CN1181194C - Method of promoting haematococcus pluvialis growing multiplication and regulating astaxanthin synthesis and accumulation in photobiological reactor - Google Patents
Method of promoting haematococcus pluvialis growing multiplication and regulating astaxanthin synthesis and accumulation in photobiological reactor Download PDFInfo
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- CN1181194C CN1181194C CNB02134387XA CN02134387A CN1181194C CN 1181194 C CN1181194 C CN 1181194C CN B02134387X A CNB02134387X A CN B02134387XA CN 02134387 A CN02134387 A CN 02134387A CN 1181194 C CN1181194 C CN 1181194C
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- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 48
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention relates to an adjustable method for culturing and promoting the growth proliferation of pluvial growing haematococcus, improving the population density, and controlling the synthesis and the accumulation of the astaxanthin of secondary metabolite in cells by a photobiologic reactor of an all weather enclosed large-scale operation system. The adjustable method comprises the following steps: the bottom, the circumference and a closed cover of the reactor of a main body are made of transparent materials and are externally provided with environmental conditions manually adjusted, such as an adjustable illumination device, a speed adjusting paddle wheel stirrer, a rolling water wheel, a sterile air blowing device, an O2 gas system, a brackish-fresh water nutrient solution preparing system, a temperature adjusting system, a cleaning and sterilization system, etc.; thereby, the growth proliferation of the pluvial growing haematococcus is induced and controlled, the population density is improved, and the synthesis and the accumulation of the astaxanthin of the secondary metabolite in the cells of the pluvial growing haematococcus are controlled. Haematococcus cells with the astaxanthin can be obtained by the procedures, such as separation, haematococcus cell obtaining, low temperature drying, etc.
Description
Technical field
The present invention relates to a kind of regulatable round-the-clock closed extensive operational system bioreactor cultivation and promotion haematococcus pluvialis growing multiplication and improve the method that the secondary metabolite astaxanthin synthesizes and accumulates in population density and the regulating cell.
Background technology
Haematocoocus Pluvialls (Haematococcus pluvialis) is a kind of unicell green alga, cell ovalize or avette, and size is not from waiting to the 20-30 micron about 5 microns, sometimes even bigger; Two isometric flagellums are arranged, and the swing of flagellum promotes cell and freely moves about; Tool space between cell walls and protoplasma, be full of gelatinoid therebetween, have tenuigenin to connect silk between cell walls and protoplastis and link to each other, nourishing and generating is cell fission, formation 2,4,8,16 even more a plurality of daughter cell, monogony forms glue colony or chlamydospore when environment is bad.The Haematocoocus Pluvialls that is in the growing multiplication stage is green; 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 pluvialis cell will be tending towards slowly and make motor cell lose flagellum, form glue colony or chlamydospore, simultaneously, promoting that secondary metabolite is synthetic in the cell, mainly is a large amount of accumulation of astaxanthin, makes the color of haematococcus pluvialis cell form brick-red rapidly.Influence secondary metabolite composition and growing amount in physiology, biomass, output and the cell of Haematocoocus Pluvialls, relate generally to kind of algae and good culture condition (as the degree of purification of environment, temperature, illumination, dissolved oxygen, nutritive medium composition, fluid shear power etc.).So that having the algae kind now, genetically engineered and the transformation of genetic engineering means obtain the excellent engineering bacterial strain by physics or chemistry, improve the light saturation value of algae kind, the inhibition of anti-light, and degeneration-resistant, contamination resistance, can obtain the fast and strong algae kind of astaxanthin accumulation ability of high temperature resistant, cell proliferation.There are some researches show, through the bioreactor of optimization and good culture condition, can impel the 4-5% of the content of astaxanthin in the Haematocoocus Pluvialls up to 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 biological pollution and Haematocoocus Pluvialls culturing process population density are low make low cause expensive of yield per unit to the principal element that hinders Haematocoocus Pluvialls scale operation.The Haematocoocus Pluvialls cultivated of bio-reactor 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 breadboard bench-scale testing.
Summary of the invention
The purpose of this invention is to provide a kind of regulatable round-the-clock closed extensive operational system bioreactor cultivation and promote that population density is bred and improved to haematococcus pluvialis cell growth and the interior secondary metabolite astaxanthin of regulating cell synthesizes and the method for accumulation.
