CN1392250A - Method of regulating haematococcus pulvialis population density and synthesing and accumulating astaxanthin in photoreactor - Google Patents
Method of regulating haematococcus pulvialis population density and synthesing and accumulating astaxanthin in photoreactor Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention relates to the process of regualting haemtococcus pluvialis population density and synthesizing and accumulating astaxanthin as intracellular secondary catabolite in a photobiological reactor system. The process includes establishing a photobiological reactor system with main reactor made of transparent material, outside controllable lighting facility, speed regulating vane wheel stirrer, water rolling wheel, ventilating and oxygen supplying unit, nutritious liquid compounding and temperature controlling unit and sterilizing unit; antering environment conditions to regulate haemtococcus pluvialis population density and synthesize, accumulate and collect astaxanthin; low temperature drying; etc. The natural astaxanthin is a kind of carotinoid with important physiological functions and very strong antioxidant activity.
Description
Technical field
The open extensive operational system bioreactor that the present invention relates to a kind of optimization is regulated the method that the secondary metabolite astaxanthin synthesizes and accumulates in haematococcus pulvialis population density and the cell.
Background technology
(Haemtococcus is a kind of unicellular miniature green alga Ag.) to haematococcus pulvialis, and its cell is greatly about the 19-63 micron.Haematococcus pulvialis motor cell ovalize or avette has two isometric flagellums.Tool space between cell walls and protoplasma, be full of gelatinoid therebetween, having tenuigenin to connect silk between cell walls and protoplastis links to each other, nourish and generate and be cell fission, formation 2,4,8 even more a plurality of daughter cell, monogony forms glue colony or chlamydospore when environment is bad, syngenesis is syngamy.Under normal growth conditions, present green, contained pigment is based on chlorophyll and xenthophylls in its cell, when the change of envrionment conditions is grown when unfavorable to it, as lacking nitrogen, phosphorus and doubly charged ion magnesium or under the strong illumination condition, the growth of haematococcus pulvialis cell will be tending towards slowly and make motor cell lose flagellum, form glue colony or chlamydospore, simultaneously, promoting secondary metabolite synthetic, mainly is a large amount of accumulation of astaxanthin, makes the color of haematococcus pulvialis cell form brick-red rapidly.Studies show that, influence physiology, biomass, output and the secondary metabolite composition and the growing amount of haematococcus pulvialis, relate generally to the kind of haematococcus pulvialis and good culture condition (as temperature, illumination, dissolved oxygen, nutritive medium composition, fluid shear power etc.).By the change of envrionment conditions haematococcus pulvialis is carried out physiology and induce the component that commercial value is arranged that can produce suitable high density, as the synthetic and accumulation of astaxanthin.Obtain the excellent engineering bacterial strain by the existing haematococcus pulvialis algae kind of genetic engineering means transformation, improve the light saturation value of haematococcus pulvialis algae kind, the inhibition of anti-light, and degeneration-resistant, contamination resistance, or the optimization by culture process can increase the content of cell internal object product astaxanthin.There are some researches show, through the bioreactor of optimization and good culture condition, the content that can impel astaxanthin in the haematococcus pulvialis is up to more than 4% of dry cell weight, and the content that adopts bacterium or yeast etc. to cultivate to generate astaxanthins the highest not enough dry cell weight 0.8%.
At present, to be that culturing process haematococcus pulvialis population density is low make low cause expensive of yield per unit to the principal element that hinders haematococcus pulvialis scale operation.The haematococcus pulvialis cultivated of bio-reactor closed system of forming by duct type bioreactor or small stationary system or airlift bioreactor on a small scale, the defective that exists is that duct wall is easily covered by the frustule adhesion and causes between incubation period light transmission to descend and clean difficulty, though adopted dissimilar paddle wheels to mix, reducing the bridging effect between the frustule and to improve the utilization of illumination as far as possible, but only adapt to the laboratory bench-scale testing.
