CN105586262B - Flue gas CO2The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation - Google Patents
Flue gas CO2The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation Download PDFInfo
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- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 65
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 65
- 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 65
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 65
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 65
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 239000003546 flue gas Substances 0.000 title claims abstract description 48
- 241000168517 Haematococcus lacustris Species 0.000 title claims abstract description 38
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- 239000002028 Biomass Substances 0.000 claims description 16
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 6
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- 230000003698 anagen phase Effects 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 3
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
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- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
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- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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Abstract
The present invention relates to biologically actie health product development technique, it is desirable to provide flue gas CO2The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation.Flue gas CO2Domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation comprising steps of it is 6% high concentration CO that haematococcus pluvialis liquid, which is successively used volumetric concentration,2Gas, the high concentration CO that volumetric concentration is 10%2Gas domestication culture；Then the strain of target algae is obtained with the domestication culture of coal-fired plant flue gas iteration again；By target algae strain coal-fired plant flue gas, successively the Low light intensity of 3000~3500Lux, 7500~10000Lux high intensity of illumination under cultivate after, algae solution centrifugation and freeze-drying are obtained into haematococcus pluvialis dry algae powder.Invention significantly improves growth rate and astaxanthin yield, for growth rate than improving 47% under air conditions, content astaxanthin improves 25%.
Description
Technical field
The present invention relates to biologically actie health product development technique field, in particular to flue gas CO2Domestication promotes rain life red
The method of ball algae growth and astaxanthin accumulation.
Background technique
Astaxanthin is the strongest antioxidant of nature being currently known, and inoxidizability is ascorbic 6000 times, dimension
1000 times, 200 times of tea polyphenols, 50 times of grape seed extract of raw element E.Astaxanthin can be enhanced the anti-aging of body, disappear
Therefore scorching, uvioresistant and immune function are used for the multiple functions product such as health care product, cosmetics, food and feed additive
In.In natural astaxanthin source, the content astaxanthin highest of haematococcus pluvialis, account for about its dry weight 1-5% (Minxi Wana,
2014), also, the structure of its astaxanthin-containing is consistent with astaxanthin structure needed for organism, this is synthesizing astaxanthin and other
What natural astaxanthin cannot compare, it is acknowledged as the best biological source of natural astaxanthin, is current investment astaxanthin business
Change unique algae of application.However, haematococcus pluvialis growing is slow, specific growth rate be about 0.15/d-0.64/d (Orosa,
2005；Kaewpintong,2006；Imamoglu,2010).Slow growth rate brings other algae invasions, protozoan
The various problems such as pollution are main bugbears present in haematococcus pluvialis large-scale production.
Due to low concentration CO in air2Mass transfer rate in water is lower, and the energy consumption of both culturing microalgae 40% is used for air pump
Air is persistently provided and maintains micro algae growth, and utilizes flue gas high concentration CO2Cultivating microalgae, not only can be by the energy of both culturing microalgae
Investment reduces by 70% (Lam et al, 2012), can also promote micro algae growth and astaxanthin accumulation.By stacked rectangular capsules, even
Continuous tubular reactor etc., haematococcus pluvialis is to 2%-5% high concentration CO2Carbon sequestration efficiency can reach 13.55%to 49.15%,
Biomass and astaxanthin yield significantly improve (Kang, 2010；Lee, 2015；Giannelli, 2015；Kwak, HS, 2015).
