CN106467896A - A kind of kelvin of the high PH of tolerance intends chlorella and its culture application - Google Patents
A kind of kelvin of the high PH of tolerance intends chlorella and its culture application Download PDFInfo
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Abstract
The invention discloses a kind of kelvin plan chlorella of the high PH of tolerance and its culture application, Chlorella mutant strain FSH-Y3 is intended by the kelvin of ultraviolet mutagenesis and the method acquisition high PH of tolerance improving PH acclimation and screening step by step, its Classification And Nomenclature isParachlorella kessleri, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 26th, 2014, deposit number is CGMCC No.9238.The kelvin of selection-breeding of the present invention is intended chlorella FSH-Y3 and can preferably be absorbed carbon dioxide under high pH value, fast-growth is bred, solve existing kelvin plan chlorella to grow under neutral PH, and under neutral pH condition, there is a problem of that little, the fixing carbon dioxide efficiency of Solubilities of Carbon Dioxide is low.
Description
Technical field
The invention belongs to field of microorganism engineering is and in particular to a kind of kelvin of the high PH of tolerance intends chlorella and its culture application.
Background technology
With the development of World Economics, using and consuming of substantial amounts of fossil energy, lead to the shortage of the energy and going from bad to worse of environment, particularly CO2Sharply increase that the greenhouse effect causing is increasingly severe, the growth cycle of microalgae is short, photosynthetic efficiency is high, CO2Fixed efficiency is high, up to more than 10 times of terrestrial plant under certain condition, not only can reduce CO2Discharge, also reduces toxigenic capacity simultaneously；Except CO2Outward, the compositions such as some SOx, NOx in waste gas are cleaned process also with the metabolism of microalgae, effectively reduce noxious gas emission, be the regenerative resource most possibly meeting fuel needed for world's transport at present hence with microalgae grease as the biodiesel of raw material production.
More for the research of the oil-producing microalgaes such as chlorella, scenedesmus at present.CN20110144545.6 discloses one plant of Scenedesmus algal strain, and the growth of this algae strain can be characterized in that lipid-producing is higher than great majority point algae strain at present, this algae strain application includes CO using synthetic medium or the growth of appropriately processed waste water2Fixation, useless water purification, oils and fatss, protein, pigment, starch, polysaccharide, the production of nucleic acid.CN20120154470.4 disclose one plant of rich oil marine microalgae micro- intend ball algae (Nannochloropsis
gaditana) algae strain and its application, this algae strain can in the environment of pH=4.5 normal growth, its fat content is up to 35%.CN20111019480.X discloses one plant of microalgae algae strain（Mychonases sp .）And its for producing the application of biodiesel, the polyunsaturated fatty acid of high added value can be produced using this algae strain, including linolenic acid C18:3 and nervonic acid C24:1, it obtains the side-product of high added value while obtaining biodiesel.CN102703326A discloses a kind of high CO2The microalgae of toleration and fixed rate and its selection, but the algae strain that provided of this patent is not directed to the fat content of this algae strain.Or above-mentioned patent is unable to efficient utilization CO2Produce oils and fatss, or fat content is not high enough in the biomass obtaining.Particularly in actual applications, as CO in environment2When volume fraction is more than 5v%, the growth of most of microalgae will be suppressed, and carbon sequestration efficiency is low；General microalgae suitable growth in neutral conditions, is unfavorable for the growth of microalgae under conditions of slant acidity or meta-alkalescence, and microalgae utilizes CO simultaneously2It is usually the HCO to be dissolved in water3 -Ionic speciess exist, and carbon dioxide dissolubility under neutral environment is low, is unfavorable for that algae absorbs.
