CN105586262A - Method for promoting growth of Haematococcus pluvialis and accumulation of astaxanthin by flue gas CO2 domestication - Google Patents
Method for promoting growth of Haematococcus pluvialis and accumulation of astaxanthin by flue gas CO2 domestication Download PDFInfo
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Abstract
The invention relates to a bioactive health product development technique, and aims to provide a method for promoting growth of Haematococcus pluvialis and accumulation of astaxanthin by flue gas CO2 domestication. The method comprises the following steps: carrying out domestication culture on a Haematococcus pluvialis liquid sequentially with 6 vol% high-concentration CO2 gas and 10 vol% high-concentration CO2 gas; carrying out iterative domestication culture by using coal-fired power plant flue gas to obtain a target alga strain; and culturing sequentially under the low illumination intensity of 3000-3500Lux and under the high illumination intensity of 7500-10000Lux, and carrying out centrifugation and freeze-drying on the alga solution to obtain the Haematococcus pluvialis dry alga powder. The method obviously enhances the growth rate and astaxanthin yield, wherein the growth rate is enhanced by 47% as compared with that under air conditions, and the astaxanthin content is enhanced by 25%.
Description
Technical field
The invention relates to biologically actie health product development technique field, particularly flue gas CO2Domestication promotes the raw red ball of rainThe method of algae growth and astaxanthin accumulation.
Background technology
Astaxanthin is the current known the strongest antioxidant of nature, and its non-oxidizability is ascorbic 6000 times,1000 times of vitamin E, 200 times of Tea Polyphenols, 50 times of grape seed extract. Astaxanthin can strengthen bodyAnti-ageing, anti-inflammatory, uvioresistant and immunologic function, therefore, be used to health products, cosmetics, food and feed and addIn the several functions products such as agent. In nature astaxanthin source, the content astaxanthin of haematococcus pluvialis is the highest, accounts for its dry weight1-5% (MinxiWana, 2014), and, the required astaxanthin structure of the structure of its astaxanthin-containing and organismUnanimously, this is that synthesizing astaxanthin and other natural astaxanthins can not be compared, and is acknowledged as the best life of natural astaxanthinThing source is unique algae of dropping at present astaxanthin commercial applications. But haematococcus pluvialis growing is slow, than lifeLong speed is about 0.15/d-0.64/d (Orosa, 2005; Kaewpintong, 2006; Imamoglu, 2010). Raw slowlyLong speed is brought the various problems such as other algae invasion, protozoan pollution, is to exist in haematococcus pluvialis large-scale productionMain bugbear.
Due to low concentration CO in air2Mass transfer rate in water is lower, and the energy consumption of both culturing microalgae 40% is held for air pumpContinue and provide air to maintain micro algae growth, and utilize flue gas high concentration CO2Cultivating microalgae, not only can be by the energy of both culturing microalgaeSource is dropped into and is reduced by 70% (Lametal, 2012), can also promote micro algae growth and astaxanthin accumulation. React by flat boardDevice, continuous tubular reactor etc., haematococcus pluvialis is to 2%-5% high concentration CO2Solid carbon efficiencies can reach 13.55%to49.15%, living beings and astaxanthin yield significantly improve (Kang, 2010; Lee, 2015; Giannelli, 2015;Kwak, HS, 2015). With 5%CO2Cultivate haematococcus pluvialis for carbon source and the output of astaxanthin can be increased to 175.7mg/l,2.7 times (Kang etc., 2005) that the heterotrophism taking acetate as carbon source is cultivated. In addition the CO of high concentration,2CanReplace the synthetic and accumulation of nitrogen stress culture medium and acetate culture medium induction astaxanthin, reduce costs. To distilled water discontinuousProperty passes into pure carbon dioxide and can impel astaxanthin accumulation, and in its content and nitrogen stress culture medium, content astaxanthin is close(Imamoglu, 2009). But, at high concentration (12～15%) CO of coal-fired plant flue gas2Under condition, rain is rawHaematococcus growth can be suppressed. The people such as Cheng (2016) utilize high concentration CO2Cultivate the mutagenesis of haematococcus pluvialis core prominentBecome algae strain and find, work as CO2Concentration is increased to 6% by air, and biological productivity has improved 82%. But, work as CO2Concentration is increased to 10% when above, and the growth of haematococcus pluvialis is suppressed. The people such as Yin (2015) are by 20%CO2LogicalEnter in biofilm reactor, to cultivate haematococcus pluvialis and find that its biomass compares 1.5%CO2Reduce by 15%, content astaxanthinReduce by 40%, will directly cause the prolongation of industrial production time and the reduction of productivity effect, so, fire coal how to be utilizedPower-plant flue gas CO2The growth rate and the astaxanthin yield that improve haematococcus pluvialis are still a technical barrier, if can be to some extentBreakthrough will be to improving production of astaxanthin efficiency and reducing production costs significant.
