the medium are as follows. 1. High-density heterotrophic culture of microalgae in bioreactor
  • the purpose of this step is to rapidly obtain a large number of algal cells for the accumulation of astaxanthin during the light-inducing phase.
  • the microalgae heterotrophic culture can be carried out by adding an organic carbon source (e.g., sodium acetate or the like) to various culture media well known in the art.
  • an organic carbon source e.g., sodium acetate or the like
  • the heterotrophic medium used in the present invention contains a nitrogen source, an organic carbon source, a plant growth hormone, a small amount of an inorganic salt, a trace element, and water.
  • Such media include C medium (Ichimura, T. 1971 Sexual cell division and conjugation-papilla formation in sexual reproduction of Closterium strigosum. In Proceedings of the Seventh International Seaweed Symposium, University of Tokyo Press, Tokyo, p. 208-214 .), MCM medium (Borowitzka et al, 1991), BG-11 medium (Boussiba and Vonshak, 1991), BBM medium (Nichols and Bold, 1969), BA medium (Barbera et al., 1993).
  • KM medium (Kobayashi et al., 1991), Z8 medium (Renstrom et al., 1981), A9 medium (Lee and Pirt, 1981), OHM medium (Fa'bregas et al., 2000), KMl Medium (Usha et al. 1999) (Garc'ia-Malea et al, 2005), HK2 medium (Chen et al., 1997), HK3 medium (Gong and Chen, 1998), and the like.
  • the C medium used in the present invention consists essentially of KN0 3 , CaN0 3 , sodium acetate and a small amount of inorganic salts, trace elements and water, on the basis of which some plant growth hormones are added.
  • composition of the present invention contains, in addition to the main components,
  • KN0 3 , CaN0 3 , sodium acetate and a small amount of inorganic salts, trace elements and water may also contain some basic properties or new properties for the composition (ie, maintaining the microalgae cell density in a shorter culture period) High levels, while the active substance content is significantly increased compared to conventional heterotrophic cultures) components that have no substantial effect.
  • the term "consisting of” as used herein means that the composition of the present invention consists of the specific components indicated, without other components, but may carry impurities in a usual range.
  • the components of the medium can be varied within a certain range without greatly affecting the density and quality of the microalgae cells. Therefore, the amounts of these components should not be strictly limited by the examples.
  • a small amount of inorganic salts such as magnesium sulfate, calcium chloride, ferrous sulfate, and phosphate may be added to the medium, and a small amount of trace elements such as Mn, Zn, B, I, M, Cu, Co, etc., and the addition of plant growth hormone, include a single hormone or a combination of hormones.
  • the trace element component may be selected from the group consisting of H 3 B0 3 , ZnS0 4 -7H 2 0, MnCl 2 H 2 0, NH 4 ) 6 Mo 7 0 24 4H 2 0, CuS0 4 -5H 2 0 and CO. ⁇ ;N0 3 ) One or more of 2 _6H 2 0 .
  • Inorganic salts and trace elements The amount used can be determined based on conventional knowledge.
  • the heterotrophic medium employed in the present invention consists essentially of the following components:
  • the medium used for heterotrophy consists essentially of the following components: sodium acetate 0.1 to 5.0 g/L, NaN0 3 0.05 to 1.5 ⁇ / liter CaCl 2 *7H 2 0 0.05 ⁇ 1.5 g / liter, KH 2 PO 4 0.01 ⁇ 1.5 g / liter, MgS0 4 '7H 2 0 0.01 ⁇ 1.0 g / liter, FeS0 4 '7H 2 0 0.01 ⁇ 0.05 g / liter, auxin 0.001-35 mg / liter, trace elements 0.5 ⁇ 4 ml and water.
  • the plant growth hormone in the heterotrophic medium comprises: 2,4-dichlorophenoxyacetic acid 0.001-5 mg/L, benzylaminopurine 0.001-5 mg/L, exogenous gibberellic 0.001 -5 mg/L, 3-butyric acid 0.001-5 mg/L, naphthaleneacetic acid 0.001-5 mg/L and/or brassinolide 0.001-5 mg/L.
  • the plant growth hormone in the heterotrophic medium comprises: benzylaminopurine 0.001-5 mg/L and 3-indolebutyric acid 0.001-5 mg/L.
  • the pH of the medium can be adjusted to 4.0-10.0 by a conventional means such as an acid or a base, and autoclaved at 115 to 125 ° C for 15 to 30 minutes.
  • Heterotrophic culture can be carried out in a variety of ways, including batch, fed-batch, semi-continuous and continuous culture.
  • the corresponding prepared medium is added to the bioreactor, and water is added to the working volume, usually with a charging coefficient of 0.6-0.8, and then steam-sterilized (121 °C). , maintain for about 20 minutes), when the temperature drops to 20 ⁇ 35 °C, access the microalgae algae according to the working volume of 1 ⁇ 15% to start heterotrophic culture.
  • the feeding is started, and the pH is kept constant within a certain range by controlling the continuous flow of the feed medium, such as 7.0-8.0, and in a preferred embodiment, the pH is controlled at 7.5.
  • the feed medium includes an organic carbon source (such as sodium acetate), a nitrogen source (such as CaN0 3 , KN0 3 ), a plant growth hormone, and an inorganic salt, and the supplemental nutrient salt is concentrated after the corresponding culture.
  • Organic carbon source, nitrogen source, plant growth hormone, inorganic salt, etc. in the feed medium should be added such that the concentration of the corresponding component in the algae solution is the same as or similar to the concentration at the beginning of the heterotrophic culture, so that the carbon source and nitrogen in the algae solution
  • concentration of source, plant growth hormone and inorganic salts can promote the continued growth of the microalgae. It is of course also possible to appropriately adjust the corresponding components in the feed medium according to the actual growth of the microalgae to increase or decrease the concentration of one or some of the components, thereby promoting the continued growth of the microalgae.
  • the phosphorus content is controlled within the range of 0.01-0.5 mM (usually 0.01-0.4 mM, 0.05-0.3 mM, 0.05-0.2 mM, 0.05-0.1 mM, etc.) to ensure stable concentration of these substances in the algae solution. . More preferably, the magnesium content in the algae liquid is simultaneously monitored, and the magnesium content in the feed medium is appropriately adjusted to control the magnesium content in the algae liquid to 0.00001-O.OOlmM (usually 0.00001-0.0008 mM, Within the range of 0.00003-0.0005 mM, etc.
  • the control conditions are changed, and the nutrients such as carbon, nitrogen and/or phosphorus in the culture solution are basically consumed, and the heterotrophic culture stage is finished.
  • the concentration of nutrients such as carbon, nitrogen and/or phosphorus in the medium is low.
  • the concentration of carbon and nitrogen is less than 0.1 mM, less than 0.05 mM, less than O.OlmM, even lower, or even zero; the concentration of phosphorus is less than 0.005 mM, less than 0.003 mM, less than 0.001 mm, or even Lower, even zero.
  • the culture conditions must be strictly controlled to allow the microalgae to grow normally.
  • the control temperature is 20 to 35 ° C, for example, 25 to 30 ° C
  • the air saturation concentration of the dissolved oxygen of not less than 5% is controlled by adjusting the aeration and stirring, and the pH is not higher than 9.0.
  • the dissolved oxygen is not less than 10% and not more than 30% of the air saturation concentration
  • the pH is constantly controlled at 7.5-8.0
  • the aeration is less than 0.3 wm
  • the agitation is less than 200 rpm.
  • Heterotrophic can be carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling.
  • a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling.
  • One of the purposes of this step is to reduce the density of algae cells, so that the astaxanthin-producing microalgae transferred to light-induced culture can efficiently absorb light energy and improve the efficiency of light energy utilization.
  • the second purpose is to adjust the nutrient composition in the induced culture medium. , causing nutritional stress, in order to facilitate the rapid accumulation of astaxanthin.
  • High-density algae liquid obtained by heterotrophic culture in the open reactor, it should be free of organic carbon source, which can avoid excessive growth of bacteria in the light-induced phase; but when induced by closed photobioreactor, The organic carbon source is included to promote the increase of the amount of cells.
  • the dilution operation should be carried out, and the high-density algae solution is diluted with a dilution medium to maintain the cell density at 0.1-20 g/L and the pH 4.0 ⁇ 10.0.
  • the high density algal fluid is diluted with water and a medium free of organic carbon source to maintain a cell density of 0.1 to 10 grams per liter and a pH of 5.0 to 8.0.
  • the algal fluid is diluted to maintain a cell density of 1 to 8 grams per liter and the pH is adjusted to 5.0 to 8.0.
  • the cell density is maintained at 1.0 to 5.0 g/l, C0 2 is passed and the pH is adjusted to 5.0 to 8.0.
  • the light-inducing medium contains or consists of a carbon source, a nitrogen source, a plant growth hormone, an inorganic salt and water, and does not contain or contain less organic carbon source than the heterotrophic medium, and the culture process can also pass into the C0. 2 .
  • the high density algae cells obtained by heterotrophic culture are suitably diluted with an initial medium free of organic carbon sources and nitrogen and phosphorus deficient.
  • the dilution medium contains: MgS (V7H 2 O 0.01 to 0.1 g/L, NaH 2 PO 4 0.01 to 0.1 g/L, KC1 0.1 to 1 g/L, CaCl) 2 0.01 to 0.2 g / liter, FeS0 4 '7H 2 0 0.01 ⁇ 0.06 g / liter, EDTA 0.020 ⁇ 0.052 g / liter, and auxin 0.001 - 35 mg / liter.