Method of the present invention comprises sets up regulatable round-the-clock closed extensive operational system bioreactor, reach all around and capping employing light transmissive material and external regulatable illumination facilities at the main body reactor bottom, speed governing paddle wheel agitator and volume water wheels, sterile air air-blowing facility and O
2Gas system, allotment of degree of saltiness water nutrition liquid and temperature adjusting system, clean and sterilizing and bacterial system, by the change of envrionment conditions carry out physiology induction regulating controlling haematococcus pluvialis growing multiplication and improve population density and the regulation and control haematococcus pluvialis cell in the secondary metabolite astaxanthin synthetic and accumulation, separate operations such as gathering in the crops frustule and cryodrying.
Method concrete steps of the present invention are as follows:
One, set up adjustable round-the-clock closed extensive operational system bioreactor: transformation and optimization the thin waterway bioreactor of the little algae use of industry cultivation usually become closed photo bioreactor, or the high round-the-clock closed extensive operational system bioreactor of design light utilization efficiency, comprising at its bioreactor support type main body base, the main body reactor (tank) of closed adjustable blowdown system is set, regulatable illumination facilities is set, speed governing paddle wheel agitator and volume water wheels are set, are provided with the sterile air facility and the O of porting
2Gas system, the degree of saltiness water nutrition liquid that is provided with reflux is allocated and the temperature adjusting system, and cleaning is with sterilizing and bacterial system and separate results frustule 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 450-700cm, is generally 550-650cm.Also round-the-clock closed extensive operational system bioreactor can be comprised that operations such as the allotment of degree of saltiness water nutrition liquid, control light, temperature control, cleaning, sterilization, sterilization, stirring, air-blowing, separation results frustule and drying realize long-term semicontinuous production by computer control.
Two, regulatable round-the-clock closed extensive operational system bioreactor (hereinafter to be referred as the bioreactor) bottom of transformation and optimization reaches all around and capping employing light transmissive material and external adjustable illumination facilities on the basis of thin waterway photo-bioreactor system: light transmissive material can be selected the water white transparency toughened glass or the special glass of high tensile force resistance and compressive resistance for use, thickness is 0.8-3cm, be generally 1.5-2cm enclosure portion light transmissive material and can select commercially available transparent plastic film or matrix material transparent film for use, thickness is 0.01-0.6mm, is generally 0.03-0.3mm; Transparent film is as main body reactor capping blended rubber band paper sealing label or tackiness agent sealing label; The enclosure portion light transmissive material also can be selected light transmissive materials such as synthetic glass for use; Reach around the main body reactor illuminating fluorescent lamp, incandescent light, sun lamp or the energy-saving spot lamp of many adjustable brightness are installed up and down, every lamp power is 40-2000w, is generally 40-300w; Can be used singly or in combination; Haematococcus pluvialis growing multiplication and raising population density regulation and control stage intensity of illumination are the 800-1800 lux, are generally the 1000-1500 lux; The interior secondary metabolite astaxanthin of regulating cell synthesizes and accumulation stage intensity of illumination is the 1900-3200 lux, is generally the 2000-2500 lux; Intensity of illumination and periodicity of illumination can be regulated and control 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: speed governing blade wheel agitator can make main body reactor degree of saltiness water nutrition liquid be in slight flow state and keep the Modulatory character of degree of saltiness water nutrition liquid composition and dissolved oxygen concentration and pH value, makes the growth of frustule be in suspended state; Haematocoocus Pluvialls, can effectively reduce the bridging effect between the frustule and improve the utilization of illumination with 0.01-0.08m/s flow velocity circulation as degree of saltiness water nutrition liquid under suitable fluid shear power effect.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; Bioreactor volume water wheels are installed on a side of main body reactor, the moving flow velocity of regulation and control degree of saltiness water nutrition liquid recycle stream.
Four, be provided with the sterile air facility and the O of porting
2Gas system: adopt the air compressor machine of industrial use and be treated as sterile air through the oil strain drainage bacteriological filtration of routine; O
2Gas adopts the industrial oxygen bottle, and adopts the filtration method degerming.Bioreactor is by variable valve and be distributed in main body reactor bottom duct type micropore pod apertures and blast sterile air and oxygen.The haematococcus pluvialis growing multiplication stage sterile air amount of blasting is 0.02-2m
3/ m is generally 0.03-0.2m
3/ m; The interior secondary metabolite astaxanthin of regulation and control haematococcus pluvialis cell synthesizes and accumulation stage sterile air air-blowing amount is 0.01-0.08m
3/ m is generally 0.02-0.05m
3/ m; O
2Gas is regulated by the concentration of the required dissolved oxygen of degree of saltiness water nutrition liquid.