Summary of the invention
Main purpose of the present invention is to overcome deficiency of the prior art, provides one to utilize coal-fired plant flue gas CO2Domestication improves the method for haematococcus pluvialis growing speed and content astaxanthin. For solving the problems of the technologies described above, solution of the present inventionCertainly scheme is:
Flue gas CO is provided2Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation, in coal-fired plant flue gas, contains12～15% high volumetric concentration CO2, described flue gas CO2Domestication promotes the side of haematococcus pluvialis growing and astaxanthin accumulationMethod specifically comprises the steps:
(1) by 10～20% inoculum concentration by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium,Then postvaccinal culture medium is placed in to photosynthetic reactor, then is 6% height to passing into volumetric concentration in photosynthetic reactorConcentration C O2Gas is that 60ml/min, intensity of illumination are 25～28 DEG C of 3000～3500Lux, temperature at gas flowCondition under, cultivate 4～6 days;
Then, by the above-mentioned CO taking volumetric concentration as 6%2Algae strain after cultivation, with 10～20% inoculum concentration transfer intoIn new liquid B G-11 culture medium, then pass into volumetric concentration and be 10% high concentration CO2Gas, at gas flowFor 60ml/min, intensity of illumination are under the condition of 25～28 DEG C of 3000～3500Lux, temperature, cultivate 4～6 days;
(2) get in step (1) at the CO taking volumetric concentration as 10%2In gas, cultivate the algae liquid after 4～6 days, withIn the liquid B G-11 culture medium that 10～20% inoculum concentration is transferred new, then postvaccinal culture medium is placed in photosynthetic anti-Answer in device, then pass into CO in photosynthetic reactor2Volumetric concentration is 12～15% coal-fired plant flue gas, at gas flowFor 60ml/min, intensity of illumination are that 3000～3500Lux, temperature are under the condition of 25～28 DEG C, Continuous Cultivation 5～10In generation, obtain the target algae strain of Fast Growth under coal-fired plant flue gas condition;
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 training with 10～20% inoculum concentrationSupport in base, then postvaccinal culture medium is placed in to photosynthetic reactor, then passes into CO in photosynthetic reactor2Volume is denseDegree is that 12～15% coal-fired plant flue gas promotes algae strain growth, gas flow be 60ml/min, intensity of illumination 3000～3500Lux, temperature are under the condition of 25～28 DEG C, and training objective algae strain 7～10 days can be by measuring frustule numberCalculate growth rate;
(4) step (3) is cultivated to the target algae strain after finishing, be placed under high intensity of illumination 7500～10000Lux and continueContinuous cultivation, and continue to pass into CO2Volumetric concentration is that 12～15% coal-fired plant flue gas is coerced the astaxanthin improving in algae strainContent, condition of culture is: gas flow is that 120ml/min, cultivation temperature are 25～28 DEG C, cultivate after 10～15 days,By after centrifugal algae liquid and freeze drying, obtain haematococcus pluvialis dry algae powder, can measure the biology of evaluating haematococcus pluvialis powderMatter dry weight and content astaxanthin.
