CN106701585A - Cultivation method of haematococcus pluvialis - Google Patents
Cultivation method of haematococcus pluvialis Download PDFInfo
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- CN106701585A CN106701585A CN201510437152.2A CN201510437152A CN106701585A CN 106701585 A CN106701585 A CN 106701585A CN 201510437152 A CN201510437152 A CN 201510437152A CN 106701585 A CN106701585 A CN 106701585A
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Abstract
The invention relates to the biological field of microalgae and discloses a cultivation method of haematococcus pluvialis. The method comprises the following steps: (1) Cultivation in the swarm cell stage is carried out; (2) When OD680 value of a solution of algae reaches 0.01-10 during cultivation, at least part of the solution of algae is separated by filtration or separated by gravity settling so as to obtain a separated clear liquid I and swarm cells, and the separated clear liquid I returns back to the step (1) for reuse; (3) The swarm cells obtained in the step (2) undergo cultivation in the aplanospore stage; (4) After cultivating to completely red, separation is carried out to obtain a separated clear liquid II and aplanospore, and the separated clear liquid II returns back to the step (3) for reuse; and (5) the aplanospore is dried so as to obtain the haematococcus pluvialis product. According to the cultivation method of haematococcus pluvialis, the cultivation process is stable to operate; quality control is easy; all the culture solutions can be recycled during the cultivation process; the cultivation cost is low, and problems such as discharge of waste liquid and environmental pollution will not be generated.
Description
Technical field
The present invention relates to microalgae biological field, in particular it relates to a kind of cultural method of haematococcus pluvialis.
Background technology
Haematococcus pluvialis (Haematococcus pluvialis) are a kind of monoplast green algas, and its propagation is relied on
In the division of cell.When environment is suitable, haematococcus biomass is continuously increased, and belongs to green swarm cell
Stage；When by stress from outside, such as illumination, temperature, pH value change, itself forms spore,
Accumulation grease and astaxanthin, frustule become red, belong to the aplanospore stage.
Haematococcus pluvialis are rich in grease and astaxanthin, and fat content can account for the 30-40% of dry cell weight, can
As the potential source of bioenergy；Content astaxanthin can account for the 1.5-3.0% of dry cell weight, be natural
The mostly important source of astaxanthin.
But, large-scale culture and the production of existing haematococcus pluvialis still suffer from very big difficulty, mainly
Reason includes：(1) haematococcus pluvialis cultivation yield per unit area it is low, easily by microorganism pollution, grease and
Content astaxanthin is unstable；(2) haematococcus pluvialis green swarm cell stage and aplanospore growth phase
Growth conditions it is different, production cost is high.
CN102766578A discloses a kind of culture production method of haematococcus pluvialis, and specifically discloses：
In sealed shell of tank, haematococcus pluvialis green swarm cell is inoculated into culture medium, using artificial
LED light source irradiation culture, can shorten cultivation cycle, increase haematococcus pluvialis cell concentration in algae solution.
But the process that haematococcus are converted to aplanospore, accumulation astaxanthin and grease is not related to.
CN102827766A discloses the optical-biological reaction for scale haematococcus breeding production astaxanthin
Device, but it is only used for haematococcus " turning the red stage ", not including haematococcus green swarm cell growth cultivation
Cultivation stage.
At present, it is domestic general to use one-step method culture haematococcus pluvialis, i.e., green swarm cell stage and not
The zoospore stage is carried out in same reactor, main purpose be in order to obtain the astaxanthin of high content,
Seldom it is related to the production of grease, and the greatest problem cultivated in same reactor is after harvesting
Nutrient solution can not reuse, as discharge of wastewater, cause aquaculture cost high, the wasting of resources and ring
Pollute in border.For example, CN1966660A disclose large-scale farming haematococcus production astaxanthin device and
Method, whole culture apparatus is by photo-bioreactor system, aerating device, nutrient solution infusion device and quiet
Cell collection device is constituted.Mutually flowed by nutrient solution, continuous charge causes that haematococcus grow and division
Basic synchronization；Extensive haematococcus are carried out using the floating dust control device and natural water that are set on fixed mount
Culture, makes photo-bioreactor system drop to required water depth, solves haematococcus culture and turns
Two phase temperatures, illumination of encapsulated astaxanthin contradiction different with nutritional condition.
CN1680539A discloses a kind of pneumatic lifting photobiological reaction of haematococcus pluvialis High Density Cultivation
Device, including culture filling, water treatment facilities, lighting device and gas supply device, mainly by controlling light
Realize that the culture of haematococcus and being accumulated in same reactor for astaxanthin complete according to conditions such as intensity.
CN1392244A discloses a kind of method for cultivating haematococcus production astaxanthin, including culture medium is matched somebody with somebody
Side, one-step method production process, culture medium recycle and using carbon dioxide regulation pH value induction it is red
The formation of ball algae spore and the method for astaxanthin accumulation.
The content of the invention
The invention aims to overcome drawbacks described above of the prior art, there is provided a kind of haematococcus pluvialis
Cultural method, the culture and the culture in aplanospore stage in the swarm cell stage of the method are different
Carried out in reactor, breeding process stable operation, quality control is easy, and all trainings in breeding process
Nutrient solution can be recycled, and aquaculture cost is low and will not produce the problem of discharging of waste liquid and environmental pollution.
