CN103184156A - Scenedesmus algal strain and its use - Google Patents

Scenedesmus algal strain and its use Download PDF

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CN103184156A
CN103184156A CN2011104487183A CN201110448718A CN103184156A CN 103184156 A CN103184156 A CN 103184156A CN 2011104487183 A CN2011104487183 A CN 2011104487183A CN 201110448718 A CN201110448718 A CN 201110448718A CN 103184156 A CN103184156 A CN 103184156A
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algae
strain
enn2201a
grid
feed
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CN103184156B (en
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邓平
罗少敬
陈传红
吴洪
马建源
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ENN Science and Technology Development Co Ltd
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ENN Science and Technology Development Co Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

Belonging to the fields of microorganisms and biological energy, the invention relates to a scenedesmus algal strain and its use. Specifically, the invention relates to a scenedesmus algal strain ENN2201A (Desmodesmus sp.). With a preservation number of CGMCC No.5150, the strain is preserved on August 17, 2011 in China General Microbiological Culture Collection Center. The scenedesmus algal strain provided in the invention can grow in a wide range of temperature, has a fast growth speed and is easy to collect, and can effectively generate fatty acids, carotenoids and other substances, thus having the potential of application in sewage treatment, biodiesel production, and feed or food industry, etc.

Description

A kind of grid algae algae strain and uses thereof
Technical field
The invention belongs to microorganism and bioenergy field, relate to a kind of grid algae algae strain and uses thereof.
Background technology
Along with a large amount of uses of fossil oils such as oil, Sweet natural gas, coal, global gas concentration lwevel sharply raises, and causes climate warming, the increase of acid rain region area, extreme weather phenomenon to take place frequently.Problems such as environmental pollution are serious day by day.And along with the lasting consumption of fossil oil, energy reserves also sharply reduces.Therefore the new forms of energy of seeking a kind of Sustainable development of green become the focus of various countries scientist research.
In clean reproducible energy, be subjected to people with the research of the little algae production of energy biofuel and pay close attention to widely.Biofuel character and fossil diesel fuel approach, and are one of the most potential large liquid biofuels.Little algae photosynthetic efficiency height absorbs a large amount of CO in the process of growth 2, can reduce CO effectively 2Discharging.There is algae slightly can utilize moiety in the waste stream, converts it into the biological substance of self, so can be effectively used to the processing of trade effluent, waste material.
The grid algae is planktonic algae common in the fresh water, and utmost point happiness is bred in nutritious hydrostatic.Wherein numerous species has stronger patience to organic pollutant.The grid algae has certain effect in self purification of water body and sewage purification, be the dominant species in the organic sewage oxidation pond biophase.It can be attached in the water on the organism fragment and other waterplant bodies simultaneously with bacterium, forms gelatinous layer, adsorb organic compound.When the grid algae carries out photosynthesis, produce oxygen on the one hand for the needs of bacterial degradation organic matter, also can directly utilize organicly as carbon source and nitrogenous source on the other hand, organism is degraded rapidly, thus purifying water body.The grid frustule contains rich in protein and VITAMIN, and the grid algae propagation is fast, is of high nutritive value, and can be used as artificial a large amount of material of cultivating.Abundant fatty acid can be accumulated in the part grid frustule, by manual operation, biofuel can be converted into.
Application number is to be separated to the new chlorella of a strain in 200780020794.6 the patent, and can Application Areas to a plurality of fields, comprise the processing of discarded carbonic acid gas in environment or the industrial production, production and the wastewater treatment of bioenergy.
Application number is to utilize self-separation grid algae in Treating Municipal Sewage in 201010111834 the patent, and coupling produce the device of diesel oil can be for the production of little algae raw material of biofuel.
However, still need and want new algae strain, its thermal adaptation scope is wide, in the winter time, all can carry out outdoor breeding summer; Fast growth and be easy to collect; And contain a certain amount of high added value product, can be applied to feed or food service industry, range of application is more extensive.
Summary of the invention
The inventor is through deep research and performing creative labour, obtained the strain of a kind of grid algae algae, and the inventor is surprised to find, this grid algae algae strain can be grown in wide temperature range (for example 9-43 ℃), fast growth and be easy to collect, and can effectively produce materials such as lipid acid, carotenoid, have the potentiality of using in sewage disposal, production of biodiesel and feed or food service industry etc.Following invention is provided thus:
One aspect of the present invention relates to a kind of grid algae algae strain ENN2201A (Desmodesmus sp.), its deposit number is CGMCC No.5150, preservation date is on August 17th, 2011, and depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center.
Described grid algae algae strain ENN2201A (Desmodesmus sp.), its nucleotide sequence comprises the nucleotide sequence shown in the SEQ ID NO:3.