Intending Chlorella is by Krienitz,et al. a new genus of proposition, he is by the small subunit ribosome group of kelvin chlorella section to Chlorella（SSU rDNA）Carry out Phylogenetic Analysis, propose the kelvin chlorella in Chlorella（C. kessleri）It is divided into Chlorella, is renamed as kelvin and intends chlorella, with plan chlorella（P. beijerinckii）, Hu Si intend chlorella（P.hussii）Together constitute a new genus and intend Chlorella.The form intended between Chlorella and Chlorella is very much like, only has nuance in terms of the ultrastructure of cell wall, and compared with Chlorella, the electron-lucent degree that kelvin intends chlorella cells wall is relatively low.But obvious difference on 18S rDNA and ITS sequence.
Report with regard to kelvin plan chlorella is fewer both at home and abroad at present, and Jiang Lili et al. exists（The screening of one plant of oil-producing microalgae and Molecular Identification, aquatile journal, 606-612 page of the 4th phase in 2013）One plant of oil-producing microalgae HY-6 of report, is spherical unicellular, has 1 obvious pyrenoids, cup-shaped chromatoplast Zhousheng, thus tentatively judging that this algae strain may belong to Chlorella（Chlorella）Or plan Chlorella（Parachlorella）；18S rDNA and ITS Phylogenetic Analysis show that HY-6 is divided into two different evolution branch from Chlorella, but intend chlorella with kelvin（Parachlorella kessleri）Sibship nearer, and there is higher supporting rate of bootstrapping, be therefore accredited as kelvin and intend chlorella（P.kessleri）, total fat unit volume yield is up to 50.8mg/ (L.d), is one plant of potential oil-producing microalgae of tool.But, this kelvin is intended chlorella and is only suitable for growth under neutral PH, is not directed to the ability that this kelvin chlorella tolerates high PH.
Content of the invention
It is an object of the invention to provide a kind of kelvin of the high PH of tolerance intends chlorella and its culture application.The kelvin of selection-breeding of the present invention is intended chlorella and can preferably be absorbed carbon dioxide under high pH value, fast-growth is bred, solve existing kelvin plan chlorella to grow under neutral PH, and under neutral pH condition, there is a problem of that little, the fixing carbon dioxide efficiency of Solubilities of Carbon Dioxide is low.
The kelvin that the present invention tolerates high PH intends chlorella, is that kelvin intends Chlorella mutant strain FSH-Y3, its Classification And Nomenclature isParachlorella
kessleri, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 26th, 2014, deposit number is CGMCC No. 9238.
The kelvin that the present invention provides intends chlorella FSH-Y3, and frustule is spherical, oval under the microscope, inside has the chromatoplast of a Zhousheng, cup-shaped or lamellar；Asexual propagation, each cell can produce 2,4,8 or 16 autospores, blast cell rupture when ripe, and spore escapes, and is new individual after growing up.
The pH value that the kelvin plan chlorella FSH-Y3 that the present invention provides can tolerate is 10～12, and the growth rate under the conditions of this pH value intends fast 2～5 times of chlorella than original kelvin.
The 18S rDNA gene sequencing analysis result that the kelvin that the present invention provides intends chlorella FSH-Y3 algae strain is shown in sequence table.According to sequence alignment, FSH-Y3 algae strain of the present invention is had differences with the 18S rDNA data of the plan chlorella algae strain of announcement.
The kelvin that the present invention provides intends chlorella FSH-Y3, can increase the dissolubility of carbon dioxide when high PH grows, and increases and intends the fixation to carbon dioxide in chlorella photosynthesis, and the reduction of discharging to carbon dioxide has remarkable effect.Kelvin intends the growth needs absorbing carbon dioxide of chlorella, and in general, the dissolubility of carbon dioxide and the Acidity of Aikalinity of solution have certain relation, and carbon dioxide dissolubility in alkaline solution significantly raises.Therefore, obtained kelvin intends the environment that chlorella FSH-Y3 can tolerate high pH value, and can dissolve more carbon dioxide under high pH value environment, improves the fixed efficiency to carbon dioxide for the algae strain, significant to the greenhouse effect controlling carbon dioxide pollution.