In the present invention, described photosynthetic reactor adopts the cylinder photosynthetic reactor that volume is 600ml.
In the present invention, the composition of described liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid.
In the present invention, in described step (3), the measure and calculation method of growth rate is:
Get 2mL algae liquid every 24h, in algae liquid, add 0.2mL Shandong brother's reagent, concussion shakes up, and film-making, at opticsMicro-Microscopic observation, utilizes blood counting chamber counting, calculates frustule and counts N (individual/mL) recycling formula μ=(lnN2-lnN1)/(t2-t1) calculate cell average growth rate, as growth rate;
Wherein, μ is cell average growth rate; N1Be t1It time frustule number (individual/mL), N2Be t2My godTime frustule number (individual/mL).
In the present invention, in described step (4), the assay method of living beings dry weight M (g/L) is:
Get 10mL algae liquid with 8000rmp centrifugal 10 minutes, remove supernatant, the algae mud twice obtaining with distilled water flushing,Again the centrifugal dehydration of algae mud is placed at-70 DEG C after freezing 24 hours, puts into low-temperature vacuum drying instrument dry 24 hours,Gained algae powder is weighed and calculated living beings dry weight M (g/L).
In the present invention, in described step (4), the assay method of content astaxanthin adopts high performance liquid chromatography (HPLC).Be specially: get 10mL algae liquid, centrifugal 10 minutes of 8000rmp, removes supernatant, and algae mud is placed in to-70 DEG C freezing 24After hour, put into low-temperature vacuum drying instrument dry 24 hours; The algae powder of freeze drying gained is placed in to 15ml dispersion machineIn test tube, add 5mL methyl alcohol-carrene mixed solution (25:75, v/v), grind extraction 2 at 5500rmp homogeneousMinute, gained mixed liquor centrifugal 5 minutes at 8000rmp, gets supernatant, is repeatedly extracted to algae-residue colourless; To repeatedly extractThe supernatant that contains astaxanthin of getting rear retention mixes, and is settled to 25mL with methyl alcohol-carrene mixed solution; Get5mL mixed liquor, adds 1mL0.05mol/LNaOH-methanol solution, carries out soap under room temperature, half-light, nitrogen environmentChange reaction (at least 8 hours); Product is settled to 5mL with Nitrogen evaporator, and with 0.45 μ m membrane filtration, filtrate is usedMeasure content astaxanthin in high performance liquid chromatography (HPLC); HPLC condition of work is as follows: mobile phase is A water, BMethyl alcohol, C isopropyl alcohol, D acetonitrile, gradient is: 0min, 20%A, 50%B, 30%D; 5min, 20%A,50%B, 30%D; 15min, 60%B, 10%C, 30%D; Flow velocity is 1ml/min; Get the shrimp under 476nm wavelengthBlue or green plain absworption peak area, calculates astaxanthin concentration D according to the content astaxanthin calibration curve corresponding with peak areaast(mg/L)；In dry algae powder, the computational methods of content astaxanthin are as follows: Cast(mg/g)＝Dast(mg/L)/M (g/L), wherein M (g/L)For the living beings dry weight after the oven dry of algae liquid.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention adopts coal-fired plant flue gas high concentration (12～15%) CO2Domestication haematococcus pluvialis, has significantly improved growthSpeed and astaxanthin yield, its growth rate is than having improved 47% under air conditions, and content astaxanthin has improved 25%. ThisOwing to utilizing flue gas high concentration CO2Make the crucial solid carbon enzyme Rubisco up-regulated of photosynthesis of haematococcus pluvialis, shortCalvin cycle and photosynthetic growth efficiency are entered; Light is caught the protein-bonded expression of chlorophyll a/b and is risen, and has improved lightEnergy utilization ratio; Chlorophyll combined coefficient improves, ATP synzyme up-regulated, thus improve growth rate. ThisBright in the chemical activators stage, flue gas high concentration CO2Sufficient carbon source is provided for grease is synthetic, for astaxanthin esterification andStorage provides advantage.