  • the plant growth hormone in the dilution medium comprises: 2,4-dichloro Phenoxyacetic acid 0.001-5 mg / liter, benzylamino hydrazine 0.001-5 mg / liter, exogenous gibberellin 0.001-5 mg / liter, 3- ⁇ ⁇ 0.001 -5 mg / liter, naphthalene acetic acid 0.001-5 Mg/L and/or Brassino 0.001-5 mg/L.
  • the plant growth hormone in the dilution medium comprises: benzylaminopurine ⁇ 0.001-5 mg / liter and 3-mercaptobutyric acid 0.001-5 mg / liter.
  • the medium used for dilution does not need to be autoclaved. After preparation, adjust the pH to 5.0 ⁇ 9.0 to use.
  • the purpose of this step is to allow the astaxanthin-producing microalgae to receive sufficient light to rapidly accumulate astaxanthin by rapid induction of algae cells by light induction, and to appropriately increase the concentration of algae cells in the culture solution.
  • the resulting dilution is transferred to a light-inducing device for light-induced culture or a semi-solid adherent method such as coating the microalgae cells on a solid film surface for light induction.
  • the temperature is controlled at 5 to 50 ° C, the light intensity is 0.1 to 150 klx, continuous light or intermittent illumination, the light-induced culture period is 1 to 480 hours, and the aeration is 0.1 to 2.0 wm.
  • the photobioreactors described therein include all closed photobioreactors (bottle shakers, tubing, flat plates, columns, film pouches and slings, etc.) and all open photobioreactors (runway cells) , round pools and bubbling basins, etc.).
  • the culture temperature can be controlled within the range of 15 to 35 ° C, for example, 18 to 35 ° C, 20 to 35 ° C, 20 to 30 ° C, and the like.
  • the light intensity is l ⁇ 70klx, for example, 1 ⁇ 60, 1 ⁇ 50, 1 ⁇ 40, 1 ⁇ 30, 1 ⁇ 20, l ⁇ 10klx, etc., depending on the specific production situation.
  • the aeration amount can be controlled to 0.1 to 2.0 wm, for example, 0.2 to 1.8, 0.5 to 1.5, 0.8 to 1.5, 1.0 to 1.5 wm, or the like.
  • a certain concentration of C0 2 is introduced to provide an inorganic carbon source and to control the pH, for example, 0.5% to 10% of C0 2 .
  • the culture temperature is controlled at 10 to 50 ° C
  • the light intensity is 1 to 10 klx
  • the aeration is 0.05 to 2.0 wm.
  • the light-induced culture period is 8 to 480 hours.
  • the light-induced culture period may be 8 to 240 hours, 8 to 120 hours, 8 to 72 hours, 8 to 48 hours, 8 to 24 hours; or, the light-induced culture period can be 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, or 24 to 60 hours, 24 to 48 Hours vary.
  • the light-inducing medium is selected from a modified Haematococcus pluvialis photoautotrophic medium, including a dilution medium as described above.
  • the "light-induced culture period" includes the entire light-induced culture process, for example, the outdoor-inducing light-induced culture period includes the time when there is no light at night.
  • lighting time refers to the time during which light-induced culture of microalgae is carried out using the light intensity described herein. That is, the time does not include the time when there is no light at night. In some embodiments, the illumination time of the light-induced culture step is
  • 8 to 120 hours for example, 8 to 72 hours, 8 to 36 hours, 8 to 24 hours, 8 to 18 hours, 8 to 12 hours, 12 to 36 hours, 12 to 24 hours, and any length within the above range .
  • the light-inducing culture step of the present application also includes a light-induced culture step in the range of 8 to 120 hours of illumination.
  • Light-induced culture can be carried out by artificial light, or light-induced culture can be carried out outdoors by natural light.
  • the concentration of astaxanthin in the culture solution reaches a maximum
  • the light-induced culture is terminated, the algal cells are harvested for separation and extraction of astaxanthin or the algal cells are directly collected for algal powder preparation. 4. Algae cell harvesting, astaxanthin extraction and comprehensive utilization of algae
  • microalgae are sedimented or centrifuged to obtain a wet algae body.
  • Methods for harvesting algae cells include, but are not limited to, sedimentation, high-speed centrifugation, flocculation, air flotation or filtration; algae cell wall breaking methods include, but are not limited to, algae autolysis, high pressure homogenization, enzymatic hydrolysis, aqueous pyrolysis, etc. Wet method of breaking the wall.
  • the microalgae was extracted from astaxanthin by a conventional organic solvent extraction method. First, an organic solvent is added to the algal mud for extraction, and then the supernatant and the algal body precipitate are obtained by stirring and centrifuging, and the supernatant is concentrated under reduced pressure, stirred and added with water, and filtered to obtain astaxanthin crystals.
  • the other components in the supernatant may be gradually separated and extracted to obtain a fatty acid, lutein or the like, or the mixture of all the components in the supernatant may be directly spray-dried to obtain a microalgal powder.
  • the microalgae is subjected to separation and extraction of astaxanthin using a supercritical co 2 extraction technique.
  • the obtained microalgae liquid is concentrated and directly spray-dried to obtain microalgae powder.
  • the microalgae obtained by the culture can be comprehensively utilized, and various active ingredients such as polyunsaturated fatty acids, proteins, chlorophyll, and polysaccharides can be extracted.
  • active ingredients such as polyunsaturated fatty acids, proteins, chlorophyll, and polysaccharides can be extracted.
  • the order of extraction of the active ingredient is not particularly limited, but it is usually the premise that the step of first extraction cannot cause loss of the component to be extracted later.
  • Determination of dry weight of algae cells Take 50 ml of culture medium during microalgae culture, centrifuge at 8000 rpm for 10 minutes, wash the algae after centrifugation 3 times with deionized water, and transfer to a weighing bottle (Wi (g)). Dry in a 105 °C oven to constant weight W 2 (g).
  • the feed medium includes nutrient salts such as organic carbon sources (such as sodium acetate), nitrogen sources (eg, CaN0 3 , KN0 3 ), plant growth hormones, and inorganic salts, and the additional nutrient salts are concentrated after the corresponding
  • the medium is used to promote the growth of the microalgae, and the contents of carbon, nitrogen, phosphorus and magnesium in the fermentation broth are monitored in time, and the contents of the four substances in the feed medium are appropriately adjusted (carbon: 0.5-50 mM, nitrogen: 0.5-10 mM, Phosphorus: 0.01-0.5 mM, magnesium: O.OOOOl-O.OOlmM), to ensure the concentration of the four substances in the fermentation broth is stable.
  • the control conditions are changed, and the three nutrients of carbon, nitrogen and phosphorus in the culture solution are basically consumed, and the heterotrophic culture stage is finished.
  • other operations and experimental conditions are the same, and only the medium does not contain phytohormone.
  • unoptimized control only the pH in the fermentation broth is monitored, and the pH is kept constant at 7-8 by the feed medium. The contents of other substances such as carbon, nitrogen, phosphorus and magnesium are not controlled, and hormonal substances are not controlled. Also not added, other experimental conditions and operations are the same.
  • the high-density algae solution 8.5 g/L in the heterotrophic culture was placed in 1 L, diluted to 1.3 g/L with a light-inducing medium, and added to the above-mentioned photoinduction medium, and transferred to a 2 L column photobioreactor. Perform light-induced culture.
  • Light-induced culture conditions The temperature is maintained at 28 ⁇ 38 °C, the air flow is lwm, natural light, and the light intensity per side is about 75klx.
  • Fig. 2 shows the light-induced culture process of Haematococcus pluvialis algae in the heterotrophic culture solution in which all three nutrients of carbon, nitrogen and phosphorus have been consumed in an outdoor 2L column photobioreactor.
  • the dry weight of the cells reached 1.92 g/L, and the astaxanthin increased from 2.67 mg/g D CW at the initial induction to 22.56 mg/g D CW (the astaxanthin content increased by about 8.5 times), and the astaxanthin was induced by light for 3 days.
  • the yield was 82.24 mg / L / d (for the current microalgae photoautotrophic production of astaxanthin yield of 3.57 times the highest yield of 23.04 mg / L / d) (see Figure 2).
  • Figure 3 shows the light-induced culture process of Haematococcus pluvialis in an outdoor 2L column photobioreactor when the three nutrients of carbon, nitrogen and phosphorus have not been consumed in the heterotrophic culture solution.
  • the dry weight of the cells reached 2.12 g/L
  • astaxanthin increased from 2.67 mg/g Dcw at the initial induction to 6.51 mg/g Dcw (the astaxanthin content only increased by about 2.4 times)
  • the shrimps were induced by light for 3 days.
  • the yield of chlorophyll was 22.51 mg/L/d (only 27% of the algae-inducing effect of the three nutrients of carbon, nitrogen and phosphorus after heterotrophic end). It is concluded that carbon and nitrogen in the heterotrophic culture solution Whether the three nutrients of phosphorus are consumed or not is very important for the improvement of astaxanthin yield during the induction process.