TTAGCCATGCATGTCTAAGTATAAACTGCTTATACTGTGAAACTGCGAATGGCTC
ATTAAATCAGTTATAGTTTATTTGGTGGTACCTTCTTACTCGGAATAACCGTAAGAAAT
TTAGAGCTAATACGTGCGTAAATCCCGACTTCTGGAAGGGACGTATATATTAGATAAAA
GGCCGACCGGGCTCTGCCCGACCCGCGGTGAATCATGATATCTTCACGAAGCGCATGGC
CTTGTGCCGGCGCTGTTCCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAG
AGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGC
CTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGA
TACGGGGAGGTAGTGACAATAAATAACAATACCGGGCATTTCATGTCTGGTAATTGGAA
TGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAG
CCGCGGTA ATTCCAGCTCCA ATAGCGTATATTTA AGTTGTTGCAGTTAAAAAGCTCGTA
GTTGGATTTCGGGTGGGTTTCAGCGGTCCGCCTATGGTGAGTACTGCTGTGGCCTTCCT
TACTGTCGGGGACCTGCTTCTGGGCTTCATTGTCCGGGACAGGGATTCGGCATGGTTAC
TTTGAGTAAATTGGAGTGTTCAAAGCAGGCTTACGCCGTGAACATTTTAGCATGGAATA
ACATGATAGGACTCTGCCCTATTCTGTTGGCCTGTAGGAGTGGAGTAATGATTAAGAGG
AACAGTCGGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGA
CGAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGG
GGCTCGAAGACGATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGAT
TGGCGGACGTTTTTGCATGACTCCGTCAGCACCTTGAGAGAAATCAAAGTTTTTGGGTT
CCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCA
GGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACAT
AGGAAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG
TTCTTAGTTGGTGGGTTGTCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCT
TTAAATAGTCACTGTCGCTTTTTGCGGCTGGCTTTTGACTTCTTAGAGGGACAGTTGGC
GTTTAGTCAACGGAAGTATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGG
CCGCACGCGCGCTACACTGATGCATTCAACAAGCCTATCCCTAGCCGAAAGGCTCGGGT
AATCTTTGAAACTGCATCGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACGAG
GAATGCCTAGTAAGCGCAATTCATCAGATTGCGTTGATTACGTCCCTGCCCTTTGTACA
CACCGCCCGTCGCTCCTACCGATTGGGTGTGCTGGTGAAGTGTTCGGATTGGCAATTAT
CGGTGGCAACACCGTCGATTGCCGAGAAGTTCATTAAACCCTCCCACCTAGAGAAGGAG
AAGTCG (SEQ ID NO：3)
Described grid algae algae strain ENN2201A (Desmodesmus sp.), its nucleotide sequence comprises the nucleotide sequence shown in the SEQ ID NO:6.
TGGGTTGGAAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA
TCATTGAATATGCAAACCACAACACGCACTCTTTATTTGTGTTCGACGTTAGGTCAACA
CGCGCAAGCGTGTGGCCTACTAACCTACACACCATTGACCAACCATTTATCAAACCAAA
CTCTGAAGCTTTGGCTGCCGTTAACCGGCAGTTCTAACAAAGAACAACTCTCAACAACG
GATATCTTGGCTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGTGAATT
GCAGAATTCCGTGAACCATCGAATCTTTGAACGCATATTGCGCTCGACTCCTCGGAGAA
GAGCATGTCTGCCTCAGCGTCGGTTTACACCCTCACCCCTCTTCCTTAACAGGAGGCGC
CTGTCGTGCTTGCTTAAGCCGGCAGCAGGGGTGGATCTGGCTCTCCCAATCGGATTCAC
TCTGGTTGGGTTGGCTGAAGCACAGAGGCTTAAACTGGGACCCAATTCGGGCTCAACTG
GATAGGTAGCAACACCCTCGGGTGCCTACACGAAGTTGTGTCTGAGGACCTGGTTAGGA
GCCAAGCAGGAAACGCGTCTCTGGCGCGTACTCTGTATTCGACCTGAGCTCAGGCAAGG
CTACCCGCTGAACTTAAGCATATCAATAAGCGGGAGGAACCCCTTTT (SEQ ID NO：6)
The inventor is triumphant rivers water sampling in the Deyang City Zhongjiang County, adds the BG11 substratum of 1/3 volume, is 40 μ mol/m at 25 ℃, intensity of illumination 2.s cultivated about 5 days under the condition, get this water sample and observe in microscopically, judge the main species that exist in the water sample.Adjusting cell concn and be about 1000/ml, get 100 μ l water samples and coat on the solid agar plate of BG11 substratum, is 40 μ mol/m at 25 ℃, intensity of illumination 2.s cultivated about 10 days under the condition about, can see that single algae falls to growing, fall to being inoculated in the BG11 substratum with transfering loop or aseptic toothpick picking list algae and cultivate.Repeat this process, the algae strain up to obtaining pure culture is called " ENN2201A " to its numbering among the present invention.
Provided concrete separation screening process among the embodiment 1.
On form level and molecular level, the classification evaluation has been carried out in the algae strain that obtains among the embodiment 2, and analyzed and compare.According to the result of identification of morphology and Molecular Identification, determine that ENN2201A is grid algae section (Scenedesmaceae), chain belt Trentepohlia (Desmodesmus).
Another aspect of the present invention relates to the cultural method of described grid algae algae strain, it comprise in following (1)-(4) each or multinomial:
(1) described substratum is the algae substratum, for example BG11, BBM, CT, SE, HB111, HB 119 or MA;
(2) culture temperature is 9-43 ℃; Be preferably 15-40 ℃; More preferably 25-30 ℃;
(3) pH is 7-9;
(4) illumination is 200-1500 μ mol/m 2/ s.
About culture temperature, best culture temperature is 25-30 ℃, is equaling to be higher than 9 ℃, also can grow during still less than 15 ℃, and the speed of growth can be subjected to certain influence.Be higher than 40 ℃ and less than 43 ℃, continue can grow under the 2-4h, see embodiment 8, influenced but the speed of growth is understood.
Grid algae algae strain of the present invention (ENN2201A) has wide range of applications, and includes but not limited to sewage disposal, production of biodiesel and feed or food service industry etc.
Another aspect of the present invention relates to the purposes of described grid algae algae strain in handling or purifying waste water; Particularly, described sewage is trade effluent or city domestic sewage, and described waste gas is industrial gaseous waste.