Brief description of the drawings
Fig. 1 is process chart of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing and detailed description of the invention, the present invention is described in further detail:
As shown in Figure 1, flue gas CO2Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation specifically to comprise followingStep:
(1) by inoculum concentration 10～20% by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium, soAfter postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then pass into volume in photosynthetic reactorConcentration is 6% high concentration CO2Gas, gas flow be 60ml/min, intensity of illumination be 3000～3500Lux,Under 25～28 DEG C of conditions of temperature, cultivate 4～6 days; By above-mentioned 6%CO2The algae strain of cultivating is with 10～20% inoculum concentrationIn the liquid B G-11 culture medium of transferring new, then pass into volumetric concentration and be 10% high concentration CO2Gas, at gasBody flow is that 60ml/min, intensity of illumination are under 3000～3500Lux, 25～28 DEG C of conditions of temperature, cultivates 4～6 days.
(2) get above-mentioned 10%CO2In gas, cultivate the algae liquid after 4～6 days, transfer into new with 10～20% inoculum concentrationLiquid B G-11 culture medium in, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml,In photosynthetic reactor, pass into CO again2Volumetric concentration is 12～15% coal-fired plant flue gas, at gas flow is60ml/min, intensity of illumination are that 3000～3500Lux, temperature are under 25～28 DEG C of conditions, 5～10 generations of Continuous Cultivation,Obtain the target algae strain of Fast Growth under coal-fired plant flue gas condition.
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 training with 10～20% inoculum concentrationSupport in base, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then to photosynthetic reactorIn pass into CO2Volumetric concentration is 12～15% coal-fired plant flue gas promotion algae strain growth, at gas flow is60ml/min, intensity of illumination 3000～3500Lux, temperature are under the condition of 25～28 DEG C, training objective algae strain 7～10My god, calculate growth rate by measuring frustule number.
The measure and calculation method of growth rate is: get 2mL algae liquid every 24h, in algae liquid, add 0.2mL Shandong brother examinationAgent, concussion shakes up, and film-making, at optical microphotograph Microscopic observation, utilizes blood counting chamber counting, and it is (individual that calculating frustule is counted N/ mL) recycling formula μ=(lnN2-lnN1)/(t2-t1) calculate cell average growth rate, as growth rate; Wherein,μ is cell average growth rate; N1Be t1It time frustule number (individual/mL), N2Be t2It time frustuleNumber (individual/mL). Chemicals used and reagent are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and reagent grade is for dividingAnalyse pure.
(4) step (3) is cultivated to the target algae strain after finishing, be placed under high intensity of illumination 7500～10000Lux and continueContinuous cultivation, and continue to pass into CO2Volumetric concentration is that 12～15% coal-fired plant flue gas is coerced the astaxanthin improving in algae strainContent, condition of culture is: gas flow is that 120ml/min, cultivation temperature are 25～28 DEG C, cultivate after 10～15 days,Centrifugal algae liquid and freeze drying is obtained to haematococcus pluvialis dry algae powder, measure evaluate haematococcus pluvialis powder living beings dry weight andContent astaxanthin.
The assay method of living beings dry weight M (g/L) is: get 10mL algae liquid with 8000rmp centrifugal 10 minutes, goClear liquid, the algae mud twice obtaining with distilled water flushing, then the centrifugal dehydration of algae mud is placed at-70 DEG C after freezing 24 hours,Put into low-temperature vacuum drying instrument dry 24 hours, gained algae powder is weighed and calculated living beings dry weight M (g/L).