In the method for existing culture haematococcus pluvialis, swarm cell stage and aplanospore stage are same
Carried out in individual reactor, and haematococcus pluvialis swarm cell do not separated, traced it to its cause mainly：
In view of the form and survival property of haematococcus pluvialis swarm cell, those skilled in the art generally believe root
Originally the haematococcus pluvialis in swarm cell stage cannot be separated, and once to the rain in swarm cell stage
Raw haematococcus are separated, and very likely injuring haematococcus pluvialis swarm cell can even influence rain life red
The survival of ball algae swarm cell, and the present inventor has been surprisingly found that under study for action, rain can be given birth into red
Ball algae swarm cell is separated by filtration or gravity settling separation, is then carried out in different reactors
The culture in aplanospore stage, the breeding process will not only cause wound to haematococcus pluvialis swarm cell
Evil, and stable operation, quality control are easy, and all nutrient solutions can be reclaimed in breeding process
Utilize, aquaculture cost is low and will not produce the problem of discharging of waste liquid and environmental pollution.
Therefore, to achieve these goals, the invention provides a kind of cultural method of haematococcus pluvialis,
The method includes：
(1) culture in haematococcus pluvialis swarm cell stage is carried out；
(2) as the OD of culture to algae solution680It is worth during for 0.01-10, at least part of algae solution is separated,
Obtain separating clear liquid I and haematococcus pluvialis swarm cell, will separate during clear liquid I is back to step (1) and return
With；
(3) the haematococcus pluvialis swarm cell that step (2) is obtained is carried out the culture in aplanospore stage；
(4) after culture to turn completely it is red after separated, obtain separating clear liquid I I and haematococcus pluvialis not
Zoospore, will separate clear liquid I I and is back to reuse in step (3)；
(5) haematococcus pluvialis aplanospore is dried and obtains haematococcus pluvialis product；
In step (2), the mode of the separation is to be separated by filtration or gravity settling separation.
The method of the present invention can realize the continuous cultivation of haematococcus pluvialis swarm cell, and then realize that rain is given birth to
The semicontinuous cultivation of haematococcus aplanospore, breeding process stable operation, quality control easily, and is being supported
All nutrient solutions can be recycled during growing, and aquaculture cost is low and will not produce discharging of waste liquid and ring
The problem of border pollution.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
Specific embodiment of the invention is described in detail below.It should be appreciated that this place is retouched
The specific embodiment stated is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The invention provides a kind of cultural method of haematococcus pluvialis, methods described includes：
(1) culture in haematococcus pluvialis swarm cell stage is carried out；
(2) as the OD of culture to algae solution680It is worth during for 0.01-10, at least part of algae solution is separated,
Obtain separating clear liquid I and haematococcus pluvialis swarm cell, will separate during clear liquid I is back to step (1) and return
With；
(3) the haematococcus pluvialis swarm cell that step (2) is obtained is carried out the culture in aplanospore stage；
(4) after culture to turn completely it is red after separated, obtain separating clear liquid I I and haematococcus pluvialis not
Zoospore, will separate clear liquid I I and is back to reuse in step (3)；
(5) haematococcus pluvialis aplanospore is dried and obtains haematococcus pluvialis product；
In step (2), the mode of the separation is to be separated by filtration or gravity settling separation.
In the inventive method, under preferable case, step (1) is carried out in swarm cell breeding device, trip
Kinetocyte breeding device is Race-way photobioreactor or closed photo bioreactor.For open light
There is no particular limitation for bioreactor and closed photo bioreactor, can be respectively commonly used in the art
Various Race-way photobioreactors and closed photo bioreactor.Such as open optical-biological reaction
Device can be track optical biological reactor or box-type bioreactor；Race-way photobioreactor
Material can be metal, cement, unorganic glass, lucite or plastics, plastics can include poly- second
Alkene, polypropylene, polyvinyl chloride, makrolon etc..Preferably, Race-way photobioreactor is transparent
Polypropylene casing or cement pit.Closed photo bioreactor can be bubble type bioreactor, gas
Lift-type bioreactor, stirred photobioreactor etc.；The material of closed photo bioreactor can
Think unorganic glass, lucite or transparent plastic, transparent plastic can include polyethylene, polypropylene,
Polyvinyl chloride, makrolon etc..Preferably, closed photo bioreactor for self-cleaning bubble type it is poly-
Vinyl chloride bioreactor.
In the inventive method, for haematococcus pluvialis algae kind and nutrient solution, there is no particular limitation, Ke Yifen
Various haematococcus pluvialis algae kinds and nutrient solution that Wei be not commonly used in the art, this by those skilled in the art public affairs
Know, will not be repeated here.
In the inventive method, it will be understood by those skilled in the art that being trained in actual haematococcus pluvialis
During supporting, the pH value of cultivation temperature, intensity of illumination and nutrient solution is often uncontrollable accurate at one
Point value, but fluctuated within the scope of one.Under preferable case, in step (1), culture
Condition includes：Temperature is 15-25 DEG C, and intensity of illumination is 1000-5000Lux, and pH value is 6-8.Wherein,
The condition of culture also includes：The carbon source being passed through in incubation is the gas containing carbon dioxide, gas
The content of middle carbon dioxide is 1-20 weight %, preferably 2-10 weight %, more preferably 3-8 weight
Amount %.When illumination is carried out, light source can be natural light, or artificial light sources (such as LED light source),
Preferably natural light.Gas containing carbon dioxide can be that high-purity carbon dioxide or industrial process are discharged
Gas containing carbon dioxide, industrial process discharge the gas containing carbon dioxide can include power plant and
The gas containing carbon dioxide of refinery.Preferably, the gas containing carbon dioxide is high-purity dioxy
Change carbon or refinery hydrogen preparing tail gas.It will be understood by those skilled in the art that the gas containing carbon dioxide
Body needs to carry out purification removing solid particulate matter and cooled down before swarm cell breeding device is entered into.