Discover, and natural astaxanthin (3,3 '-dihydroxyl-4,4 '-diketo-β-Hu Luobusu) the important physical function is arranged, its anti-oxidant activity is higher more than 10 times than other carotenoid, and is higher 500 times than vitamin-E, is to have the active carotenoid of strong anti-oxidation.Pharmaceutical research proves; astaxanthin is to the cancellation singlet oxygen; remove free radical; stop lipid peroxidation and protection body to escape injury and have remarkable effect; have very strong immunoregulatory activity simultaneously and help to suppress some by the disease that free radical caused, obvious to the generation effect of preventing cancer.
Summary of the invention
The extensive operational system bioreactor that the purpose of this invention is to provide a kind of optimization is regulated the method that the secondary metabolite astaxanthin synthesizes and accumulates in haematococcus pulvialis population density and the cell.
Method of the present invention comprises sets up the open extensive operational system bioreactor of optimizing, regulatable envrionment conditions is provided, in bioreactor bottom and both sides adopt light transmissive material and external regulatable illumination facilities, speed governing paddle wheel agitator and volume water wheels, air-blowing facility and O
2Gas system, nutritive medium allotment and temperature adjusting system, sterilizing and bacterial system carries out physiology by the change of envrionment conditions and induces the operations such as synthetic and accumulation, results haematococcus pulvialis cell and cryodrying of regulating haematococcus pulvialis population density and regulation and control haematococcus pulvialis secondary metabolite astaxanthin.
Method concrete steps of the present invention are as follows:
One, set up the open extensive operational system bioreactor of optimizing: transformation is also optimized thin waterway bioreactor or the high extensive operational system bioreactor of design light utilization efficiency that little algae use is cultivated in industry usually, comprising support type main body base, the main body reactor (tank) of adjustable blowdown system is set, regulatable illumination facilities is set, speed governing paddle wheel agitator and volume water wheels, air-blowing facility and O are set
2Gas system is provided with nutritive medium allotment and temperature adjusting system, sterilizing and bacterial system and cryodrying facility.The main body reactor is rectangle, and length and width are 200-1500cm * 100-300cm, are generally 220-500cm * 110-150cm; The degree of depth is 250-500cm, is generally 350-450cm.Also the extensive operational system bioreactor all operations system that optimizes can be comprised that nutritive medium allotment, control light, temperature control, sterilization, stirring, air-blowing, cell harvesting, separation, drying etc. realize long-term semicontinuous production by computer control.
Two, adopt light transmissive material and external adjustable illumination facilities in bioreactor bottom and both sides: light transmissive material can be selected the water white transparency toughened glass of high tensile force resistance and compressive resistance for use, and thickness is 0.8-3cm, is generally 1.5-2cm; Reach balanced illuminating fluorescent lamp, incandescent light, sun lamp or the energy-saving spot lamp that many adjustable brightness are installed in side outside bioreactor up and down, every lamp power is 40-2000w, is generally 40-300w; Can be used singly or in combination; Adjustable intensity of illumination is the 800-3800 lux, is generally the 1500-2500 lux.Intensity of illumination and periodicity of illumination can be controlled according to natural daylight intensity of illumination and the quantity by lamp and power and light application time.
Three, speed governing paddle wheel agitator and volume water wheels: the speed governing blade wheel stirs the cell suspension that can make in the reactor, but middling speed to the shearing force that produces at a high speed easily makes frustule injured.Haematococcus pulvialis, as the rate loop of nutritive medium with 0.01-0.08m/s, can effectively reduce the bridging effect between the frustule and improve the utilization of illumination under suitable fluid shear power effect in bioreactor the inside.Adopt the adjustable speed paddle wheel agitator of industrial use, rotating speed is 10-400r/m, is generally 20-60r/m; The volume water wheels are the volume water wheels of industrial use, and rotating speed is 10-300r/m, is generally 20-80r/m, and the volume water wheels are installed on a side of photoproduction sundries.
Four, air-blowing facility and O
2Gas system: kind pure culture of haematococcus pulvialis algae and population density adjusting stage blast sterile air and help to improve algae cell density and promote the frustule division.Adopt the pneumatic plant of industrial use and be treated as sterile air, O through the oil strain drainage bacteriological filtration of routine
2Gas adopts the industrial oxygen bottle; O
2Gas adopts the filtration method degerming.By variable valve and be distributed in bioreactor bottom porous pod apertures and blast sterile air or oxygen.The sterile air amount of blasting is 0.05-5m
3/ m is generally 0.08-0.3m
3/ m.O
2Gas is regulated by the concentration of the required dissolved oxygen of nutritive medium and is blasted.
