CN104662162A - Method using micro-algae for high-efficiency production of astaxanthin - Google Patents

Abstract

Disclosed in the present invention is a method for the culture of micro-algae for the production of astaxanthin. The method comprises steps of heterotrophic culture of micro-algae, dilution, light-induced culture, collection of algae cells and extraction of astaxanthin.

Description

Method using micro-algae for high-efficiency production of astaxanthin

A kind of utilization microalgae efficiently produces the new method and technique field of astaxanthin

The invention belongs to technical field of microalga biology, it is related to a kind of method for cultivating microalgae production astaxanthin.Background technology

Astaxanthin (Astaxanthin), chemical name is 3,3'- dihydroxy -4,4'- diketos-β, β '-carrotene, and molecular formula is C₄₀H₅₂O₄, relative molecular mass is 596.86, also known as astaxanthin, ASX or Astaxanthin, is a kind of keto-acid carotenoid.Color and luster is pink, has fat-soluble, water insoluble, is soluble in the organic solvents such as chloroform, acetone, benzene and carbon disulfide.The chemical constitution of astaxanthin is to be linked by 4 isoprene units with conjugated double bond pattern, and two ends constitute six-membered ring structure by 2 iso-amylene units again, its chemical constitution See Figure.Due in the chemical constitution of astaxanthin containing conjugation unsaturated double-bond system one long, because of structure.

Astaxanthin is one kind of carotenoid, is also the highest level product of class Hu Luosu synthesis, and beta carotene, lutein, canthaxanthin, lycopene etc. is all the intermediate product of carotenogenesis.Therefore, in nature, astaxanthin has most strong inoxidizability.Natural astaxanthin is that the mankind have found antioxidant most strong in nature so far, and its antioxidation activity is described as " super oxidant " considerably beyond existing antioxidant.Astaxanthin is with a wide range of applications, and serves not only as the feed addictive and human food's additive of aquaculture, also has very big application potential in fields such as medicine, cosmetics and nutrient and healthcare products.

Comparatively speaking, there is obvious advantage using microalgae production astaxanthin.Haematococcus pluvialis contain the astaxanthin for accounting for dry cell weight 1-5%, are the natural species of content astaxanthin highest in nature.First, astaxanthin mainly exists in microalgae in the form of monoesters, and its structure is transconfiguration, and the cis-structure bioavilability compared with chemical synthesis is high；Secondly, the growth cycle of microalgae is short, production equipment floor space is small, and product quality and yield are stablized relatively；Finally, microalgae（Such as haematococcus pluvialis）Inherently a kind of product of high value, containing substantial amounts of protein, grease, polysaccharide isoreactivity composition, can realize the comprehensive utilization of microalgae cell with these materials of separation and Extraction.

So far, the training mode for producing astaxanthin using microalgae mainly has two kinds of light autotrophy and heterotrophism.

The shortcoming of microalgae light autotrophy culture is that microalgae cell growth is slow, cell density is low and astaxanthin low yield.(cell is produced the 6.8g/L that the maximum cell dry weight of current light autotrophy culture microalgae reaches for Ranjbar etc. in 16 liters of bubbling column reactors Rate is 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. ) .Blue or green element highest volume productivity is cultivated 23.04mg/L/d (Ranjbar R, Inoue R, Katsuda T, Yamaji H, the Katoh S that haematococcus pluvialis are obtained by Ranjbar etc. in 1L airlift photobioreactors under worm: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 that Olaizola etc. cultivates reached 390mg/m in the outdoor Photoreactors of 25,000L²/ d, but its cell yield is only 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. ) .In addition, the visible maximum cell yield of document is the 0.58g/L/d and outdoor 220L tubular reactors that Garcia-Malea MC etc. are reached in 1.8L bubblings column reactor indoors（Gas-lifting type）In the 0.68g/L/d that reaches, above is being obtained under continuous condition of culture, the condition is unfavorable for accumulation (Garcia-Malea MC, Acien FG, Fernandez JM, Ceron MC, the Molina E of astaxanthin: Continuous production of green cells of Haematococcus pluvialis: Modeling of the irradiance effect. Enzyme and Microbial Technology 2006, 38(7):981-989. ) .

At present, according to different existing forms during haematococcus pluvialis light autotrophy, microalgae light autotrophy process is typically divided into two stages of microdisk electrode and astaxanthin accumulation.Previous stage（Microdisk electrode）The culture of haematococcus pluvialis is substantially carried out, makes its fast-growth, the stage, existing training mode there are two kinds：I.e. so-called continuous culture and Semi-continuous cultivation.Continuously culture refers to carry out microdisk electrode under the conditions of stable nutrient solution, haematococcus pluvialis is continuously produced under conditions of the physiological property of the constant speed of growth of holding and stabilization；Semi-continuous cultivation then refers to after culture frustule reaches finite concentration, and part algae solution is taken out daily and is transferred to stressful environmental, and supplements the new nutrient solution of equivalent and continues to cultivate.Second stage（Astaxanthin accumulation）It is, by a series of coercions such as bloom photograph, high temperature, high salt, nutrition salt-hungers, to promote haematococcus pluvialis to be changed into akinete under severe living environment, to reach the purpose of accumulation astaxanthin.In the two stages, the nutrition and environmental condition needed for microalgae are different, and research both at home and abroad at present is concentrated mainly in terms of the selection of the condition in the two stages and control and the influence of envirment factor.Under normal circumstances, the light autotrophy culture first stage does not accumulate astaxanthin, purpose is to increase cell number and weight, (the now 1.5g/L of cell density 0.5 when reaching latter stage exponential phase of growth, 20 50 ten thousand/ml of cell number), due to nitrogen, the consumption of the nutritive salt such as phosphorus, light autotrophic cell is directly transferred to second stage without the operation such as dilution, now it is aided with strong light, high temperature, the culture medium that the stress conditions such as high salt and addition nitrogen phosphorus lack, promote the accumulation of astaxanthin, this phase cell number is not further added by, sometimes with the severe degree increase of stress conditions, cell number declines, but due to cellular spore and expand, cell weight is slowly increased, compared with coercing cultivation terminates when starting, cell weight in unit volume nutrient solution increases by 24 times, reach about 2 3g/L.Astaxanthin accumulation stage culture medium and light autotrophy culture medium are incomplete same, and the latter N, P are abundant and require reasonable mixture ratio between each element（Required carbon, nitrogen phosphate and sulfur, the element such as sodium, calcium, potassium, magnesium）, the former only needs to add calcium salt Deng a small number of salts substances and general lack of nitrogen, phosphorus.

The physical environment factor and nutrition for influenceing haematococcus pluvialis light autotrophy culture mainly include the parameters such as temperature, light intensity, pH value, dissolved oxygen and nutrient concentrations.Domestic and foreign literature has considerable report to this, as shown in table 1.

The different phase of table 1 is to envirment factor demand

There are some researches show although there is difference between haematococcus pluvialis different lines, its vegetative cell optimum growth temperature is

15 25 °C, most suitable light intensity be the η ι of 30 50 μ η ι ο 1-²S-optimal pH, to alkalescence, mixotrophic growth is carried out using NaAc to be neutral.And many environmental conditions such as bloom photograph, high temperature, nutritive salt (nitrogen, phosphorus) are hungry, salt stress (NaCl, NaAc etc.) and oxidative pressure (active oxygen, oxygen radical and dissolved oxygen) all can induce the accumulation of intracellular astaxanthin, they are referred to as inductive condition or stress conditions, without exception be all cell growth and division rejection condition, and with synergy.