The present invention tolerates the selection of the kelvin plan chlorella of high PH, including herein below：
（1）Prepare the BG11 culture medium that kelvin intends chlorella growth；
（2）Inoculate original kelvin and intend chlorella, through illumination and dark cycle induction, chlorella is intended to original kelvin and activates；
（3）Take the logarithm trophophase kelvin intend chlorella carry out ultraviolet mutagenesis, then carry out light culture；
（4）Algae solution after light culture is carried out illumination cultivation with being applied on solid medium after fresh medium dilution；
（5）Rapidly single algae falls to being inoculated into carry out in BG11 culture fluid primary dcreening operation for picking growth, and the PH of culture fluid is 8.0～9.0；
（6）By step（5）The algae solution of primary dcreening operation, is inoculated in the culture fluid improving PH step by step and carries out domestication culture, and each PH improves 0.5～1.0, and screening obtains the algae strain tolerating high PH.
In step（1）In, the BG11 preparing culture medium is divided in the conical flask of 50～250mL, often bottled liquid measure is 20～100mL, with ventilated membrane sealing, wraps kraft paper.Above-mentioned culture medium is sterilized, in 121 DEG C of temperature, is sterilized 20～30 minutes under pressure 0.1MPa.
In step（2）In, the volume ratio by inoculation liquid and culture fluid is 1:10～1:5 inoculations, operation is carried out at aseptic, shakes up culture fluid, wrapped with ventilated membrane after inoculation.Described illumination and dark cycle induction are to carry out in 22～32 DEG C of constant temperature illumination reaction shaking table, and brightness reaction time is 24h, and the brightness time, ratio was for 14:10；Shaking speed is 80～160rpm, cultivates 4～7 days, and the original kelvin obtaining activation intends chlorella algae solution.
In step（3）In, the algae solution 5～10mL of trophophase of taking the logarithm is placed in sterile petri dish and carries out ultraviolet mutagenesis, and algae solution spreads a thin layer in culture dish bottom surface, and culture dish is placed in aseptic darkroom, opens ware lid.Under magnetic stirring, with the ultra violet lamp mutation of 30w, irradiation time is 10～20min, and uviol lamp is 15～25cm with the distance of culture dish, and irradiating fatality rate is 60%～80%.Cover ware lid after irradiation, carry out light culture with the good culture dish of lighttight paper bag, the cultivation temperature of dark reaction is 20～30 DEG C, and incubation time is 24～72h.
In step（4）In, by step（3）Algae solution after dark reaction dilutes 10～10000 times with BG11 fresh culture, draws 0.1～0.3mL and is applied to BG11 solid agar flat board（Agar content is 1%～2%）On.Illumination box is cultivated, cultivation temperature is 20～30 DEG C, intensity of illumination is 1000～5000Lux, brightness reaction time is 24h, the brightness time, ratio was for 14:10, incubation time is 10～15d.Select grow on flat board than larger, color is deep, rapidly single algae drops into row screening for growth.
In step（5）In, cultivate in constant temperature illumination shaking table, shaking speed is 60～180rpm, intensity of illumination is 1000～5000Lux, brightness reaction time is 24h, cultivation temperature is 20～30 DEG C, the brightness time, ratio was for 14:10, incubation time is 10～15d days.After culture terminates, measure the OD of algae solution680Value, due to having good linear relationship with biomass cells dry weight, according to OD680The screening to carry out algae kind for the size, select the algae strain that OD value is than the larger high PH of tolerance, carry out acclimation and screening.
In step（6）In, with 1:10～1:5 ratio is by step（5）In the algae that selects fall to being inoculated in culture medium.Carry out domestication culture by the way of improving pH value step by step, initial p H is 8.0～9.0, and each PH improves 0.5～1.0, often improves and once cultivates 24～96h under the same conditions, select the algae solution that OD value is than the larger high PH of tolerance, obtain the algae strain that tolerance PH is 10～12.
The kelvin of the present invention intends chlorella in fixing CO2In application.This algae strain is resistant to the up to CO of 40v%2Concentration, has higher CO2Fixed efficiency.