Five, bioreactor is provided with the allotment of degree of saltiness water nutrition liquid and the temperature adjusting system of reflux: allotment of degree of saltiness water nutrition liquid and temperature adjusting system are made up of the interlayer receiver of the industrial use of configuration cold water circulating refrigerating device, heating unit, nutritive medium recycle pump, reflux line, measuring and controlling temp facility and belt stirrer.Every group of receiver is 2 to 3, and the receiver capacity is 100-10000L, is generally 1000-6000L; Agitator speed is 20-300r/m, is generally 60-80r/m.
The Haematocoocus Pluvialls degree of saltiness water nutrition liquid that is in the growth and proliferation of cell stage is formed: degree of saltiness water proportioning is clear water 30-70%, is generally 45-65%; Seawater 25-75% is generally 35-55%; Also can adopt artificial allotment seawater; Ratio of nutrient solution is saltpetre 0.1-0.42g/L, is generally 0.2-0.3g/L; Sodium-acetate 0.3-0.65g/L is generally 0.45-0.55g/L; Can also add nutritive salt such as urea, sal epsom, potassium primary phosphate, can be one or more, and add-on is 0.01-1g/L; Degree of saltiness water nutrition liquid dissolved oxygen concentration is the 40-55% saturation ratio; Haematococcus pluvialis growing multiplication stage optimum pH is neutral; Frustule growing multiplication stage optimum temperuture is 14-27 ℃, is generally 20-24 ℃; The culture cycle that frustule ramp propagation is in logarithmic phase is 48-96h, is generally 60-72h.
Carry out the physiology induction regulating controlling by the change of bad border condition and promote in the haematococcus pluvialis cell that the synthetic and the suitableeest culture temperature of accumulation stage of secondary metabolite astaxanthin is 21-32 ℃, be generally 26-29 ℃, optimum pH is a slight alkalinity; Culture cycle is 50-80h, is generally 60-72h.
Six, cleaning and sterilizing and bacterial system: should be before putting into production with the thorough cleaning and sterilizing of bioreactor, all should remove the transparent film of enclosure portion and the transparent film that more renews during each the cleaning, it is clean pollution-free that degree of saltiness water nutrition liquid allocation process should keep, and clear water and seawater can adopt the ozonizer of industrial use to carry out sterilization in the degree of saltiness water; Nutritive medium can adopt the heating method sterilization, and Heating temperature is 70-80 ℃, and time 15-25 minute, or 95-100 ℃, time 5-15 minute; Also can adopt the chemical sterilization method to carry out sterilization.After can adopting the heating method sterilization, the degree of saltiness water nutrition liquid that recycles comes into operation again.
Seven, results haematococcus pluvialis cell and cryodrying: centrifugal is the method for at present most widely used results frustule, adopts industrial continous way screenings tripping device 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 pluvialis cell 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 providing regulatable envrionment conditions to promote Haematocoocus Pluvialls ramp propagation and improving population density and promote the synthetic of secondary metabolite astaxanthin in the cell and accumulate most important in the closed extensive operational system.Cultivate Haematocoocus Pluvialls because the variable of abiotic factor is too many, the synthetic and accumulation that promotes cell internal object product as the influence and the strain environment of the multiple factors such as biological pollution, intensity of illumination, dissolved oxygen, temperature and nutritive medium composition and become very complicated.Illumination is the important factor of haematococcus pluvialis growing.The illuminance that is suitable for most haematococcus pluvialis growing multiplication is medium tenacity illumination 1000-1600 lux; Strengthen intensity of illumination in the 1900-3200 lux, can promote the synthetic and accumulation of the biological astaxanthin of secondary metabolism in the haematococcus pluvialis cell; To suppress the growth of Haematocoocus Pluvialls 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 growing multiplication of Haematocoocus Pluvialls and obviously improves the cell volume productive rate.The bridging effect each other of culturing process illumination deficiency and frustule will cause frustule growth to be suppressed and increased the chance that other biological pollutes.The thin waterway bioreactor that the paddle wheel that adopts in the little algae of outdoor artificial culture stirs, textural the illumination of accepting front lighting, to light make full use of and the regulation and control of illumination are limited to, a guardian technique difficult problem that round the clock and rain or shine day becomes the synthetic of secondary metabolite astaxanthin in large scale culturing Haematocoocus Pluvialls and the regulating cell and accumulate with the big influence of normal daylight intensity of illumination difference particularly.