The data presentation of embodiment 8, grid algae algae of the present invention strain can purify waste water effectively particularly trade effluent, for example wastewater from chemical industry.
The purposes of described grid algae algae strain in preparation biofuel and/or grease that relate in one aspect to again of the present invention.
Term " biofuel " includes but not limited to: by animal-plant oil or its waste oil or by fatty acid methyl ester and/or the fatty-acid ethyl ester of algae oil and fat preparation; Perhaps by animal-plant oil or its waste oil or by the lipid acid mono alkyl ester of algae oil and fat preparation.
The data presentation of embodiment 4, grid algae algae of the present invention strain can generate lipid acid effectively.
The purposes of described grid algae algae strain in the preparation feed that relate in one aspect to again of the present invention; Particularly, described feed is that the described feed of fishery products feed, animal and fowl fodder or feed for pet can all or part ofly be made of grid algae algae of the present invention strain.Grid algae algae of the present invention strain can also can be used as the additive preparation feed separately as feed.Can directly use frond, namely the algae powder adds in the feed, also can add behind the broken wall.The preparation method of feed can reference, for example: Zhou Anguo chief editor, feed handbook, agriculture press .2002; Qi Guanghai etc. write, feed-formulating technique handbook, Chinese agriculture press, 2000; Han Changri, Wu Weixiong, the fodder additives manufacturing technology, the 2011-02 of scientific and technical literature press, etc.
The purposes of described grid algae algae strain in preparation carotenoid that relate in one aspect to again of the present invention; Particularly, described carotenoid is selected from any one or more in zeaxanthin, canthaxanthin and the β-Hu Luobusu.
The data presentation of embodiment 7, grid algae algae of the present invention strain can generate carotenoid effectively.
The present invention also provides following method.
Of the present invention relating in one aspect to again a kind ofly handled or purified waste water or the method for waste gas, comprises the step of using grid algae algae of the present invention strain; Particularly, described sewage is trade effluent or city domestic sewage, and described waste gas is industrial gaseous waste.
Of the present inventionly relate in one aspect to a kind of method for preparing biofuel and/or grease again, comprise the step of using grid algae algae of the present invention strain.
Of the present inventionly relate in one aspect to a kind of method for preparing feed again, comprise the step of using grid algae algae of the present invention strain.
Of the present inventionly relate in one aspect to a kind of method for preparing carotenoid again, comprise the step of using grid algae algae of the present invention strain; Particularly, described carotenoid is selected from any one or more in zeaxanthin, canthaxanthin and the β-Hu Luobusu.
The beneficial effect of the invention
1) this algae kind comfort zone is wide, and culturing at low temperatures also has preferable performance, can effectively increase the annual fate of culturing.
2) this algae kind settling property is good, can effectively reduce the cost that frond is collected, and has higher commercial application prospect.
3) this algae kind fast growth, oil-containing and carotenoid have many-sided commercial application effect.
Description of drawings
Fig. 1: ENN2201A microscopically form.Line segment among figure A and the B represents 10 microns.
Fig. 2: ENN2201A algae 18S sequence B last result.
Fig. 3: the ENN2201A evolutionary tree is analyzed (18S).
Fig. 4: ENN2201A algae ITS sequence B last result.
Fig. 5: the ENN2201A evolutionary tree is analyzed (ITS).
Fig. 6: ENN2201A is growth result in triangular flask.
Fig. 7: ENN2201A, No. 1 algae strain, the fatty acid content of No. 2 algae strains and the evaluation result of dry weight.
Fig. 8: ENN2201A is growth result under low temperature environment.Growth velocity calculation formula: v=(m1-m2)/(t1-t2) m: algae liquid dry weight, t: incubation time.Annotate: during inoculation, three algae strain starting point concentrations were respectively 1: 0.914,2: 0.812,1.288, and gap is not very big, can special great influence not arranged to subsequent growth.
Fig. 9: the ENN2201A algae kind that will be in logarithmic phase is inoculated in the plate-type reactor of 50 * 3 * 50cm, with the BG11 culture medium culturing.And, inoculate the two strains grid algae algae strain faster of growing with the same terms and compare.
Figure 10: ENN2201A growth each substances content in the carotenoid in the time of 16 days.Account for the dry weight ratio: a left side for zeaxanthin, in for canthaxanthin, right be β-Hu Luobusu.
The growing state of Figure 11: ENN2201A in tap water and waste water.
Biomaterial preservation explanation:
Grid algae algae strain ENN2201A (the classification called after Desmodesmus sp. of suggestion), it is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 17th, 2011, preserving number is CGMCC No.5150, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Embodiment
Be described in detail below in conjunction with the embodiment of the present invention of embodiment.It will be understood to those of skill in the art that the following examples only are used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment, according to the described technology of the document in this area or condition (for example with reference to works such as J. Sa nurse Brookers, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: the strain of screening produce oil grid algae algae
The triumphant rivers water sampling in the Deyang City Zhongjiang County adds the BG11 substratum of 1/3 volume, is 40 μ mol/m at 25 ℃, intensity of illumination 2.s cultivated about 5 days under the condition, get this water sample and observe in microscopically, judge the main species that exist in the water sample.Adjusting cell concn and be about 1000/ml, get 100 μ l water samples and coat on the solid agar plate of BG11 substratum, is 40 μ mol/m at 25 ℃, intensity of illumination 2.s cultivated about 10 days under the condition about, can see that single algae falls to growing, fall to being inoculated in the BG11 substratum with transfering loop or aseptic toothpick picking list algae and cultivate.Repeat this process, up to the algae strain that obtains pure culture.The pure lines algae liquid that grows is cultivated in triangular flask, column reactor respectively, and results of regular determination dry weight and fat content are therefrom selected fast growth, algae strain that fat content is high.By this method, obtain an algae strain called after ENN2201A.