Five, nutritive medium allotment and temperature adjusting system: nutritive medium allotment and temperature adjusting system are made up of the interlayer receiver that is equipped with cold water circulating refrigerating device, heating unit, nutritive medium recycle pump, measuring and controlling temp facility and belt stirrer of industrial use.The receiver capacity is 30-10000L, is generally 1000-6000L; Agitator speed is 20-300r/m, is generally 60-80r/m.
Nutritive medium is formed: clear water 20-70% is generally 30-45%; Seawater 30-80% is generally 55-70%; Also can adopt artificial allotment seawater; Saltpetre 0.1-0.4g/L is generally 0.2-0.28g/L; Sodium-acetate 0.3-0.6g/L is generally 0.45-0.53g/L; Can also add nutritive salt such as urea, sal epsom, potassium primary phosphate, add-on is 0.01-0.1g/L.Haematococcus pulvialis algae kind is the 40-55% saturation ratio through pure culture and the required dissolved oxygen concentration of population density adjusting stage nutritive medium; The haematococcus pulvialis pH value of suitable growth is neutral to slight alkalinity.
Six, sterilizing and bacterial system: the open extensive movement system bioreactor of optimization is cleaning and sterilizing regularly, it is clean pollution-free that the nutritive medium allocation process keeps, clear water and seawater can adopt the water electrolytic ozone producer of industrial use to carry out sterilization, the nutritive medium that employing recycles can adopt the heating method sterilization, Heating temperature is 70-80 ℃, time 8-25 minute, or 95-100 ℃, time 2-15 minute; Also can adopt the chemical sterilization method to carry out sterilization.
Seven, carry out physiology by the change of envrionment conditions and induce the synthetic and accumulation of regulating haematococcus pulvialis population density and regulation and control haematococcus pulvialis secondary metabolite astaxanthin: it is the 800-1800 lux that kind pure culture of haematococcus pulvialis algae and population density are regulated the stage intensity of illumination, is generally the 1000-1500 lux; Solution temperature is 16-31 ℃, is generally 24-28 ℃; Nutrient solution concentration is: saltpetre 0.1-0.4g/L is generally 0.2-0.28g/L; Sodium-acetate 0.3-0.6g/L is generally 0.45-0.53g/L.The culture cycle of frustule logarithmic phase is 72-108h, is generally 80-96h.
The interior secondary metabolite astaxanthin of haematococcus pulvialis cell synthesizes and accumulation stage intensity of illumination is the 1900-3200 lux, is generally the 2000-2500 lux; Solution temperature is 21-32 ℃, is generally 26-30 ℃; Nutrient solution concentration is: saltpetre 0.01-0.05g/L is generally 0.02-0.035g/L; Sodium-acetate 0.8-2g/L is generally 1-1.7g/L.Promote that secondary metabolite astaxanthin culture cycle synthetic and accumulation is 50-80h in the frustule, be generally 60-72h.
Eight, results haematococcus pulvialis cell and cryodrying: centrifugal is the method for at present most widely used results frustule, adopts industrial continous way screenings separation centrifuges usually, and rotating speed is 3000-30000r/m, is generally 8000-15000r/m.Also can adopt other separating devices or filtration method results frustule.Isolating haematococcus pulvialis is adopted the cryodrying facility of industrial use, carry out drying as equipment such as vacuum lyophilization, vacuum-drying, low temperature warm air drying, low temperature spray dryings, drying temperature is 38-85 ℃, is generally 43-48 ℃.Require the dried product exhibited water content to be lower than 3%, be generally below 1%.