Become closed photo bioreactor by transforming and optimizing the thin waterway photo-bioreactor system, whole intensity of illumination around the bottom of main body reactor reaches and in the capping employing light transmissive material raising bioreactor, improving whole culture condition is the envrionment conditions of regulatable high purification, utilize daylight illumination and external regulatable source of artificial light irradiation to make frustule can receive medium above intensity illumination round the clock equably, higher to the efficiency of light energy utilization, both can promote haematococcus pluvialis cell growth propagation, can regulate and control the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pluvialis cell again, the haematococcus pluvialis cell volumetric production is up to more than the 4-6g/L, wherein content astaxanthin can be more than the 4-5% up to dry cell weight, can overcome again simultaneously between the on-stream period of open extensive operational system bioreactor round the clock and rain or shine day influence and other assorted algaes and biofouling and topsoil.Because main body reactor enclosure portion light transmissive material is changed convenient, it is convenient more quick that the main body reactor monolith is cleaned, and helps realizing by the long-term semi-continuous production of computer control.Therefore, setting up regulatable round-the-clock closed extensive operational system bioreactor cultivates than thin waterway bioreactor and promotes haematococcus pluvialis cell growth propagation and improve in population density and the regulating cell the synthetic and accumulation of secondary metabolite astaxanthin and have more superiority.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment one:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 500 * 150 * 65cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 2cm, it is the transparent film blended rubber band paper sealing label of 0.2mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 20 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 50r/m, and degree of saltiness water nutrition liquid circulates with the 0.04m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 3 capacity 4000L of temperature adjusting system disposition, agitator speed is 80r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 65%, seawater 35%, saltpetre 0.21g/L, sodium-acetate 0.52g/L, sal epsom 0.04g/L, potassium primary phosphate 0.04g/L, urea 0.9g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 100 ℃ of heat-sterilizations 5 minutes; Frustule growing multiplication temperature is 20 ℃; Intensity of illumination is the 1300-1700 lux; The sterile air amount of blasting is 0.3m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 43% saturation ratio; The suitableeest frustule growing multiplication pH value is 6.8, and culture cycle is 60h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 28 ℃; Intensity of illumination is the 2300-2800 lux; The sterile air amount of blasting is 0.1m
3/ m; Optimum pH is 8.2; Culture cycle is 72h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 10000r/m, and then through 45 ℃ of vacuum drying treatment, makes the frustule water content be lower than 1%.By analysis, the frustule content astaxanthin is up to 3.9% of dry cell weight.
Embodiment two:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 220 * 110 * 55cm deeply, around reaching, the bottom adopts the thick water white transparency special glass of 1.5cm, it is the transparent film blended rubber band paper sealing label of 0.1mm that enclosure portion adopts thickness, and reaching the power that adjustable brightness is installed up and down around the main body reactor is 20 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 20r/m in the reactor; Volume water wheels rotating speed is 30r/m, and degree of saltiness water nutrition liquid is with the flow velocity circulation of 0.02m/s; The receiver of the allotment of degree of saltiness water nutrition liquid and 2 capacity 1200L of temperature adjusting system disposition, agitator speed is 60r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 40%, seawater 60%, saltpetre 0.2g/L, sodium-acetate 0.6g/L, potassium primary phosphate 0.4g/L, sal epsom 0.2g/L, urea 1g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 95 ℃ of heat-sterilizations 10 minutes; Frustule growing multiplication temperature is 23 ℃; Intensity of illumination is the 1100-1500 lux; The sterile air amount of blasting is 0.3m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 53% saturation ratio; The suitableeest frustule growing multiplication pH value is neutral, and culture cycle is 48h, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 28.5 ℃; Intensity of illumination is the 2400-2700 lux; The sterile air amount of blasting is 0.1m
3/ m; Optimum pH is 8; Culture cycle is 72h, and it is brick-red that frustule is.Device is gathered in the crops haematococcus pluvialis cell after filtration, and then through 45 ℃ of vacuum drying treatment, makes the frustule water content be lower than 1%.By analysis, the frustule content astaxanthin is up to 4.1% of dry cell weight.
Embodiment three:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 200 * 100 * 65cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 1cm, it is the transparent film blended rubber stick sealing label of 0.2mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 7 of 40W fluorescent lamps, 5 of 100W sun lamps; Speed governing blade wheel agitator speed is 10r/m in the reactor; Volume water wheels rotating speed is 10r/m, and degree of saltiness water nutrition liquid circulates with the 0.03m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 2 capacity 800L of temperature adjusting system disposition, agitator speed is 20r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 68%, seawater 32%, saltpetre 0.4g/L, sodium-acetate 0.4g/L, urea 0.3g/L, sal epsom 0.04g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 98 ℃ of heat-sterilizations 8 minutes; Frustule growing multiplication temperature is 24 ℃; Intensity of illumination is the 800-1600 lux; The sterile air amount of blasting is 0.05m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 51% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 6.6, and culture cycle is 60h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 28 ℃; Intensity of illumination is the 2400-2900 lux; The sterile air amount of blasting is 0.08m
3/ m; Optimum pH is 8.3; Culture cycle is 68h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 12000r/m, and then through 42 ℃ of vacuum drying treatment, makes the frustule water content be lower than 1.5%.By analysis, the frustule content astaxanthin is up to 4.3% of dry cell weight.