With 5%CO2It can be using acetate as carbon source by the output increased of astaxanthin to 175.7mg/l for carbon source culture haematococcus pluvialis
Heterotrophic culture 2.7 times (Kang etc., 2005).In addition, the CO of high concentration2It can replace nitrogen stress culture medium and acetate culture
Base induces the synthesis and accumulation of astaxanthin, reduces cost.Astaxanthin can be promoted by being passed through pure carbon dioxide to distilled water intermittent
Accumulation, content are close with content astaxanthin in nitrogen stress culture medium (Imamoglu, 2009).But in coal-fired plant flue gas
High concentration (12~15%) CO2Under the conditions of, haematococcus pluvialis growing will receive inhibition.Cheng et al. (2016) utilizes high concentration
CO2Haematococcus pluvialis core mutagenesis mutation algae strain discovery is cultivated, CO is worked as2Concentration is improved by air to 6%, and biological productivity improves
82%.But work as CO2When concentration is improved to 10% or more, the growth of haematococcus pluvialis is suppressed.Yin et al. (2015) will
20%CO2It is passed through culture haematococcus pluvialis in biofilm reactor and finds its biomass ratio 1.5%CO215% is reduced, astaxanthin
Content reduces 40%, will result directly in the extension of industrial production time and the reduction of productivity effect, so, how to utilize fire coal
Power-plant flue gas CO2The growth rate and astaxanthin yield for improving haematococcus pluvialis are still a technical problem, if can be dashed forward
It is broken to be of great significance to improving production of astaxanthin efficiency and reducing production cost.
Summary of the invention
It is a primary object of the present invention to overcome deficiency in the prior art, a kind of utilization coal-fired plant flue gas CO is provided2
The method that domestication improves haematococcus pluvialis growing rate and content astaxanthin.In order to solve the above technical problems, solution of the invention
Scheme is:
Flue gas CO is provided2The method for promoting haematococcus pluvialis growing and astaxanthin accumulation is tamed, is contained in coal-fired plant flue gas
There is 12~15% high volumetric concentration CO2, the flue gas CO2The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation
Specifically include the following steps:
(1) by 10~20% inoculum concentration by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, so
The culture medium after inoculation is placed in photosynthetic reactor afterwards, then is passed through the high concentration that volumetric concentration is 6% into photosynthetic reactor
CO2Gas, gas flow is 60ml/min, intensity of illumination is 3000~3500Lux, 25~28 DEG C of temperature under conditions of, training
It supports 4~6 days；
Then, by the above-mentioned CO for being 6% with volumetric concentration2After culture algae strain, with 10~20% inoculum concentration transfer into
In new liquid B G-11 culture medium, the high concentration CO that volumetric concentration is 10% is then passed to2Gas is 60ml/ in gas flow
Min, intensity of illumination are 3000~3500Lux, under conditions of 25~28 DEG C of temperature, are cultivated 4~6 days；
(2) it takes in step (1) in the CO for being 10% with volumetric concentration2Algae solution after being cultivated 4~6 days in gas, with 10~
In the liquid B G-11 culture medium that 20% inoculum concentration is transferred new, then the culture medium after inoculation is placed in photosynthetic reactor,
CO is passed through into photosynthetic reactor again2Volumetric concentration be 12~15% coal-fired plant flue gas, gas flow be 60ml/min,
It under conditions of intensity of illumination is 3000~3500Lux, temperature is 25~28 DEG C, continuously cultivated for 5~10 generations, that is, obtains in fire coal
The target algae strain of fast-growth under the conditions of power-plant flue gas；
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture with 10~20% inoculum concentration
In base, then the culture medium after inoculation is placed in photosynthetic reactor, then CO is passed through into photosynthetic reactor2Volumetric concentration is 12
~15% coal-fired plant flue gas promotes algae strain growth, gas flow be 60ml/min, 3000~3500Lux of intensity of illumination,
Under conditions of temperature is 25~28 DEG C, training objective algae strain 7~10 days, growth rate can be calculated by measurement frustule number；
(4) it by the target algae strain after step (3) culture, is placed under high 7500~10000Lux of intensity of illumination and continues to train
It supports, and is continually fed into CO2The coal-fired plant flue gas stress that volumetric concentration is 12~15% improves the content astaxanthin in algae strain, training
Support condition are as follows: gas flow 120ml/min, cultivation temperature are 25~28 DEG C, culture 10~15 days after, by algae solution centrifugation and it is cold
Be lyophilized it is dry after contain to get the biomass dry weight and astaxanthin for haematococcus pluvialis dry algae powder, being capable of measuring evaluation haematococcus pluvialis powder
Amount.