The used BG11 substratum of the present invention is all shown in following table 1.
Table 1:BG11 culture medium prescription
NaNO 3 1.5g/l
K 2HPO 4·3H 2O 0.04g/l
MgSO 4·7H 2O 0.075g/l
CaCl 2·2H 2O 0.036g/l
Citric acid 0.006g/l
FeCl 3·6H 2O 0.00315g/l
Na 2EDTA·2H 2O 0.00436g/l
Na 2CO 3 0.02g/l
A5 Trace element* 1ml
* the moiety of A5 Trace element (being added in the 1000 ml deionized waters) is as follows:
Embodiment 2: identification of morphology
The above-mentioned algae strain ENN 2201A that separates is observed under 1000 power microscopes, the results are shown in Figure 1.Frond is unicellular or typing colony form exists, and unicellular spherical in shape or avette, typing colony is made up of 2-4 cell, colony's cell straight line arranged side by side becomes row, each cell two ends up and down is extensively round, and cell walls is level and smooth, and the two ends up and down of each cell of colony respectively have 1 bandy lunge.The long 8-14 μ of individual cells m, wide 4-6 μ m.Chromatoplast Zhousheng, one piece of tool pyrenoids.It is green that cell is generally, and the later stage reddens.Preliminary evaluation is the grid algae.
Embodiment 3: Molecular Identification 1 (18S)
1. genome extracts
The centrifugal 5min of 6,000rpm collects the ENN 2201A frustule of cultivating 4 days; The cell of getting after 200mg grinds adds 600 μ l CTAB damping fluids, homogenate; The phenol that adds 600 μ l again in the reaction system: chloroform (volume ratio 1: 1) extracting, the centrifugal 15min of 14,000rpm, get supernatant, add 0.6 times of volume isopropanol precipitating of supernatant, be dissolved in the 100 μ l sterilization distilled water its concentration of UV spectrophotometer measuring and purity after 75% washing with alcohol.
Adopt eukaryote 18S amplification universal primer (primer is synthetic synthetic by the living worker in Shanghai bio-engineering corporation) the little algae genome 18S gene fragment of amplification, primer sequence is as follows:
Primer 1:5 '-CCTGGTTGATCCTGCCAG-3 ' (SEQ ID NO:1)
Primer 2: 5 '-TTGATCCTTCTGCAGGTTCA-3 ' (SEQ ID NO:2)
Getting the total DNA of 1 μ i is template, and reaction conditions is as follows: 95 ℃ of pre-sex change 5min, and 94 ℃ of sex change 50s then, 56 ℃ of annealing 50s, 72 ℃ are extended 1min 30s, reacts 30 circulations, last 72 ℃ of extension 10min.1.0% agarose gel electrophoresis detects its amplified production.Obtain the 18S sequence of ENN2201A.Hereinafter listed 18S sequence is the gene order that order-checking obtains, shown in following SEQ ID NO:3:
TTAGCCATGCATGTCTAAGTATAAACTGCTTATACTGTGAAACTGCGAATGGCTC
ATTAAATCAGTTATAGTTTATTTGGTGGTACCTTCTTACTCGGAATAACCGTAAGAAAT
TTAGAGCTAATACGTGCGTAAATCCCGACTTCTGGAAGGGACGTATATATTAGATAAAA
GGCCGACCGGGCTCTGCCCGACCCGCGGTGAATCATGATATCTTCACGAAGCGCATGGC
CTTGTGCCGGCGCTGTTCCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGATAG
AGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGC
CTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGA
TACGGGGAGGTAGTGACAATAAATAACAATACCGGGCATTTCATGTCTGGTAATTGGAA
TGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAG
CCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTA
GTTGGATTTCGGGTGGGTTTCAGCGGTCCGCCTATGGTGAGTACTGCTGTGGCCTTCCT
TACTGTCGGGGACCTGCTTCTGGGCTTCATTGTCCGGGACAGGGATTCGGCATGGTTAC
TTTGAGTAAATTGGAGTGTTCAAAGCAGGCTTACGCCGTGAACATTTTAGCATGGAATA
ACATGATAGGACTCTGCCCTATTCTGTTGGCCTGTAGGAGTGGAGTAATGATTAAGAGG
AACAGTCGGGGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGA
CGAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGG
GGCTCGAAGACGATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGAT
TGGCGGACGTTTTTGCATGACTCCGTCAGCACCTTGAGAGAAATCAAAGTTTTTGGGTT
CCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCA
GGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACAT
AGGAAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCG
TTCTTAGTTGGTGGGTTGTCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCT
TTAAATAGTCACTGTCGCTTTTTGCGGCTGGCTTTTGACTTCTTAGAGGGACAGTTGGC
GTTTAGTCAACGGAAGTATGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGG
CCGCACGCGCGCTACACTGATGCATTCAACAAGCCTATCCCTAGCCGAAAGGCTCGGGT
AATCTTTGAAACTGCATCGTGATGGGGATAGATTATTGCAATTATTAGTCTTCAACGAG
GAATGCCTAGTAAGCGCAATTCATCAGATTGCGTTGATTACGTCCCTGCCCTTTGTACA
CACCGCCCGTCGCTCCTACCGATTGGGTGTGCTGGTGAAGTGTTCGGATTGGCAATTAT
CGGTGGCAACACCGTCGATTGCCGAGAAGTTCATTAAACCCTCCCACCTAGAGAAGGAG
AAGTCG (SEQ ID NO：3)
2. the nucleotide sequence homology search of cloning
The 18S sequence login GenBank database of the ENN2201A that amplification is obtained carries out the BLAST comparison, and the result shows to be with the highest two sequences of its matching degree and fraction of coverage and do not identify sequence.Come the 3rd be to be the Scenedesmuscommunis gene for 18S small subunit rRNA sequence of X73994.1 ACCESSION number, both matching degrees are 99%, fraction of coverage is 99% (Fig. 2).Mate 1699 pairs of bases (99%), have 1 gap (0%).