Embodiment four:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 500 * 150 * 65cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 2cm, it is the matrix material transparent film blended rubber band paper sealing label of 0.18mm that enclosure portion adopts thickness, and reaching the power that adjustable brightness is installed up and down around the main body reactor is 27 of 100W sun lamps; Speed governing blade wheel agitator speed is 30r/m in the reactor; Volume water wheels rotating speed is 50r/m, and degree of saltiness water nutrition liquid circulates with the 0.04m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 3 capacity 4000L of temperature adjusting system disposition, agitator speed is 80r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 65%, seawater 35%, saltpetre 0.21g/L, sodium-acetate 0.5g/L, urea 0.4g/L, potassium primary phosphate 0.01g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 70 ℃ of heat-sterilizations 25 minutes; Frustule growing multiplication temperature is 17 ℃; Intensity of illumination is the 1600-1750 lux; The sterile air amount of blasting is 0.3m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 55% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 6.9, and culture cycle is 96h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 28 ℃; Intensity of illumination is the 2300-2500 lux; The sterile air amount of blasting is 0.1m
3/ m; Optimum pH is 8; Culture cycle is 72h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 8000r/m, and then handles through 80 ℃ of low temperature spray dryings, makes the frustule water content be lower than 1%.Devices such as the nutritive medium allotment of above bioreactor configuration, control light, temperature control, cleaning, sterilization, sterilization, stirring, air-blowing, separation, drying are realized semicontinuous production by computer control.By analysis, the frustule content astaxanthin is up to 4.2% of dry cell weight.
Embodiment five:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 1500 * 300 * 70cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 3cm, it is the transparent film blended rubber stick sealing label of 0.6mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 20 of 300W incandescent light, 50 of 30W energy-saving spot lamps; Speed governing blade wheel agitator speed is 80r/m in the reactor; Volume water wheels rotating speed is 100r/m, and degree of saltiness water nutrition liquid circulates with the 0.06m/s flow velocity; The allotment of degree of saltiness water nutrition liquid and 3 capacity 10000L of temperature adjusting system disposition receiver, agitator speed is 60r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 70%, seawater 30%, saltpetre 0.33g/L, sodium-acetate 0.55g/L, sal epsom 0.03g/L, potassium primary phosphate 0.08g/L, urea 0.8g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 95 ℃ of heat-sterilizations 10 minutes; Frustule growing multiplication temperature is 20 ℃; Intensity of illumination is the 1100-1600 lux; The sterile air amount of blasting is 1m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 54% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 6.9, and culture cycle is 60h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 29 ℃; Intensity of illumination is the 2300-2500 lux; The sterile air amount of blasting is 0.16m
3/ m; Optimum pH is 8.1; Culture cycle is 72h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 20000r/m, and then through 43 ℃ of vacuum drying treatment, makes the frustule water content be lower than 1%.By analysis, the frustule content astaxanthin is up to 4.4% of dry cell weight.
Embodiment six:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 1000 * 200 * 68cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 2.5cm, it is the transparent film blended rubber band paper sealing label of 0.65mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 18 of 150W sun lamps, 12 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 30r/m in the reactor; Volume water wheels rotating speed is 60r/m, and degree of saltiness water nutrition liquid circulates with the 0.04m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 3 capacity 7000L of temperature adjusting system disposition, agitator speed is 100r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 56%, seawater 44%, saltpetre 0.38g/L, sal epsom 0.6g/L, sodium-acetate 0.38g/L, urea 0.9g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 98 ℃ of heat-sterilizations 6 minutes; Frustule growing multiplication temperature is 21 ℃; Intensity of illumination is the 1200-1600 lux; The sterile air amount of blasting is 0.9m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 49% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 7.2, and culture cycle is 54h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition: culture temperature is 28 ℃; Intensity of illumination is the 2400-2500 lux; The sterile air amount of blasting is 0.13m
3/ m; Optimum pH is 8.2; Culture cycle is 72h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 11000r/m, and then through 45 ℃ of vacuum drying treatment, makes the frustule water content be lower than 1%.By analysis, the frustule content astaxanthin is up to 4.5% of dry cell weight.