  • the heterotrophic and feed medium used contains:
  • the light-inducing medium used contains:

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Abstract

本发明涉及一种利用微藻高效生产虾青素的新方法，该方法包括微藻异养培养、稀释、光诱导培养、藻细胞采收以及虾青素提取等步骤。本发明方法充分发挥了微藻在异养阶段快速生长的优势以及由异养培养所获得的大量藻细胞在光诱导阶段大量积累虾青素的优势，可极大地提高微藻生产虾青素的效率，实现低成本、高效率及大规模培养微藻生产虾青素，不仅为解决源于微藻的虾青素大规模产业化提供重要的技术手段，而且为虾青素的广泛应用提供了充足的原料保障。

Description

一种利用微藻高效生产虾青素的新方法 技术领域
本发明属于微藻生物技术领域， 涉及一种培养微藻生产虾青素的方法。 背景技术
虾青素 (Astaxanthin), 化学名称为 3，3'-二羟基 -4,4'-二酮基 -β,β'-胡萝卜素， 分子式为 C40H52O4, 相对分子质量为 596.86， 又名虾黄质、 虾黄素或龙虾壳色素， 是一种酮式类胡 萝卜素。 色泽为粉红色， 具脂溶性， 不溶于水， 易溶于氯仿、 丙酮、 苯和二硫化碳等有机 溶剂。虾青素的化学结构是由 4个异戊二烯单位以共轭双键型式连结，两端又有 2个异戊 烯单位组成六节环结构，其化学结构见下图。 由于虾青素的化学结构中含有一个长的共轭 不饱和双键系统， 因 构。
Figure imgf000002_0001
虾青素是类胡萝卜素的一种， 也是类胡萝素合成的最高级别产物， β-胡萝卜素、 叶黄 素、 角黄素、 番茄红素等都是类胡萝卜素合成的中间产物。 因此， 在自然界中， 虾青素具 有最强的抗氧化性。天然虾青素是迄今为止人类发现自然界中最强的抗氧化剂，其抗氧化 活性远远超过现有的抗氧化剂， 被誉为 "超级氧化剂"。虾青素具有广泛的应用价值， 不仅 可以用作水产养殖的饲料添加剂和人类食品添加剂，在药品、化妆品和营养保健品等领域 也具有很大的应用潜力。
相比较而言， 利用微藻生产虾青素具有明显的优势。 雨生红球藻含有约占细胞干重 1-5%的虾青素， 是自然界中虾青素含量最高的天然物种。 首先， 虾青素在微藻中主要是 以单酯的形式存在， 其结构为反式结构， 较化学合成的顺式结构生物利用度高； 其次， 微 藻的生长周期短、 生产设备占地面积小， 产品质量和产量相对稳定； 最后， 微藻（如雨生 红球藻等）本身就是一种高价值的产品， 含有大量的蛋白质、 油脂、 多糖等活性成分， 可 以分离提取这些物质， 实现微藻细胞的综合利用。
至今， 利用微藻产虾青素的培养模式主要有光自养和异养两种。
微藻光自养培养的缺点是微藻细胞生长慢、细胞密度低及虾青素产率低。 目前光自养 培养微藻的最大细胞干重为 Ranjbar等在 16升鼓泡柱式反应器中达到的 6.8g/L (细胞产 率为 0.2g/L/d) ( Ranjbar R， Inoue R， Shiraishi H， Katsuda T， Katoh S: High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor. Biochemical Engineering Journal 2008, 39(3):575-580. ) 。 虫下青素最 高体积产率为 Ranjbar 等在 1L 气升式光生物反应器中培养雨生红球藻所获得的 23.04mg/L/d ( Ranjbar R， Inoue R， Katsuda T， Yamaji H， Katoh S: High efficiency production of astaxanthin in an airlift photobioreactor. Journal of Bioscience and Bioengineering 2008， 106(2):204-207. ) ， 虾青素的最高面积产率为 Olaizola等在 25，000L户外光反应器中培养 所达到的 390mg/m2/d，但其细胞产率仅为 0.052g/L/d( Olaizola M: Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. Journal of Applied Phycology 2000 , 12(3):499-506. ) 。 此外， 文献可见的最大细胞产率为 Garcia-Malea MC等在室内 1.8L鼓泡柱式反应器中达到的 0.58g/L/d和室外 220L管式反应 器 （气升式） 中达到的 0.68g/L/d， 以上是在连续培养条件下得到的， 该条件并不利于虾 青素的积累 ( Garcia-Malea MC, Acien FG, Fernandez JM, Ceron MC, Molina E: Continuous production of green cells of Haematococcus pluvialis: Modeling of the irradiance effect. Enzyme and Microbial Technology 2006, 38(7):981-989. ) 。
目前，根据雨生红球藻光自养时的不同存在形态，一般将微藻光自养过程分为微藻培 养和虾青素积累两个阶段。前一阶段（微藻培养）主要进行雨生红球藻的培养， 使其快速 生长， 该阶段现有的培养模式有两种： 即所谓的连续培养和半连续培养。连续培养是指在 稳定的培养液条件下进行微藻培养，使雨生红球藻在保持恒定的生长速度和稳定的生理特 性的条件下连续生产；半连续培养则指在培养藻细胞达到一定浓度后，每天取出部分藻液 转入胁迫环境， 并补充等量的新培养液继续培养。第二阶段（虾青素积累）是通过诸如高 光照、 高温、 高盐、 营养盐饥饿等一系列胁迫手段， 促使雨生红球藻在恶劣的生存环境下 转变为厚壁孢子， 以达到积累虾青素的目的。在这两个阶段中， 微藻所需的营养及环境条 件不同，目前国内外研究主要集中在这两个阶段的条件选择和控制以及环境因子的影响等 方面。通常情况下，光自养培养第一阶段并不积累虾青素， 目的在于增加细胞数目及重量， 当到达指数生长期末期时 (此时细胞密度 0.5〜1.5g/L， 细胞数目 20〜50万 /ml)， 由于氮、 磷 等营养盐的消耗，光自养细胞不经稀释等操作而直接转入第二阶段，此时辅以强光、高温、 高盐等胁迫条件且添加氮磷缺乏的培养基，促进虾青素的积累，此阶段细胞数目不再增加， 有时随着胁迫条件的恶劣程度增加， 细胞数目下降， 但由于细胞孢子化及膨大， 细胞重量 缓慢增加， 胁迫培养结束与开始时相比， 单位体积培养液中的细胞重量增加 2〜4倍， 达 到约 2〜3g/L。虾青素积累阶段培养基和光自养培养基不完全相同， 后者 N、 P丰富且要求 各元素间配比合理 （必需碳、 氮、 磷、 硫， 钠、 钙、 钾、 镁等元素） ， 前者仅需添加钙盐 等少数盐类物质且一般缺乏氮、 磷。
影响雨生红球藻光自养培养的物理环境因子及营养主要包括温度、 光强、 pH值、 溶 解氧及营养盐含量等参数。 国内外文献对此已有相当多的报道， 如表 1所示。
表 1 不同阶段对环境因子需求
Figure imgf000004_0001
已有研究表明，雨生红球藻不同品系间虽然存在差别，但其营养细胞最适生长温度为
15〜25 °C， 最适光强为 30〜50μηιο1 ηι - 2 s— 最适 pH为中性至微碱性， 可利用 NaAc进行 混合营养生长。 而高光照、 高温、 营养盐 (氮、 磷)饥饿、 盐胁迫 (NaCl、 NaAc等)和氧化 压力 (活性氧、 氧自由基和溶解氧)等许多环境条件都可诱导细胞内虾青素的积累， 它们统 称为诱导条件或胁迫条件，无一例外的都是细胞生长和分裂的抑制条件，并具有协同作用。
传统的光自养两阶段培养系统无法克服诸如产量低、易污染、受季节变化影响大、 占 地面积大、 成本高等难题。
光自养培养的细胞密度不高，在于雨生红球藻对培养的理化生条件要求相当苛刻，无 法使其长期处于营养细胞的状态。虽然孢子状态的细胞自身重量能缓慢增加，但它不再进 行营养生殖， 细胞群体数目不能迅速增加。 因而限制了光自养时所能达到的最大细胞量。 雨生红球藻对环境变化非常敏感，指数生长期短，在营养生长期内抵抗细菌和原生动物污 染的能力很差，而在极端环境下则又失去繁殖能力，不易建立稳定、高效的培养技术体系。 因此，培养雨生红球藻生产虾青素，在藻种、光生物反应器的设计、细胞高密度培养条件、 虾青素积累的生态调控技术等方面具有相当的难度。
目前， 国际上成功的生产模式都采用了两阶段方式， 即先采用封闭式光生物反应器培 养系统实现细胞的高密度营养生长、且可克服污染问题，再采用常规的开放池系统在胁迫 条件下使细胞积累虾青素。当前世界上只有美国的 Cyanotech和 Aquasearch等几家大公司 能够实现雨生红球藻的规模化培养。