What come the 4th of comparison result is to be the Scenedesmus abundans gene for 18S small subunit rRNA sequence of X73995.1 ACCESSION number, and the matching degree of it and ENN2201A 18S sequence is 98%, and fraction of coverage is 99% (Fig. 2).Mate 1692 pairs of bases (99%), have 3 gap (0%).
3. evolutionary tree is analyzed
Utilize the BLAST instrument in the ncbi database that the 18S partial sequence of algae strain ENN 2201A is carried out the sequence similarity analysis.The 18S partial sequence of selected part homologous sequence and this algae strain is utilized clustalw2 software package (European Molecular Bioglogy Laboratory and European information biology institute, http://www.ebi.ac.uk/Tools/msa/clustalw2/) carries out sequence homology evolution comparison, form a multiple sequence coupling permutation matrix.Then, use Mega4.0 software (Tamura K etc., 2007), adopt continuous (Neighbor-joining) algorithm in ortho position, the data set bootstraps value of bootstrapping is that 1000 constructing systems are grown evolutionary tree.
According to 18S sequence B last result, choose 6 strains wherein, be respectively Desmodesmus communis (Scenedesmus communis), Scenedesmus abundans, Scenedesmaceae sp.MLO-4, Desmodesmus costato-granulatus (Scenedesmus costato-granulatus), Coccoid scenedesmid sp.Tow 9/21P-13w (Scenedesmaceae sp.Tow 9/21 P-13w) and Scenedesmus subspicatus strain UTEX 2532 (Desmodesmus subspicatus), add Chlamydomonas reinhardtii Chlamydomonas reinhardtii again, the 18S sequence of two chlorella Chlorella vulgaris strain CCAP 211/113 and Chlorella vulgaris strain KMMCC FC-16 makes up evolutionary tree (Fig. 3) jointly.
The evolutionary tree result shows that the sibship of ENN2201A and Scenedesmus subspicatus strain UTEX 2532 is nearest, with Desmodesmus communis, Scenedesmusabundans is in same branch, and other Scenedesmus algae is divided into another (Fig. 3).As chlamydomonas (chlamydomonas), chlorella (chlorella) is equidistant far away apart from other kinds.
Embodiment 4: Molecular Identification 2 (ITS)
1. genome extracts
The centrifugal 5min of 6,000rpm collects the ENN 2201A frustule of cultivating 4 days; The cell of getting after 200mg grinds adds 600 μ l CTAB damping fluids, homogenate; The phenol that adds 600 μ l again in the reaction system: chloroform (volume ratio 1: 1) extracting, the centrifugal 15min of 14,000rpm, get supernatant, add 0.6 times of volume isopropanol precipitating of supernatant, be dissolved in the 100 μ l sterilization distilled water its concentration of UV spectrophotometer measuring and purity after 75% washing with alcohol.
The ITS sequence amplification adopts eukaryote ITS amplification universal primer (primer is given birth to worker bio-engineering corporation by Shanghai and synthesized), and primer sequence is as follows:
Primer 3:5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' (SEQ ID NO:4)
Primer 4:5 '-GCATATCAATAAGCGGAGGA-3 ' (SEQ ID NO:5)
Getting the total DNA of 1 μ l is template; Amplification condition is as follows: 94 ℃ of sex change 5min, 94 ℃ of 40s then, 56 ℃ of 40s, 30 circulations of 72 ℃ of 2min, last 72 ℃ of 10min.1.0% agarose gel electrophoresis detects its amplified production, and pcr amplification obtains about 692bp segment.The SEQ ID NO:6 that obtains is the ITS partial sequence of ENN 2201A.Sequence is as shown in the SEQ ID NO:6:
TGGGTTGGAAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA
TCATTGAATATGCAAACCACAACACGCACTCTTTATTTGTGTTCGACGTTAGGTCAACA
CGCGCAAGCGTGTGGCCTACTAACCTACACACCATTGACCAACCATTTATCAAACCAAA
CTCTGAAGCTTTGGCTGCCGTTAACCGGCAGTTCTAACAAAGAACAACTCTCAACAACG
GATATCTTGGCTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGTGAATT
GCAGAATTCCGTGAACCATCGAATCTTTGAACGCATATTGCGCTCGACTCCTCGGAGAA
GAGCATGTCTGCCTCAGCGTCGGTTTACACCCTCACCCCTCTTCCTTAACAGGAGGCGC
CTGTCGTGCTTGCTTAAGCCGGCAGCAGGGGTGGATCTGGCTCTCCCAATCGGATTCAC
TCTGGTTGGGTTGGCTGAAGCACAGAGGCTTAAACTGGGACCCAATTCGGGCTCAACTG
GATAGGTAGCAACACCCTCGGGTGCCTACACGAAGTTGTGTCTGAGGACCTGGTTAGGA
GCCAAGCAGGAAACGCGTCTCTGGCGCGTACTCTGTATTCGACCTGAGCTCAGGCAAGG
CTACCCGCTGAACTTAAGCATATCAATAAGCGGGAGGAACCCCTTTT (SEQ ID NO：6)
2. the search of the nucleotide sequence of cloning (SEQ ID NO:6) homology:
The ITS partial sequence and the 5.8S sequence login GenBank database that obtain E are carried out the BLAST comparison, the result shows that with ACCESSION number be the internal transcribed spacer 1 of the desmodesmus armatus var. of DQ414547.1,5.8S ribosomal RNA gene and internal transcribed spacer 2 sequence have higher similarity and reach 97%, fraction of coverage is also up to 96% (Fig. 4).