The design of bioreactor is for regulating the haematococcus pulvialis population density and promoting the synthetic of haematococcus pulvialis secondary metabolite astaxanthin and accumulate most important in the open extensive operational system.The cultivation haematococcus pulvialis is because the variable of abiotic factor is too many, as the control of the multiple factors such as intensity of illumination, temperature and nutritive medium composition, the synthetic and accumulation and the very complexity that becomes of strain environment promotion cell internal object product.Illumination is the important factor of haematococcus pulvialis growth.The illuminance that is suitable for most the haematococcus pulvialis growth is medium tenacity illumination 1000-1500 lux; To suppress the growth of haematococcus pulvialis greater than the intensity of illumination of 3500 luxs, the splitted cell also transfers dormant state to by nourishing and growing; Continuous illumination round the clock helps the growth of haematococcus pulvialis and obviously improves the cell volume productive rate.The thin waterway bioreactor that the paddle wheel that adopts in the little algae of outdoor artificial culture stirs, textural the illumination irradiation of accepting front lighting, to light make full use of and the regulation and control of illumination are limited to, particularly cultivating the illumination deficiency night becomes a large scale culturing haematococcus pulvialis guardian technique difficult problem., illumination deficiency and frustule bridging effect each other, frustule growth increased the chance that other biological pollutes because being suppressed.The present invention finds, transform and optimization thin waterway photo-bioreactor system, or the high bioreactor of design light utilization efficiency, bottom and both sides at bioreactor, adopt light transmissive material to improve whole illumination exposure intensity in the bioreactor, utilizing daylight illumination and external regulatable source of artificial light strong illumination and regulatable envrionment conditions to carry out physiology induces, make frustule can receive illumination equably round the clock, higher to the efficiency of light energy utilization, both can promote haematococcus pulvialis fissiparity speed and form the high-density population to use as pure strain, can improve the frustule volume productivity again, also can reach the synthetic and accumulation that logarithmic phase finishes back regulation and control haematococcus pulvialis secondary metabolite astaxanthin at frustule, haematococcus pulvialis cell volume output is up to more than the 4-6g/L, wherein content astaxanthin can have stable output up to keep simultaneously more than the 4-5% of dry cell weight turning round throughout the year, and is easy to overcome other assorted algaes and biofouling and topsoil between the on-stream period of open extensive operational system bioreactor.Therefore, the open extensive operational system bioreactor through optimizing has more superiority than thin waterway bioreactor.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment one:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 500 * 150 * 45cm deeply, the thick water white transparency toughened glass of 2cm is adopted in bottom and both sides, the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 30 of 100W sun lamps, 20 of 40W energy-saving spot lamps; Speed governing blade wheel agitator speed is 30r/m in the reactor; Volume water wheels rotating speed is 60r/m; Nutritive medium and the receiver capacity 4000L of temperature adjusting system, agitator speed is 80r/m.Nutritive medium is formed: clear water 45%, and seawater 55%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.23g/L, sodium-acetate 0.51g/L; Sal epsom 0.05g/L, potassium primary phosphate 0.04g/L, urea 0.1g/L; Culture temperature is 23 ℃; Intensity of illumination is the 1000-1500 lux; The sterile air amount of blasting is 0.3m
3/ m; Control nutritive medium dissolved oxygen concentration is 40% saturation ratio; Nutritive medium pH value is 6.8, and culture cycle is 90h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 5g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.032g/L, sodium-acetate 1.4g/L; Culture temperature is 28 ℃; Intensity of illumination is the 2000-2500 lux; The sterile air amount of blasting is 0.1m
3/ m; Nutritive medium pH value is 8; Culture cycle is 72h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 10000r/m, and then through 45 ℃ of vacuum drying treatment, frustule water content 1%.By analysis, content astaxanthin is up to 3.5% of dry cell weight.Nutritive medium behind the results frustule can be through the heating method sterilization for recycling, and Heating temperature is 100 ℃, 2 minutes time.
Embodiment two:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 220 * 110 * 35cm deeply, the thick water white transparency toughened glass of 1.5cm is adopted in bottom and both sides, and the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 12 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 20r/m in the reactor; Volume water wheels rotating speed is 20r/m; Nutritive medium and the receiver capacity 1000L of temperature adjusting system, agitator speed is 60r/m.Nutritive medium is formed: clear water 30%, and seawater 70%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.2g/L, sodium-acetate 0.45g/L, potassium primary phosphate 0.01g/L, sal epsom 0.02g/L; Culture temperature is 24 ℃; Intensity of illumination is the 1000-1200 lux; The sterile air amount of blasting is 0.3m
3/ m; Control nutritive medium dissolved oxygen concentration is 53% saturation ratio; Nutritive medium pH value is neutral, and culture cycle is 72h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 4g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.02g/L, sodium-acetate 1g/L; Culture temperature is 28 ℃; Intensity of illumination is the 2000-2200 lux; The sterile air amount of blasting is 0.08m
3/ m; Nutritive medium pH value is 8.2; Culture cycle is 68h.Through industrial filter plant results haematococcus pulvialis cell, and then through 43 ℃ of vacuum lyophilizations processing, frustule water content 0.9%.By analysis, content astaxanthin is up to 4.1% of dry cell weight.