尽管雨生红球藻能够生存的温度范围比较广，但是适宜该藻生长和虾青素累积的温度 范围相差较大， 对于雨生红球藻生长而言， 15〜25 °C是比较适宜的； 对于红球藻虾青素累 积而言， 25〜35 °C较为理想。
培养微藻生产虾青素产业之所以能够在美国夏威夷等少数地区形成，重要原因之一就 是能够相对容易地控制培养过程中的温度， 夏威夷地区地处热带、光线充足、温度相对较 高， 对于虾青素累积过程来说， 温度是非常适宜的。尽管这种较高的气温不利于雨生红球 藻的快速生长繁殖， 但是该地区有特殊的地理条件， 可以很方便地获得降温用的冷海水， 因此控制温度培养雨生红球藻也不是问题。事实上 Cyanotech和 Aquasearch等公司就是采 用了从深海 （600m) 抽取冷海水， 对雨生红球藻培养过程进行经济有效的温度控制。 受 地理条件限制， 该方法到目前为止还不适合中国国情。
雨生红球藻生长条件相对温和，很多种敌害生物比如轮虫、原生动物和其它微型藻类 都能在雨生红球藻培养基中生长繁殖，生物污染防治成为该藻规模化培养时很难克服的问 题。 早期实验表明， 开放池培养过程中， 大约 4〜5 天左右培养液中就会出现吞食红球藻 的轮虫， 随后导致整个培养失败。而如果藻细胞完全转化为不动孢子则其抵抗敌害生物的 能力大大增强。
雨生红球藻生长缓慢、易受污染，且生长适宜温度较低等特点使得雨生红球藻的大规 模高密度培养受到限制。为进行藻细胞的大量生产， 国外学者普遍采用封闭式光生物反应 器。国外学者关于利用封闭式光生物反应器培养雨生红球藻的研究报道已有不少，且主要 集中在柱式、平板式和管式等 3大类型，其研究重点已从反应器的应用转移至反应器的结 构改进以及培养参数 (通气速率、 质量传输速率研究等)的优化。 但存在反应器温度及光强 难以控制， 反应器清洗及放大困难， 维护成本高等一系列问题。 因此有必要开展利用现有 成熟的发酵工业设备进行雨生红球藻高密度培养方面的研究。
另一方面，微藻异养培养虽然存在细胞内虾青素及叶绿素等色素含量低等缺点，但其 优点是微藻能够在发酵罐中进行高密度培养，细胞生长速率较快。异养培养能够获得高细 胞密度和高细胞生长速度， 文献报道的最高藻细胞干重为 7g/L ( Hata N， Ogbonna JC， Hasegawa Y， Taroda H， Tanaka H: Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture. Journal of Applied Phycology 2001， 13(5):395-402 ) ， 细胞产率为 0.3g/L/d， 但其虾青素产率较低 （仅为 4.4mg/L/d) ， 光胁迫 8天后虾青素含量 1.85%。异养培养在装液 2.3L的发酵罐中进行。光自养培养则在室内的 玻璃容器 （直径 16cm， 装液 900ml， 液位深度 5.5cm) 中的进行， 自上而下照射人工光， 液面处光照强度 （950μηιο1 ηι- 2 s— 。 控制温度为 30°C。 通过磁力搅拌 （lOOrpm) 来实现 混合， 向藻液所通气体为含 5%C02的空气， 通气量为 0.22wm。 它虽采用了异养-光自养 的两段模式， 但主要存在以下 4方面问题：
( 1 ) 该文献开展了补料分批 （fed-batch) 、 重复补料分批 （repeated fed-batch) 培养 的研究，所获得的最大细胞干重 7g/L是在补料分批培养时得到，但异养阶段延滞期较长， 平均细胞生长速率低 （约为 0.3g/L/d) 。
( 2 ) 为防止异养培养基中的醋酸钠等有机物在光自养时滋生大量细菌， 该工艺采用 在放出前一天进行饥饿处理的方法以消耗醋酸钠，但尚未考虑培养液中氮、磷的含量是否 消耗完， 细胞经此种处理后再进行光自养， 会造成细胞死亡和虾青素积累缓慢。
( 3 ) 异养过程， 选择的培养基为普通的 basal medium, 未添加促进生长的植物生长 激素类物质， 培养过程通过间歇流加未经优化的补料培养基来控制 pH为 7.5-8.0， 这一方 法未考虑雨生红球藻异养与普通光自养在营养需求上存在的差异，造成培养基对细胞的培 养效果不佳， 细胞生长缓慢； 此外， 间歇补料造成 pH震荡和培养基各成分浓度存在较大 波动 （如补料培养基组分不当， 在培养后期造成氮、 磷、 镁 3种元素缺乏） ， 极易对细胞 生长造成不良影响；特别是培养后期为补充上述 3种元素，而需要打开罐体进行额外的补 料操作， 大幅提高了培养物的染菌风险。
(4) 异养转为光自养时采取的是放罐藻液不添加培养基、 不进行稀释而直接进行光 自养培养， 这样会存在二个问题： 1 ) 一是细胞会大量死亡： 由于藻液未进行稀释到较低 的密度而维持异养培养结束时的细胞密度 5.5g/L，高密度光自养时由于藻细胞的自遮蔽效 应， 大量细胞无法受到充分的光照， 以致于藻细胞大量死亡， 由初始光自养时的 65万个 细胞 /ml降为光自养结束时的 21万个细胞 /ml， 藻细胞数量损失约达 70%; 2)二是细胞内 的虾青素含量提高幅度有效： 由于未经稀释而直接采用原液进行光自养培养，而雨生红球 藻异养和自养对于营养需求不一样，导致藻细胞中虾青素含量升高幅度不大，经过 8天诱 导， 藻细胞中的虾青素含量从 0.57%升高至 1.85%。
由上述可见， 该文献报道的最高藻细胞干重为 7g/L， 细胞平均生长速率为 0.3g/L/d; 虾青素产率较低（仅为 4.4mg/L/d)，虾青素含量不高（光胁迫 8天后虾青素含量仅 1.85%)。 该方法与传统的光自养两阶段培养产虾青素相比不具有优势。因此，有必要寻找微藻产虾 青素的高效培养方法。
微藻除异养培养和光自养培养模式外， 还有一种不常用的培养模式即混合营养培养。 但该培养模式只能在可蒸汽灭菌的封闭式光生物反应器中进行，且培养过程必须保证绝对 的无菌， 同时需要光源的合理配置， 这在实际生产中无法实现。 因此， 利用混合营养模式 培养微藻生产虾青素不具有产业化价值。
由上述可见，无论是采用光自养培养模式还是异养培养模式，较低的胞内虾青素含量 和虾青素产率， 同时加上微藻大规模培养较高的成本，制约了应用微藻培养来生产虾青素 的产业化进程。因此，有必要探索新的微藻培养工艺或方法使虾青素产率及含量大幅度提 高， 同时又使微藻大规模培养的成本大幅度下降，这样才能够满足利用培养微藻大规模生 产虾青素的要求。
综合上述几种产虾青素微藻培养模式的优缺点，本发明设计了一种用于产虾青素微藻 培养的 "异养-稀释-光诱导"串联培养模式， 其流程如下： （1 ) 首先利用生物反应器异养 培养可产虾青素的微藻以获得高密度细胞； （2) 待培养液中有机碳源和氮源等营养几乎 消耗完毕后， 及时用不含有机碳源的培养基稀释藻液； （3 ) 经光诱导， 使藻细胞内的虾 青素快速大量积累。 该模式中的异养阶段是在摇瓶、机械搅拌式、气升式、鼓泡式等可异 养培养的生物反应器中进行， 目的是为了在短时间内获得较高密度的藻细胞；光诱导阶段 可在任何可用于微藻光自养培养的系统中进行，目的是通过光诱导作用提高藻细胞内虾青 素含量；异养阶段与光诱导阶段分别独立进行，异养阶段放出的藻液视其细胞密度及其中 的营养成分高低、户外光照强度高低等因素，考虑是否用光诱导培养基进行稀释后再转入 光诱导培养阶段。通过稀释作用可以确保光诱导阶段的藻细胞能获得充足的光照， 同时使 其中的营养盐成分变低造成营养胁迫， 从而实现胞内虾青素含量的快速提升。
本发明将微藻培养法生产虾青素的过程分成为以快速获得高密度细胞为目的藻细胞 异养生长培养、以降低藻细胞密度和造成营养胁迫为目的的稀释、和以提高微藻胞内虾青 素含量同时进一步提高微藻细胞量为目的光诱导培养三个阶段， 即异养培养、稀释和光诱 导培养。通过异养培养可在短时间内获得大量的可积累虾青素的微藻细胞，藻液稀释后转 入光诱导培养， 藻体内虾青素含量迅速提升至初始的数倍以上。 本发明具有如下优势：
( 1 ) 异养培养能使雨生红球藻长期处于营养繁殖的阶段， 异养阶段细胞产率高， 在 一个具体的实施例中， 平均细胞产率 1.53g/L/d， 最高可达 5.74g/L/d， 异养阶段终细胞密 度可高达 26.01g/L， 所以光诱导的藻细胞密度很高 （2〜10g/L) ， 是常规光自养培养藻细 胞密度 （约 0.2〜2g/L) 的 5〜10倍；
(2) 异养放出的藻细胞用诱导培养基进行稀释后可以直接进行胁迫诱导以积累虾青 素， 不需要适应期或者过渡期， 因而光诱导时间可以较短 （约 5c！〜 7d) ； 而传统的雨生红 球藻光自养培养时间很长（约 14c！〜 30d) ， 本发明中光诱导阶段与传统的光自养培养过程 中虾青素积累阶段相同的是诱导结束时细胞干重均可增加，因而此阶段单位藻液体积虾青 素体积产率较传统光自养可提高数倍以上；