In the inventive method, under preferable case, in step (2), the OD of culture to algae solution680It is worth and is
0.05-5, more preferably 0.1-1.
In the inventive method, under preferable case, in step (2), at least part of algae solution accounts for whole algae solutions
The 20-50% of volume.
In the inventive method, under preferable case, in step (2), at least part of algae solution is transferred to point
Separated from storage and transportation apparatus.For shift mode there is no particular limitation, can be respectively this
The mode of the conventional various transfers in field.Wherein, the mode of transfer is preferably force of gravity, overflow transfer
Or siphon transfer, more preferably overflow transfer.
In the inventive method, under preferable case, in step (2), the mode of separation is to be separated by filtration.
The mode being separated by filtration can be natural filtration separation, pressure filtration separation and isolated by vacuum filtration, enter one
Step is preferably natural filtration and separates.
In the inventive method, under preferable case, separate with storage and transportation apparatus as cylinder separates void tower, it is described
The ratio of height to diameter that cylinder separates void tower is 1-10:1, more preferably 1.5-5:1, and the cylinder point
Bottom from void tower is provided with filter medium.For filter medium, there is no particular limitation, can be ability
The conventional various filter mediums in domain, under preferable case, filter medium is glass sand, filter cloth or filter paper, enters one
Step is preferably glass sand；The aperture of filter medium is 1-60 μm, more preferably 10-30 μm.
In the inventive method, under preferable case, cylinder separates void tower and is provided with agitator, further excellent
Selection of land, agitator be anchor agitator, gate stirrer or helix(ribbon type) agitator, still more preferably for
Gate stirrer.
In the inventive method, it will be understood by those skilled in the art that separating the number with storage and transportation apparatus
It can be 1, or many depending on the volume of swarm cell breeding device and aplanospore breeding device
Individual parallel operation.
In the inventive method, under preferable case, clear liquid I will be separated and be back to reuse in step (1)
Mode includes：To separate after clear liquid I mixes with (fresh) nutrient solution of supplement in medium tank and return
To swarm cell breeding device.
In the inventive method, under preferable case, step (3) is carried out in aplanospore breeding device, no
Zoospore breeding device be Race-way photobioreactor or closed photo bioreactor, it is preferably open
Bioreactor.For Race-way photobioreactor and closed photo bioreactor without special
Limit, various Race-way photobioreactors commonly used in the art can be respectively and closed photo thing is anti-
Answer device.Such as Race-way photobioreactor can be track optical biological reactor or box-type photo-biological
Reactor；The material of Race-way photobioreactor can be metal, cement, unorganic glass, organic glass
Glass or plastics, plastics can be including polyethylene, polypropylene, polyvinyl chloride, makrolon etc..Preferably,
Race-way photobioreactor is transparent polypropylene casing or cement pit.Closed photo bioreactor can be with
It is bubble type bioreactor, airlift photobioreactor, stirred photobioreactor etc.；Envelope
The material of enclosed bioreactor can be unorganic glass, lucite or transparent plastic, transparent plastic
Can be including polyethylene, polypropylene, polyvinyl chloride, makrolon etc..
In the inventive method, under preferable case, in step (3), haematococcus pluvialis swarm cell with point
From clear liquid I I separation with mix in storage and transportation apparatus after be transferred to aplanospore breeding device in carry out motionless spore
The culture of sub.It is further preferred that the mode being transferred in aplanospore breeding device is shifted for siphon
Or vacuum transfer, still more preferably for vacuum is shifted.
It is no for the mode of stress haematococcus pluvialis accumulation grease and astaxanthin special in the inventive method
Restriction, can be various modes commonly used in the art.For example, in step (3), the mode of culture
Can be temperature stress culture, illumination coercing cultivation or stress agent coercing cultivation.Preferably, culture
Mode is stress agent coercing cultivation.It is further preferred that the condition of stress agent coercing cultivation includes：pH
It is 7-9 to be worth, and temperature is 24-30 DEG C, and intensity of illumination is 5000-15000Lux, and stress agent is sodium salt, and
In terms of every liter of mixing algae solution, the addition for coercing agent is every liter of nutrient solution 0.005-0.05mol, further
Preferably 0.02-0.05mol；Again it is further preferred that stress agent is sodium acid carbonate.
In the inventive method, it will be understood by those skilled in the art that the number of aplanospore breeding device
Should with the growth cycle of swarm cell haematococcus in swarm cell breeding device, the harvesting cycle, harvesting quantity,
Aplanospore haematococcus accumulate grease and the growth cycle of astaxanthin matches, and can be 1, it is also possible to
It is multiple parallel operations, preferably 2-5.
In the inventive method, under preferable case, in step (3), after transfer terminates, deionized water is used
Cleaning is separated and separates next time and shift to carry out with storage and transportation apparatus to neutrality, and cleaning fluid is stored for future use.
For example in cleaning fluid being delivered into Clear liquid tank store for future use.