The assay method of content astaxanthin adopts high performance liquid chromatography (HPLC), is specially: get 10mL algae liquid,Centrifugal 10 minutes of 8000rmp, removes supernatant, and algae mud is placed in to-70 DEG C after freezing 24 hours, puts into cryogenic vacuum dryIn dry instrument, be dried 24 hours; The algae powder of freeze drying gained is placed in to 15ml dispersion machine test tube, add 5mL methyl alcohol-Carrene mixed solution (25:75, v/v), grinds extraction 2 minutes at 5500rmp homogeneous, and gained mixed liquor is at 8000rmpCentrifugal 5 minutes, get supernatant, be repeatedly extracted to algae-residue colourless; The supernatant that contains astaxanthin of rear retention will be extracted repeatedlyLiquid mixes, and is settled to 25mL with methyl alcohol-carrene mixed solution; Get 5mL mixed liquor, add 1mL0.05mol/LNaOH-methanol solution carries out saponification (at least 8 hours) under room temperature, half-light, nitrogen environment; ProductBe settled to 5mL with Nitrogen evaporator, with 0.45 μ m membrane filtration, filtrate is measured shrimp green grass or young crops for high performance liquid chromatography (HPLC)Cellulose content; HPLC condition of work is as follows: mobile phase is A water, B methyl alcohol, C isopropyl alcohol, D acetonitrile, wash-out ladderDegree is: 0min, 20%A, 50%B, 30%D; 5min, 20%A, 50%B, 30%D; 15min, 60%B,10%C, 30%D; Flow velocity is 1ml/min; Get the astaxanthin absworption peak area under 476nm wavelength, contain according to astaxanthinMeasure the calibration curve corresponding with peak area and calculate astaxanthin concentration Dast(mg/L); The calculating of content astaxanthin in dry algae powderMethod is as follows: Cast(mg/g)＝Dast(mg/L)/M (g/L), wherein M (g/L) for algae liquid dry after living beings dry weight.Astaxanthin standard medicine used is purchased from aldrich company of Sigma (China), and reagent grade is chromatographically pure, other reagentPurchased from Chemical Reagent Co., Ltd., Sinopharm Group, reagent grade is chromatographically pure.
In the present invention, the composition of liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid. Chemicals used and reagent are all purchased from the chemistry examination of traditional Chinese medicines groupAgent Co., Ltd, reagent grade is pure for analyzing.
The following examples can make this professional professional and technical personnel's comprehend the present invention, but never in any formRestriction the present invention.
Embodiment 1
(1) by inoculum concentration 10% by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium, then willPostvaccinal culture medium is placed in the cylinder photosynthetic reactor that volume is 600ml, then passes into volumetric concentration in photosynthetic reactorIt is 6% high concentration CO2Gas is that 60ml/min, intensity of illumination are 3000Lux, 25 DEG C of bars of temperature at gas flowUnder part, cultivate 6 days; By above-mentioned 6%CO2The algae strain of cultivating is transferred into new liquid B G-11 training with 10% inoculum concentrationSupport in base, then pass into volumetric concentration and be 10% high concentration CO2Gas is 60ml/min, illumination at gas flowIntensity is under 3000Lux, 25 DEG C of conditions of temperature, cultivates 6 days;
(2) get above-mentioned 10%CO2In gas, cultivate the algae liquid after 6 days, transfer into new liquid with 10% inoculum concentrationIn BG-11 culture medium, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then to lightClose and in reactor, pass into CO2Volumetric concentration is 12% coal-fired plant flue gas, is 60ml/min, illumination at gas flowIntensity is that 3000Lux, temperature are under 25 DEG C of conditions, in 5 generations of Continuous Cultivation, obtains under coal-fired plant flue gas condition fastThe target algae strain that fast-growing is long;
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 culture medium with 10% inoculum concentrationIn, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then leads in photosynthetic reactorEnter CO2Volumetric concentration is 12% coal-fired plant flue gas promotion algae strain growth, is 60ml/min, illumination at gas flowIntensity 3000Lux, temperature are under the condition of 25 DEG C, and training objective algae strain 10 days is calculated by measuring frustule numberObtaining its growth rate is 0.37/d;
(4) step (3) is cultivated to the target algae strain after finishing, is placed under high intensity of illumination 10000Lux and continues to cultivate,And continue to pass into CO2Volumetric concentration is that 12% coal-fired plant flue gas is coerced the content astaxanthin improving in algae strain, cultivates barPart is: gas flow is that 120ml/min, cultivation temperature are 25 DEG C, cultivates after 15 days, by centrifugal algae liquid and freezing dryThe dry haematococcus pluvialis dry algae powder that obtains, recording its living beings dry weight is that 2.0g/L and content astaxanthin are 26mg/g.