3. evolutionary tree is analyzed
The results are shown in Figure 5.
Utilize the BLAST instrument in the ncbi database that the ITS partial sequence of algae strain ENN 2201A is carried out the sequence similarity analysis.The ITS partial sequence of selected part homologous sequence and this algae strain is utilized clustalw2 software package (European Molecular Bioglogy Laboratory and European information biology institute, http://www.ebi.ac.uk/Tools/msa/clustalw2/) carries out sequence homology evolution comparison, form a multiple sequence coupling permutation matrix.Then, use Mega4.0 software (Tamura K etc., 2007), adopt continuous (Neighbor-joining) algorithm in ortho position, the data set bootstraps value of bootstrapping is that 1000 constructing systems are grown evolutionary tree.
The sibship of ENN2201A and desmodesmus armatus var. is nearer as can be seen from Figure 5, is positioned in the same branch.Illustrate that ENN2201A should belong to the chain belt Trentepohlia.
According to the result of identification of morphology and Molecular Identification (18S and ITS), limiting ENN 2201A is grid algae section (Scenedesmaceae), chain belt Trentepohlia (Desmodesmus), and preliminary evaluation is Desmodesmus sp..
The triangular flask of embodiment 5:ENN 2201A algae is cultured
The algae kind that is cultured to logarithmic phase is transferred in the triangular flask (capacity is 100ml) that the aseptic BG11 substratum of 40ml is housed, places on the shaking table and cultivate.Initial inoculation OD750 is about 0.086; The about 35 μ molm-2s-1 of intensity of illumination, temperature is 25+1 ℃, rotating speed 100rpm/min, light source are fluorescent lamp.Its OD680 of periodic measurement and 750 changing conditions.The results are shown in Figure 6.
As seen from Figure 6, the ENN2201A well-grown, with bibliographical information (Ji Xiang etc., the screening of a strain scenedesmus obliquus and the optimization of growth conditions, aquatic science and technology information, 2010,37 (6), 265-268) compare, have the speed of growth faster.
Appraisal of growth and the oil and fat accumulation of embodiment 6:ENN 2201A algae
1) cultivation of grid algae algae strain ENN 2201A separates with collecting
The grid algae algae strain ENN 2201A green cell that will be in logarithmic phase is seeded in the BG11 substratum (prescription sees Table 1) for preparing by 20% inoculative proportion, uses the column reactor of 40mm internal diameter, 600mm length to carry out both culturing microalgae.Intensity of illumination control is at 150-600 μ mol/m in the culturing process 2.s in the scope.In incubation period, by the mixed gas (containing the carbonic acid gas that volume fraction is 1.5-2%) of bubbling air and carbonic acid gas in nutrient solution, with the pH value that carbon source is provided and regulates and control substratum between 7-9.Temperature adjusting is in 25-35 ℃ of scope.In the culturing process, timing sampling is measured dry weight, the results are shown in Figure 7.Cultivation proceeds to the 17th day, when algae liquid color reddens, algae liquid is collected, and the method acquisition algae mud by centrifugal or natural subsidence is carrying out vacuum lyophilization with algae mud.Other algae strains of grid algae (numbering is respectively No. 1, No. 2) of selecting this laboratory self-separation to obtain are together estimated, to compare its growing state.Three parallel samples of each algae strain inoculation are to verify its collimation.
After drying is finished, prepare to measure its oil component content.
2) lipid acid extracts:
Getting the volume that 50mg or 100mg freeze-dried algae powder be placed on tool Telfnon screw socket bottle cap is in the phial of 15-20ml, place a little magnetic bar again, add 2-4ml 10%DMSO-Methanol solution, 40 ℃ of sand baths (beaker of containing sand is placed on the thermostatically heating magnetic stirring apparatus) 5 minutes; Stir extracting 30 minutes at 4 ℃ of lower magnetic forces then, 3500 leave the heart, shift supernatant liquor in another bottle.4 ℃ of lower magnetic forces of ether, normal hexane 4-8ml that remaining algae-residue added 1: 1 again stir extracting 1 hour, and 3500 leave the heart, shift supernatant liquor in an above-mentioned bottle.Can repeat said process bleaches up to algae-residue.Adding pure water in above-mentioned merging extract, to make four (water, DMSO-Methanol, ether, normal hexane) volume ratio be 1: 1: 1: 1, the concussion phase-splitting, pipetting organic phase transfers in another phial, in stink cupboard, blow to becoming concentrated solution with nitrogen, transfer to then in the 1.5ml plastic centrifuge tube of weighing in advance, dry up to constant weight with nitrogen again.