In the present invention, the photosynthetic reactor uses volume for the cylinder photosynthetic reactor of 600ml.
In the present invention, the liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、
0.075g/L MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·
7H2O、 1.8×10-3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×
10-3g/L H3BO3, 0.001g/L EDTA, 0.006g/L citric acid.
In the present invention, in the step (3), the measure and calculation method of growth rate are as follows:
Every taking 2mL algae solution for 24 hours, the Shandong 0.2mL brother's reagent is added into algae solution, concussion shakes up, film-making, in optical microscopy
Lower observation, is counted using blood counting chamber, is calculated frustule number N (a/mL) and is recycled formula μ=(lnN2-ln N1)/(t2-
t1) cell average growth rate is calculated, as growth rate；
Wherein, μ is cell average growth rate；N1For t1It when frustule number (a/mL), N2For t2It when
Frustule number (a/mL).
In the present invention, in the step (4), the measuring method of biomass dry weight M (g/L) are as follows:
Take 10mL algae solution with 8000rmp centrifugation 10 minutes, remove supernatant, with distilled water flushing obtain algal gel twice, then
Algal gel centrifugal dehydration is placed on after being freezed 24 hours at -70 DEG C, drying 24 hours in low-temperature vacuum drying instrument is put into, to gained
Algae powder weighs and calculates biomass dry weight M (g/L).
In the present invention, in the step (4), the measuring method of content astaxanthin uses high performance liquid chromatography
(HPLC).Specifically: 10mL algae solution is taken, 8000rmp is centrifuged 10 minutes, removes supernatant, and algal gel is placed in -70 DEG C of freezings 24
After hour, it is put into low-temperature vacuum drying instrument 24 hours dry；Resulting algae powder will be freeze-dried and be placed in 15ml dispersion machine test tube
In, it is added 5mL methanol dichloromethane mixed solution (25:75, v/v), at the grinding of 5500rmp homogeneous extraction 2 minutes, gained was mixed
It closes liquid to be centrifuged 5 minutes in 8000rmp, takes supernatant, it is colourless to be extracted to algae-residue repeatedly；That retains after extracting repeatedly is green containing shrimp
The supernatant mixing of element, and 25mL is settled to methanol dichloromethane mixed solution；5mL mixed liquor is taken, 1mL is added
0.05mol/L NaOH- methanol solution carries out saponification (at least 8 hours) under room temperature, half-light, nitrogen environment；Reaction produces
Object is settled to 5mL with nitrogen evaporator, and with 0.45 μm of membrane filtration, filtrate contains for high performance liquid chromatography (HPLC) measurement astaxanthin
Amount；HPLC operating condition is as follows: mobile phase is A water, B methanol, C isopropanol, D acetonitrile, gradient are as follows: 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；It takes
Astaxanthin under 476nm wavelength absorbs peak area, calculates astaxanthin according to content astaxanthin standard curve corresponding with peak area
Concentration Dast(mg/L)；Then the calculation method of content astaxanthin is as follows in dry algae powder: Cast(mg/g)=Dast(mg/L)/M (g/L),
Wherein M (g/L) is the biomass dry weight after algae solution drying.
Compared with prior art, the beneficial effects of the present invention are:
The present invention uses coal-fired plant flue gas high concentration (12~15%) CO2Haematococcus pluvialis is tamed, life is significantly improved
Long rate and astaxanthin yield, for growth rate than improving 47% under air conditions, content astaxanthin improves 25%.This is
Due to utilizing flue gas high concentration CO2So that the photosynthesis key carbon sequestration enzyme Rubisco of haematococcus pluvialis is expressed up-regulation, promotes card
Er Wen circulation and photosynthetic growth efficiency；Light captures the protein-bonded expression quantity of chlorophyll a/b and rises, and improves light-use effect
Rate；Chlorophyll synthesis efficiency improves, and ATP synthesizes expression of enzymes up-regulation, to improve growth rate.The present invention is in chemical activators
Stage, flue gas high concentration CO2Sufficient carbon source is provided for oil synthesis, provides advantageous item for astaxanthin esterification and storage
Part.