(3 ) 相对于微藻光自养培养， 光诱导时较高的藻细胞密度使得光诱导所需的占地面 积很小 （即虾青素的面积产率高） ， 同时高细胞密度使得采收成本大幅度降低；
(4) 异养培养几乎不受气候、 天气的影响， 光诱导培养可以在玻璃房内进行， 光源 可用自然光照或人工光照， 同时光诱导的温度范围较宽 （15〜35°C ) ， 高温可以促进虾青 素的积累， 低温条件虽不促进虾青素的积累， 但由于诱导培养面积小， 仍可以通过人工升 温、 设置高盐、 高碳氮比、 强光等协同胁迫条件实现虾青素的快速积累。 因此， 采用本发 明的方法可以实现虾青素的大规模连续生产；
(5 ) 由于异养培养结束时的细胞绝大多数已经成为孢子态， 其抗逆性较强； 且经稀 释后的藻液密度仍然较高， 具有种群优势， 所以在进行光诱导时， 不易受原生动物、 杂藻 等敌害生物的污染，传统两阶段光自养培养中常见的原生动物污染及恶劣胁迫条件导致的 细胞量损失在此发明中可以降到最低。
综上所述， 本发明的 "异养-稀释-光诱导"串联培养模式合理地结合了异养和光诱导 培养两种方式的各自优势， 与其它模式相比， 具有生产效率高、培养系统的组合方式灵活 和生产成本低等优点，可充分发挥异养培养模式获得高密度藻液和光诱导阶段虾青素快速 积累的优势， 为解决源于微藻虾青素的大规模产业化问题提供了重要的技术手段。
通过对国内外已申请的相关专利分析表明，这些专利大都集中于培养的光生物反应器 及装置、 雨生红球藻光自养培养基、 虾青素的提取新方法、 雨生红球藻光自养培养基等。 涉及培养的绝大多数为光自养。 然而， 雨生红球藻 Haematococcus pluvialis) 、 小球藻 ( Chlorella zofingiensis) 等可以产虾青素微藻的 "异养-稀释-光诱导" 串联培养高产虾青 素方面的专利尚未检索到。 发明内容
本发明一方面提供一种快速培养微藻积累虾青素的 "异养-稀释-光诱导" 串联培养 的新方法，该方法包括微藻的异养培养步骤，将所获得的微藻异养培养液进行稀释的步骤， 和光诱导培养的步骤。
本发明另一方面提供一种快速提高微藻中虾青素含量的方法，该方法包括微藻异养的 步骤， 将微藻的异养培养液稀释后进行光诱导培养的步骤。
本发明同时还提供一种虾青素生产方法，该方法包括微藻异养的步骤，将微藻的异养 培养液稀释后进行光诱导培养的步骤， 以及藻细胞采收、 虾青素分离提取的步骤。
在本发明的方法中， 还可直接对异养培养获得的微藻细胞进行光诱导培养。
本发明的方法能够实现胞内虾青素的快速积累，显著提高了生产效率， 降低了生产成 本， 且提供了高品质的虾青素。
在本发明的方法中， 异养培养微藻过程中， 通过补料控制 pH恒定以及碳、氮和 /或磷 等元素稳定在一定浓度范围内， 且异养培养结束时碳、 氮和 /或磷等营养成分的浓度较低 甚至为零。
在一个实施方式中， 异养培养结束时， 培养基中碳、 氮和 /或磷等营养成分耗尽。 在一个实施方式中， 异养过程中， 通过补料将藻液的 pH控制在 4.0-10.0的范围内的 一个恒定值， 例如 pH 7.5。 通常将藻液的 pH控制在 5.0-9.0范围内的一个恒定值， 更优 选控制在 7.0-8.0的范围内。
应理解， pH值的些许变动是允许的。 例如， 可允许 pH有士 Y的变动， 其中 Y 1.0， 例如 Y 0.2、 Y^O.l o 在某些实施例中， Y=0。 因此， 在一个具体实施例中， 通过补料将 藻液的 pH控制为 Χ±Υ， 其中， 该 士 Υ 9.0。例如， 在本发明的一个实施方式中， 通过补料将藻液的 pH控制在 7.5 ± 0.3的范围之内。
异养培养微藻过程中， 通过补料控制碳、 氮和 /或磷等元素稳定在一定浓度范围内。 例如， 可通过补料将藻液中碳的含量控制在 0.5-50mM 的范围内， 氮的含量控制在 0.5-10mM的范围内， 磷的含量控制在 0.01-0.5mM的范围内。
在一具体实施例中，通过补料控制碳、氮和磷三种元素稳定在一定浓度范围内。例如， 可通过补料将藻液中碳的含量控制在 0.5-50mM的范围内， 氮的含量控制在 0.5-10mM的 范围内， 磷的含量控制在 0.01-0.5mM的范围内。
在一个具体实施例中，还包括通过补料将藻液中镁的含量控制在 0.00001 -0.00 ImM的 范围内。
在一个具体实施方式中， 所述的微藻选自雨生红球藻 Haematococcus pluvialis) ， 小球藻 ( Chlorella zofingiensis) 等。
在一个具体实施方式中， 所述微藻异养培养的步骤包括： 在生物反应器中加入 pH为 4.0〜10.0的培养基， 按工作体积的 0.1〜50%接入微藻藻种进行分批培养、 补料分批培养、 重复分批补料培养、 半连续培养或连续培养， 培养温度为 10〜40°C， 控制 pH小于 10.0， 控制溶氧在 0.1 %以上。
在一个具体实施方式中， 微藻异养培养液的稀释是采用光诱导培养基将异养获得的 藻液稀释至细胞密度为 0.1〜20克 /升、 pH为 4.0〜9.0。
在一个具体实施方式中，所述光诱导培养包括将稀释后的藻液转入光诱导装置中进行 光诱导， 连续光照或间歇光照， 培养温度为 5〜50°C， 光照强度为 0.1〜150klx， 光诱导培 养周期为 1〜480小时。
在一个具体实施方式中， 异养培养基含有氮源、有机碳源、 少量无机盐、植物生长激 素、微量元素和水或由这些成分组成； 光诱导培养基含有植物生长激素、氮源、无机盐和 水或由这些成分组成。
在一个具体实施方式中， 当产虾青素藻种为雨生红球藻时，异养所使用的培养基基本 上由以下成分组成： 醋酸钠 0.1〜5.0克 /升， NaN03 0.05〜1.5克 /升 CaCl2*7H20 0.05〜1.5克 / 升、 KH2PO40.01〜1.5克 /升、 MgS04*7H20 0.01〜1.0克 /升、 FeS04*7H20 0.01〜0.05克 /升、 植物生长激素 0.001-35毫克 /升、 微量元素 0.5〜4毫升和水。
在一个具体的实施方式中， 所述异养步骤在摇瓶、机械搅拌式、气升式或鼓泡式可异 养培养的生物反应器中进行，所述光诱导培养步骤在摇瓶或敞开式的跑道池或圆池、封闭 式的平板式光生物反应器或管道式光生物反应器或柱式光生物反应器或薄膜立袋与吊袋 光生物反应器等任何可用于微藻光自养培养的装置中进行， 光源为自然光或各种人工光。
在一个具体实施方式中， 采用超临界 co2萃取法、 有机溶剂提取法或超声辅助溶剂 提取法提取虾青素。
在一个具体实施方式中，本发明的方法还包括：对诱导后的藻细胞进行固液分离（即 采收） ， 对所获得的藻细胞进行干燥获得含有虾青素的藻粉。
在一个具体实施方式中， 本发明的方法还包括： 将提取虾青素后的藻体与其它色素 混合进行干燥制成藻粉， 或对藻体中的其它生物活性物质进行分离提取。
在一个具体实施方式中， 所述其它色素包括叶绿素。在一个具体实施方式中， 所述生 物活性物质包括蛋白质、 油脂、 叶绿素和多糖。 附图简述
图 1显示雨生红球藻在 5L生物反应器异养培养过程（含本发明异养培养过程最佳生 长过程、 只控制异养 pH但未优化初始及补料培养基策略下的生长过程、 以及只是单独控 制异养 pH和优化初始及补料培养基策略，但是未加入植物生长激素下的生长过程数据比 较） 。
图 2显示异养培养液中碳、 氮、 磷 3种营养消耗完毕的雨生红球藻藻液在户外 2L柱 式光生物反应器内光诱导培养过程。
图 3显示异养培养液中碳、氮、磷 3种营养成分未消耗完毕的雨生红球藻藻液在户外 2L柱式光生物反应器内光诱导培养过程。 具体实施方案
适用于本申请的微藻包括那些可以合成虾青素且能进行异养培养的微藻，包括但不限 于雨生红球藻 (Haematococcus pluvialis) ， 小球藻 ( Chlorella zofingiensis)等。 在优选的 实施方式中， 本发明采用雨生红球藻 Haematococcus pluvialis 来生产虾青素。
用于本发明培养基（包括异养培养基和光诱导培养基）的植物生长激素包括但不限于
2，4-二氯苯氧乙酸、 苄氨基嘌呤、 外源赤霉素、 3-吲哚丁酸、 萘乙酸和芸苔素等。 培养基 中可含有一种或多种植物生长激素。 培养基中植物生长激素的总含量可为 0.001-35毫克 / 升培养基，通常在 0.001-20毫克 /升，更通常为 0.001-15毫克 /升、 0.005-10毫克 /升、 0.01-10 毫克 /升、 0.1-5毫克 /升不等。
在具体实施例中， 各植物生长激素若存在， 其浓度可以是， 例如 2，4-二氯苯氧乙酸