Traditional light autotrophy two benches culture systems can not overcome yield poorly, easily pollution, by seasonal variations influenceed greatly, the problem such as floor space is big, cost is high.

The cell density of light autotrophy culture is not high, is that physics and chemistry carded sliver part requirement of the haematococcus pluvialis to culture is quite harsh, it is impossible to the state for making it be chronically at vegetative cell.Although the cell own wt of spore state can be slowly increased, it no longer carries out vegetative reproduction, and cell colony number can not increase sharply.The maximum cell amount that can be reached when thus limiting light autotrophy.Haematococcus pluvialis are very sensitive to environmental change, and exponential phase of growth is short, and the poor ability of bacterium and protozoan pollution is resisted within vegetative growth phase, and then lose fertility in extreme circumstances, are difficult to set up stable, efficient culture technique system.Therefore, Haematococcus pluvialis production astaxanthin is cultivated, there is suitable difficulty in terms of algae kind, the design of bioreactor, high cell densities condition of culture, the ecological regulation and control technology of astaxanthin accumulation.

At present, successful production model all employs two stage method in the world, first realize the high density nutrient growth of cell using closed photo bioreactor culture systems and pollution problem can be overcome, then Cellular Accumulation astaxanthin is made under stress conditions using conventional opening cell system.Cyanotech the and Aquasearch Deng Jijia major companies for there was only the U.S. on our times can realize the pilot scale culture of haematococcus pluvialis.

Although the temperature range that haematococcus pluvialis can survive is wider, suitably the temperature range difference of algae growth and Accumulation of Astaxanthin is larger, and for haematococcus pluvialis growing, 15 25 °C are than convenient；For haematococcus Accumulation of Astaxanthin, 25 35 °C ideal.

Cultivate why microalgae production astaxanthin industry can be formed in a small number of areas such as Hawaii, America, one of major reason is exactly that can relatively easily control the temperature in incubation, Hawaii area tropical, bright and clear, temperature are of a relatively high, for Accumulation of Astaxanthin process, temperature is particularly suitable for.Although this higher temperature, which is unfavorable for rain, gives birth to red ball The fast-growth breeding of algae, but there are special geographical conditions this area, can easily obtain the Mare Frigoris water of cooling, therefore control temperature culture haematococcus pluvialis are nor problem.In fact the company such as Cyanotech and Aquasearch is exactly to employ from deep-sea（Mare Frigoris water 600m) is extracted, cost-effective temperature control is carried out to haematococcus pluvialis incubation.Limited by geographical conditions, up to the present this method is also not suitable for China's actual conditions.

Haematococcus pluvialis growing condition is relatively mild, a variety of harmful organisms such as wheel animalcule, protozoan and other microalgaes can in Growth Medium For Haematococcus Pluvialis growth and breeding, biological pollution preventing and treating turn into the algae large-scale culture when be difficult the problem of overcoming.Earlier trials show, in open pond incubation, the wheel animalcule for haematococcus of eating just occurring in nutrient solution in about 45 days or so, subsequently result in whole culture failure.And its ability for resisting harmful organisms is greatly enhanced if frustule is fully converted to aplanospore.

Haematococcus pluvialis growing is slow, vulnerable to pollution, and growth preference temperature it is relatively low the features such as the large scale and high density culture of haematococcus pluvialis is restricted.To carry out a large amount of productions of frustule, foreign scholar generally uses closed photo bioreactor.Foreign scholar is existing many on the research report using closed photo bioreactor culture haematococcus pluvialis, and be concentrated mainly on pillar, the 3 big types such as flat and tubular type, the structure that its research emphasis has been transferred to reactor from the application of reactor is improved and the optimization of culture parameters (Ventilation Rate, quality transmission rate research etc.).But there is temperature of reactor and light intensity is difficult to control to, reactor cleaning and amplification are difficult, a series of problems, such as maintenance cost is high.Research in terms of carrying out haematococcus pluvialis High Density Cultivation it is therefore desirable to the existing ripe fermentation industry equipment of application.

On the other hand, although there is the low shortcoming of the pigment contents such as intracellular astaxanthin and chlorophyll in microalgae Heterotrophic culture, but its advantage is that microalgae can carry out High Density Cultivation in fermentation tank, and cell growth rate is very fast.Heterotrophic culture results in high-cell density and high vitro growth rates, and the highest frustule dry weight of document report 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. Journal of Applied Phycology 2001,13 (5):395-402), cell yield is 0.3g/L/d, but its astaxanthin yield is relatively low（Only 4.4mg/L/d), content astaxanthin 1.85% after light is coerced 8 days.Heterotrophic culture is carried out in dress liquid 2.3L fermentation tank.The glass container of light autotrophy culture then indoors（Diameter 16cm, fill liquid 900ml, liquid level depth 5.5cm) in progress, intensity of illumination at artificial light, liquid level is irradiated from top to bottom（950μηιο1 ηι-²s— .It is 30 °C to control temperature.Pass through magnetic agitation（LOOrpm) realize mixing, be containing 5%C0 to algae solution body of ventilating₂Air, throughput is 0.22wm.Though it employs the two stage mode of heterotrophism-light autotrophy, following 4 aspect problem is primarily present：

(1) document has carried out fed-batch（Fed-batch), repeated fed-batch（Repeated fed-batch) culture research, the maximum cell dry weight 7g/L obtained is obtained in fed-batch culture, but heterotrophism stage lag phase is longer, and average cell growth rate is low（About 0.3g/L/d).

(2) to prevent the organic matters such as sodium acetate in Heterotrophic culture base from growing a large amount of bacteriums in light autotrophy, the technique use and carries out the method for Nature enemy to consume sodium acetate releasing the previous day, but nitrogen not yet in consideration nutrient solution, phosphorus content whether Run out of, cell carries out light autotrophy again after such a processing, cell death and astaxanthin accumulation can be caused slow.

(3) heterotrophism process, the culture medium of selection is common basal medium, it is not added with promoting growing plants growth hormone material, incubation adds not optimized supplemented medium to control pH to be 7.5-8.0 by intermittent flow, this method does not consider haematococcus pluvialis heterotrophism with normal light autotrophy in difference present on nutritional need, cause culture medium not good to the culture effect of cell, cell growth is slow；In addition, fed-batch causes pH concussions and each constituent concentration of culture medium to there is larger fluctuation（As supplemented medium component is improper, after incubation the phase cause nitrogen, phosphorus, 3 kinds of elements of magnesium to lack）, easily cell growth has undesirable effect；Particularly late stage of culture is supplements above-mentioned 3 kinds of elements, and needs to open the extra feed operation of tank body progress, and the microbiological contamination risk of culture greatly improved.

(4) what heterotrophism switched to take during light autotrophy is to put tank algae solution directly to carry out light autotrophy culture without culture medium, without dilution, can so there are problems that two：1) one is that cell can mortality：Due to algae solution is not diluted relatively low density and cell density 5.5g/L at the end of maintaining Heterotrophic culture, because frustule is from shadowing effect during high density light autotrophy, a large amount of cells can not be by sufficient illumination, so that frustule mortality, by initial light autotrophy when 650,000 cell/ml be reduced to light autotrophy at the end of 210,000 cell/ml, frustule quantity loss about 70%;2) two be that intracellular content astaxanthin increase rate is effective：Light autotrophy culture is directly carried out using stoste due to not diluted, and haematococcus pluvialis heterotrophism and autotrophy are different for nutritional need, content astaxanthin elevation amplitude in frustule is caused less, to be induced by 8 days, the content astaxanthin in frustule is increased to 1.85% from 0.57%.