Detailed description of the invention
Fig. 1 is process flow chart of the invention.
Specific embodiment
Present invention is further described in detail with specific embodiment with reference to the accompanying drawing:
As shown in Figure 1, flue gas CO2It is following that domestication promotes the method for haematococcus pluvialis growing and astaxanthin accumulation to specifically include
Step:
(1) inoculum concentration 10~20% is pressed by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, then
Culture medium after inoculation is placed in volume and is the cylinder photosynthetic reactor of 600ml, then is passed through volumetric concentration into photosynthetic reactor
For 6% high concentration CO2Gas, gas flow is 60ml/min, intensity of illumination is 3000~3500Lux, temperature 25~28
Under the conditions of DEG C, cultivate 4~6 days；By above-mentioned 6%CO2The algae strain of culture is transferred with 10~20% inoculum concentration into new liquid B G-
In 11 culture mediums, the high concentration CO that volumetric concentration is 10% is then passed to2Gas is that 60ml/min, illumination are strong in gas flow
Degree is 3000~3500Lux, under the conditions of 25~28 DEG C of temperature, is cultivated 4~6 days.
(2) above-mentioned 10%CO is taken2Algae solution after cultivating 4~6 days in gas, is transferred with 10~20% inoculum concentration into new
In liquid B G-11 culture medium, the culture medium after inoculation is then placed in the cylinder photosynthetic reactor that volume is 600ml, then to light
It closes in reactor and is passed through CO2The coal-fired plant flue gas that volumetric concentration is 12~15% is 60ml/min, illumination in gas flow
It under the conditions of intensity is 3000~3500Lux, temperature is 25~28 DEG C, continuously cultivated for 5~10 generations, that is, obtains in coal-fired plant flue gas
Under the conditions of fast-growth the strain of target algae.
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture with 10~20% inoculum concentration
In base, the culture medium after inoculation is then placed in the cylinder photosynthetic reactor that volume is 600ml, then be passed through into photosynthetic reactor
CO2The coal-fired plant flue gas that volumetric concentration is 12~15% promotes algae strain growth, is that 60ml/min, illumination are strong in gas flow
Under conditions of 3000~3500Lux of degree, temperature are 25~28 DEG C, training objective algae strain 7~10 days, pass through measurement frustule
Number calculates growth rate.
The measure and calculation method of growth rate are as follows: every taking 2mL algae solution for 24 hours, the Shandong 0.2mL brother's reagent is added into algae solution,
Concussion shakes up, and film-making is observed under an optical microscope, is counted using blood counting chamber, and it is sharp again to calculate frustule number N (a/mL)
With formula μ=(lnN2-ln N1)/(t2-t1) cell average growth rate is calculated, as growth rate；Wherein, μ is flat for cell
Equal growth rate；N1For t1It when frustule number (a/mL), N2For t2It when frustule number (a/mL).Usedization
It learns drug and reagent is purchased from Sinopharm Chemical Reagent Co., Ltd., reagent grade is that analysis is pure.
(4) it by the target algae strain after step (3) culture, is placed under high 7500~10000Lux of intensity of illumination and continues to train
It supports, and is continually fed into CO2The coal-fired plant flue gas stress that volumetric concentration is 12~15% improves the content astaxanthin in algae strain, training
Support condition are as follows: gas flow 120ml/min, cultivation temperature are 25~28 DEG C, culture 10~15 days after, by algae solution centrifugation and it is cold
Jelly is dried to obtain haematococcus pluvialis dry algae powder, the biomass dry weight and content astaxanthin of measurement evaluation haematococcus pluvialis powder.