It is further excellent by cleaning fluid reuse to step (3) under preferable case in the inventive method
Selection of land, the mode of reuse includes：Cleaning fluid and haematococcus pluvialis swarm cell are being separated and storage and transportation apparatus
Being transferred to after middle mixing in aplanospore breeding device carries out the culture in aplanospore stage；Or the side of reuse
Formula includes：In the culture in aplanospore stage, cleaning fluid is transferred in aplanospore breeding device to be used for
Keep the skin wet.
In the inventive method, it will be understood by those skilled in the art that in step (4), Ke Yitong
That crosses observation by light microscope aplanospore turns red state, when aplanospore is changed into red from periphery to center
Separated during color.
In the inventive method, under preferable case, in step (4), the mode of separation is centrifugation,
It is separated by filtration or gravity settling separation, more preferably gravity settling separation.
In the inventive method, under preferable case, clear liquid I I will be separated and be back to reuse in step (3)
Mode includes：Clear liquid I I will be separated to be separated and storage and transportation apparatus by being pumped into, it is red with the rain life isolated
Ball algae swarm cell is well mixed.It is further preferred that convey separating clear liquid I I by centrifugal pump.Ability
Field technique personnel are it is understood that the reuse of separation clear liquid I I for convenience, can separate clear liquid I I
First it is stored in standby in Clear liquid tank.
In the inventive method, in step (5), haematococcus pluvialis aplanospore first can be delivered into concentration
It is dried again in tank.For dry mode, there is no particular limitation, can be commonly used in the art each
Drying mode is planted, under preferable case, dry mode is natural drying, is vacuum dried or is spray-dried,
More preferably whirlwind spray drying.
Embodiment
Below will by embodiment, the present invention will be described in detail, but the present invention is not limited only to this.
Haematococcus pluvialis are purchased from Wuhan aquatile research institute of the Chinese Academy of Sciences.
The OD value (OD values) of algae solution is determined：OD value spectrophotometric determination, to distill
Water is compared, and light absorption value of the algae solution at wavelength 680nm is determined, as the index of algae solution concentration.
BG11 culture mediums are used during culture, Media Components are shown in Table 1- tables 2, in BG11 culture mediums
NaNO3It is nitrogen source.
Table 1BG11 culture mediums
| Component | Content, mg/L |
| K2HPO4·3H2O | 40 |
| NaNO3 | 1500 |
| Na2CO3 | 20 |
| MgSO4·7H2O | 75 |
| CaCl2·2H2O | 36 |
| Citric acid | 6 |
| Ferric citrate | 6 |
| EDTA-2Na | 1 |
| Micro- A5 | 1 |
The trace element of table 2 A5
| Component | Composition, mg/L |
| H3BO3 | 2860 |
| MnCl2·4H2O | 1810 |
| ZnSO4·7H2O | 222 |
| CuSO4·5H2O | 79 |
| NaMoO4·5H2O | 390 |
| Co(NO3)2·6H2O | 50 |
Refinery hydrogen preparing tail gas is the refinery hydrogen preparing tail gas of Shijiazhuang refinery branch company of Sinopec company, dioxy
The content for changing carbon is 20-25 weight %, removes solid particulate matter and is cooled down using purification is preceding carried out.
Mixed gas are mixed to form with air so that carbon dioxide in gas mixture content is 6 after being cooled to 25 DEG C
Weight %, by controlling the charge flow rate control ph of mixed gas in haematococcus pluvialis incubation.
Closed photo bioreactor be polyvinyl chloride film bag, a diameter of 220mm, a height of 1500mm,
Volume is 35L, without inner draft tube.
Race-way photobioreactor is transparent polypropylene casing, and volume is 20L, and light-receiving area is
0.095m2。
Cultivation speed (the g/m of haematococcus pluvialis2/ d)=haematococcus pluvialis dry weight/haematococcus pluvialis are travelling thin
The culturing time of the light-receiving area of born of the same parents' cultivation stage/haematococcus pluvialis swarm cell cultivation stage.
It is lucite cylinder of the bottom of 30L with glass sand filter medium to separate with storage and transportation apparatus
Shape separates void tower, and ratio of height to diameter is 2:1, frame type stirring, with the delivery pipe that bottom is inserted into from top,
And be connected with aplanospore breeding device, a diameter of 10mm of delivery pipe, the specification of glass sand is G3, aperture
It is 15-30 μm.
Embodiment 1
The present embodiment is used for the method for illustrating to cultivate haematococcus pluvialis using closed photo bioreactor.
In the present embodiment, swarm cell breeding device and aplanospore breeding device are the reaction of closed photo thing
Device.
(1) the one-level culture of haematococcus pluvialis
Haematococcus pluvialis algae kind is inoculated into the 2000mL triangular flasks equipped with 1000mL BG11 culture mediums
In, the OD of nutrient solution after inoculation haematococcus pluvialis algae kind680Be worth is 0.015.20-22 DEG C,
It is 0.3 that algae solution to OD values are cultivated under 1800-2200Lux, pH value 6-7, standby.PH in incubation
Value is adjusted by high-purity carbon dioxide.
(2) training in haematococcus pluvialis swarm cell stage is carried out in 1 closed photo bioreactor
Support：Add 28L BG11 culture mediums and the life of the rain of one-level culture red after closed photo bioreactor sterilization
Ball algae algae kind, the initial OD of nutrient solution680Be worth is 0.08.Mixed gas are from closed photo bioreactor
Bottom disperses to enter in bubbling form by gas stone, and produces agitation, mixing, and charge flow rate is 200L/h.