From above-mentioned, the highest frustule dry weight of document report is 7g/L, and cell average growth rate is 0.3g/L/d;Astaxanthin yield is relatively low（Only 4.4mg/L/d), content astaxanthin is not high（Light coerce 8 days after content astaxanthin only 1.85%).This method does not have advantage compared with traditional light autotrophy two benches culture production astaxanthin.Therefore, it is necessary to find the high-efficient culture method that microalgae produces astaxanthin.

Microalgae is in addition to Heterotrophic culture and light autotrophy training mode, also a kind of training mode being of little use i.e. Combined hardening model.But the training mode be able to can only be being carried out in the closed photo bioreactor of steam sterilizing, and incubation must assure that absolute sterile, while needing the reasonable disposition of light source, this can not be realized in actual production.Therefore, do not have industrialization value using mixotrophism pattern culture microalgae production astaxanthin.

From above-mentioned, either use light autotrophy training mode or Heterotrophic culture pattern, relatively low intracellular content astaxanthin and astaxanthin yield, while plus the higher cost of microalgae large-scale culture, constraining using microdisk electrode to produce the industrialization process of astaxanthin.Astaxanthin yield and content are increased substantially therefore, it is necessary to explore new microdisk electrode technique or method, while make the cost of microalgae large-scale culture significantly decline again, can so meet and utilize the requirement for cultivating microalgae and mass producing astaxanthin.

The advantage and disadvantage of several production astaxanthin microdisk electrode patterns of summary, the present invention devises a kind of " heterotrophism-dilution-photoinduction " series connection training mode for being used to produce astaxanthin microdisk electrode, and its flow is as follows：（1) it can produce the microalgae of astaxanthin to obtain high-density cells first with bioreactor Heterotrophic culture；（2) treat that the nutrition such as organic carbon source and nitrogen source is almost in nutrient solution After being exhausted, in time with the culture medium dilution algae solution without organic carbon source；（3) through photoinduction, accumulate the astaxanthin rapid, high volume in frustule.The heterotrophism stage in the pattern is can be carried out in shaking flask, mechanical agitation type, gas-lifting type, bubble type etc. in the bioreactor of Heterotrophic culture, in order to obtain the frustule of higher density in a short time；The photoinduction stage can be carried out in any system available for microalgae light autotrophy culture, it is therefore an objective to improve content astaxanthin in frustule by photoinduction effect；The heterotrophism stage is independently carried out with the photoinduction stage, the algae solution that the heterotrophism stage releases regards its cell density and nutritional ingredient therein height, the high low factor of outdoor intensity of illumination, it is considered to be transferred to photoinduction cultivation stage again after whether being diluted with light inducing culture.It may insure that the frustule in photoinduction stage can obtain the illumination of abundance by diluting effect, while making nutrition salt component step-down therein cause Nutrient Stress, so as to realize the fast lifting of intracellular content astaxanthin.

The present invention by microdisk electrode method produce astaxanthin process be divided into by quickly obtain high-density cells be the culture of purpose frustule heterotrophic growth, the dilution for the purpose of reducing algae cell density and cause Nutrient Stress and using improve microalgae intracellular content astaxanthin simultaneously further improve microalgae cell amount as purpose photoinduction culture three phases, i.e. Heterotrophic culture, dilution and photoinduction culture.The substantial amounts of microalgae cell for accumulating astaxanthin can be obtained in a short time by Heterotrophic culture, being transferred to content astaxanthin in photoinduction culture, frond after algae solution dilution is promoted to rapidly more than initial several times.The present invention has following advantage：

(1) Heterotrophic culture can make haematococcus pluvialis be chronically at the stage nourished and generated, heterotrophism phase cell yield is high, in a specific embodiment, average cell yield 1.53g/L/d, reach as high as 5.74g/L/d, end in heterotrophism stage cell density may be up to 26.01g/L, so the algae cell density of photoinduction is very high（2 10g/L), it is conventional light autotrophy culture algae cell density（About 0.2 2g/L) 5 10 times；

(2) frustule that heterotrophism is released directly can carry out stress-inducing to accumulate astaxanthin after being diluted with inducing culture, it is not necessary to laundering period or transitional period, thus the photoinduction time can be shorter（About 5c！〜 7d) ；And traditional haematococcus pluvialis light autotrophy incubation time is very long（About 14c！30d), dry cell weight can increase at the end of photoinduction stage and astaxanthin accumulation stage identical in traditional light autotrophy incubation are induction in the present invention, thus this more traditional light autotrophy of stage unit algae solution volume astaxanthin volume productivity can improve more than several times；

(3) relative to microalgae light autotrophy culture, higher algae cell density causes the floor space very little needed for photoinduction during photoinduction（That is the area yield of astaxanthin is high）, while high-cell density causes harvesting cost to be greatly lowered；

(4) influence of Heterotrophic culture hardly climate, weather, photoinduction culture can be carried out in glass room, and light source can use natural lighting or artificial lighting, while the wider range of photoinduction（15 35 °C), high temperature can promote the accumulation of astaxanthin,, still can be by manually heating up, setting the collaboration stress conditions such as high salt, high carbon-nitrogen ratio, strong light to realize the Rapid Accumulation of astaxanthin though cryogenic conditions do not promote the accumulation of astaxanthin, because Fiber differentiation area is small.Therefore, the large-scale continuous production of astaxanthin can be realized using the method for the present invention；

(5) due to Heterotrophic culture at the end of the cell overwhelming majority have become spore state, its resistance is stronger；And the algae solution density after dilution is still higher, with population advantage, so when carrying out photoinduction, be difficult to be polluted by harmful organisms such as protozoan, miscellaneous algaes, in traditional two benches light autotrophy culture caused by common protozoan pollution and severe stress conditions Cell concentration loss can be preferably minimized in this invention.

In summary, " heterotrophism-dilution-photoinduction " series connection training mode of the present invention reasonably combines the respective advantage of heterotrophism and photoinduction culture two ways, compared with other patterns, with production efficiency is high, culture systems combinations are flexible and the low advantage of production cost, Heterotrophic culture pattern can be given full play to and obtain high density algae solution and the advantage of photoinduction stage astaxanthin Rapid Accumulation, the extensive Industrialization for coming from microalgae astaxanthin for solution provides important technological means.

By the Patents analysis shows to having applied both at home and abroad, these patents mostly concentrate on bioreactor and device, haematococcus pluvialis light autotrophy culture medium, the new method for extracting of astaxanthin, haematococcus pluvialis light autotrophy culture medium of culture etc..The overwhelming majority for being related to culture is light autotrophy.However, haematococcus pluvialis Haematococcus pluvialis), the patent in terms of chlorella (Chlorella zofingiensis) etc. can produce " heterotrophism-dilution-photoinduction " series connection culture high-yield astaxanthin of astaxanthin microalgae not yet retrieves.The content of the invention

One aspect of the present invention provides a kind of new method of " heterotrophism-dilution-photoinduction " series connection culture of fast culture microalgae accumulation astaxanthin, this method includes the Heterotrophic culture step of microalgae, the step of microalgae Heterotrophic culture liquid obtained is diluted, and the step of photoinduction culture.