The measuring method of biomass dry weight M (g/L) are as follows: it takes 10mL algae solution with 8000rmp centrifugation 10 minutes, removes supernatant,
The algal gel obtained with distilled water flushing twice, then after algal gel centrifugal dehydration is placed on freezing 24 hours at -70 DEG C, is put into low temperature
Drying 24 hours in instrument are dried in vacuo, weighs to gained algae powder and calculates biomass dry weight M (g/L).
The measuring method of content astaxanthin uses high performance liquid chromatography (HPLC), specifically: 10mL algae solution is taken,
8000rmp is centrifuged 10 minutes, removes supernatant, after algal gel is placed in -70 DEG C of freezings 24 hours, is put into low-temperature vacuum drying instrument
It is 24 hours dry；Resulting algae powder will be freeze-dried to be placed in 15ml dispersion machine test tube, 5mL methanol-methylene chloride mixing is added
Solution (25:75, v/v), at the grinding of 5500rmp homogeneous extraction 2 minutes, gained mixed liquor was centrifuged 5 minutes in 8000rmp, is taken
It is colourless to be extracted to algae-residue repeatedly for clear liquid；The supernatant containing astaxanthin retained after extracting repeatedly mixes, and with methanol-two
Chloromethanes mixed solution is settled to 25mL；Take 5mL mixed liquor, 1mL 0.05mol/L NaOH- methanol solution be added, in room temperature,
Saponification (at least 8 hours) are carried out under half-light, nitrogen environment；Reaction product is settled to 5mL with nitrogen evaporator, with 0.45 μm of filter membrane
Filtering, filtrate measure content astaxanthin for high performance liquid chromatography (HPLC)；HPLC operating condition is as follows: mobile phase is A water, B
Methanol, C isopropanol, D acetonitrile, gradient are as follows: 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；The astaxanthin under 476nm wavelength is taken to absorb peak area, according to shrimp
Green cellulose content standard curve corresponding with peak area calculates astaxanthin concentration Dast(mg/L)；Then content astaxanthin in dry algae powder
Calculation method is as follows: Cast(mg/g)=Dast(mg/L)/M (g/L), wherein M (g/L) is the biomass dry weight after algae solution drying.
Astaxanthin standard pharmaceutical used is purchased from Sigma-Aldrich (China), and reagent grade is chromatographically pure, and other reagents are purchased from
Sinopharm Chemical Reagent Co., Ltd., reagent grade are chromatographically pure.
In the present invention, liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、
0.075g/L MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·
7H2O、 1.8×10-3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×
10-3g/L H3BO3, 0.001g/L EDTA, 0.006g/L citric acid.Chemicals and reagent used are purchased from Chinese medicines group
Reagent Co., Ltd is learned, reagent grade is that analysis is pure.
The following examples can make the professional technician of this profession that the present invention be more fully understood, but not with any side
The formula limitation present invention.
Embodiment 1
(1) by inoculum concentration 10% by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, then will connect
Culture medium after kind is placed in the cylinder photosynthetic reactor that volume is 600ml, then being passed through volumetric concentration into photosynthetic reactor is 6%
High concentration CO2Gas, gas flow be 60ml/min, intensity of illumination 3000Lux, 25 DEG C of temperature under the conditions of, culture 6
It；By above-mentioned 6%CO2In the liquid B G-11 culture medium that the algae strain of culture is transferred new with 10% inoculum concentration, body is then passed to
The high concentration CO that product concentration is 10%2Gas, gas flow be 60ml/min, 25 DEG C of intensity of illumination 3000Lux, temperature items
Under part, cultivate 6 days；
(2) above-mentioned 10%CO is taken2Algae solution after cultivating 6 days in gas, is transferred with 10% inoculum concentration into new liquid
In BG-11 culture medium, the culture medium after inoculation is then placed in the cylinder photosynthetic reactor that volume is 600ml, then to photosynthetic anti-
It answers and is passed through CO in device2The coal-fired plant flue gas that volumetric concentration is 12%, gas flow is 60ml/min, intensity of illumination is
It is continuous to cultivate for 5 generations under the conditions of 3000Lux, temperature are 25 DEG C, that is, obtain the target of fast-growth under the conditions of coal-fired plant flue gas
Algae strain；
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture medium with 10% inoculum concentration,
Then the culture medium after inoculation is placed in volume and is the cylinder photosynthetic reactor of 600ml, then be passed through CO into photosynthetic reactor2Body
Product concentration be 12% coal-fired plant flue gas promote algae strain growth, gas flow be 60ml/min, intensity of illumination 3000Lux,
Under conditions of temperature is 25 DEG C, training objective algae strain 10 days, by measure frustule number calculate its growth rate is 0.37/
d；
(4) it by the target algae strain after step (3) culture, is placed under high intensity of illumination 10000Lux and continues to cultivate, and
It is continually fed into CO2The coal-fired plant flue gas stress that volumetric concentration is 12% improves the content astaxanthin in algae strain, condition of culture are as follows:
Gas flow is 120ml/min, cultivation temperature is 25 DEG C, and after culture 15 days, it is red that algae solution centrifugation and freeze-drying are obtained rain life
Ball algae dry algae powder, measures that its biomass dry weight is 2.0g/L and content astaxanthin is 26mg/g.