3) fatty acid analysis:
After extracting according to top method, use n-hexane dissolution, (chromatographic condition is carrier gas: nitrogen flow 1ml/min, hydrogen flowing quantity 30ml/min, air flow quantity 300ml/min to use Agilent 6820 gas chromatographs to carry out gas chromatographic analysis, injector temperature: 280 ℃, detector temperature: 280 ℃, detector type: Agilent FID, chromatographic column: Agilent DB-5 capillary chromatographic column (30m * 0.25mm, 0.25 splitting ratio μ m): 4: 1.Analytical procedure: marker method GB/T 17377-1998 (gas-chromatography is done carrier gas with nitrogen, is equivalent to the moving phase of liquid chromatography).
As seen from Figure 7, the ENN2201A fast growth, high level cadre Beijing South Maxpower Technology Co. Ltd reaches 11.552g/L.The high level cadre Beijing South Maxpower Technology Co. Ltd of No. 1 algae strain reaches 10.266g/L, and the high level cadre Beijing South Maxpower Technology Co. Ltd of No. 2 algae strains reaches 7.869g/L.By the result as can be known, ENN2201A has the speed of growth faster than other two strains grid algaes.The oil length three approaches, and high energy reaches about 54%.The proximate analysis of ENN2201A lipid acid the results are shown in Table 2.
Table 2: the sample fatty acid component table of gas Chromatographic Determination
Figure BDA0000126272130000151
Culture under the embodiment 7:ENN 2201A low temperature environment to reach with other grid algaes and compare (1)
The ENN2201A algae kind that will be in logarithmic phase is inoculated in the plate-type reactor of 50 * 3 * 50cm, BG11 (prescription sees Table 1) culture medium culturing.And, inoculate the two strains grid algae algae strain faster (No. 1 algae strain and No. 2 algae strains) of growing with the same terms and compare, the results are shown in Figure 8.
This experimental period is January in winter, daytime the variation of ambient temperature scope mainly between-7 to 10 ℃, algae liquid temp variation range is mainly between 9-15 ℃.The daylight variation range is mainly at 200-1500 μ mol/m 2Between/the s.
Growth velocity calculation formula: v=(m1-m2)/(t1-t2) m: algae liquid dry weight, t: incubation time.
As seen from Figure 8, the ENN2201A growth is better compared with other two strains algae strains under low temperature environment, still has the obvious growth advantage.With growth velocity relatively, ENN2201A improves 35.5% than No. 1 algae, improves 12.5% than No. 2 algaes.In conjunction with Fig. 7, Fig. 8 result, growth is slower than No. 1 under No. 2 algae strain normal temperature, at low temperatures faster than No. 1 algae strain.ENN2201A is under two kinds of conditions, and the speed of growth all is better than this two strains algae strain.
Culture under the embodiment 8:ENN 2201A low temperature environment to reach with other grid algaes and compare (2)
The ENN2201A algae kind that will be in logarithmic phase is inoculated in the plate-type reactor of 50 * 3 * 50cm, BG11 (prescription sees Table 1) culture medium culturing.And, inoculate the two strains grid algae algae strain faster of growing with the same terms and compare, the results are shown in down Fig. 9.
This experiment variation of ambient temperature on daytime scope is mainly 4-20 ℃, and algae liquid temp variation range is mainly between 10-25 ℃.The daylight variation range is mainly at 200-1500 μ mol/m 2Between/the s.
Growth velocity calculation formula: v=(m1-m2)/(t1-t2) m: algae liquid dry weight, t: incubation time.
As seen from Figure 9, the ENN2201A growth is better compared with other two strains algae strains under low temperature environment, still has the obvious growth advantage.With growth velocity relatively, ENN2201A exceeds 49.3% than No. 1 algae, exceeds 24% than No. 2 algaes.
The settleability research of embodiment 9:ENN 2201A
The ENN2201A that cultivated among the embodiment 5 20 days was left standstill 8 hours, and measuring dry weight before leaving standstill is 4.7g/L, leave standstill 8 hours after, in the sampling of reactor middle part, the measurement dry weight is 0.3g/L, rate of descent reaches 93.6%.Illustrate that the ENN2201A effect of settling is better, be beneficial to collection, be conducive to use in the industrialization.
Embodiment 10:ENN 2201A algae carotene carotene content is measured
Carotenoid be in the animal body must and the natural pigment that self can't synthesize.As fodder additives the existence of animal, breeding had good promoter action.Green frustule ENN2201A is inoculated in the tubular reactor of 4cm optical path with logarithmic phase, and centrifugal collection frustule behind continuous illumination in the 24 hours cultivation 16d is measured the carotenoid content in the cell.
1, the extraction of carotenoid and saponification:
(1) accurately takes by weighing 100mg algae powder, place 15ml phial with cover, add 2-4mLDMSO, put into 20 ℃ the ultrasonic 15min of ultrasonic washing instrument.
(2) take out bottle, add the extract (methylene dichloride: methyl alcohol=V25: V75), put into 20 ℃ the ultrasonic 15min of ultrasonic washing instrument of 2-4mL.
(3) take out bottle, 4 ℃ of centrifugal 5min of 3500rpm transfer to supernatant liquor in the long Glass tubing.
(4) remaining algae-residue adds the extract (methylene dichloride: methyl alcohol=V25: V75), put into 20 ℃ the ultrasonic 15min of ultrasonic washing instrument of 4ml.
(5) take out bottle, 4 ℃ of centrifugal 5min of 3500rpm transfer to supernatant liquor in the above-mentioned long Glass tubing.
(6) repeating step 3,4 bleaches until supernatant liquor and algae-residue.
(7) in the long Glass tubing of above-mentioned united extraction liquid, add the 0.107MNaOH-CH of 1/6 volume 3OH solution, mixing, towards nitrogen, sealing, lucifuge saponification 12h in 4 ℃ of refrigerators.