The method that another aspect of the present invention provides content astaxanthin in a kind of quick raising microalgae, the step of this method includes microalgae heterotrophism, the step of carrying out photoinduction culture after the Heterotrophic culture liquid of microalgae is diluted.

The present invention also provides a kind of Production method of astaxanthin, the step of this method includes microalgae heterotrophism, the step of carrying out photoinduction culture after the Heterotrophic culture liquid of microalgae is diluted, and the step of frustule harvesting, astaxanthin separation and Extraction simultaneously.

In the method for the invention, also photoinduction culture directly can be carried out to the microalgae cell that Heterotrophic culture is obtained.

The method of the present invention can realize the Rapid Accumulation of intracellular astaxanthin, significantly improve production efficiency, reduce production cost, and there is provided the astaxanthin of high-quality.

In the method for the invention, during Heterotrophic culture microalgae, by the way that control of additive raw material pH is constant and the elemental stable such as carbon, nitrogen and/or phosphorus is in the range of finite concentration, and at the end of Heterotrophic culture the nutritional ingredient such as carbon, nitrogen and/or phosphorus concentration relatively low even zero.

In one embodiment, at the end of Heterotrophic culture, the nutritional ingredient such as carbon, nitrogen and/or phosphorus exhausts in culture medium.In one embodiment, during heterotrophism, a steady state value by feed supplement by the pH controls of algae solution in the range of 4.0-10.0, such as pH 7.5.The pH of algae solution is generally controlled into a steady state value in the range of 5.0-9.0, more preferably controlled in the range of 7.0-8.0.

It should be understood that a little variation of pH value is allowed.For example, pH can be allowed to have a scholar Y variation, wherein Y 1.0, such as Y 0.2, Y^O.l o in certain embodiments, Y=0.Therefore, in a specific embodiment, it is Χ ± Υ by the pH controls of algae solution by feed supplement, wherein, the scholar Υ 9.0.For example, in an embodiment of the invention, The pH of algae solution is controlled within the scope of 7.5 ± 0.3 by feed supplement.

During Heterotrophic culture microalgae, by elemental stables such as control of additive raw material carbon, nitrogen and/or phosphorus in the range of finite concentration.For example, can be controlled the content of carbon in algae solution in the range of 0.5-50mM by feed supplement, the content of nitrogen is controlled in the range of 0.5-10mM, and the content of phosphorus is controlled in the range of 0.01-0.5mM.

In one embodiment, by control of additive raw material carbon, three kinds of elemental stables of nitrogen and phosphorus in the range of finite concentration.For example, can be controlled the content of carbon in algae solution in the range of 0.5-50mM by feed supplement, the content of nitrogen is controlled in the range of 0.5-10mM, and the content of phosphorus is controlled in the range of 0.01-0.5mM.

In a specific embodiment, in addition to by feed supplement the content of magnesium in algae solution is controlled in the range of 0.00001-0.00 ImM.

In a detailed embodiment, described microalgae is selected from haematococcus pluvialis Haematococcus pluvialis), chlorella (Chlorella zofingiensis) etc..

In a detailed embodiment, the step of microalgae Heterotrophic culture includes：The culture medium that pH is 4.0 10.0 is added in bioreactor, batch culture, fed-batch culture, repeated fed-batch culture, Semi-continuous cultivation or continuous culture are carried out by 0.1 50% access microalgae algae kind of working volume, cultivation temperature is 10 40 °C, pH is controlled to be less than 10.0, control dissolved oxygen is in 0.1 more than %.

In a detailed embodiment, the dilution of microalgae Heterotrophic culture liquid is that to use photoinduction culture medium that the algae solution that heterotrophism is obtained is diluted into cell density for 0.1 20 g/l, pH be 4.0 9.0.

In a detailed embodiment, the photoinduction culture includes the algae solution after dilution being transferred in photoinduction device carrying out photoinduction, continuous illumination or intermittent illumination, and cultivation temperature is 5 50 °C, intensity of illumination is 0.1 150klx, and photoinduction cultivation cycle is 1 480 hours.

In a detailed embodiment, Heterotrophic culture base contains nitrogen source, organic carbon source, a small amount of inorganic salts, auxin, trace element and water or is made up of these compositions；Photoinduction culture medium contains auxin, nitrogen source, inorganic salts and water or is made up of these compositions.

In a detailed embodiment, when it is haematococcus pluvialis to produce astaxanthin algae kind, culture medium used in heterotrophism is substantially consisted of the following composition：0.1 5.0 g/l of sodium acetate, NaN0₃0.05 1.5 g/l of CaCl₂*7H₂0 0.05 1.5 g/l, KH₂PO₄0.01 1.5 g/l, MgS0₄*7H₂0 0.01 1.0 g/l, FeS0₄*7H₂0 0.01 0.05 g/l, auxin 0.001-35 mg/litres, micro- 0.5 4 milliliters and water.

In a specific embodiment, the heterotrophism step can be carried out in shaking flask, mechanical agitation type, gas-lifting type or bubble type in the bioreactor of Heterotrophic culture, the photoinduction incubation step is carried out in the raceway pond or circle pond, enclosed flat plate photobioreactor or duct type bioreactor or pillar bioreactor or film of shaking flask or open type found any device available for microalgae light autotrophy culture such as bag and Pig bioreactor, and light source is natural light or various artificial lights.

In a detailed embodiment, using overcritical co₂Extraction, organic solvent extraction or ultrasonic wave added solvent Extraction method extracts astaxanthin.

In a detailed embodiment, method of the invention also includes：Separation of solid and liquid is carried out to the frustule after induction（Harvest）, the frustule obtained is dried algae powder of the acquisition containing astaxanthin.

In a detailed embodiment, method of the invention also includes：The frond extracted after astaxanthin is mixed with other pigments algae powder processed is dried, or separation and Extraction is carried out to other bioactive substances in frond.

In a detailed embodiment, other pigments include chlorophyll.In a detailed embodiment, the bioactive substance includes protein, grease, chlorophyll and polysaccharide.Brief description

Fig. 1 shows haematococcus pluvialis in 5L bioreactor Heterotrophic culture processes（Containing Heterotrophic culture process optimum growh process of the present invention, only control heterotrophism pH but the growth course that is not optimised under initial and supplemented medium strategy and simply independent control heterotrophism pH and optimization is initial and supplemented medium strategy, but the growth course data not added under auxin compare）.

Fig. 2 shows carbon, nitrogen, 3 kinds of nutrient consumptions of phosphorus are finished in Heterotrophic culture liquid haematococcus pluvialis algae solution photoinduction incubation in 2L pillars bioreactor out of doors.

Fig. 3 shows carbon, nitrogen, 3 kinds of nutritional ingredients of phosphorus are not exhausted in Heterotrophic culture liquid haematococcus pluvialis algae solution photoinduction incubation in 2L pillars bioreactor out of doors.Specific embodiment

Microalgae suitable for the application includes those and with synthesizing astaxanthin and can carry out the microalgae of Heterotrophic culture, including but not limited to haematococcus pluvialis (Haematococcus pluvialis), chlorella (Chlorella zofingiensis) etc..In a preferred embodiment, the present invention produces astaxanthin using haematococcus pluvialis Haematococcus pluvialis.