In the present embodiment, liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、
0.075g/L MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·
7H2O、 1.8×10-3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×
10-3g/L H3BO3, 0.001g/L EDTA, 0.006g/L citric acid.
Embodiment 2
(1) by inoculum concentration 15% by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, then will connect
Culture medium after kind is placed in the cylinder photosynthetic reactor that volume is 600ml, then being passed through volumetric concentration into photosynthetic reactor is 6%
High concentration CO2Gas, gas flow be 60ml/min, intensity of illumination 3200Lux, 26 DEG C of temperature under the conditions of, culture 5
It；By above-mentioned 6%CO2In the liquid B G-11 culture medium that the algae strain of culture is transferred new with 15% inoculum concentration, body is then passed to
The high concentration CO that product concentration is 10%2Gas, gas flow be 60ml/min, 26 DEG C of intensity of illumination 3200Lux, temperature items
Under part, cultivate 5 days；
(2) above-mentioned 10%CO is taken2Algae solution after cultivating 5 days in gas, is transferred with 15% inoculum concentration into new liquid
In BG-11 culture medium, the culture medium after inoculation is then placed in the cylinder photosynthetic reactor that volume is 600ml, then to photosynthetic anti-
It answers and is passed through CO in device2The coal-fired plant flue gas that volumetric concentration is 13%, gas flow is 60ml/min, intensity of illumination is
It is continuous to cultivate for 8 generations under the conditions of 3200Lux, temperature are 26 DEG C, that is, obtain the target of fast-growth under the conditions of coal-fired plant flue gas
Algae strain；
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture medium with 15% inoculum concentration,
Then the culture medium after inoculation is placed in volume and is the cylinder photosynthetic reactor of 600ml, then be passed through CO into photosynthetic reactor2Body
Product concentration be 13% coal-fired plant flue gas promote algae strain growth, gas flow be 60ml/min, intensity of illumination 3200Lux,
Under conditions of temperature is 26 DEG C, training objective algae strain 8 days, by measure frustule number calculate its growth rate is 0.43/
d；
(4) it by the target algae strain after step (3) culture, is placed under high intensity of illumination 8000Lux and continues to cultivate, and hold
It is continuous to be passed through CO2The coal-fired plant flue gas stress that volumetric concentration is 13% improves the content astaxanthin in algae strain, condition of culture are as follows: gas
Body flow is 120ml/min, cultivation temperature is 26 DEG C, and after culture 12 days, algae solution centrifugation and freeze-drying are obtained the raw red ball of rain
Algae dry algae powder, measuring its biomass dry weight is 2.4g/L and content astaxanthin 22mg/g.
In the present embodiment, liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、
0.075g/L MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·
7H2O、 1.8×10-3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×
10-3g/L H3BO3, 0.001g/L EDTA, 0.006g/L citric acid.