Cultivated under 20-24 DEG C, intensity of illumination 1800-5000Lux, pH value 6-8, pH value passes through gaseous mixture
The charge flow rate control of body.
(3) haematococcus pluvialis swarm cell is separated and transfer：The OD of nutrient solution after cultivating 7 days680Value
0.3 is reached, by 8.4L haematococcus pluvialis algae solution from the closing of step (2) by way of overflow is shifted
Separation and storage and transportation apparatus are transferred in formula bioreactor.Filtered by glass sand, obtain separating clear liquid I
With haematococcus pluvialis swarm cell, separate clear liquid I and enter mixed with the culture medium for supplementing in nutrient solution medium tank
Close, by causing that closed photo thing reacts in the closed photo bioreactor of pump input step (2)
The volume of nutrient solution is 28L in device, continues the culture in haematococcus pluvialis swarm cell stage.8.4L liquid
It is pumped up into separation and storage and transportation apparatus by Clear liquid tank, it is mixed with the haematococcus pluvialis swarm cell being filtrated to get
Close.Vacuumized after well mixed, mixed liquor enters aplanospore breeding device by delivery pipe.
(4) separate and storage and transportation apparatus washing：After vacuum transfer terminates, deionized water cleaning is added to separate
With storage and transportation apparatus to neutrality, it is standby that cleaning fluid enters Clear liquid tank.
(5) in 2 aplanospore breeding devices of parallel connection, i.e., rain is carried out in closed photo bioreactor
The accumulation of the conversion of raw haematococcus aplanospore, grease and astaxanthin：Mixed liquor vacuum is transferred to the 1st envelope
After enclosed bioreactor, in terms of every liter of mixing algae solution, 0.02mol stress agent sodium acid carbonates are added,
Carry out the accumulation of aplanospore conversion, grease and astaxanthin.Temperature is 28-32 DEG C during culture, and illumination is strong
It is 5000-15000Lux to spend, and pH value is 7-9.Mixed gas are logical from closed photo bioreactor bottom
Cross the dispersion of gas stone to enter in bubbling form, and produce agitation, mixing, the air inflow of mixed gas is 60L/h,
PH value is controlled by the charge flow rate of mixed gas.
Culture is observed under an optical microscope after 10 days, and aplanospore is changed into red from periphery to center,
Stop air inlet, standing separation obtains separating clear liquid I I and haematococcus pluvialis aplanospore, separates clear liquid I I
Into Clear liquid tank, haematococcus pluvialis aplanospore is into concentration tank and by obtaining rain after whirlwind spray drying
Raw haematococcus algae powder.
Treat to be reacted for the closed photo thing that the haematococcus pluvialis swarm cell stage cultivates in step (3)
The OD of nutrient solution in device680When value reaches 0.3, mixed liquor is transferred to the 2nd not by repeat step (3)
The closed photo bioreactor of zoospore breeding device, i.e., the 2nd.Circulate successively.
By the total volume meter of the nutrient solution of haematococcus pluvialis swarm cell cultivation stage, haematococcus pluvialis are supported
Speed is grown for 3g/m2/ d, the content of grease is 36%, the content of astaxanthin in the haematococcus pluvialis for obtaining
It is 1.5%.
Embodiment 2
The present embodiment is used for the method for illustrating to cultivate haematococcus pluvialis using Race-way photobioreactor.
In the present embodiment, swarm cell breeding device and aplanospore breeding device are open optical-biological reaction
Device.
(1) the one-level culture of haematococcus pluvialis
Haematococcus pluvialis algae kind is inoculated into the 2000mL triangular flasks equipped with 1000mL BG11 culture mediums
In, the OD of nutrient solution after inoculation haematococcus pluvialis algae kind680Be worth is 0.015.20-22 DEG C,
It is 0.3 that algae solution to OD values are cultivated under 1800-2200Lux, pH value 6-7, standby.PH in incubation
Value is adjusted by high-purity carbon dioxide.
(2) training in haematococcus pluvialis swarm cell stage is carried out in 1 Race-way photobioreactor
Support：16L BG11 culture mediums and the rain of one-level culture is added to give birth to red ball in Race-way photobioreactor
Algae algae kind, the initial OD of nutrient solution680Be worth is 0.03.Mixed gas are disperseed in bubbling form by gas stone
Into bioreactor, and agitation, mixing are produced, charge flow rate is 80L/h.In 22-25 DEG C, light
Cultivated according under intensity 1800-5000Lux, pH value 6-8, the charge flow rate control that pH value passes through mixed gas
System.
(3) haematococcus pluvialis swarm cell is separated and transfer：The OD of nutrient solution after cultivating 8 days680Value
0.28 is reached, by 4.8L haematococcus pluvialis algae solution from the opening of step (2) by way of overflow is shifted
Separation and storage and transportation apparatus are transferred in formula bioreactor.Filtered by glass sand, obtain separating clear liquid I
With haematococcus pluvialis swarm cell, separate clear liquid I and enter mixed with the culture medium for supplementing in nutrient solution medium tank
Close, by causing open optical-biological reaction in the Race-way photobioreactor of pump input step (2)
The volume of nutrient solution is 16L in device, continues the culture in haematococcus pluvialis swarm cell stage.4.8L liquid
It is pumped up into separation and storage and transportation apparatus by Clear liquid tank, it is mixed with the haematococcus pluvialis swarm cell being filtrated to get
Close.After well mixed, mixed liquor enters aplanospore breeding device by delivery pipe siphon.