For culture medium of the present invention（Including Heterotrophic culture base and photoinduction culture medium）Auxin include but is not limited to

2,4- dichlorphenoxyacetic acids, benayl aminopurine, Exogenous gibberellic acid, 3- indolebutyric acids, methyl α-naphthyl acetate and brassin etc..One or more auxins can be contained in culture medium.The total content of auxin can be 0.001-35 mg/litre culture mediums in culture medium, generally in 0.001-20 mg/litres, more typically 0.001-15 mg/litres, 0.005-10 mg/litres, 0.01-10 mg/litres, 0.1-5 mg/litres.

In a particular embodiment, if each auxin can be in the presence of, its concentration, such as 2,4- dichlorphenoxyacetic acids

0.001-5 mg/litres, benayl aminopurine 0.001-5 mg/litres, Exogenous gibberellic acid 0.001-5 mg/litres, 3- indolebutyric acid 0.001-5 mg/litres, methyl α-naphthyl acetate 0.001-5 mg/litres, brassin 0.001-5 mg/litres.The concentration of each auxin is respectively preferably, such as 0.01-4 mg/litres, 0.1-4 mg/litres, 0.3-4 mg/litres, 0.3-3 mg/litres, 0.5-2.5 mg/litres not. The auxin can be obtained from commercially available approach, be then directly appended to it is known in the art be used for Heterotrophic culture and photoinduction culture can be so that in synthesizing astaxanthin and the culture medium for the microalgae that can carry out Heterotrophic culture, the example of this kind of culture medium be as described below.1. high Density Heterotrophic of the microalgae in bioreactor

The purpose of this step is in order to quickly obtain a large amount of frustules, so that the photoinduction stage accumulates astaxanthin.

Can be using various culture medium addition organic carbon sources well known in the art（Such as sodium acetate）To carry out microalgae Heterotrophic culture.The Heterotrophic culture base for being commonly used for the present invention contains nitrogen source, organic carbon source, auxin, a small amount of inorganic salts, trace element and water.

This kind of culture medium includes C culture mediums (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 culture mediums (Borowitzka et al, 1991), BG-11 culture mediums (Boussiba and Vonshak, 1991), BBM culture mediums（Nichols and Bold, 1969), BA culture mediums (Barbera et al., 1993) KM culture mediums（Kobayashi et al., 1991), Z8 culture mediums (Renstrom et al., 1981), A9 culture mediums (Lee and Pirt, 1981), OHM culture mediums（Fa'bregas et al., 2000), KMl culture mediums (Usha et al. 1999) (Garc'ia-Malea et al, 2005), HK2 culture mediums (Chen et al., 1997), HK3 culture mediums (Gong and Chen, 1998) etc..

C culture mediums used in the present invention are substantially by KN0₃、 CaN0₃, sodium acetate and a small amount of inorganic salts, trace element and water composition, some auxins are with the addition of on this basis.

Term used herein " substantially by ... constitute " represent in the composition of the present invention except containing key component such as

KN0₃、 CaN0₃, outside sodium acetate and a small amount of inorganic salts, trace element and water, can also include some fundamental characteristics or new characteristic for composition（Microalgae can be maintained to reach higher level in shorter cultivation cycle inner cell density, while activity substance content has a more substantial increase compared with conventional Heterotrophic culture）The component not influenceed substantially.Term used herein " by ... constitute " represent that the composition of the present invention is made up of pointed concrete component, without other components, but impurity of the content in usual scope can be carried.

In the culture medium, each component of culture medium can change without there is very big materially affect to microalgae cell density and quality within the specific limits.Therefore, the consumption of these components should not by embodiment strict limitation.As known to those skilled in the art, a small amount of inorganic salts can be also added in culture medium, such as magnesium sulfate, calcium chloride, ferrous sulfate and phosphate, and a small amount of trace element such as Mn, Zn, B, I, M, Cu, Co, and the addition of auxin, include the combination of single hormone or a variety of hormones.

In the present invention, micro- component may be selected from H₃B0₃、 ZnS0₄-7H₂0、 MnCl₂ H₂0、 NH₄)₆Mo₇0₂₄4H₂0、 CuS0₄-5H₂0 and CO<;N0₃)₂_6H₂One or more in 0.Inorganic salts and trace element Consumption can be determined according to Conventional wisdom.

In one embodiment, Heterotrophic culture base of the present invention is substantially consisted of the following composition：

In a detailed embodiment, when it is haematococcus pluvialis to produce astaxanthin algae kind, culture medium used in heterotrophism is substantially consisted of the following composition：0.1 5.0 g/l of sodium acetate, NaN0₃0.05 1.5 g/l of CaCl₂*7H₂0 0.05 1.5 g/l, KH₂PO₄0.01 1.5 g/l, MgS0₄'7H₂0 0.01 1.0 g/l, FeS0₄'7H₂0 0.01 0.05 g/l, auxin 0.001-35 mg/litres, micro- 0.5 4 milliliters and water.

In an embodiment, the auxin in Heterotrophic culture base contains：2,4- dichlorphenoxyacetic acid 0.001-5 mg/litres, benayl aminopurine 0.001-5 mg/litres, Exogenous gibberellic acid 0.001-5 mg/litres, 3- indolebutyric acid 0.001-5 mg/litres, methyl α-naphthyl acetate 0.001-5 mg/litres and/or brassin 0.001-5 mg/litres.

In one embodiment, the auxin in Heterotrophic culture base contains：Benayl aminopurine 0.001-5 mg/litres and 3- indolebutyric acid 0.001-5 mg/litres.

After culture medium is prepared according to above-mentioned formula, the pH of the culture medium can be adjusted to 4.0-10.0 with conventional meanses such as acid or alkali, and in 115 125 °C of lower autoclavings 15 30 minutes.Can using in batches, fed-batch, it is semicontinuous and it is continuous culture etc. various ways implement Heterotrophic culture.

When Heterotrophic culture uses fed-batch process, the culture medium accordingly prepared is added in bioreactor, benefit adds water to working volume, usual coefficient is 0.6 0.8, then steam sterilizing（121 °C, maintain about 20 minutes）, when temperature is down to 20 35 °C, Heterotrophic culture is started by 1 15% access microalgae algae kind of working volume.

In Heterotrophic culture, that is, start feed supplement, by controlling the continuous stream of supplemented medium to add that pH is constant within the specific limits, such as 7.0-8.0, in a preferred embodiment, pH is controlled 7.5.

Supplemented medium includes organic carbon source（Such as sodium acetate）, nitrogen source（Such as CaN0₃、 KN0₃), the nutritive salt such as auxin and inorganic salts, the nutritive salt added is the above-mentioned corresponding culture medium Jing after Nong Shrink, promotes microalgae continued growth.The addition such as organic carbon source, nitrogen source, auxin, inorganic salts should cause the concentration of corresponding composition in algae solution same or like with concentration when initially starting Heterotrophic culture in supplemented medium, the concentration of carbon source in algae solution, nitrogen source, auxin and inorganic salts is promoted microalgae continued growth.Certainly also appropriate adjustment can be made to the corresponding composition in supplemented medium according to the actual growing state of microalgae, to increase or decrease the concentration of certain or some compositions, so as to promote microalgae continued growth.