The kelvin of the present invention intends application in producing microalgae grease for the chlorella.This algae strain PH be 10～12 growth conditionss under, cell total lipid content can account for more than the 40% of dry cell weight, can carry out the production of biodiesel.
The kelvin of the present invention is intended chlorella and is produced microalgae grease fixing CO simultaneously under high PH2In application.This algae strain can be fixed carbon dioxide under the growth conditionss that PH is 10～12 and carry out the illumination autophyting growth rich grease-contained biomass of acquisition, this algae strain can utilize the fresh water culture medium culturing such as BG11, SE, intensity of illumination 1500～the 20000Lux of culture, temperature is 15～40 DEG C, initial p H of culture fluid is 8.0～9.0, and each PH improves 0.5～1.0, until improving to 10～12, culture enters stable phase in 7 days, harvests frustule.After testing, cell total lipid content accounts for more than the 40% of dry cell weight.
Compared with existing algae strain, the present invention can bring following beneficial effect：
1st, ultraviolet mutagenesis and the method selection-breeding acquisition kelvin plan chlorella improving PH acclimation and screening step by step are passed through, the kelvin that acquisition tolerates high PH intends Chlorella mutant strain.Compared with original algae strain, original algae strain poor growth when PH reaches more than 10, or it is even dead to stop growing, and the kelvin of selection-breeding of the present invention intends PH that chlorella is resistant to up to 10～12, is that growth rate improves 2～5 times under conditions of 10～12 in pH value.
2nd, the kelvin of the present invention is intended chlorella and can be increased the dissolubility of carbon dioxide under high pH value, increase the fixation to carbon dioxide in photosynthesis, preferably absorb carbon dioxide, reduction of discharging to carbon dioxide has remarkable effect, solve existing kelvin plan chlorella to grow under neutral PH, and under neutral pH condition, there is a problem of that little, the fixing carbon dioxide efficiency of Solubilities of Carbon Dioxide is low.Meanwhile, when high pH value culture kelvin intends chlorella, kelvin can be effectively suppressed to intend the growth of miscellaneous bacteria during chlorella growth.
3rd, the inventive method acclimation and screening by the way of improving pH value step by step tolerates the algae kind of high PH, avoid and once improve the damage that PH screening brings algae kind, cause the failure of mutagenesis screening it is ensured that the reliability tamed step by step and the hereditary stability that improve mutation algae strain.With OD680Value as the measurement index of Biomass and growth rate, has easily feature directly perceived it is ensured that higher within a short period of time screening amount.
Biomaterial preservation explanation
The kelvin that the present invention provides intends Chlorella mutant strain（Parachlorella
kessleri）FSH-Y3, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；Deposit number：CGMCC
No. 9238；Preservation date：On May 26th, 2014.
Specific embodiment
Below by embodiment, the present invention is described in further detail.In the present invention, v% is volume fraction.
Embodiment 1 kelvin is intended Chlorella mutant strain FSH-Y3 and is separated and screen
It is that the wild kelvin separated by way of plate streaking from the water sample that Fushun City of Liaoning Province Hun River gathers in November, 2011 intends chlorella that the original kelvin of the present invention intends chlorella algae strain, and numbering is FSH-3.Original kelvin plan chlorella algae strain FSH-3 is carried out with ultraviolet mutagenesis and domestication culture selection-breeding tolerates kelvin plan Chlorella mutant strain FSH-Y3 of high PH.
The acquisition pattern that kelvin of the present invention intends Chlorella mutant strain FSH-Y3 is specific as follows：
（1）Prepare BG11 culture medium, the BG11 preparing culture medium is divided in 250mL shaking flask, often bottled liquid measure is 100mL, with ventilated membrane sealing, kraft paper is wrapped, and is placed in 121 DEG C, sterilizes 30 minutes under 0.1MPa.Described BG11 culture medium forms as shown in table 1, table 2.