In the present embodiment, the composition of liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid.
Embodiment 2
(1) by inoculum concentration 15% by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium, then willPostvaccinal culture medium is placed in the cylinder photosynthetic reactor that volume is 600ml, then passes into volumetric concentration in photosynthetic reactorIt is 6% high concentration CO2Gas is that 60ml/min, intensity of illumination are 3200Lux, 26 DEG C of bars of temperature at gas flowUnder part, cultivate 5 days; By above-mentioned 6%CO2The algae strain of cultivating is transferred into new liquid B G-11 training with 15% inoculum concentrationSupport in base, then pass into volumetric concentration and be 10% high concentration CO2Gas is 60ml/min, illumination at gas flowIntensity is under 3200Lux, 26 DEG C of conditions of temperature, cultivates 5 days;
(2) get above-mentioned 10%CO2In gas, cultivate the algae liquid after 5 days, transfer into new liquid with 15% inoculum concentrationIn BG-11 culture medium, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then to lightClose and in reactor, pass into CO2Volumetric concentration is 13% coal-fired plant flue gas, is 60ml/min, illumination at gas flowIntensity is that 3200Lux, temperature are under 26 DEG C of conditions, in 8 generations of Continuous Cultivation, obtains under coal-fired plant flue gas condition fastThe target algae strain that fast-growing is long;
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 culture medium with 15% inoculum concentrationIn, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then leads in photosynthetic reactorEnter CO2Volumetric concentration is 13% coal-fired plant flue gas promotion algae strain growth, is 60ml/min, illumination at gas flowIntensity 3200Lux, temperature are under the condition of 26 DEG C, and training objective algae strain 8 days calculates by measuring frustule numberIts growth rate is 0.43/d;
(4) step (3) is cultivated to the target algae strain after finishing, is placed under high intensity of illumination 8000Lux and continues to cultivate,And continue to pass into CO2Volumetric concentration is that 13% coal-fired plant flue gas is coerced the content astaxanthin improving in algae strain, cultivates barPart is: gas flow is that 120ml/min, cultivation temperature are 26 DEG C, cultivates after 12 days, by centrifugal algae liquid and freezing dryThe dry haematococcus pluvialis dry algae powder that obtains, recording its living beings dry weight is 2.4g/L and content astaxanthin 22mg/g.
In the present embodiment, the composition of liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid.
Embodiment 3
(1) by inoculum concentration 20% by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium, then willPostvaccinal culture medium is placed in the cylinder photosynthetic reactor that volume is 600ml, then passes into volumetric concentration in photosynthetic reactorIt is 6% high concentration CO2Gas is that 60ml/min, intensity of illumination are 3500Lux, 28 DEG C of bars of temperature at gas flowUnder part, cultivate 4 days; By above-mentioned 6%CO2The algae strain of cultivating is transferred into new liquid B G-11 training with 20% inoculum concentrationSupport in base, then pass into volumetric concentration and be 10% high concentration CO2Gas is 60ml/min, illumination at gas flowIntensity is under 3500Lux, 28 DEG C of conditions of temperature, cultivates 4 days;
(2) get above-mentioned 10%CO2In gas, cultivate the algae liquid after 4 days, transfer into new liquid with 20% inoculum concentrationIn BG-11 culture medium, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then to lightClose and in reactor, pass into CO2Volumetric concentration is 15% coal-fired plant flue gas, is 60ml/min, illumination at gas flowIntensity is that 3500Lux, temperature are under 28 DEG C of conditions, in 10 generations of Continuous Cultivation, obtains under coal-fired plant flue gas conditionThe target algae strain of Fast Growth;
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 culture medium with 20% inoculum concentrationIn, then postvaccinal culture medium is placed in to the cylinder photosynthetic reactor that volume is 600ml, then leads in photosynthetic reactorEnter CO2Volumetric concentration is 15% coal-fired plant flue gas promotion algae strain growth, is 60ml/min, illumination at gas flowIntensity 3500Lux, temperature are under the condition of 28 DEG C, and training objective algae strain 7 days calculates by measuring frustule numberIts growth rate is 0.38/d;
(4) step (3) is cultivated to the target algae strain after finishing, is placed under high intensity of illumination 7500Lux and continues to cultivate,And continue to pass into CO2Volumetric concentration is that 15% coal-fired plant flue gas is coerced the content astaxanthin improving in algae strain, cultivates barPart is: gas flow is that 120ml/min, cultivation temperature are 28 DEG C, cultivates after 10 days, by centrifugal algae liquid and freezing dryThe dry haematococcus pluvialis dry algae powder that obtains, measures to such an extent that its living beings dry weight is that 2.7g/L and content astaxanthin are 24mg/g.