Embodiment three:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 200 * 100 * 35cm deeply, the thick water white transparency toughened glass of 1cm is adopted in bottom and both sides, and the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 9 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 10r/m in the reactor; Volume water wheels rotating speed is 10r/m; Nutritive medium and the receiver capacity 800L of temperature adjusting system, agitator speed is 20r/m.Nutritive medium is formed: clear water 55%, and seawater 45%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.1g/L, sodium-acetate 0.3g/L, urea 0.1g/L, sal epsom 0.05g/L; Culture temperature is 21 ℃; Intensity of illumination is the 800-1100 lux; The sterile air amount of blasting is 0.05m
3/ m; Control nutritive medium dissolved oxygen concentration is 51% saturation ratio; Nutritive medium pH value is 6.6, and culture cycle is 92h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 4g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.035g/L, sodium-acetate 1.7g/L; Culture temperature is 29 ℃; Intensity of illumination is the 1900-2100 lux; The sterile air amount of blasting is 0.05m
3/ m; Nutritive medium pH value is 8.1; Culture cycle is 69h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 30000r/m, and then handles frustule water content 0.95% through 40 ℃ of vacuum lyophilizations.By analysis, content astaxanthin is up to 3.9% of dry cell weight.Above operating system is comprised that nutritive medium allotment, control light, temperature control, sterilization, stirring, air-blowing, cell harvesting, separation, drying etc. realize long-term semicontinuous production by computer control.Nutritive medium behind the results frustule can be through the heating method sterilization for recycling, and Heating temperature is 70 ℃, 25 minutes time.
Embodiment four:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 500 * 150 * 45cm deeply, the thick water white transparency toughened glass of 2cm is adopted in bottom and both sides, and the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 40 of 100W sun lamps; Speed governing blade wheel agitator speed is 30r/m in the reactor; Volume water wheels rotating speed is 60r/m; Nutritive medium and the receiver capacity 4000L of temperature adjusting system, agitator speed is 80r/m.Nutritive medium is formed: clear water 45%, and seawater 55%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.28g/L, sodium-acetate 0.53g/L, urea 0.05g/L; Culture temperature is 25 ℃; Intensity of illumination is the 1000-1500 lux; The sterile air amount of blasting is 0.3m
3/ m; Control nutritive medium dissolved oxygen concentration is 55% saturation ratio; Nutritive medium pH value is 6.9, and culture cycle is 104h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 5.2g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.035g/L, sodium-acetate 1.7g/L; Culture temperature is 29 ℃; Intensity of illumination is the 2000-2500 lux; The sterile air amount of blasting is 0.1m
3/ m; Nutritive medium pH value is 8; Culture cycle is 80h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 10000r/m, and then handles frustule water content 0.8% through 38 ℃ of vacuum lyophilizations.By analysis, content astaxanthin is up to 4.8% of dry cell weight.