Table 1 BG11 culture medium
* in table 2 table 1 A5+Co solution composition
（2）Aseptically, draw original kelvin plan chlorella algae solution FSH-3 about 10mL to be inoculated in the shaking flask after above-mentioned sterilizing, shaking flask is placed in culture in constant temperature illumination shaking table.The rotating speed of shaking table is set to 120rpm, and temperature is 25 DEG C, and setting brightness cultivation cycle is 24h, and light dark reaction time ratio is for 14:10, intensity of illumination is 5000Lux.
（3）After illumination shaking table is cultivated 5 days, algae solution enters exponential phase, now draws algae solution 10mL in the culture dish after sterilizing, makes algae solution in culture dish bottom tiling a thin layer, it is placed under magnetic stirring and be irradiated under uviol lamp, uviol lamp power is 30w, and irradiation distance is 15cm, irradiation time 15min, ware lid is covered after having irradiated, good with lighttight paper bag, light culture 2 days at 25 DEG C, fatality rate is 75%.
（4）The algae solution that dark reaction is cultivated dilutes 10000 times with fresh medium, draws 0.2ml and is applied to the BG11 agar solid plate that PH is 11（Agar content is 1.5%）On cultivated, cultivation temperature is 25 DEG C, and intensity of illumination is 5000Lux, and culture is carried out in illumination box, and brightness reaction time is 24h, and the brightness time, ratio was for 14:10, incubation time 15 days.
（5）Select the algae that flat board upper volume is larger, color is greener drop into row screening, its single algae is fallen be inoculated in equipped with volume of culture be 20mL, pH value be 9 BG11 culture medium 50mL shaking flask in cultivated.Cultivation temperature is 25 DEG C, and shaking speed is 120rpm, and intensity of illumination is 5000Lux, and light dark period is 24h, and the brightness time, ratio was for 14:10, after cultivating 10 days, measure the OD of algae solution in shaking flask680Value, according to OD680Size, select the algae strain that OD value is than larger tolerance PH9 and carry out acclimation and screening.
（6）By the algae filtering out strain by 1:10 ratio accesses and carries out domestication culture in the shaking flask of the 250mL of BG11 culture fluid that the PH containing 100mL is 10, and cultivation temperature is 25 DEG C, and shaking speed is 120rpm, and intensity of illumination is 3000Lux, and light dark period is 24h, and the brightness time, ratio was for 14:10, after cultivating 3 days, reject yellowish algae solution in the dust, measure the OD of algae solution in remaining shaking flask680Value, according to OD680Size, select the algae strain that OD value is than larger tolerance PH10 and tame further.PH is improved to 10.5,11.0,11.5, tames further under above-mentioned the same terms, select OD value than larger algae strain, this algae strain tolerates the target algae strain FSH-Y3 of high PH.
The comparison of the original algae strain of embodiment 2 and mutation algae strain growth rate under different PH
The algae solution of the algae solution of the exponential phase of the algae strain FSH-Y3 of screening and the exponential phase of original algae kind FSH-3 is accessed in the shaking flask equipped with BG11 culture medium according to a certain percentage, initial OD after inoculation680Value is identical, is 0.2.It is passed through CO from reactor bottom2Content is the gas of 5v%, cultivates 7 days under different pH values, measures OD680, as the index weighing the speed of growth, the result of gained is as shown in table 3 for value.
OD value under different PH compares the original algae strain of table 3 with mutation algae strain
From table 3, mutation algae strain is inconspicuous with the rising OD value changes of pH value, and the rapid decline of original algae strain OD value, poor growth, in addition dead.
The comparison of the growth result of embodiment 3 FSH-Y3 and FSH-3
The algae solution of FSH-Y3 and FSH-3 of exponential phase is seeded in BG11 culture medium respectively cultivated, culture is in the homemade column reactor of laboratory（Internal diameter is 40mm, highly for 500mm）In carry out, postvaccinal density is adjusted to OD680For 0.2.According to test needs, assemble the CO of different content2Gas, is then passed through from reactor bottom.In incubation, intensity of illumination is 5000Lux, and cultivation temperature is 28 DEG C, and pH value controls 11, and periodicity of illumination is 24h, and the brightness time, ratio was for 14:10, incubation time is 7 days, and culture collects frustule after terminating, and vacuum lyophilization under the conditions of -60 DEG C measures algae dried bean noodles weight to constant weight, calculates yield of biomass.Result is as shown in table 4.