In the present embodiment, the composition of liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid.
Finally, it should be noted that above what enumerate is only specific embodiments of the invention. Obviously, the present invention does not limitIn above embodiment, can also there be a lot of distortion. Those of ordinary skill in the art can be from content disclosed by the inventionAll distortion of directly deriving or associating, all should think protection scope of the present invention.
Claims (6)
1. flue gas CO2Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation, in coal-fired plant flue gas, contains12～15% high volumetric concentration CO2, it is characterized in that described flue gas CO2Domestication promotes haematococcus pluvialis growing and shrimpThe method of blue or green element accumulation specifically comprises the steps:
(1) by 10～20% inoculum concentration by haematococcus pluvialis liquid inoculation in 400ml liquid B G-11 culture medium,Then postvaccinal culture medium is placed in to photosynthetic reactor, then is 6% height to passing into volumetric concentration in photosynthetic reactorConcentration C O2Gas is that 60ml/min, intensity of illumination are 25～28 DEG C of 3000～3500Lux, temperature at gas flowCondition under, cultivate 4～6 days;
Then, by the above-mentioned CO taking volumetric concentration as 6%2Algae strain after cultivation, with 10～20% inoculum concentration transfer intoIn new liquid B G-11 culture medium, then pass into volumetric concentration and be 10% high concentration CO2Gas, at gas flowFor 60ml/min, intensity of illumination are under the condition of 25～28 DEG C of 3000～3500Lux, temperature, cultivate 4～6 days;
(2) get in step (1) at the CO taking volumetric concentration as 10%2In gas, cultivate the algae liquid after 4～6 days, withIn the liquid B G-11 culture medium that 10～20% inoculum concentration is transferred new, then postvaccinal culture medium is placed in photosynthetic anti-Answer in device, then pass into CO in photosynthetic reactor2Volumetric concentration is 12～15% coal-fired plant flue gas, at gas flowFor 60ml/min, intensity of illumination are that 3000～3500Lux, temperature are under the condition of 25～28 DEG C, Continuous Cultivation 5～10In generation, obtain the target algae strain of Fast Growth under coal-fired plant flue gas condition;
(3) the target algae strain of taking the logarithm growth period, is inoculated into 400ml liquid B G-11 training with 10～20% inoculum concentrationSupport in base, then postvaccinal culture medium is placed in to photosynthetic reactor, then passes into CO in photosynthetic reactor2Volume is denseDegree is that 12～15% coal-fired plant flue gas promotes algae strain growth, gas flow be 60ml/min, intensity of illumination 3000～3500Lux, temperature are under the condition of 25～28 DEG C, and training objective algae strain 7～10 days can be by measuring frustule numberCalculate growth rate;
(4) step (3) is cultivated to the target algae strain after finishing, be placed under high intensity of illumination 7500～10000Lux and continueContinuous cultivation, and continue to pass into CO2Volumetric concentration is that 12～15% coal-fired plant flue gas is coerced the astaxanthin improving in algae strainContent, condition of culture is: gas flow is that 120ml/min, cultivation temperature are 25～28 DEG C, cultivate after 10～15 days,By after centrifugal algae liquid and freeze drying, obtain haematococcus pluvialis dry algae powder, can measure the biology of evaluating haematococcus pluvialis powderMatter dry weight and content astaxanthin.