0.001-5毫克 /升， 苄氨基嘌呤 0.001-5毫克 /升， 外源赤霉素 0.001-5毫克 /升， 3-吲哚丁酸 0.001-5毫克 /升， 萘乙酸 0.001-5毫克 /升， 芸苔素 0.001-5毫克 /升。 各植物生长激素的浓 度各自优选为， 例如 0.01-4毫克 /升、 0.1-4毫克 /升、 0.3-4毫克 /升、 0.3-3毫克 /升、 0.5-2.5 毫克 /升不等。 可从市售途径获得所述植物生长激素，然后直接添加到本领域已知的用于异养培养和 光诱导培养可以合成虾青素且能进行异养培养的微藻的培养基中，这类培养基的例子如下 文所述。 1. 微藻在生物反应器中的高密度异养培养
此步骤的目的是为了快速获得大量藻细胞， 以供光诱导阶段积累虾青素。
可采用本领域熟知的各种培养基添加有机碳源 （如醋酸钠等） 来进行微藻异养培养。 通常， 用于本发明的异养培养基含有氮源、 有机碳源、 植物生长激素、 少量无机盐、 微量 元素和水。
这类培养基包括 C培养基 ( Ichimura, T. 1971 Sexual cell division and conjugation-papilla formation in sexual reproduction of Closterium strigosum. In Proceedings of the Seventh International Seaweed Symposium, University of Tokyo Press, Tokyo , p. 208-214. )， MCM 培养基 (Borowitzka et al, 1991)， BG-11培养基 (Boussiba and Vonshak, 1991)， BBM培 养基 （Nichols and Bold, 1969), BA 培养基 (Barbera et al.,1993). KM培养基 （Kobayashi et al.， 1991)， Z8培养基 (Renstrom et al. , 1981)， A9培养基 (Lee and Pirt, 1981)， OHM 培 养基 （Fa'bregas et al.，2000)，KMl 培养基 (Usha et al. 1999) (Garc'ia-Malea et al, 2005)， HK2 培养基 (Chen et al.， 1997)， HK3 培养基 (Gong and Chen, 1998)等。
本发明所用的 C培养基基本上由 KN03、 CaN03、 醋酸钠以及少量无机盐、 微量元素 和水组成， 在此基础上添加了一些植物生长激素。
本文所用的术语 "基本上由 ......组成"表示本发明的组合物中除了含有主要组分如
KN03、 CaN03、 醋酸钠以及少量无机盐、 微量元素和水外， 还可包含一些对于组合物的 基本特性或新的特性（即可维持微藻在较短的培养周期内细胞密度达到较高的水平， 同时 活性物质含量与常规异养培养相比有较大幅度提高）没有实质上影响的组分。本文所用的 术语"由 ......组成"表示本发明的组合物由所指出的具体组分组成， 没有其他组分， 但是 可以带有含量在通常范围内的杂质。
在该培养基中，培养基的各组分可在一定范围内变化而不会对微藻细胞密度和品质有 很大的实质影响。 因此， 这些组分的用量不应受实施例的严格限制。如本领域技术人员所 熟知的， 培养基中还可加入少量无机盐， 例如硫酸镁、 氯化钙、硫酸亚铁和磷酸盐等， 以 及少量微量元素如 Mn、 Zn、 B、 I、 M、 Cu、 Co等， 以及植物生长激素的添加， 包括单 一激素或者多种激素的组合。
在本发明中， 微量元素组分可选自 H3B03、 ZnS04-7H20、 MnCl2 H20、 NH4)6Mo70244H20、 CuS04-5H20和 CO<;N03)2_6H20中的一种或多种。无机盐和微量元素 的用量可根据常规知识确定。
在一具体实施例中， 本发明所采用的异养培养基基本上由以下成分组成：
在一个具体实施方式中， 当产虾青素藻种为雨生红球藻时，异养所使用的培养基基本 上由以下成分组成： 醋酸钠 0.1〜5.0克 /升， NaN03 0.05〜1.5克 /升 CaCl2*7H20 0.05〜1.5克 / 升、 KH2PO40.01〜1.5克 /升、 MgS04'7H20 0.01〜1.0克 /升、 FeS04'7H20 0.01〜0.05克 /升、 植物生长激素 0.001-35毫克 /升、 微量元素 0.5〜4毫升和水。
在一具体实施方式中，异养培养基中的植物生长激素含有： 2，4-二氯苯氧乙酸 0.001-5 毫克 /升、 苄氨基嘌呤 0.001-5毫克 /升、 外源赤霉素 0.001-5毫克 /升、 3-吲哚丁酸 0.001-5 毫克 /升、 萘乙酸 0.001-5毫克 /升和 /或芸苔素 0.001-5毫克 /升。
在一具体实施例中， 异养培养基中的植物生长激素含有： 苄氨基嘌呤 0.001-5 毫克 / 升和 3-吲哚丁酸 0.001-5毫克 /升。
在根据上述配方配制培养基后， 可用常规手段如酸或碱将所述培养基的 pH 调为 4.0-10.0, 并在 115〜125 °C下高压灭菌 15〜30分钟。 可采用分批、 补料分批、 半连续和连 续培养等多种方式实施异养培养。
当异养培养采用补料分批培养方式时， 将相应配制好的培养基加入到生物反应器中， 补加水至工作体积， 通常装料系数为 0.6〜0.8， 然后蒸汽灭菌 （121 °C， 维持约 20分钟） ， 当温度降至 20〜35 °C时， 按工作体积的 1〜15%接入微藻藻种开始异养培养。
在异养培养时， 即开始补料， 通过控制补料培养基的连续流加将 pH恒定在一定范围 内， 如 7.0-8.0， 在优选的实施例中， pH控制在 7.5。
补料培养基包括有机碳源（如醋酸钠） 、 氮源（如 CaN03、 KN03 ) 、 植物生长激素、 和无机盐等营养盐， 补加的营养盐是经浓縮后的上述相应培养基， 促使微藻继续生长。补 料培养基中有机碳源、氮源、植物生长激素、无机盐等添加应使得藻液中相应成分的浓度 与最初开始异养培养时的浓度相同或相近， 使藻液中碳源、氮源、植物生长激素和无机盐 的浓度能够促使微藻继续生长。当然也可根据微藻的实际生长情况对补料培养基中的相应 成分做出适当调整， 以增加或降低某种或某些成分的浓度， 从而促使微藻继续生长。
补料的同时及时监测培养液中碳、 氮和 /或磷的含量， 从而适当调整这些物质在补料 培养基中的含量， 以将藻液中碳的含量控制在 0.5-50mM (通常为 1.0-40mM、 1.0-30mM、 1.0-20mM、 1.0-lOmM 等） 的范围内， 氮的含量控制在 0.5-10mM (通常为 0.5-8mM、 0.5-6mM、 1.0-6mM、 1.0-5.0mM 等） 的范围内， 磷的含量控制在 0.01-0.5mM (通常为 0.01-0.4mM、 0.05-0.3mM、 0.05-0.2mM、 0.05-O. lmM等） 的范围内， 从而保证藻液中这 些物质浓度稳定。更优选的， 同时监测藻液中镁的含量， 并适当调整补料培养基中镁的含 量， 以将藻液中镁的含量控制在 0.00001-O.OOlmM (通常在 0.00001-0.0008mM、 0.00003-0.0005mM等） 的范围内。
流加至一定阶段， 微藻细胞密度达到所需值时， 更改控制条件， 使培养液中碳、氮和 /或磷等营养基本消耗完毕， 异养培养阶段结束。 通常， 异养培养结束时， 培养基中碳、 氮和 /或磷等营养成分的浓度较低。 例如碳和氮的浓度低于 0.1mM、 低于 0.05mM、 低于 O.OlmM, 甚至更低， 甚至为零； 磷的浓度低于 0.005mM、低于 0.003mM、低于 O.OOlmM, 甚至更低， 甚至为零。
无论采用何种培养方式，在培养过程中，须严格控制适合的培养条件使微藻正常生长。 通常， 控制温度为 20〜35 °C， 例如 25〜30°C， 通过调节通气和搅拌控制溶氧不低于 5%的 空气饱和浓度， pH不高于 9.0。 在优选的实施例中， 溶氧不低于 10%不高于 30%的空气 饱和浓度， pH恒定控制在 7.5-8.0， 通气量小于 0.3wm， 搅拌小于 200 rpm。
异养可以在摇瓶、 机械搅拌式、 气升式、 鼓泡式等可异养培养的生物反应器中进行。
2. 高浓度藻液的稀释
此步骤的目的之一是为了降低藻细胞密度，使转入光诱导培养的产虾青素微藻高效地 吸收光能， 提高光能利用效率； 目的之二是为了调整诱导培养液中营养成分， 造成营养胁 迫， 以利于虾青素的快速积累。
异养培养获得的高密度藻液（在敞开式反应器诱导时， 宜不含有机碳源， 这样可避免 光诱导阶段滋生过多的杂菌； 但在封闭式光生物反应器诱导时， 可含有机碳源， 促进细胞 量增加）应进行稀释操作， 用稀释专用培养基对高密度的藻液进行稀释， 使细胞密度维持 在 0.1〜20克 /升， pH为 4.0〜10.0。 在某些实施例中， 用水和不含有机碳源的培养基对高密 度的藻液进行稀释， 使细胞密度维持在 0.1〜10克 /升， 调节 pH至 5.0〜8.0。 在其它实施例 中， 稀释藻液， 使细胞密度维持在 1〜8克 /升， 调节 pH至 5.0〜8.0。 在优选的实施例中， 使细胞密度维持在 1.0〜5.0克 /升， 通入 C02并调节 pH至 5.0〜8.0。