Embodiment seven:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 800 * 150 * 65cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 2cm, it is the transparent film blended rubber band paper sealing label of 0.45mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 8 of 300W sun lamps, 13 of 40W fluorescent lamps; Speed governing blade wheel agitator speed is 60r/m in the reactor; Volume water wheels rotating speed is 30r/m, and degree of saltiness water nutrition liquid circulates with the 0.06m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 3 capacity 6000L of temperature adjusting system disposition, agitator speed is 200r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 70%, seawater 30%, saltpetre 0.33g/L, sodium-acetate 0.45g/L, urea 0.7g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 96 ℃ of heat-sterilizations 10 minutes; Frustule growing multiplication temperature is 23 ℃; Intensity of illumination is the 1300-1600 lux; The sterile air amount of blasting is 0.7m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 52% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 7.1, and induction regulating controlling promotes that secondary metabolite astaxanthin culture cycle synthetic and accumulation is 80h in the cell, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.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 pluvialis cell, this stage culture condition; Culture temperature is 28.5 ℃; Intensity of illumination is the 2400-2600 lux; The sterile air amount of blasting is 0.18m
3/ m; Optimum pH is 8.4; Culture cycle is 72h, and it is brick-red that frustule is.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 15000r/m, and then handles through 40 ℃ of vacuum lyophilizations, makes the frustule water content be lower than 1%.By analysis, the frustule content astaxanthin is up to 4.5% of dry cell weight.
Embodiment eight:
A kind of regulatable round-the-clock closed extensive operational system bioreactor of on the basis of thin waterway photo-bioreactor system, transforming and optimizing, main body reactor (tank) length and width are 900 * 160 * 65cm deeply, around reaching, the bottom adopts the thick water white transparency toughened glass of 2.5cm, it is the transparent film blended rubber band paper sealing label of 0.5mm that enclosure portion adopts thickness, reaching the power that adjustable brightness is installed up and down around the main body reactor is 10 of 200W sun lamps, 20 of 30W electricity-saving lamps; Speed governing blade wheel agitator speed is 50r/m in the reactor; Volume water wheels rotating speed is 30r/m, and degree of saltiness water nutrition liquid circulates with the 0.05m/s flow velocity; The receiver of the allotment of degree of saltiness water nutrition liquid and 3 capacity 8000L of temperature adjusting system disposition, agitator speed is 200r/m.The culture condition in frustule growing multiplication and raising population density regulation and control stage is that degree of saltiness water nutrition liquid is formed: clear water 68%, seawater 32%, saltpetre 0.27g/L, sal epsom 0.01g/L, sodium-acetate 0.33g/L, urea 0.85g/L; Clear water and seawater can be through the ozonizer sterilizations; Nutritive medium adopts through 96 ℃ of heat-sterilizations 4 minutes; Frustule growing multiplication temperature is 23 ℃; Intensity of illumination is the 1400-1600 lux; The sterile air amount of blasting is 0.8m
3/ m; Degree of saltiness water nutrition liquid dissolved oxygen concentration is 54% saturation ratio; The pH value of the suitableeest frustule growing multiplication is 6.8, and culture cycle is 60h, and the frustule growing multiplication is in logarithmic phase, and it is green that frustule is.Carry out the physiology induction regulating controlling by the change of bad border condition and promote the synthetic and accumulation of secondary metabolite astaxanthin in the haematococcus pluvialis cell, this stage culture condition: culture temperature is 28.5 ℃; Intensity of illumination is the 2500-2600 lux; The sterile air amount of blasting is 0.1m
3/ m; Optimum pH is 8; Culture cycle is 65h.Through continous way screenings tripping device results haematococcus pluvialis cell, rotating speed is 13000r/m, and then handles through 55 ℃ of warm air dryings, makes the frustule water content be lower than 1.2%.By analysis, the frustule content astaxanthin is up to 4.2% of dry cell weight.