(4) separate and storage and transportation apparatus washing：After siphon transfer terminates, deionized water cleaning is added to separate
With storage and transportation apparatus to neutrality, it is standby that cleaning fluid enters Clear liquid tank.
(5) in 2 aplanospore breeding devices of parallel connection, i.e., rain is carried out in Race-way photobioreactor
The accumulation of the conversion of raw haematococcus aplanospore, grease and astaxanthin：Mixed liquor siphon is transferred to the 1st and opens
After putting formula bioreactor, in terms of every liter of mixing algae solution, 0.03mol stress agent sodium acid carbonates are added,
Carry out the accumulation of aplanospore conversion, grease and astaxanthin.Temperature is 28-32 DEG C during culture, and illumination is strong
It is 5000-15000Lux to spend, and pH value is 7-9.Mixed gas disperse to enter in bubbling form by gas stone
Race-way photobioreactor, and agitation, mixing are produced, the air inflow of mixed gas is 40L/h, pH
Value is controlled by the charge flow rate of mixed gas.
Culture is observed under an optical microscope after 10 days, and aplanospore is changed into red from periphery to center,
Stop air inlet, standing separation obtains separating clear liquid I I and haematococcus pluvialis aplanospore, separates clear liquid I I
Into Clear liquid tank, haematococcus pluvialis aplanospore is into concentration tank and red by obtaining rain life after vacuum drying
Ball algae powder.
Treat the open optical-biological reaction for the culture of haematococcus pluvialis swarm cell stage in step (3)
The OD of nutrient solution in device680When value reaches 0.28, mixed liquor is transferred to the 2nd by repeat step (3)
Aplanospore breeding device, i.e. Race-way photobioreactor.Circulate successively.
By the total volume meter of the nutrient solution of haematococcus pluvialis swarm cell cultivation stage, haematococcus pluvialis are supported
Speed is grown for 2.7g/m2/ d, the content of grease is 35%, the content of astaxanthin in the haematococcus pluvialis for obtaining
It is 1.6%.
Embodiment 3
The present embodiment is used for the method for illustrating to cultivate haematococcus pluvialis using combined type bioreactor.
In the present embodiment, swarm cell breeding device is closed photo bioreactor, aplanospore breeding device
It is Race-way photobioreactor.
(1) the one-level culture of haematococcus pluvialis
Haematococcus pluvialis algae kind is inoculated into the 2000mL triangular flasks equipped with 1000mL BG11 culture mediums
In, the OD of nutrient solution after inoculation haematococcus pluvialis algae kind680Be worth is 0.015.20-22 DEG C,
It is 0.3 that algae solution to OD values are cultivated under 1800-2200Lux, pH value 6-7, standby.PH in incubation
Value is adjusted by high-purity carbon dioxide.
(2) training in haematococcus pluvialis swarm cell stage is carried out in 1 closed photo bioreactor
Support：28L BG11 culture mediums and the rain of one-level culture is added to give birth to red ball in closed photo bioreactor
Algae algae kind, the initial OD of nutrient solution680Be worth is 0.075.Mixed gas are disperseed with bubbling shape by gas stone
Formula enters bioreactor, and produces agitation, mixing, and charge flow rate is 200L/h.20-24 DEG C,
Cultivated under intensity of illumination 1800-5000Lux, pH value 6-8, the charge flow rate that pH value passes through mixed gas
Control.
(3) haematococcus pluvialis swarm cell is separated and transfer：The OD of nutrient solution after cultivating 7 days680Value
0.31 is reached, by 8.4L haematococcus pluvialis algae solution from the closing of step (2) by way of overflow is shifted
Separation and storage and transportation apparatus are transferred in formula bioreactor.Filtered by glass sand, obtain separating clear liquid I
With haematococcus pluvialis swarm cell, separate clear liquid I and enter mixed with the culture medium for supplementing in nutrient solution medium tank
Close, by causing that closed photo thing reacts in the closed photo bioreactor of pump input step (2)
The volume of nutrient solution is 28L in device, continues the culture in haematococcus pluvialis swarm cell stage.8.4L liquid
It is pumped up into separation and storage and transportation apparatus by Clear liquid tank, it is mixed with the haematococcus pluvialis swarm cell being filtrated to get
Close.After well mixed, mixed liquor enters aplanospore breeding device by delivery pipe siphon.
(4) separate and storage and transportation apparatus washing：After siphon transfer terminates, deionized water cleaning is added to separate
With storage and transportation apparatus to neutrality, it is standby that cleaning fluid enters Clear liquid tank.
(5) in 2 aplanospore breeding devices of parallel connection, i.e., rain is carried out in Race-way photobioreactor
The accumulation of the conversion of raw haematococcus aplanospore, grease and astaxanthin：Mixed liquor siphon is transferred to the 1st and opens
After putting formula bioreactor, in terms of every liter of mixing algae solution, 0.05mol stress agent sodium acid carbonates are added,
Carry out the accumulation of aplanospore conversion, grease and astaxanthin.Temperature is 28-32 DEG C during culture, and illumination is strong
It is 5000-15000Lux to spend, and pH value is 7-9.Mixed gas disperse to enter in bubbling form by gas stone
Bioreactor, and agitation, mixing are produced, the air inflow of mixed gas is 40L/h, and pH value passes through
The charge flow rate control of mixed gas.