The different CO of table 42The effectiveness comparison of content
From the results, it was seen that the kelvin through acclimation and screening intends chlorella FSH-Y3 than initial algae strain FSH-3 to CO2There are more preferable toleration, Biomass good stability with PH.
SEQUENCE
LISTING
<110>
Sinopec Group
China Petroleum and Chemical Corporation Fushun Petrochemical Research Institute
<120>
A kind of kelvin of the high PH of tolerance intends chlorella and its culture application
<130>
New patent application
<170> PatentIn version 3.5
<211> 1421
<212> DNA
<213>Parachlorella kessleri
<400> 1
gtagtcatat gcttgtctca
aagattaagc catgcatgtc taagtataaa ctgctttata
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ctgtgaaact gcgaatggct
cattaaatca gttatagttt atttgatggt accttactac
120
cggataaccg tagtaattct
agagctaata cgtgcgtaaa tcccgacttc tggaagggac
180
gtatttatta gatttaaggc
cgacccggct ctgccggtcc cacggtgaat catgataact
240
tcacgaatcg catggccttg
cgccggcgat gtttcattca aatttctgcc ctatcaactt
300
tcgatggtag gatagaggcc
taccatggtg gtaacgggtg acggaggatt agggttcgat
360
tccggagagg gagcctgaga
aacggctacc acatccaagg aaggcagcag gcgcgcaaat 420
tacccaatcc tgacacaggg
aggtagtgac aataaataac aataccgggc cttttcaggt
480
ctggtaattg gaatgagtac
aatctaaacc ccttaacgag gatcaattgg agggcaagtc
540
tggtgccagc agccgcggta
attccagctc caatagcgta tatttaagtt gctgcagtta
600
aaaagctcgt agttggattt
cgggcggggc ctgccggtcc gccgtttcgg tgtgcactgg
660
ccgggcccgc cttgttgccg
gggacgggct cctgggcttc actgtccggg actcggagtc
720
ggcgctgtta ctttgagtaa
attagagtgt tcaaagcagg cctacgctct gaatacatta
780
gcatggaata acacgatagg
actctggcct atcctgttgg tctgtaggac cggagtaatg
840
attaagaggg acagtcgggg
gcattcgtat ttcgatgtca gaggtgaaat tcttggattt
900
tcgaaagacg aactactgcg
aaagcatttg ccaaggatgt tttcattaat caagaacgaa
960
agttgggggc tcgaagacga
ttagataccg tcctagtctc aaccataaac gatgccgact
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agggatcggc ggatgtttct
tcgatgactc cgccggcacc ttatgagaaa tcaaagtttt
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caccaggcgt ggagcctgcg
gcttaatttg actcaacacg ggaaaactta ccaggtccag
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acatagtgag gattgacaga
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cctcgggggc cggcagactt cttagaggga ctattggcga
1380
ctagccaatg gaagcatgag
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c
1421
Claims (10)
1. a kind of kelvin of the high PH of tolerance intends chlorella, is that kelvin intends Chlorella mutant strain FSH-Y3, its Classification And Nomenclature isParachlorella kessleri, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 26th, 2014, deposit number is CGMCC No. 9238.
2. according to described in claim 1 kelvin intend chlorella it is characterised in that：Frustule is spherical, oval under the microscope for this algae strain, inside has the chromatoplast of a Zhousheng, cup-shaped or lamellar；Asexual propagation, each cell can produce 2,4,8 or 16 autospores, blast cell rupture when ripe, and spore escapes, and is new individual after growing up.