Embodiment 3
(1) by inoculum concentration 20% by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, then will connect
Culture medium after kind is placed in the cylinder photosynthetic reactor that volume is 600ml, then being passed through volumetric concentration into photosynthetic reactor is 6%
High concentration CO2Gas, gas flow be 60ml/min, intensity of illumination 3500Lux, 28 DEG C of temperature under the conditions of, culture 4
It；By above-mentioned 6%CO2In the liquid B G-11 culture medium that the algae strain of culture is transferred new with 20% inoculum concentration, body is then passed to
The high concentration CO that product concentration is 10%2Gas, gas flow be 60ml/min, 28 DEG C of intensity of illumination 3500Lux, temperature items
Under part, cultivate 4 days；
(2) above-mentioned 10%CO is taken2Algae solution after cultivating 4 days in gas, is transferred with 20% inoculum concentration into new liquid
In BG-11 culture medium, the culture medium after inoculation is then placed in the cylinder photosynthetic reactor that volume is 600ml, then to photosynthetic anti-
It answers and is passed through CO in device2The coal-fired plant flue gas that volumetric concentration is 15%, gas flow is 60ml/min, intensity of illumination is
It is continuous to cultivate for 10 generations under the conditions of 3500Lux, temperature are 28 DEG C, that is, obtain the mesh of fast-growth under the conditions of coal-fired plant flue gas
Mark algae strain；
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture medium with 20% inoculum concentration,
Then the culture medium after inoculation is placed in volume and is the cylinder photosynthetic reactor of 600ml, then be passed through CO into photosynthetic reactor2Body
Product concentration be 15% coal-fired plant flue gas promote algae strain growth, gas flow be 60ml/min, intensity of illumination 3500Lux,
Under conditions of temperature is 28 DEG C, training objective algae strain 7 days, by measure frustule number calculate its growth rate is 0.38/
d；
(4) it by the target algae strain after step (3) culture, is placed under high intensity of illumination 7500Lux and continues to cultivate, and hold
It is continuous to be passed through CO2The coal-fired plant flue gas stress that volumetric concentration is 15% improves the content astaxanthin in algae strain, condition of culture are as follows: gas
Body flow is 120ml/min, cultivation temperature is 28 DEG C, and after culture 10 days, algae solution centrifugation and freeze-drying are obtained the raw red ball of rain
Algae dry algae powder, measure its biomass dry weight is 2.7g/L and content astaxanthin is 24mg/g.
In the present embodiment, liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、
0.075g/L MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·
7H2O、1.8×10-3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×
10-3g/L H3BO3, 0.001g/L EDTA, 0.006g/L citric acid.
Finally it should be noted that the above enumerated are only specific embodiments of the present invention.It is clear that the invention is not restricted to
Above embodiments can also have many variations.Those skilled in the art can directly lead from present disclosure
Out or all deformations for associating, it is considered as protection scope of the present invention.