Embodiment five:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 1500 * 300 * 500cm deeply, the thick water white transparency toughened glass of 3cm is adopted in bottom and both sides, the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 15 of 800W incandescent light, 60 of 30W energy-saving spot lamps; Speed governing blade wheel agitator speed is 200r/m in the reactor; Volume water wheels rotating speed is 100r/m; Nutritive medium and the receiver capacity 10000L of temperature adjusting system, agitator speed is 30r/m.Nutritive medium is formed: clear water 35%, and seawater 65%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.26g/L, sodium-acetate 0.52g/L, sal epsom 0.03g/L, potassium primary phosphate 0.08g/L, urea 0.09g/L; Culture temperature is 26 ℃; Intensity of illumination is the 1100-1600 lux; The sterile air amount of blasting is 4m
3/ m; Control nutritive medium dissolved oxygen concentration is 54% saturation ratio; Nutritive medium pH value is neutral, and culture cycle is 96h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 5.4g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.033g/L, sodium-acetate 1.6g/L; Culture temperature is 30 ℃; Intensity of illumination is the 2200-2600 lux; The sterile air amount of blasting is 0.4m
3/ m; Nutritive medium pH value is 7.8; Culture cycle is 68h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 28000r/m, and then handles frustule water content 1.6% through 70 ℃ of low temperature spray dryings.By analysis, content astaxanthin is up to 4.2% of dry cell weight.Above operating system is comprised that nutritive medium allotment, control light, temperature control, sterilization, stirring, air-blowing, cell harvesting, separation, drying etc. realize long-term semicontinuous production by computer control.
Embodiment six:
Set up the open extensive operational system of optimizing and set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 1000 * 200 * 45cm deeply, the thick water white transparency toughened glass of 2.5cm is adopted in bottom and both sides, the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 20 of 150W sun lamps, 42 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 30r/m in the reactor; Volume water wheels rotating speed is 60r/m; Nutritive medium and the receiver capacity 5000L of temperature adjusting system * 2, agitator speed is 100r/m.Nutritive medium is formed: clear water 20%, and seawater 80%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.1g/L, sodium-acetate 0.3g/L, urea 0.09g/L; Culture temperature is 25 ℃; Intensity of illumination is the 1200-1500 lux; The sterile air amount of blasting is 0.9m
3/ m; Control nutritive medium dissolved oxygen concentration is 49% saturation ratio; Nutritive medium pH value is 7.2, and culture cycle is 72h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 3.2g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.02g/L, sodium-acetate 0.8g/L; Culture temperature is 29 ℃; Intensity of illumination is the 2400-2800 lux; The sterile air amount of blasting is 0.8m
3/ m; Nutritive medium pH value is 7.8; Culture cycle is 108h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 15000r/m, and then handles frustule water content 2% through 60 ℃ of low temperature warm air dryings.By analysis, content astaxanthin is up to 4% of dry cell weight.
Embodiment seven:
Set up the open extensive operational system bioreactor of optimizing, main body reactor (tank) length and width are 800 * 150 * 45cm deeply, the thick water white transparency toughened glass of 2cm is adopted in bottom and both sides, and the power that the main body reactor reaches the adjustable brightness of side equalizing device up and down is 30 of 2000W sun lamps; Speed governing blade wheel agitator speed is 60r/m in the reactor; Volume water wheels rotating speed is 30r/m; Nutritive medium and the receiver capacity 6000L of temperature adjusting system, agitator speed is 200r/m.Nutritive medium is formed: clear water 33%, and seawater 67%, nutritive salt such as saltpetre, sodium-acetate are adjusted according to different growth phase physiological requirements.Clear water and seawater can be through the ozonizer sterilizations.Pure culture of algae kind and population density are regulated the good culture condition of stage through optimizing: nutrient solution concentration saltpetre 0.25g/L, sodium-acetate 0.45g/L, urea 0.06g/L; Culture temperature is 26 ℃; Intensity of illumination is the 1300-1500 lux; The sterile air amount of blasting is 0.7m
3/ m; Control nutritive medium dissolved oxygen concentration is 52% saturation ratio; Nutritive medium pH value is 7.3, and culture cycle is 76h.Good culture condition promotes that haematococcus pulvialis reaches logarithmic phase, and the frustule volumetric production is up to 4g/L.Carry out the physiology induction regulating controlling by the change of envrionment conditions and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pulvialis cell, this stage culture condition is: nutrient solution concentration saltpetre 0.033g/L, sodium-acetate 1.5g/L; Culture temperature is 28.5 ℃; Intensity of illumination is the 2500-2900 lux; The sterile air amount of blasting is 0.7m
3/ m; Nutritive medium pH value is 8.1; Culture cycle is 100h.Separate centrifugal results haematococcus pulvialis cell through the continous way screenings, rotating speed is 2000r/m, and then handles frustule water content 1.8% through 60 ℃ of low temperature warm air dryings.By analysis, content astaxanthin is up to 3.9% of dry cell weight.Above operating system is comprised that nutritive medium allotment, control light, temperature control, sterilization, stirring, air-blowing, cell harvesting, separation, drying etc. realize long-term semicontinuous production by computer control.