Culture is observed under an optical microscope after 10 days, and aplanospore is changed into red from periphery to center,
Stop air inlet, standing separation obtains separating clear liquid I I and haematococcus pluvialis aplanospore, separates clear liquid I I
Into Clear liquid tank, haematococcus pluvialis aplanospore is into concentration tank and by obtaining rain after whirlwind spray drying
Raw haematococcus algae powder.
Treat to be reacted for the closed photo thing that the haematococcus pluvialis swarm cell stage cultivates in step (3)
The OD of nutrient solution in device680When value reaches 0.31, mixed liquor is transferred to the 2nd by repeat step (3)
Aplanospore breeding device, i.e. Race-way photobioreactor.Circulate successively.
By the total volume meter of the nutrient solution of haematococcus pluvialis swarm cell cultivation stage, haematococcus pluvialis are supported
Speed is grown for 3.1g/m2/ d, the content of grease is 39%, the content of astaxanthin in the haematococcus pluvialis for obtaining
It is 1.5%.
Embodiment 4
The present embodiment is used for the method for illustrating to cultivate haematococcus pluvialis using combined type bioreactor.
In the present embodiment, swarm cell breeding device is Race-way photobioreactor, aplanospore breeding device
It is closed photo bioreactor.
(1) the one-level culture of haematococcus pluvialis
Haematococcus pluvialis algae kind is inoculated into the 2000mL triangular flasks equipped with 1000mL BG11 culture mediums
In, the OD of nutrient solution after inoculation haematococcus pluvialis algae kind680Be worth is 0.015.20-22 DEG C,
It is 0.3 that algae solution to OD values are cultivated under 1800-2200Lux, pH value 6-7, standby.PH in incubation
Value is adjusted by high-purity carbon dioxide.
(2) training in haematococcus pluvialis swarm cell stage is carried out in 1 Race-way photobioreactor
Support：16L BG11 culture mediums and the rain of one-level culture is added to give birth to red ball in Race-way photobioreactor
Algae algae kind, the initial OD of nutrient solution680Be worth is 0.025.Mixed gas are disperseed with bubbling shape by gas stone
Formula enters bioreactor, and produces agitation, mixing, and charge flow rate is 80L/h.22-25 DEG C,
Cultivated under intensity of illumination 1800-5000Lux, pH value 6-8, the charge flow rate that pH value passes through mixed gas
Control.
(3) haematococcus pluvialis swarm cell is separated and transfer：The OD of nutrient solution after cultivating 7 days680Value
0.29 is reached, by 4.8L haematococcus pluvialis algae solution from the opening of step (2) by way of overflow is shifted
Separation and storage and transportation apparatus are transferred in formula bioreactor.Filtered by glass sand, obtain separating clear liquid I
With haematococcus pluvialis swarm cell, separate clear liquid I and enter mixed with the culture medium for supplementing in nutrient solution medium tank
Close, by causing open optical-biological reaction in the Race-way photobioreactor of pump input step (2)
The volume of nutrient solution is 16L in device, continues the culture in haematococcus pluvialis swarm cell stage.4.8L liquid
It is pumped up into separation and storage and transportation apparatus by Clear liquid tank, it is mixed with the haematococcus pluvialis swarm cell being filtrated to get
Close.After well mixed, mixed liquor enters aplanospore breeding device by delivery pipe.
(4) separate and storage and transportation apparatus washing：After vacuum transfer terminates, deionized water cleaning is added to separate
With storage and transportation apparatus to neutrality, it is standby that cleaning fluid enters Clear liquid tank.
(5) in 2 aplanospore breeding devices of parallel connection, i.e., rain is carried out in closed photo bioreactor
The accumulation of the conversion of raw haematococcus aplanospore, grease and astaxanthin：Mixed liquor vacuum is transferred to the 1st envelope
After enclosed bioreactor, in terms of every liter of mixing algae solution, 0.02mol stress agent sodium acid carbonates are added,
Carry out the accumulation of aplanospore conversion, grease and astaxanthin.Temperature is 28-32 DEG C during culture, and illumination is strong
It is 5000-15000Lux to spend, and pH value is 7-9.Mixed gas are logical from closed photo bioreactor bottom
Cross the dispersion of gas stone to enter in bubbling form, and produce agitation, mixing, the air inflow of mixed gas is 60L/h,
PH value is controlled by the charge flow rate of mixed gas.
Culture is observed under an optical microscope after 10 days, and aplanospore is changed into red from periphery to center,
Stop air inlet, standing separation obtains separating clear liquid I I and haematococcus pluvialis aplanospore, separates clear liquid I I
Into Clear liquid tank, haematococcus pluvialis aplanospore is into concentration tank and red by obtaining rain life after vacuum drying
Ball algae powder.
Treat the open optical-biological reaction for the culture of haematococcus pluvialis swarm cell stage in step (3)
The OD of nutrient solution in device680When value reaches 0.29, mixed liquor is transferred to the 2nd by repeat step (3)
Aplanospore breeding device, i.e. closed photo bioreactor.Circulate successively.
By the total volume meter of the nutrient solution of haematococcus pluvialis swarm cell cultivation stage, haematococcus pluvialis are supported
Speed is grown for 2.6g/m2/ d, the content of grease is 38%, the content of astaxanthin in the haematococcus pluvialis for obtaining
It is 1.8%.