可采用各种已知的稀释培养基来稀释藻液。通常， 光诱导培养基含有碳源、氮源、植 物生长激素、无机盐和水或由这些成分组成，相对于异养培养基不含有或含有较少有机碳 源， 培养过程还可通入 C02。
在一个优选的具体方案中，异养培养获得的高密度藻细胞宜用不含有机碳源且氮和磷 缺乏的初始培养基进行适当稀释。
在一具体实施方式中， 稀释培养基 （光诱导培养基） 含有： MgS(V7H2O 0.01〜0.1克 /升、 NaH2PO40.01〜0.1克 /升、 KC1 0.1〜1克 /升、 CaCl2 0.01〜0.2克 /升、 FeS04'7H20 0.01〜0.06 克 /升、 EDTA 0.020〜0.052克 /升、 和植物生长激素 0.001-35毫克 /升。
在一具体实施例中， 稀释培养基 （光诱导培养基） 中的植物生长激素含有： 2，4-二氯 苯氧乙酸 0.001-5毫克 /升、 苄氨基嘌呤 0.001-5毫克 /升、 外源赤霉素 0.001-5毫克 /升、 3- 吲哚丁酸 0.001-5毫克 /升、 萘乙酸 0.001-5毫克 /升和 /或芸苔素 0.001-5毫克 /升。
在一具体实施例中， 稀释培养基（光诱导培养基）中的植物生长激素含有： 苄氨基嘌 呤 0.001-5毫克 /升和 3-吲哚丁酸 0.001-5毫克 /升。
稀释采用的培养基无需高压灭菌， 配制好后调节 pH至 5.0〜9.0即可使用。
应理解， 在某些实施例中， 不需要对异养培养所得藻细胞进行稀释， 而直接对其实施 光诱导培养， 这取决于异养培养的密度、异养培养液成分与实际诱导条件（如光强、温度 等） 间的合理匹配。 3. 光诱导培养
该步骤的目的是让产虾青素微藻接受充足的光照，通过光诱导使藻细胞快速大量合成 积累虾青素， 同时使培养液中的藻细胞浓度适当提高。
如上所述高密度微藻培养液经稀释后，将所得稀释液转入光诱导装置中进行光诱导培 养或者将微藻细胞涂覆在固体膜表面等半固态的贴壁法进行光诱导。温度控制在 5〜50°C， 光照强度为 0.1〜150klx， 连续光照或间歇光照， 光诱导培养周期为 1〜480小时， 通气量为 0.1〜2.0 wm。 其中所述的光生物反应器包括所有的封闭式光生物反应器 （摇瓶、 管道式、 平板式、 柱式、 薄膜立袋与吊袋等）和所有的开放式光生物反应器（跑道池、 圆池和鼓泡 式大盆等） 。
通常， 培养温度可控制在 15〜35 °C的范围内， 例如 18〜35 °C、 20〜35 °C、 20〜30°C等。 通常， 光照强度为 l〜70klx， 例如， 1〜60、 1〜50、 1〜40、 1〜30、 1〜20、 l〜10klx等， 可视 具体生产情况而定。通常，如通过气体造成藻液充分混合，则通气量可控制为 0.1〜2.0 wm, 例如， 0.2〜1.8、 0.5〜1.5、 0.8〜1.5、 1.0〜1.5wm等。 同时， 通入一定浓度的 C02以提供无 机碳源和控制 pH， 例如， 0.5%-10%的 C02。 在其它实施方式中， 培养温度控制在 10〜50 °C， 光照强度为 l〜10klx， 通气量为 0.05〜2.0wm。
在其它实施例中， 光诱导培养周期为 8〜480 小时， 例如， 根据实际的天气情况， 光 诱导培养周期可以为 8〜240小时、 8〜120小时、 8〜72小时、 8〜48小时、 8〜24小时不等； 或者， 光诱导培养周期可以为 12〜72小时、 12〜60小时、 12〜48小时、 12〜36小时、 12〜24 小时不等或 24〜60小时、 24〜48小时不等。
光诱导培养基选用改良的雨生红球藻光自养培养基， 包括如上文所述的稀释培养基。 在本申请中， "光诱导培养周期"包括了整个光诱导培养过程， 例如， 户外培养时光诱导培 养周期包括夜晚没有光照的时间。
在本申请中， "光照时间"指使用本申请所述光照强度对微藻实施光诱导培养的时间， 即该时间不包括夜晚没有光照的时间。 在一些实施例中， 光诱导培养步骤的光照时间为
8〜120小时， 例如 8〜72小时、 8〜36小时、 8〜24小时、 8〜18小时、 8〜12小时、 12〜36小 时、 12〜24小时不等， 以及上述范围内的任意时长。
因此， 本申请的光诱导培养步骤也包括光照时间为 8〜120 小时范围内的光诱导培养 步骤。可采用人工光照的方式进行光诱导培养，也可在户外利用自然光照的方式进行光诱 导培养。
在具体实施方式中， 当培养液中虾青素浓度达到最高时， 结束光诱导培养， 收获藻细 胞进行虾青素的分离提取或直接采收藻细胞进行藻粉制备。 4. 藻细胞采收、 虾青素分离提取及藻体综合利用
光诱导培养结束后， 对微藻进行沉降或离心采收， 获得湿藻体。藻细胞的采收方法包 括但不限于沉降、 高速离心、 絮凝， 气浮或过滤等技术； 藻细胞破壁方法包括但不限于藻 体自溶、 高压匀浆、 酶水解、 水相热解等湿法破壁方法。
采用传统的有机溶剂提取法对微藻进行虾青素的提取。首先将有机溶剂加入到藻泥中 进行萃取，然后进行搅拌离心获得上清液和藻体沉淀，对上清液进行减压浓縮、搅拌加水、 过滤获得虾青素晶体。
上清液中的其他成分可逐步分离提取获得脂肪酸、叶黄素等，或直接将上清液中的所 有成分与藻体沉淀混合喷雾干燥获得微藻粉。
在一个较佳的方案中， 采用超临界 co2萃取技术对微藻进行虾青素的分离提取。 在 一个更佳的方案中， 将获得的微藻液浓縮后直接喷雾干燥获得微藻粉。
本发明中，可对培养所得的微藻进行综合利用，提取其中的多不饱和脂肪酸、蛋白质、 叶绿素、多糖等各种活性成分。活性成分的提取顺序并无特殊限制， 但通常要满足先提取 的步骤不能导致后提取的成分损失这一前提。
本文中涉及到藻细胞干重和虾青素含量测定方法如下：
藻细胞干重测定： 在微藻培养过程中取培养液 V毫升， 8000 rpm离心 10分钟， 将离 心后的藻体用去离子水洗涤 3次， 转移至称量瓶 （Wi (克） ） 中， 在 105 °C烘箱中烘干 至恒重 W2 (克） 。 藻体干重 Cx可根据下式计算： Cx (克 /升） = ( W2-Wi ) /V/1000。
虾青素测定：采用高效液相色谱法（HPLC )，具体操作步骤见文献（J.P.Yuan, F. Chen, Chromatographic separation and purification of trans-astaxanthin from the extracts of Haematococcus pluvialis, J. Agric. Food Chem. 46 (1998) 3371-3375 ) 。 实施例 在 5L生物反应器中加入下述异养培养基和水至 2.5L后进行蒸汽灭菌，然后当温度降 到 25°C时接入雨生红球藻， 开始异养培养。 过程中通过调节通气和搅拌控制溶氧不低于 5%的空气饱和浓度。
在异养培养时， 即开始补料， 通过控制补料培养基的连续流加将 pH恒定在 7-8。 补 料培养基包括有机碳源 （如醋酸钠） 、 氮源 （如， CaN03、 KN03) 、 植物生长激素、 和 无机盐等营养盐， 补加的营养盐是经浓縮后的上述相应培养基， 促使微藻继续生长， 同时 及时监测发酵液中碳、 氮、 磷、 镁的含量， 适当调整该 4 类物质在补料培养基含量 (碳: 0.5-50mM、 氮： 0.5-10mM、 磷： 0.01-0.5mM、 镁： O.OOOOl-O.OOlmM), 以保证发酵液 中该 4类物质浓度稳定。 流加至一定阶段， 微藻细胞密度达到所需值时， 更改控制条件， 使培养液中碳、氮、磷 3种营养基本消耗完毕， 异养培养阶段结束。 而在未加激素的情况 下， 其他操作和实验条件皆相同， 仅培养基中不含有植物生长激素。而在未优化控制的情 况下， 仅监测发酵液中的 pH， 通过补料培养基保持 pH恒定在 7-8， 其他物质如碳、 氮、 磷、 镁的含量不做控制， 并且激素类物质亦不添加， 其他实验条件和操作相同。
结果见图 1。在异养培养结束时， 控制 pH和补料策略， 且加入植物激素的异养培养， 其细胞干重达到 26 g/1; 而只控制 pH和补料策略但不加入植物激素的异养培养， 其细胞 干重达到 8.7 g/l; 而未优化补料策略且不加入植物激素的异养培养， 其细胞干重仅为 4.2 g/lo 因此， 控制补料策略优化且加入植物激素的异养培养， 与单纯控制 pH但未优化补料 策略且未加植物生长激素的异养培养相比， 细胞密度提高了 6.2倍； 而与只控制 pH和补 料策略但不加入植物激素的异养培养相比， 细胞密度提高了 2.1倍。
将异养培养过程中的高密度藻液 8.5g/L带放 1L， 用光诱导培养基稀释到 1.3g/L， 并 加入上述光诱导培养基， 转入到 2L柱式光生物反应器中进行光诱导培养。 光诱导培养条 件： 温度维持在 28〜38°C， 空气流量为 lwm， 自然光照， 每侧光强约为为 75klx。