Claims (9)
1. the open extensive operational system bioreactor of an optimization is regulated the method that the secondary metabolite astaxanthin synthesizes and accumulates in haematococcus pulvialis population density and the cell, it is characterized in that setting up the open extensive operational system bioreactor of optimization, regulatable envrionment conditions is provided, in bioreactor bottom and both sides adopt light transmissive material and external regulatable illumination facilities, speed governing paddle wheel agitator and volume water wheels, air-blowing facility and O
2Gas system, nutritive medium allotment and temperature adjusting system, sterilizing and bacterial system, carry out physiology by the change of envrionment conditions and induce synthetic and accumulation, results haematococcus pulvialis cell and the cryodrying of regulating haematococcus pulvialis population density and regulation and control haematococcus pulvialis secondary metabolite astaxanthin, obtain isolating haematococcus pulvialis cell.
2. in accordance with the method for claim 1, it is characterized in that the concrete steps of this method are as follows:
(1), transforms and optimizes usually thin waterway bioreactor or the high open extensive operational system bioreactor of design light utilization efficiency that the little algae of industry cultivation is used, comprising support type main body base, the main body reactor of adjustable blowdown system is set, regulatable illumination facilities is set, speed governing paddle wheel agitator and volume water wheels, air-blowing facility and O are set
2Gas system is provided with nutritive medium allotment and temperature adjusting system, sterilizing and bacterial system and cryodrying facility.
(2), the main body reactor is rectangle, length and width are 200-1500cm * 100-300cm, the degree of depth is 250-500cm.
(3), light transmissive material can select the water white transparency toughened glass of high tensile force resistance and compressive resistance for use, thickness is 0.8-3cm; Reach balanced illuminating fluorescent lamp, incandescent light, sun lamp or the energy-saving spot lamp that many adjustable brightness are installed in side outside bioreactor up and down, every lamp power is 40-2000w; Can use separately or compound action; Adjustable intensity of illumination is the 800-3800 lux.
(4), adopt the adjustable speed paddle wheel agitator of industrial use, rotating speed is 10-400r/m; The volume water wheels are the volume water wheels of industrial use, and rotating speed is 10-300r/m, and the volume water wheels are installed on a side of photoproduction sundries.
(5), adopt the pneumatic plant of industrial use and be treated as sterile air through the oil strain drainage bacteriological filtration of routine; O
2Gas adopts industrial oxygen bottle, O
2Gas adopts the filtration method degerming.By variable valve and be distributed in bioreactor bottom porous pod apertures and blast sterile air or oxygen.The sterile air amount of blasting is 0.05-5m
3/ m.O
2Gas is regulated by the concentration of the required dissolved oxygen of nutritive medium and is blasted.
(6), nutritive medium allotment and temperature adjusting system are made up of the interlayer receiver that is equipped with cold water circulating refrigerating device, heating unit, nutritive medium recycle pump, measuring and controlling temp facility and belt stirrer of industrial use.The receiver capacity is 30-10000L; Agitator speed is 20-300r/m.
(7), nutritive medium is formed: clear water 20-70%; Seawater 30-80%; Also can adopt artificial allotment seawater; Saltpetre 0.1-0.4g/L; Sodium-acetate 0.3-0.6g/L; Can also add nutritive salt such as urea, sal epsom, potassium primary phosphate, add-on is 0.01-0.1g/L.The required dissolved oxygen concentration of nutritive medium is the 40-55% saturation ratio.The haematococcus pulvialis pH value of suitable growth is neutral to slight alkalinity.
(8), the open extensive movement system bioreactor of You Huaing cleaning and sterilizing regularly, it is clean pollution-free that the nutritive medium allocation process keeps, clear water and seawater can adopt the water electrolytic ozone producer of industrial use to carry out sterilization, the nutritive medium that employing recycles can adopt the heating method sterilization, Heating temperature is 70-80 ℃, time 8-25 minute, or 95-100 ℃, time 2-15 minute; Also can adopt the chemical sterilization method to carry out sterilization.