2. flue gas CO according to claim 12Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation,It is characterized in that, described photosynthetic reactor adopts the cylinder photosynthetic reactor that volume is 600ml.
3. flue gas CO according to claim 12Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation,It is characterized in that, the composition of described liquid B G-11 culture medium consists of: 1.5g/LNaNO3、0.2g/LNa2CO3、0.075g/LMgSO4·7H2O、0.4g/LK2HPO4、0.036g/LCaCl2·2H2O、2.2×10-4g/LZnSO4·7H2O、1.8×10-3g/LMnCl2·4H2O、2.1×10-5g/LNaMoO4、0.8×10-5g/LCuSO4·5H2O、2.8×10-3g/LH3BO3, 0.001g/LEDTA, 0.006g/L citric acid.
4. flue gas CO according to claim 12Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation,It is characterized in that, in described step (3), the measure and calculation method of growth rate is:
Get 2mL algae liquid every 24h, in algae liquid, add 0.2mL Shandong brother's reagent, concussion shakes up, and film-making, at opticsMicro-Microscopic observation, utilizes blood counting chamber counting, calculates frustule and counts N, recycling formula μ=(lnN2-lnN1)/(t2-t1)Calculate cell average growth rate, as growth rate;
Wherein, μ is cell average growth rate; N1Be t1It time frustule number, N2Be t2It time frustuleNumber.
5. flue gas CO according to claim 12Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation,It is characterized in that, in described step (4), the assay method of living beings dry weight M is:
Get 10mL algae liquid with 8000rmp centrifugal 10 minutes, remove supernatant, the algae mud twice obtaining with distilled water flushing,Again the centrifugal dehydration of algae mud is placed at-70 DEG C after freezing 24 hours, puts into low-temperature vacuum drying instrument dry 24 hours,Gained algae powder is weighed and calculated living beings dry weight M.
6. flue gas CO according to claim 12Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation,It is characterized in that, in described step (4), the assay method of content astaxanthin adopts high performance liquid chromatography.
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| CN105925487A (en) * | 2016-07-14 | 2016-09-07 | 中国科学院上海高等研究院 | Culture method for improving sugar content of micro-algal cells |
| CN106480155A (en) * | 2016-12-23 | 2017-03-08 | 山东金晶生物技术有限公司 | A kind of method being suitable for promoting Haematococcus pluvialis production astaxanthin under the high temperature conditions |
| CN106480155B (en) * | 2016-12-23 | 2020-05-26 | 山东金晶生物技术有限公司 | Method suitable for promoting haematococcus pluvialis to produce astaxanthin under high-temperature condition |
| CN106479898A (en) * | 2016-12-29 | 2017-03-08 | 青岛藻蓝生物有限公司 | A kind of culture medium of Haematocoocus Pluvialls and its application |
| CN107365826A (en) * | 2017-09-18 | 2017-11-21 | 深圳市德和生物科技有限公司 | A kind of method of regulating astaxanthin accumulation |
| CN112973434A (en) * | 2021-03-08 | 2021-06-18 | 青岛大学 | Phase change solvent reinforced microalgae fixed coal-fired flue gas CO2And resource conversion method |
| CN112973434B (en) * | 2021-03-08 | 2023-08-11 | 青岛大学 | Phase-change solvent-reinforced microalgae-immobilized coal-fired flue gas CO 2 And a method of converting into resources |
| CN114480367A (en) * | 2022-01-28 | 2022-05-13 | 浙江大学 | Electrochemical promotion of high-concentration CO in nannochloropsis oculata fixed smoke2Method (2) |
| CN114480367B (en) * | 2022-01-28 | 2024-02-09 | 浙江大学 | Electrochemical promotion of high concentration CO in nannochloropsis immobilization flue gas 2 Is a method of (2) |
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