图 2显示了异养培养液中碳、氮、磷 3种营养成分全部消耗完毕的雨生红球藻藻液在 户外 2L柱式光生物反应器内光诱导培养过程，光诱导培养 3天后，细胞干重达到 1.92g/L， 虾青素从诱导初期的 2.67mg/gDCW上升至 22.56mg/gDCW (虾青素含量提高约 8.5倍） ， 以光诱导 3天计的虾青素的产率为 82.24mg/L/d (为目前微藻光自养生产虾青素最高产率 23.04mg/L/d的 3.57倍 )(见图 2)。
图 3显示了在异养培养液中碳、氮、磷 3种营养成分尚未消耗完毕的情况下， 雨生红 球藻藻液在户外 2L柱式光生物反应器内光诱导培养过程， 结果显示诱导 3天后， 细胞干 重达到 2.12g/L， 虾青素从诱导初期的 2.67mg/gDcw上升至 6.51mg/gDcw (虾青素含量仅 提高约 2.4倍） ， 以光诱导 3天计的虾青素的产率为 22.51 mg/L/d (仅为异养结束碳、 氮、 磷 3种营养成分全部消耗完毕的藻液诱导效果的 27%)。 由此得出， 异养培养液中碳、氮、 磷 3种营养成分是否消耗完毕， 对于诱导过程中虾青素产率的提高十分重要。
所用的异养及补料培养基含有：
醋酸钠 0.1〜5.0克 /升， NaN03 0.05〜1.5 克 /升 CaCl2'7H20 0.05〜1.5 克 /升、 KH2P04 0.01〜1.5克 /升、 MgS04*7H20 0.01〜1.0克 /升、 FeS04*7H20 0.01〜0.05克 /升、 苄氨基嘌呤 0.001-5毫克 /升、 3-吲哚丁酸 0.001-5毫克 /升、 微量元素 0.5〜4ml和水。
所用的光诱导培养基含有：
MgS04-7H20 0.01〜0.1克 /升、 NaH2P040.01〜0.1克 /升、 KC1 0.1〜1克 /升， CaCl2 0.01〜0.2 克 /升、 FeSO4'7H2O 0.01〜0.06克 /升、 EDTA 0.020〜0.052克 /升、 苄氨基嘌呤 0.001-5毫克 / 升、 3-吲哚丁酸 0.001-5毫克 /升。 尽管上面已经描述了本发明的具体例子，但是有一点对于本领域技术人员来说是明显 的， 即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。 因此， 所附 权利要求覆盖了所有这些在本发明范围内的变动。

Claims

1 . 一种生产虾青素或提高微藻中虾青素含量的方法， 其特征在于， 该方法包括： ( 1 ) 异养培养微藻， 和
( 2 ) 稀释所获得的微藻异养培养液， 然后向稀释藻液中添加诱导培养基后进行 光诱导培养； 或直接对异养培养获得的微藻细胞进行光诱导培养；
从而制备虾青素， 或提高微藻中虾青素的含量。
2. 一种生产虾青素的方法， 其特征在于， 该方法包括：
( 1 ) 异养培养微藻， 其中， 培养过程中， 通过补料控制 pH恒定以及碳、 氮和 / 或磷等元素稳定在一定浓度范围内， 且异养培养结束时， 培养基中碳、 氮和 /或磷等 营养成分的浓度较低甚至为零；
( 2 ) 稀释所获得的微藻异养培养液， 然后向稀释藻液中添加诱导培养基后进行 光诱导培养； 或直接对异养培养获得的微藻细胞进行光诱导培养， 以及
( 3 ) 采收藻细胞， 和分离提取虾青素。
3 . 一种提高微藻中虾青素含量的方法， 其特征在于， 该方法包括：
( 1 ) 异养培养微藻， 其中， 培养过程中， 通过补料控制 pH恒定以及碳、 氮和 / 或磷等元素稳定在一定浓度范围内， 且异养培养结束时， 培养基中碳、 氮和 /或磷等 营养成分的浓度较低甚至为零， 和
( 2 ) 稀释所获得的微藻异养培养液， 然后向稀释藻液中添加诱导培养基后进行 光诱导培养； 或直接对异养培养获得的的微藻细胞进行光诱导培养；
从而提高微藻中虾青素的含量。
4. 如权利要求 1一 3 中任一项所述的方法， 其特征在于， 所述的微藻选自可以 合成虾青素且能进行异养培养的微藻， 如雨生红球藻 Haematococcus pluvialis 、 小 球藻 ( Chlorella zofingiensis ) 等。
5 . 如权利要求 1一 4 中任一项所述的方法， 其特征在于， 所述微藻异养培养的 步骤包括： 在可异养培养的生物反应器中加入 pH为 4.0〜10.0的培养基， 按工作体积 的 0.1〜50%接入微藻藻种进行分批培养、 补料分批培养、 重复分批补料培养、 半连续 培养或连续培养， 培养温度为 10〜40 °C， 控制 pH小于 10.0， 控制溶氧在 0.1 %以上。
6. 如权利要求 1一 5 中任一项所述的方法， 其特征在于， 所述微藻异养培养液 的稀释是采用光诱导培养基或水将异养获得的藻液稀释至细胞密度为 0.1〜20克 /升、 pH为 4.0〜10.0。
7. 如权利要求 1一 6 中任一项所述的方法， 其特征在于， 所述光诱导培养包括 将稀释后的藻液或异养培养的微藻细胞直接转入光诱导装置中进行光诱导或者将微 藻细胞依附在固体膜表面等半固态的贴壁法进行光诱导， 光诱导温度为 5〜50°C， 连 续光照或间歇光照， 光照强度为 0.1〜150klx， 光诱导培养周期为 1〜480小时。
8. 如权利要求 1一 7 中任一项所述的方法， 其特征在于， 所述异养培养基含有 氮源、 有机碳源 （包括但不限于醋酸钠） 、 植物生长激素、 无机盐、 微量元素和水， 或由所述氮源、 有机碳源 （包括但不限于醋酸钠） 、 植物生长激素、 无机盐、 微量元 素和水组成； 所述光诱导培养基含有植物生长激素、 氮源、 无机盐和水， 或由植物生 长激素、 氮源、 无机盐和水组成。
9. 如权利要求 1一 8中任一项所述的方法， 其特征在于， 所述异养步骤在摇瓶、 搅拌式或气升式或鼓泡式发酵罐、带内部光源或外部光源的生物反应器中进行； 所述 光诱导培养在摇瓶、 跑道池、 圆池、 平板式光生物反应器、 管道式光生物反应器、 柱 式光生物反应器、球形光生物反应器、 薄膜立袋与吊袋等任何可用于微藻光自养或光 诱导培养的装置中进行，或者将微藻细胞涂覆在固体膜表面等半固态的贴壁法进行光 诱导； 光源可以为自然光或人工光。
10. 如权利要求 1一 9中任一项所述的方法， 其特征在于， 所述方法还包括： 对 诱导后的藻细胞进行固液分离 （即采收） ， 对所获得的藻细胞进行干燥获得含有虾青 素的藻粉。
11 . 如权利要求 1一 10 中任一项所述的方法， 其特征在于， 所述方法还包括： 对藻细胞中的虾青素进行提取，对提取虾青素后的藻体和其他色素混合后进行干燥制 成藻粉， 或对藻体中的其它生物活性物质进行分离提取。
12. 如权利要求 1一 1 1 中任一项所述的方法， 其特征在于， 采用超临界 C02萃 取法、 有机溶剂提取法或超声辅助溶剂提取法提取虾青素。
13. 一种异养培养基， 所述培养基含有氮源、有机碳源（包括但不限于醋酸钠）、 植物生长激素、 无机盐、 微量元素和水， 或由所述氮源、 有机碳源 （包括但不限于醋 酸钠） 、 植物生长激素、 无机盐、 微量元素和水组成， 用于可以合成虾青素且能进行 异养培养的微藻的异养培养。
14. 一种光诱导培养基， 所述培养基含有植物生长激素、 氮源、 无机盐和水， 或由植物生长激素、 氮源、 无机盐和水组成， 用于可以合成虾青素且能进行异养培养 的微藻的光诱导培养。
PCT/CN2013/084262 2012-07-27 2013-09-26 一种利用微藻高效生产虾青素的新方法 WO2014015841A2 (zh)

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EP13823422.4A EP2878676A4 (en) 2012-07-27 2013-09-26 PROCESS WITH MICROALGEN FOR THE HIGHLY EFFICIENT MANUFACTURE OF ASTAXANTHINE
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US14/417,166 US20150252391A1 (en) 2012-07-27 2013-09-26 Method using microalgae for high-efficiency production of astaxanthin
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BR112015001637A BR112015001637A2 (pt) 2012-07-27 2013-09-26 método usando microalgas para produção com eficiência elevada de astaxantina
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