(9), pure culture of haematococcus pulvialis algae kind and population density adjusting stage intensity of illumination is the 800-1800 lux; Solution temperature is 16-31 ℃.Nutrient solution concentration is: saltpetre 0.1-0.4g/L; Sodium-acetate 0.3-0.6g/L can also add nutritive salt such as urea, sal epsom, potassium primary phosphate, and add-on is 0.01-0.1g/L.Culture cycle is 72-108h.
(10), synthetic and accumulation stage of secondary metabolite astaxanthin is the 1900-3200 lux according to intensity in the haematococcus pulvialis cell; Solution temperature is 21-32 ℃.Nutrient solution concentration is: saltpetre 0.01-0.05g/L; Sodium-acetate 0.8-2g/L.Culture cycle is 50-80h.
(11), adopt industrial continous way screenings separation centrifuges, rotating speed is 3000-30000r/m.Also can adopt other separating devices or filtration method results frustule.Isolating haematococcus pulvialis is adopted the cryodrying facility of industrial use, carry out drying as equipment such as vacuum lyophilization, vacuum-drying, low temperature warm air drying, low temperature spray dryings, drying temperature is 38-85 ℃.
3. in accordance with the method for claim 2, it is characterized in that the main body reactor is rectangle in the step (2), length and width are 220-500cm * 110-150cm; The degree of depth is 350-450cm.
4. in accordance with the method for claim 2, it is characterized in that adjustable intensity of illumination is the 1500-2500 lux in the step (3).
5. in accordance with the method for claim 2, it is characterized in that adopting adjustable speed paddle wheel agitator in the step (4), rotating speed is 20-60r/m; Volume water wheels rotating speed is 20-80r/m.
6. in accordance with the method for claim 2, it is characterized in that the sterile air amount of blasting is 0.08-0.3m in the step (5)
3/ m.
7. in accordance with the method for claim 2, it is characterized in that nutritive medium is formed in the step (7): clear water is 30-45%; Seawater is 55-70%; Saltpetre is 0.2-0.28g/L; Sodium-acetate is 0.45-0.53g/L.
8. in accordance with the method for claim 2, it is characterized in that intensity of illumination is the 1000-1500 lux in the step (9); Solution temperature is 24-28 ℃; Nutrient solution concentration is: saltpetre 0.2-0.28g/L; Sodium-acetate is 0.45-0.53g/L; Culture cycle is 80-96h.
9. in accordance with the method for claim 2, it is characterized in that intensity of illumination is the 2000-2500 lux in the step (10); Solution temperature is 26-30 ℃; Nutrient solution concentration is: saltpetre 0.02-0.035g/L; Sodium-acetate is 1-1.7g/L.Culture cycle is 60-72h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101639690B (en) * | 2009-06-19 | 2012-05-02 | 新奥科技发展有限公司 | System and method for controlling reaction of alga |
| CN106906138A (en) * | 2017-05-05 | 2017-06-30 | 宁波浮田生物技术有限公司 | A kind of haematococcus pluvialis high concentration induces the device and method of astaxanthin accumulation |
| WO2021057711A1 (en) * | 2019-09-23 | 2021-04-01 | 山东拜昂生物技术有限公司 | Method for producing astaxanthin by heterotrophic culture of haematococcus pluvialis |
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2002
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101639690B (en) * | 2009-06-19 | 2012-05-02 | 新奥科技发展有限公司 | System and method for controlling reaction of alga |
| CN106906138A (en) * | 2017-05-05 | 2017-06-30 | 宁波浮田生物技术有限公司 | A kind of haematococcus pluvialis high concentration induces the device and method of astaxanthin accumulation |
| CN106906138B (en) * | 2017-05-05 | 2023-08-29 | 宁波浮田生物技术有限公司 | Device and method for inducing astaxanthin accumulation in haematococcus pluvialis at high concentration |
| WO2021057711A1 (en) * | 2019-09-23 | 2021-04-01 | 山东拜昂生物技术有限公司 | Method for producing astaxanthin by heterotrophic culture of haematococcus pluvialis |
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