The content of carbon, nitrogen and/or phosphorus in nutrient solution is monitored while feed supplement in time, so as to suitably adjust content of these materials in supplemented medium, it (is usually 1.0-40mM, 1.0-30mM, 1.0-20mM, 1.0-lOmM etc. that the content of carbon in algae solution, which is controlled in 0.5-50mM,）In the range of, the content of nitrogen controls in 0.5-10mM (to be usually 0.5-8mM, 0.5-6mM, 1.0-6mM, 1.0-5.0mM etc.）In the range of, the content of phosphorus controls in 0.01-0.5mM (to be usually 0.01-0.4mM, 0.05-0.3mM, 0.05-0.2mM, 0.05-O. lmM etc.）In the range of, so as to ensure that the material concentration of these in algae solution is stable.It is furthermore preferred that simultaneously monitoring algae solution in magnesium content, and suitably adjustment supplemented medium in magnesium content, by algae solution magnesium content control 0.00001-O.OOlmM (generally 0.00001-0.0008mM, 0.00003-0.0005mM etc.）In the range of.

Stream adds to certain phase, when microalgae cell density reaches desirable value, changes control condition, the nutrition such as carbon in nutrient solution, nitrogen and/or phosphorus is exhausted substantially, the Heterotrophic culture stage terminates.Generally, at the end of Heterotrophic culture, the concentration of the nutritional ingredient such as carbon, nitrogen and/or phosphorus is relatively low in culture medium.The concentration of such as carbon and nitrogen is even lower less than 0.1mM, less than 0.05mM, less than O.OlmM, and even zero；The concentration of phosphorus is even lower less than 0.005mM, less than 0.003mM, less than O.OOlmM, and even zero.

No matter which kind of training method is used, in incubation, must strictly control suitable condition of culture to make microalgae normal growth.Generally, it is 20 35 °C to control temperature, and such as 25 30 °C, by adjusting the saturation of the air concentration that ventilation and mixing control dissolved oxygen are not less than 5%, pH is not higher than 9.0.In a preferred embodiment, dissolved oxygen is not less than 10% not higher than 30% saturation of the air concentration, and pH constant controls are in 7.5-8.0, and throughput is less than 0.3wm, and stirring is less than 200 rpm.

Heterotrophism can be carried out in shaking flask, mechanical agitation type, gas-lifting type, bubble type etc. in the bioreactor of Heterotrophic culture.

2. the dilution of high concentration algae solution

The first purpose of this step is in order to reduce algae cell density, the production astaxanthin microalgae for being transferred to photoinduction culture is efficiently absorbed luminous energy, improves optical energy utilization efficiency；The second purpose is in order to adjust nutritional ingredient in induction broth, cause Nutrient Stress, in favor of the Rapid Accumulation of astaxanthin.

The high density algae solution that Heterotrophic culture is obtained（When open type reactor is induced, preferably without organic carbon source, the photoinduction stage can be so avoided to grow excessive miscellaneous bacteria；But when closed photo bioreactor is induced, organic carbon source can be contained, promote cell concentration increase）Operation should be diluted, highdensity algae solution is diluted with dilution special culture media, cell density is maintained 0.1 20 g/l, pH is 4.0 10.0.In certain embodiments, highdensity algae solution is diluted with the culture medium without organic carbon source with water, cell density is maintained 0.1 10 g/l, adjust pH to 5.0 8.0.In other embodiments, algae solution is diluted, cell density is maintained 18 g/l, pH to 5.0 8.0 is adjusted.In a preferred embodiment, cell density is maintained 1.0 5.0 g/l, be passed through C0₂And pH is adjusted to 5.0 8.0.

Algae solution can be diluted using various known diluted mediums.Generally, photoinduction culture medium contains carbon source, nitrogen source, auxin, inorganic salts and water or is made up of these compositions, is not contained relative to Heterotrophic culture base or containing less organic carbon source, incubation can also be passed through C0₂。

In a preferred concrete scheme, the high density frustule that Heterotrophic culture is obtained preferably suitably is diluted with the initial medium lacked without organic carbon source and nitrogen and phosphorus.

In an embodiment, diluted medium（Photoinduction culture medium）Contain： MgS(V7H₂0.01 0.1 g/l of O, NaH₂PO₄0.01 0.1 g/l, 0.1 1 g/l of KC1, CaCl₂0.01 0.2 g/l, FeS0₄'7H₂0 0.01 0.06 g/l, 0.020 0.052 g/l of EDTA and auxin 0.001-35 mg/litres.

In one embodiment, diluted medium（Photoinduction culture medium）In auxin contain：2,4- dichloros Phenoxy acetic acid 0.001-5 mg/litres, benayl aminopurine 0.001-5 mg/litres, Exogenous gibberellic acid 0.001-5 mg/litres, 3- indolebutyric acid 0.001-5 mg/litres, methyl α-naphthyl acetate 0.001-5 mg/litres and/or brassin 0.001-5 mg/litres.

In one embodiment, diluted medium（Photoinduction culture medium）In auxin contain：Benayl aminopurine 0.001-5 mg/litres and 3- indolebutyric acid 0.001-5 mg/litres.

The culture medium used is diluted without autoclaving, and pH to 5.0 9.0 is adjusted after preparing and be can be used.

It should be understood that in certain embodiments, it is not necessary to frustule obtained by Heterotrophic culture is diluted, and directly implements photoinduction culture to it, this depends on the density of Heterotrophic culture, Heterotrophic culture liquid composition and actual inductive condition（Such as light intensity, temperature）Between Proper Match.3. photoinduction culture

The purpose of the step is to allow production astaxanthin microalgae to receive sufficient illumination, makes frustule rapid, high volume dynamic accumulation astaxanthin by photoinduction, while properly increasing the frustule concentration in nutrient solution.

Gained dilution is transferred to the adherent method that photoinduction culture is carried out in photoinduction device or microalgae cell is coated in into the semisolids such as solid film surface and carries out photoinduction by high density micro algae culturing liquid as described above after dilution.Temperature control is at 5 50 °C, and intensity of illumination is 0.1 150klx, continuous illumination or intermittent illumination, and photoinduction cultivation cycle is 1 480 hours, and throughput is 0.1 2.0 wm.Wherein described bioreactor includes all closed photo bioreactors（Shaking flask, duct type, flat, pillar, film found bag and Pig etc.）With all Race-way photobioreactors（Raceway pond, circle pond and bubble type big basin etc.）.

Generally, cultivation temperature is can be controlled in the range of 15 35 °C, such as 18 35 °C, 20 35 °C, 20 30 °C.Generally, intensity of illumination is l 70klx, for example, 1 60,1 50,1 40,1 30,1 20, l 10klx etc., depending on the visual specific condition of production.Generally, such as algae solution is caused to be sufficiently mixed by gas, then throughput is controllable to 0.1 2.0 wm, for example, 0.2 1.8,0.5 1.5,0.8 1.5,1.0 1.5wm etc..Meanwhile, it is passed through certain density C0₂To provide inorganic carbon source and control pH, for example, 0.5%-10% C0₂.In other embodiments, cultivation temperature control is at 10 50 °C, and intensity of illumination is l 10klx, and throughput is 0.05 2.0wm.

In other embodiments, photoinduction cultivation cycle is 8 480 hours, for example, according to actual weather condition, photoinduction cultivation cycle can be 8 240 hours, 8 120 hours, 8 72 hours, 8 48 hours, 8 24 hours；Or, photoinduction cultivation cycle can for 12 72 hours, 12 60 hours, 12 48 hours, 12 36 hours, 12 24 hours not etc. or 24 60 hours, 24 48 hours.

Photoinduction culture medium is from the haematococcus pluvialis light autotrophy culture medium improved, including diluted medium as described above.In this application, " photoinduction cultivation cycle " includes whole photoinduction incubation, for example, the outdoor Fiber differentiation cycle in culture time, which includes night, does not have the time of illumination.