Claims (5)
1. a regulatable round-the-clock closed extensive operational system bioreactor is cultivated and is promoted haematococcus pluvialis growing multiplication and improve the method for the synthetic and accumulation of secondary metabolite astaxanthin in population density and the regulating cell, it is characterized in that this method be included in common industry cultivate the regulatable round-the-clock closed extensive operational system bioreactor main body reactor bottom transforming on the basis of the thin waterway photo-bioreactor system that little algae uses and optimize and around and capping employing light transmissive material and external regulatable illumination facilities, speed governing paddle wheel agitator and volume water wheels, sterile air air-blowing facility and O
2Gas system, degree of saltiness water nutrition liquid allotment and temperature adjusting system, clean and artificial regulatable envrionment conditions induction regulating controlling haematococcus pluvialis growing multiplication such as sterilizing and bacterial system and improve population density and the regulation and control haematococcus pluvialis cell in the secondary metabolite astaxanthin synthesize and accumulate, separate operations such as gathering in the crops frustule and cryodrying; Haematococcus pluvialis growing multiplication and raising population density regulation and control stage intensity of illumination are the 800-1800 lux; The interior secondary metabolite astaxanthin of regulating cell synthesizes and accumulation stage intensity of illumination is the 1900-3200 lux; Haematocoocus Pluvialls is under suitable fluid shear power effect, and degree of saltiness water nutrition liquid is with 0.01-0.08m/s flow velocity circulation, can effectively reduce the bridging effect between the frustule and improve the utilization of illumination; The haematococcus pluvialis growing multiplication stage sterile air amount of blasting is 0.02-2m
3/ m; The interior secondary metabolite astaxanthin of regulation and control haematococcus pluvialis cell synthesizes and accumulation stage sterile air air-blowing amount is 0.01-0.08m
3/ m; The Haematocoocus Pluvialls degree of saltiness water nutrition liquid that is in the growth and proliferation of cell stage is formed: degree of saltiness water proportioning is clear water 30-70%, seawater 25-75%; Ratio of nutrient solution is saltpetre 0.1-0.42g/L, sodium-acetate 0.3-0.65g/L; Degree of saltiness water nutrition liquid dissolved oxygen concentration is the 40-55% saturation ratio; Haematococcus pluvialis growing multiplication stage optimum pH is neutral; Frustule growing multiplication stage optimum temperuture is 14-27 ℃; The culture cycle that frustule ramp propagation is in logarithmic phase is 48-96h; Carry out the physiology induction regulating controlling and promote that the interior secondary metabolite astaxanthin of haematococcus pluvialis cell synthesizes and the suitableeest culture temperature of accumulation stage is 21-32 ℃; Optimum pH is a slight alkalinity; Culture cycle is 50-80h.
2. a promotion haematococcus pluvialis growing multiplication as claimed in claim 1 and improve the method for the synthetic and accumulation of secondary metabolite astaxanthin in population density and the regulating cell is characterized in that the concrete steps of this method are as follows:
(1), the industrial usually thin waterway bioreactor of cultivating little algae use of transformation and optimization becomes closed photo bioreactor, or the high round-the-clock closed extensive operational system bioreactor of design light utilization efficiency, comprising at its bioreactor support type main body base, the main body reactor of closed adjustable blowdown system is set, regulatable illumination facilities is set, speed governing paddle wheel agitator is set and rolls up water wheels, be provided with the sterile air facility and the O of porting
2Gas system is provided with the degree of saltiness water nutrition liquid allotment and the temperature adjusting system of reflux, and cleaning is with sterilizing and bacterial system and be provided with and separate facilities such as gathering in the crops frustule and dehumidification system; Also round-the-clock closed extensive operational system bioreactor can be comprised that operations such as the allotment of degree of saltiness water nutrition liquid, control light, temperature control, cleaning, sterilization, sterilization, stirring, air-blowing, separation results frustule and drying realize long-term semicontinuous production by computer control;
(2), bioreactor main body reactor is rectangle, length and width are 200-1500cm * 100-300cm, the degree of depth is 450-700cm;
(3), light transmissive material can select the water white transparency toughened glass or the special glass of high tensile force resistance and compressive resistance for use, thickness is 0.8-3cm, the enclosure portion light transmissive material can be selected commercially available transparent plastic film or matrix material transparent film for use, thickness is 0.01-0.6mm; Reach around bioreactor main body reactor illuminating fluorescent lamp, incandescent light, sun lamp or the energy-saving spot lamp of many adjustable brightness are installed up and down, every lamp power is 40-2000w; Can be used singly or in combination;
(4), adopt the adjustable speed paddle wheel agitator of industrial use, rotating speed is 10-400r/m, and the volume water wheels are the volume water wheels of industrial use, and rotating speed is 10-300r/m, the volume water wheels are installed on a side of bioreactor main body reactor, the moving flow velocity of regulation and control degree of saltiness water nutrition liquid recycle stream;
(5), adopt the air compressor machine of industrial use and be treated as sterile air through the oil strain drainage bacteriological filtration of routine; O
2Gas adopts the industrial oxygen bottle, and adopts the filtration method degerming.By variable valve and be distributed in bioreactor main body reactor bottom duct type micropore pod apertures and blast sterile air and oxygen; O
2Gas is regulated by the concentration of the required dissolved oxygen of degree of saltiness water nutrition liquid;
(6), allotment of degree of saltiness water nutrition liquid and the temperature adjusting system that uses for bioreactor is made up of the interlayer receiver of the industrial use of disposing cold water circulating refrigerating device, heating unit, nutritive medium recycle pump, reflux line, measuring and controlling temp facility and belt stirrer.Every group of receiver is 2 to 3, and the receiver capacity is 100-10000L; Agitator speed is 20-300r/m;
(7), can adopt the ozonizer of industrial use to carry out sterilization for clear water and seawater in the degree of saltiness water of bioreactor use; Nutritive medium can adopt the heating method sterilization, and Heating temperature is 70-80 ℃, and time 15-25 minute, or 95-100 ℃, time 5-15 minute; Also can adopt the chemical sterilization method to carry out sterilization.After can adopting the heating method sterilization, the degree of saltiness water nutrition liquid that recycles comes into operation again;
(8), results haematococcus pluvialis cell adopt industrial continous way screenings tripping device, rotating speed is 3000-30000r/m; Isolating haematococcus pluvialis cell is adopted the cryodrying facility of industrial use, and as equipment such as vacuum lyophilization, vacuum-drying, low temperature warm air drying, low temperature spray dryings, drying temperature is 38-85 ℃; The dried product exhibited water content is lower than 3%.