Claims (5)
1. flue gas CO2The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation, in coal-fired plant flue gas containing 12~
15% high volumetric concentration CO2, which is characterized in that the flue gas CO2Domestication promotes haematococcus pluvialis growing and astaxanthin accumulation
Method specifically include the following steps:
(1) by 10~20% inoculum concentration by haematococcus pluvialis liquid inoculation into 400ml liquid B G-11 culture medium, then will
Culture medium after inoculation is placed in photosynthetic reactor, then the high concentration CO that volumetric concentration is 6% is passed through into photosynthetic reactor2Gas
Body, gas flow is 60ml/min, intensity of illumination is 3000~3500Lux, 25~28 DEG C of temperature under conditions of, culture 4~
6 days；
Then, by the above-mentioned CO for being 6% with volumetric concentration2Algae strain after culture, is transferred with 10~20% inoculum concentration into new liquid
In BG-11 culture medium, the high concentration CO that volumetric concentration is 10% is then passed to2Gas is 60ml/min, illumination in gas flow
Intensity is 3000~3500Lux, under conditions of 25~28 DEG C of temperature, is cultivated 4~6 days；
(2) it takes in step (1) in the CO for being 10% with volumetric concentration2Algae solution after being cultivated 4~6 days in gas, with 10~20%
In the liquid B G-11 culture medium that inoculum concentration is transferred new, then the culture medium after inoculation is placed in photosynthetic reactor, then to light
It closes in reactor and is passed through CO2The coal-fired plant flue gas that volumetric concentration is 12~15% is that 60ml/min, illumination are strong in gas flow
It under conditions of degree is 3000~3500Lux, temperature is 25~28 DEG C, continuously cultivated for 5~10 generations, that is, obtains in coal-burning power plant's cigarette
The target algae strain of fast-growth under the conditions of gas；
(3) the target algae strain of logarithmic growth phase, is inoculated into 400ml liquid B G-11 culture medium with 10~20% inoculum concentration,
Then the culture medium after inoculation is placed in photosynthetic reactor, then is passed through CO into photosynthetic reactor2Volumetric concentration is 12~15%
Coal-fired plant flue gas promote algae strain growth, gas flow is 60ml/min, 3000~3500Lux of intensity of illumination, temperature are
Under conditions of 25~28 DEG C, training objective algae strain 7~10 days, growth rate can be calculated by measurement frustule number；
(4) it by the target algae strain after step (3) culture, is placed under high 7500~10000Lux of intensity of illumination and continues to cultivate,
And it is continually fed into CO2The coal-fired plant flue gas stress that volumetric concentration is 12~15% improves the content astaxanthin in algae strain, cultivates item
Part are as follows: gas flow 120ml/min, cultivation temperature are 25~28 DEG C, after culture 10~15 days, algae solution are centrifuged and are freezed
To get haematococcus pluvialis dry algae powder is arrived after drying, it is capable of measuring the biomass dry weight and content astaxanthin of evaluation haematococcus pluvialis powder；
Wherein, the photosynthetic reactor uses volume for the cylinder photosynthetic reactor of 600ml.
2. flue gas CO according to claim 12The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation, it is special
Sign is, the liquid B G-11 culture medium at being grouped as are as follows: 1.5g/L NaNO3、0.2g/L Na2CO3、0.075g/L
MgSO4·7H2O、0.4g/L K2HPO4、0.036g/L CaCl2·2H2O、2.2×10-4g/L ZnSO4·7H2O、1.8×10- 3g/L MnCl2·4H2O、2.1×10-5g/L NaMoO4、0.8×10-5g/L CuSO4·5H2O、2.8×10-3g/L H3BO3、
0.001g/L EDTA, 0.006g/L citric acid.
3. flue gas CO according to claim 12The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation, it is special
Sign is, in the step (3), the measure and calculation method of growth rate are as follows:
Every taking 2mL algae solution for 24 hours, the Shandong 0.2mL brother's reagent is added into algae solution, concussion shakes up, and film-making is seen under an optical microscope
It examines, is counted using blood counting chamber, calculate frustule number N, recycle formula μ=(lnN2-ln N1) /(t2-t1) calculate carefully
Born of the same parents' average growth rate, as growth rate；
Wherein, μ is cell average growth rate；N1For t1It when frustule number, N2For t2It when frustule number.
4. flue gas CO according to claim 12The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation, it is special
Sign is, in the step (4), the measuring method of biomass dry weight M are as follows:
Take 10mL algae solution with 8000rmp centrifugation 10 minutes, remove supernatant, the algal gel obtained with distilled water flushing twice, then by algae
Mud centrifugal dehydration is placed on freezed 24 hours at -70 DEG C after, be put into low-temperature vacuum drying instrument it is 24 hours dry, to gained algae
Powder weighs and calculates biomass dry weight M.
5. flue gas CO according to claim 12The method that domestication promotes haematococcus pluvialis growing and astaxanthin accumulation, it is special
Sign is, in the step (4), the measuring method of content astaxanthin uses high performance liquid chromatography.
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| 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 |
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| 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 |
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