The cultural method of haematococcus pluvialis of the invention, the culture in swarm cell stage and aplanospore stage
Culture carried out in different reactors, breeding process stable operation, quality control easily, and support
All nutrient solutions can be recycled during growing, and aquaculture cost is low and will not produce discharging of waste liquid and ring
The problem of border pollution.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned reality
The detail in mode is applied, in range of the technology design of the invention, can be to technical side of the invention
Case carries out various simple variants, and these simple variants belong to protection scope of the present invention.
It is further to note that each particular technique described in above-mentioned specific embodiment is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition wanted, the present invention is no longer separately illustrated to various possible combinations.
Additionally, can also be combined between a variety of implementation methods of the invention, as long as its
Without prejudice to thought of the invention, it should equally be considered as content disclosed in this invention.
Claims (19)
1. a kind of cultural method of haematococcus pluvialis, it is characterised in that methods described includes：
(1) culture in haematococcus pluvialis swarm cell stage is carried out；
(2) as the OD of culture to algae solution680It is worth during for 0.01-10, at least part of algae solution is separated,
Obtain separating clear liquid I and haematococcus pluvialis swarm cell, will separate during clear liquid I is back to step (1) and return
With；
(3) the haematococcus pluvialis swarm cell that step (2) is obtained is carried out the culture in aplanospore stage；
(4) after culture to turn completely it is red after separated, obtain separating clear liquid I I and haematococcus pluvialis not
Zoospore, will separate clear liquid I I and is back to reuse in step (3)；
(5) haematococcus pluvialis aplanospore is dried and obtains haematococcus pluvialis product；
In step (2), the mode of the separation is to be separated by filtration or gravity settling separation.
2. method according to claim 1, wherein, in step (1), the condition of the culture
Including：Temperature is 15-25 DEG C, and intensity of illumination is 1000-5000Lux, and pH value is 6-8.
3. method according to claim 1, wherein, in step (2), culture to algae solution
OD680It is 0.05-5 to be worth, preferably 0.1-1.
4. method according to claim 1, wherein, in step (2), at least part of algae
Liquid accounts for the 20-50% of whole algae solution volumes.
5. method according to claim 1, wherein, in step (2), by least part of algae solution
Separation is transferred to be separated with storage and transportation apparatus.
6. method according to claim 5, wherein, in step (2), the mode of the transfer
For force of gravity, overflow transfer or siphon are shifted, preferably overflow transfer.
7. method according to claim 1 or 5, wherein, in step (2), the separation
To be separated by filtration, preferably natural filtration is separated mode.
8. method according to claim 5, wherein, the separation is cylinder with storage and transportation apparatus
Void tower is separated, the ratio of height to diameter that the cylinder separates void tower is 1-10:1, preferably 1.5-5:1, and it is described
The bottom that cylinder separates void tower is provided with filter medium.
9. method according to claim 8, wherein, the filter medium be glass sand, filter cloth or
Filter paper, preferably glass sand；The aperture of the filter medium is 1-60 μm, preferably 10-30 μm.
10. method according to claim 8, wherein, the cylinder separates void tower and is provided with to be stirred
Device is mixed, the agitator is preferably anchor agitator, gate stirrer or helix(ribbon type) agitator, further
Preferably gate stirrer.
11. methods according to claim 5, wherein, in step (3), haematococcus pluvialis trip
Kinetocyte with separate clear liquid I I it is described separate with mix in storage and transportation apparatus after be transferred to aplanospore cultivate
The culture in aplanospore stage is carried out in device.
12. methods according to claim 11, wherein, it is transferred in aplanospore breeding device
Mode is that siphon transfer or vacuum are shifted, preferably vacuum transfer.
13. methods according to claim 1, wherein, in step (3), the side of the culture
Formula is temperature stress culture, illumination coercing cultivation or stress agent coercing cultivation, preferably coerces agent stress
Culture.
14. methods according to claim 13, wherein, the condition of the stress agent coercing cultivation
Including：PH value is 7-9, and temperature is 24-30 DEG C, and intensity of illumination is 5000-15000Lux, coerces agent
It is sodium salt, and in terms of every liter of mixing algae solution, the addition for coercing agent is every liter of nutrient solution 0.005-0.05mol,
Preferably 0.02-0.05mol；It is further preferred that stress agent is sodium acid carbonate.
15. methods according to claim 11, wherein, in step (3), after transfer terminates,
Separation is cleaned with deionized water to separate next time and shift to carry out with storage and transportation apparatus to neutrality, and by cleaning fluid
Store for future use.
16. methods according to claim 15, wherein, by cleaning fluid reuse to step (3),
Preferably, the mode of the reuse includes：Cleaning fluid is separated with haematococcus pluvialis swarm cell described
With mix in storage and transportation apparatus after be transferred in aplanospore breeding device the culture that carries out the aplanospore stage；Or
The mode of reuse described in person includes：In the culture in aplanospore stage, cleaning fluid is transferred to motionless spore
It is used to keep the skin wet in sub- breeding device.
17. methods according to claim 1, wherein, in step (4), the side of the separation
Formula is centrifugation, is separated by filtration or gravity settling separation, preferably gravity settling separation.
18. methods according to claim 1, wherein, in step (5), the dry side
To spontaneously dry, being vacuum dried or be spray-dried, preferably whirlwind is spray-dried formula.
19. methods according to claim 1, wherein, step (1) is in swarm cell breeding device
In carry out, the swarm cell breeding device is that Race-way photobioreactor or closed photo thing react
Device；Step (3) is carried out in aplanospore breeding device, and the aplanospore breeding device is open light
Bioreactor or closed photo bioreactor.
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