3. according to described in claim 1 kelvin intend chlorella it is characterised in that：The pH value that this algae strain can tolerate is 10～12, and the growth rate under the conditions of this pH value intends fast 2～5 times of chlorella than original kelvin.
4. the kelvin of the high PH of tolerance described in claims 1 to 3 any claim intends the selection of chlorella it is characterised in that including herein below：
（1）Prepare the BG11 culture medium that kelvin intends chlorella growth；
（2）Inoculate original kelvin and intend chlorella, through illumination and dark cycle induction, chlorella is intended to original kelvin and activates；
（3）Take the logarithm trophophase kelvin intend chlorella carry out ultraviolet mutagenesis, then carry out light culture；
（4）Algae solution after light culture is carried out illumination cultivation with being applied on solid medium after fresh medium dilution；
（5）Rapidly single algae falls to being inoculated into carry out in BG11 culture fluid primary dcreening operation for picking growth, and the PH of culture fluid is 8.0～9.0；
（6）By step（5）The algae solution of primary dcreening operation, is inoculated in the culture fluid improving PH step by step and carries out domestication culture, and each PH improves 0.5～1.0, and screening obtains the algae strain tolerating high PH.
5. in accordance with the method for claim 4 it is characterised in that：In step（3）In, the algae solution 5～10mL of trophophase of taking the logarithm is placed in sterile petri dish and carries out ultraviolet mutagenesis；Under magnetic stirring, with the ultra violet lamp mutation of 30w, irradiation time is 10～20min, and uviol lamp is 15～25cm with the distance of culture dish, and irradiating fatality rate is 60%～80%；Cover ware lid after irradiation, carry out light culture with the good culture dish of lighttight paper bag, the cultivation temperature of dark reaction is 20～30 DEG C, and incubation time is 24～72h.
6. in accordance with the method for claim 4 it is characterised in that：In step（6）In, with 1:10～1:5 ratio is by step（5）In the algae that selects fall to being inoculated in culture medium, carry out domestication culture by the way of improving pH value step by step, initial p H is 8.0～9.0, and each PH improves 0.5～1.0, often improve and once cultivate 24～96h under the same conditions.
7. intend chlorella in fixing CO according to the kelvin described in claim 12In application.
8. intend chlorella according to the kelvin described in claim 1 and produce the application in microalgae grease.
9. intend chlorella according to the kelvin described in claim 1 and produce microalgae grease fixing CO simultaneously under high PH2In application.
10. according to the application described in claim 9 it is characterised in that：This algae strain PH be 10～12 growth conditionss under fix carbon dioxide and carry out illumination autophyting growth obtain rich grease-contained biomass, using the fresh water culture medium culturing such as BG11 or SE, intensity of illumination 1500～the 20000Lux of culture, temperature is 15～40 DEG C, initial p H of culture fluid is 8.0～9.0, and each PH improves 0.5～1.0, until improving to 10～12, culture enters stable phase in 7 days, harvests frustule.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541466A (en) * | 2017-08-02 | 2018-01-05 | 海南大学 | A kind of chlorella of resistance to strong light of salt tolerant high temperature resistant for discharging aroma type smell and its application |
| CN109576315A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | A method of microalgae grease is produced using flue gas |
| CN109576314A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | A kind of method that mixed culture prepares microalgae grease |
| CN109576158A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | The grease-contained chlorella of one plant of richness and its culture application |
| CN109876642A (en) * | 2017-12-06 | 2019-06-14 | 中国石油化工股份有限公司 | A kind of method and device handling nitrogen-containing oxide flue gas |