3. a promotion haematococcus pluvialis growing multiplication as claimed in claim 2 and improve the method for the synthetic and accumulation of secondary metabolite astaxanthin in population density and the regulating cell is characterized in that main body reactor length and width are 220-500cm * 110-150cm; The degree of depth is 550-650cm; The enclosure portion light transmissive material can be selected commercially available transparent plastic film or matrix material transparent film for use, and thickness is 0.03-0.3mm; Reach around the main body reactor illuminating fluorescent lamp, incandescent light, sun lamp or the energy-saving spot lamp of many adjustable brightness are installed up and down, every lamp power is 40-300w; Adopt the adjustable speed paddle wheel agitator of industrial use, rotating speed is 20-60r/m; The volume water wheels are the volume water wheels of industrial use, and rotating speed is 20-80r/m; Every group of receiver is 2 to 3, and the receiver capacity is 1000-6000L; Agitator speed is 60-80r/m; Degree of saltiness water nutrition liquid is formed: clear water is 45-65%; Seawater is 35-55%; Saltpetre is 0.2-0.3g/L; Sodium-acetate is 0.45-0.55g/L.
4. a promotion haematococcus pluvialis growing multiplication as claimed in claim 2 and improve the method for the synthetic and accumulation of secondary metabolite astaxanthin in population density and the regulating cell, it is characterized in that haematococcus pluvialis growing multiplication and improving population density regulation and control stage intensity of illumination is the 1000-1500 lux; The suitableeest culture temperature is 20-24 ℃; The sterile air amount of blasting is 0.03-0.2m
3/ m; The culture cycle that frustule ramp propagation is in logarithmic phase is 60-72h.
5. a promotion haematococcus pluvialis growing multiplication as claimed in claim 2 and improve the method for the synthetic and accumulation of secondary metabolite astaxanthin in population density and the regulating cell is characterized in that in the regulating cell that the synthetic and accumulation stage intensity of illumination of secondary metabolite astaxanthin is the 1000-1500 lux; Intensity of illumination is the 2000-2500 lux; The suitableeest culture temperature is 26-29 ℃; The sterile air amount of blasting is 0.02-0.05m
3/ m; Optimum pH is a slight alkalinity; The interior secondary metabolite astaxanthin of regulating cell synthesizes and the accumulation culture cycle is 60-72h; Results frustule tripping device rotating speed is 8000-15000r/m; The frustule drying temperature is 43-48 ℃; The dried product exhibited water content is lower than below 1%.
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| CN104232720B (en) * | 2014-08-26 | 2017-10-20 | 山东金晶生物技术有限公司 | A kind of method for inducing Haematococcus pluvialis production astaxanthin |
| CN107099458B (en) * | 2017-04-06 | 2021-04-23 | 深圳市德和生物科技有限公司 | Biological reaction kettle and method for promoting haematococcus pluvialis proliferation and red-forcing |
| 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 |
| CN109170855A (en) * | 2018-09-07 | 2019-01-11 | 福建品鉴食品有限公司 | A kind of haematococcus pluvialis instant cubilose and preparation method thereof |
| CN109825546B (en) * | 2019-04-08 | 2020-12-18 | 睿藻生物科技（苏州）有限公司 | Method for producing astaxanthin by haematococcus pluvialis |
| US20220356504A1 (en) * | 2019-09-23 | 2022-11-10 | Bioalgo (Wf) Co., Ltd. | Method for producing astaxanthin by heterotrophic culture of haematococcus pluvialis |
| CN110684644B (en) * | 2019-11-08 | 2020-11-24 | 安徽德宝生物科技有限公司 | Photobioreactor for algae cultivation |
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