In this application, " light application time " refers to the time for implementing photoinduction culture to microalgae using herein described intensity of illumination, I.e. the time does not have the time of illumination including night.In certain embodiments, the light application time of photoinduction incubation step is

8 120 hours, such as 8 72 hours, 8 36 hours, 8 24 hours, 8 18 hours, 8 12 hours, 12 36 hours, 12 24 hours not, and any duration in above range.

Therefore, it is the photoinduction incubation step in the range of 8 120 hours that the photoinduction incubation step of the application, which also includes light application time,.Photoinduction culture can be carried out by the way of artificial lighting, also photoinduction culture can be carried out using the mode of natural lighting out of doors.

In a specific embodiment, when astaxanthin concentration reaches highest in nutrient solution, photoinduction culture is terminated, the separation and Extraction or directly harvesting frustule that harvest frustule carries out astaxanthin carry out algae powder preparation.4. frustule harvesting, astaxanthin separation and Extraction and frond comprehensive utilization

After photoinduction culture terminates, harvesting is settled or centrifuged to microalgae, obtains wet frond.The collecting method of frustule includes but is not limited to sedimentation, high speed centrifugation, flocculation, the technology such as air supporting or filtering；Frustule wall-breaking method includes but is not limited to the Wet-process wall breaking methods such as frond self-dissolving, high-pressure homogenization, enzyme hydrolysis, aqueous phase pyrolysis.

The extraction of astaxanthin is carried out to microalgae using traditional organic solvent extraction.Add organic solvents into algal gel and extracted first, be then stirred centrifugation and obtain supernatant and frond precipitation, Jian Ya Nong Shrink, stirring are carried out to supernatant and adds water, filter acquisition astaxanthin crystal.

Other compositions in supernatant progressively separation and Extraction can obtain aliphatic acid, lutein etc., or directly dry and obtain microalgae powder all the components in supernatant and frond precipitation mixed atomizing.

In one preferably scheme, using overcritical co₂Abstraction technique carries out the separation and Extraction of astaxanthin to microalgae.In one more preferably scheme, Direct spraying after the microalgae Ye Nong Shrink of acquisition is dried and obtains microalgae powder.

In the present invention, the microalgae obtained by culture can be comprehensively utilized, extract the various active components such as polyunsaturated fatty acid therein, protein, chlorophyll, polysaccharide.The sequence of extraction of active component has no specifically limited, but generally to meet component damages this premise extracted after the step of first extracting can not cause.

Frustule dry weight is referred to herein and content astaxanthin assay method is as follows：

Frustule dry weight is determined：V milliliters of nutrient solution is taken during microdisk electrode, 8000 rpm are centrifuged 10 minutes, the frond after centrifugation is washed with deionized 3 times, measuring cup is transferred to（Wi (gram））In, dried in 105 °C of baking ovens to constant weight W₂(gram）.Frond dry weight Cx can be calculated according to following formula：Cx (g/l） = ( W₂-Wi ) /V/1000。

Astaxanthin is determined：Using high performance liquid chromatography（HPLC), concrete operation step is shown in document（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 ) .Embodiment Following Heterotrophic culture bases and water are added in 5L bioreactors to steam sterilizing is carried out after 2.5L, haematococcus pluvialis are then accessed when temperature drops to 25 °C, start Heterotrophic culture.During by adjust ventilation and mixing control dissolved oxygen be not less than 5% saturation of the air concentration.

In Heterotrophic culture, that is, start feed supplement, it is by controlling the continuous stream of supplemented medium to add that pH is constant in 7-8.Supplemented medium includes organic carbon source（Such as sodium acetate）, nitrogen source（Such as, CaN0₃、 KN0₃), the nutritive salt such as auxin and inorganic salts, the nutritive salt added is the above-mentioned corresponding culture medium Jing after Nong Shrink, promote microalgae continued growth, monitor carbon, nitrogen, phosphorus, the content of magnesium in zymotic fluid in time simultaneously, suitably adjust the 4 class material in supplemented medium content (carbon:0.5-50mM, nitrogen：0.5-10mM, phosphorus：0.01-0.5mM, magnesium：), O.OOOOl-O.OOlmM to ensure that the class material concentration of this in zymotic fluid 4 is stable.Stream adds to certain phase, when microalgae cell density reaches desirable value, changes control condition, carbon in nutrient solution, nitrogen, 3 kinds of nutrition of phosphorus is exhausted substantially, the Heterotrophic culture stage terminates.And not plus in the case of hormone, other operations and experiment condition are all identical, only auxin is not contained in culture medium.And in the case where being not optimised control, pH only in monitoring zymotic fluid, constant in 7-8 by supplemented medium holding pH, other materials such as carbon, nitrogen, phosphorus, the content of magnesium are not controlled, and hormonal substance is also without other experiment conditions are identical with operation.

As a result Fig. 1 is seen.At the end of Heterotrophic culture, control pH and feeding strategy, and the Heterotrophic culture of plant hormone is added, its dry cell weight reaches 26 g/1;And only control pH and feeding strategy but be added without the Heterotrophic culture of plant hormone, its dry cell weight reaches 8.7 g/l;And be not optimised feeding strategy and be added without the Heterotrophic culture of plant hormone, its dry cell weight be only 4.2 g/lo therefore, control feeding strategy optimization and add the Heterotrophic culture of plant hormone, with merely control pH but be not optimised feeding strategy and not plus auxin Heterotrophic culture compared with, cell density improves 6.2 times；And compared with only controlling pH and feeding strategy but being added without the Heterotrophic culture of plant hormone, cell density improves 2.1 times.

High density algae solution 8.5g/L bands during Heterotrophic culture are put into 1L, 1.3g/L is diluted to light inducing culture, and add above-mentioned photoinduction culture medium, progress photoinduction culture in 2L pillar bioreactors is transferred to.Photoinduction condition of culture：Temperature maintains 28 38 °C, and air mass flow is lwm, and natural lighting is about 75klx by force per sidelight.

Fig. 2 shows haematococcus pluvialis algae solution that carbon in Heterotrophic culture liquid, nitrogen, 3 kinds of nutritional ingredients of phosphorus are all exhausted photoinduction incubation in 2L pillars bioreactor out of doors, after photoinduction culture 3 days, dry cell weight reaches 1.92g/L, 2.67mg/gD of the astaxanthin from induction initial stage_CWRise to 22.56mg/gD_CW(content astaxanthin improves about 8.5 times）, the yield for the astaxanthin counted for 3 days using photoinduction is 82.24mg/L/d (being 3.57 times of current microalgae light autotrophy production astaxanthin maximum output 23.04mg/L/d) (see Fig. 2).

In the case that Fig. 3 shows that carbon, nitrogen, 3 kinds of nutritional ingredients of phosphorus are not yet exhausted in Heterotrophic culture liquid, haematococcus pluvialis algae solution photoinduction incubation in 2L pillars bioreactor out of doors, as a result after showing induction 3 days, dry cell weight reaches 2.12g/L, and astaxanthin rises to 6.51mg/gDcw from the 2.67mg/gDcw at the initial stage of induction, and (content astaxanthin only improves about 2.4 times）, the yield for the astaxanthin counted for 3 days using photoinduction is 22.51 mg/L/d (being only that heterotrophism terminates the 27% of the algae solution inducing effect that carbon, nitrogen, 3 kinds of nutritional ingredients of phosphorus are all exhausted).It follows that carbon in Heterotrophic culture liquid, nitrogen, Whether 3 kinds of nutritional ingredients of phosphorus are exhausted, and the raising for astaxanthin yield in Induction Process is particularly significant.