| CN109880856A (en) * | 2017-12-06 | 2019-06-14 | 中国石油化工股份有限公司 | A kind of method of open production microalgae grease |
| CN109971647A (en) * | 2017-12-28 | 2019-07-05 | 深圳华大生命科学研究院 | Chlorella and application thereof and preparation method |
| CN114686392A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Normal-temperature preservation method of nitrifying bacteria |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412965A (en) * | 2008-11-17 | 2009-04-22 | 武汉钢铁(集团)公司 | Preparation of microalgae for stabilizing carbon dioxide |
| CN104611229A (en) * | 2014-12-22 | 2015-05-13 | 黄河三角洲京博化工研究院有限公司 | A microalgae strain having a high grease content and a preparing method thereof |
| CN104611227B (en) * | 2013-11-05 | 2017-05-17 | 中国石油化工股份有限公司 | Scenedesmus obliquus with tolerance to high pH and breeding method thereof |
-
2015
- 2015-08-17 CN CN201510503294.4A patent/CN106467896B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412965A (en) * | 2008-11-17 | 2009-04-22 | 武汉钢铁(集团)公司 | Preparation of microalgae for stabilizing carbon dioxide |
| CN104611227B (en) * | 2013-11-05 | 2017-05-17 | 中国石油化工股份有限公司 | Scenedesmus obliquus with tolerance to high pH and breeding method thereof |
| CN104611229A (en) * | 2014-12-22 | 2015-05-13 | 黄河三角洲京博化工研究院有限公司 | A microalgae strain having a high grease content and a preparing method thereof |
Non-Patent Citations (3)
| Title |
|---|
| DANA IVÁNOVÁ ET AL.: "DETERMINATION OF THE FUNCTIONAL GROUPS IN ALGAE PARACHLORELLA KESSLERI BY POTENTIOMETRIC TITRATIONS", 《NOVA BIOTECHNOLOGICA ET CHIMICA》 * |
| XIULING LI ET AL.: "The Microalga Parachlorella kessleri––A Novel Highly Efficient Lipid Producer", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 张虎等: "pH 对小球藻Chlorella sp. XQ-200419 光合作用、生长和产油的影响", 《水生生物学报》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541466A (en) * | 2017-08-02 | 2018-01-05 | 海南大学 | A kind of chlorella of resistance to strong light of salt tolerant high temperature resistant for discharging aroma type smell and its application |
| CN107541466B (en) * | 2017-08-02 | 2019-07-05 | 海南大学 | It is a kind of discharge aroma type smell the resistance to strong light of salt tolerant high temperature resistant chlorella and its application |
| CN109576158A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | The grease-contained chlorella of one plant of richness and its culture application |
| CN109576314A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | A kind of method that mixed culture prepares microalgae grease |
| CN109576315A (en) * | 2017-09-28 | 2019-04-05 | 中国石油化工股份有限公司 | A method of microalgae grease is produced using flue gas |
| CN109576315B (en) * | 2017-09-28 | 2021-12-31 | 中国石油化工股份有限公司 | Method for producing microalgae grease by using flue gas |
| CN109576158B (en) * | 2017-09-28 | 2022-02-08 | 中国石油化工股份有限公司 | Oil-rich chlorella and culture application thereof |
| CN109576314B (en) * | 2017-09-28 | 2022-03-04 | 中国石油化工股份有限公司 | Method for preparing microalgae grease through mixed culture |
| CN109876642A (en) * | 2017-12-06 | 2019-06-14 | 中国石油化工股份有限公司 | A kind of method and device handling nitrogen-containing oxide flue gas |
| CN109880856A (en) * | 2017-12-06 | 2019-06-14 | 中国石油化工股份有限公司 | A kind of method of open production microalgae grease |
| CN109880856B (en) * | 2017-12-06 | 2022-09-09 | 中国石油化工股份有限公司 | Open type microalgae grease production method |
| CN109971647A (en) * | 2017-12-28 | 2019-07-05 | 深圳华大生命科学研究院 | Chlorella and application thereof and preparation method |
| CN109971647B (en) * | 2017-12-28 | 2022-07-05 | 深圳华大生命科学研究院 | Chlorella and its use and preparation method |
| CN114686392A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | Normal-temperature preservation method of nitrifying bacteria |
| CN114686392B (en) * | 2020-12-31 | 2023-07-04 | 中国石油化工股份有限公司 | Normal temperature preservation method of nitrifying bacteria |
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