Heterotrophism and supplemented medium used contains：

0.1 5.0 g/l of sodium acetate, NaN0₃0.05 1.5 g/l of CaCl₂'7H₂0 0.05 1.5 g/l, KH₂P0₄0.01 1.5 g/l, MgS0₄*7H₂0 0.01 1.0 g/l, FeS0₄*7H₂0 0.01 0.05 g/l, benayl aminopurine 0.001-5 mg/litres, 3- indolebutyric acid 0.001-5 mg/litres, micro- 0.5 4ml and water.

Photoinduction culture medium used contains：

MgS0₄-7H₂0 0.01 0.1 g/l, NaH₂P0₄0.01 0.1 g/l, 0.1 1 g/l of KC1, CaCl₂0.01 0.2 g/l, FeSO₄'7H₂0.01 0.06 g/l of O, 0.020 0.052 g/l of EDTA, benayl aminopurine 0.001-5 mg/litres, 3- indolebutyric acid 0.001-5 mg/litres.Although the specific example of the present invention is described above, there is any will be apparent to practitioners skilled in the art, i.e., the present invention can be made various changes and be changed under the premise without departing from the spirit and scope of the present invention.Therefore, appended claims cover all these variations within the scope of the present invention.

Claims (1)

1. A kind of methods for producing astaxanthin or improving content astaxanthin in microalgae of 1, it is characterised in that this method includes：(1) Heterotrophic culture microalgae, and

   (2) obtained microalgae Heterotrophic culture liquid is diluted, then photoinduction culture is carried out after addition inducing culture into dilution algae solution；Or photoinduction culture directly is carried out to the microalgae cell that Heterotrophic culture is obtained；

   So as to prepare astaxanthin, or improve the content of astaxanthin in microalgae.

   2. a kind of method for producing astaxanthin, it is characterised in that this method includes：

   (1) Heterotrophic culture microalgae, wherein, in incubation, by the way that control of additive raw material pH is constant and the elemental stable such as carbon, nitrogen and/or phosphorus is in the range of finite concentration, and at the end of Heterotrophic culture, the concentration of the nutritional ingredient such as carbon, nitrogen and/or phosphorus relatively low even zero in culture medium；

   (2) obtained microalgae Heterotrophic culture liquid is diluted, then photoinduction culture is carried out after addition inducing culture into dilution algae solution；Or photoinduction culture directly is carried out to the microalgae cell that Heterotrophic culture is obtained, and

   (3) frustule, and separation and Extraction astaxanthin are harvested.

   A kind of methods for improving content astaxanthin in microalgae of 3, it is characterised in that this method includes：

   (1) Heterotrophic culture microalgae, wherein, in incubation, by the way that control of additive raw material pH is constant and the elemental stable such as carbon, nitrogen and/or phosphorus is in the range of finite concentration, and at the end of Heterotrophic culture, the concentration of the nutritional ingredient such as carbon, nitrogen and/or phosphorus relatively low even zero in culture medium, and

   (2) obtained microalgae Heterotrophic culture liquid is diluted, then photoinduction culture is carried out after addition inducing culture into dilution algae solution；Or photoinduction culture directly is carried out to the microalgae cell that Heterotrophic culture is obtained；

   So as to improve the content of astaxanthin in microalgae.

   4. the method as any one of claim 11, it is characterized in that, described microalgae, which is selected from, with synthesizing astaxanthin and can carry out the microalgae of Heterotrophic culture, such as haematococcus pluvialis Haematococcus pluvialis, chlorella (Chlorella zofingiensis).

   Methods of 5, as any one of claim 11, it is characterised in that include the step of the microalgae Heterotrophic culture：The culture medium that pH is 4.0 10.0 can be being added in the bioreactor of Heterotrophic culture, batch culture, fed-batch culture, repeated fed-batch culture, Semi-continuous cultivation or continuous culture are carried out by 0.1 50% access microalgae algae kind of working volume, cultivation temperature is 10 40 °C, pH is controlled to be less than 10.0, control dissolved oxygen is in 0.1 more than %.

   6. the method as any one of claim 11, characterized in that, the dilution of the microalgae Heterotrophic culture liquid is that to use photoinduction culture medium or water that the algae solution that heterotrophism is obtained is diluted into cell density for 0.1 20 g/l, pH be 4.0 10.0.

   7. the method as any one of claim 11, it is characterised in that the photoinduction culture includes Algae solution or the microalgae cell of Heterotrophic culture after dilution is directly transferred to the adherent method that photoinduction is carried out in photoinduction device or microalgae cell is attached into the semisolids such as solid film surface and carries out photoinduction, photoinduction temperature is 5 50 °C, continuous illumination or intermittent illumination, intensity of illumination is 0.1 150klx, and photoinduction cultivation cycle is 1 480 hours.

   8. the method as any one of claim 11, it is characterised in that the Heterotrophic culture base contains nitrogen source, organic carbon source（Including but not limited to sodium acetate）, auxin, inorganic salts, trace element and water, or by the nitrogen source, organic carbon source（Including but not limited to sodium acetate）, auxin, inorganic salts, trace element and water composition；The photoinduction culture medium contains auxin, nitrogen source, inorganic salts and water, or is made up of auxin, nitrogen source, inorganic salts and water.

   9. the method as any one of claim 11, it is characterised in that the heterotrophism step is carried out in shaking flask, stirring-type or gas-lifting type or bubbling fermentation tank, the bioreactor with internal light source or external light source；The photoinduction culture is carried out in shaking flask, raceway pond, circle pond, flat plate photobioreactor, duct type bioreactor, pillar bioreactor, spherical bioreactor, film found any device that can be used for microalgae light autotrophy or photoinduction culture such as bag and Pig, or the adherent method that microalgae cell is coated in into the semisolids such as solid film surface carries out photoinduction；Light source can be natural light or artificial light.

   10. the method as any one of claim 11, it is characterised in that methods described also includes：Separation of solid and liquid is carried out to the frustule after induction（Harvest）, the frustule obtained is dried algae powder of the acquisition containing astaxanthin.

   Methods of 11, as any one of claim 11, it is characterised in that methods described also includes：Astaxanthin in frustule is extracted, to algae powder processed is dried after extracting the frond after astaxanthin and the mixing of other pigments, or separation and Extraction is carried out to other bioactive substances in frond.

   12. the method as any one of claim 11, it is characterised in that use overcritical C0₂Extraction, organic solvent extraction or ultrasonic wave added solvent extraction method extract astaxanthin.

   13. a kind of Heterotrophic culture base, the culture medium contains nitrogen source, organic carbon source（Including but not limited to sodium acetate）, auxin, inorganic salts, trace element and water, or by the nitrogen source, organic carbon source（Including but not limited to sodium acetate）, auxin, inorganic salts, trace element and water composition, for can with synthesizing astaxanthin and can carry out Heterotrophic culture microalgae Heterotrophic culture.

   14. a kind of photoinduction culture medium, the culture medium contains auxin, nitrogen source, inorganic salts and water, or be made up of auxin, nitrogen source, inorganic salts and water, for can with synthesizing astaxanthin and can carry out Heterotrophic culture microalgae photoinduction culture.