(8) in above-mentioned saponification liquor, add the 3-5ml sherwood oil, the vibration mixing, after be incorporated in the isopyknic distilled water of saponification liquor, behind the standing demix, draw supernatant liquid, transfer in the phial.
(9) remaining liq continues to add the sherwood oil extracting, and is complete until the whole extractings of pigment.
(10) above-mentioned phial is dried up in nitrogen, the back, is carried out HPLC and is detected analysis through the organic membrane filtration of the syringe-type of 0.22 μ m with the moving phase dissolving.
2, carotenoid detects:
After extracting according to top method, (detector: UV/VIS detects wavelength: 450nm to use the Agilent1100 high performance liquid chromatograph; Chromatographic column: 4.6mm * 250mm kromasilC18; Column temperature: 25 ℃; Flow velocity: 0.8mL/min; Moving phase: methyl alcohol: acetonitrile=50: 50; Analytical procedure: marker method; Sample size: 20 μ l), each substances content such as Figure 10 in its carotenoid.
This detects the algae powder is the result who obtains under the common breed, carotenoid is not carried out the specific aim inducing culture.
Embodiment 11:ENN 2201A waste water and gas is cultured
The ENN2201A algae kind that will be in logarithmic phase is seeded in 50 * 10 * 50cm plate-type reactor, and two experimental group are set: first group is used tap water to culture according to table 1 preparation BG11 substratum, and second group is used chemical plant wastewater to add 1.5g/L NaNO 3, 0.04g/LK 2HPO 43H 2O, 0.00315g/l FeCl 36H 2O, 0.00436g/l Na 2EDTA2H 2O, 1ml/L A5 Trace elemen preparation substratum is cultured.The situation analysis of chemical plant wastewater water quality the results are shown in Table 3.
Table 3: chemical plant wastewater water quality situation analysis
Figure BDA0000126272130000181
Figure BDA0000126272130000191
By table 3 as seen, the waste water various ion concentration of chemical plant emission is far above the theoretical value of BG11 substratum various ion concentration.Illustrate that this waste water of chemical plant emission is enough to satisfy the micro algae growth nutritional need fully, can use behind the interpolation nitrogen phosphorus.
Utilize natural condition to carry out grown cultures, natural condition are as follows between incubation period: culture cycle is 10 days, and the day temperature variation range is mainly between 23-43 ℃.The illumination variation scope sunny side of 9:00-17:00 is mainly at 200-1300 μ mol/m 2Between/the s.Feed the waste gas (content sees Table 4) of 2-5% daytime, carbon source to be provided and to regulate and control pH value between 7-9.Be interrupted sampling and measuring dry weight (the results are shown in Figure 11) in the culturing process.
As seen from Figure 11, the strain of ENN2201A algae can normal growth in tap water and trade effluent.The algae strain of proof present patent application protection can use industrial gaseous waste waste water to culture, and can obviously save cost, is conducive to the industrialization actual production.And in the chemical plant wastewater component, the part concentration of component is far above the medium standard component.It is wide to illustrate that this algae strain adapts to component concentration ranges, applied range.
Table 4: chemical plant emission waste gas component
Figure BDA0000126272130000192
Figure BDA0000126272130000201
Annotate: butt generally refers to the dry air of unit mass or dry gas to be the character such as humidity, specific heat, specific volume, enthalpy of the wet air represented of benchmark or humid gas.Be benchmark when representing moisture in the wet solid with the unit mass anhydrous solid, be also referred to as butt.
In the experiment, only added nitrogen in the waste water, phosphorus is cultured, algal grown must absorb other inorganic salt and CO 2, this can only be from waste water, thereby waste water is had cleaning action.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Figure IDA0000126272230000021
Figure IDA0000126272230000031
Figure IDA0000126272230000041

Claims (12)

1. a grid algae algae strain ENN2201A (Desmodesmus sp.), its deposit number is CGMCC No.5150, and preservation date is on August 17th, 2011, and depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. grid algae algae strain ENN2201A according to claim 1 (Desmodesmus sp.), its nucleotide sequence comprises the nucleotide sequence shown in the SEQ ID NO:3.
3. grid algae algae strain ENN2201A according to claim 1 and 2 (Desmodesmus sp.), its nucleotide sequence comprises the nucleotide sequence shown in the SEQ ID NO:6.
4. the cultural method of the described grid algae of claim 1 algae strain, it comprises in following (1)-(4) at least one:
(1) described substratum is the algae substratum, for example BG11, BBM, CT, SE, HB111, HB 119 or MA;
(2) culture temperature is 9-43 ℃; Be preferably 15-4o ℃; More preferably 25-30 ℃;
(3) pH is 7-9;
(4) illumination is 200-1500 μ mol/m 2/ s.
The strain of the described grid algae of claim 1 algae handle or purify waste water and/or waste gas in purposes; Particularly, described sewage is trade effluent or city domestic sewage, and described waste gas is industrial gaseous waste.
6. the purposes of the described grid algae of claim 1 algae strain in preparation biofuel and/or grease.
7. the strain of the described grid algae of claim 1 algae is in the purposes of preparation in the feed; Particularly, described feed is fishery products feed, animal and fowl fodder or feed for pet.
8. the strain of the described grid algae of claim 1 algae is in the purposes of preparation in the carotenoid; Particularly, described carotenoid is selected from least a in zeaxanthin, canthaxanthin and the β-Hu Luobusu.
9. handle or purify waste water or the method for waste gas for one kind, comprise that right to use requires the step of 1 described grid algae algae strain; Particularly, described sewage is trade effluent or city domestic sewage, and described waste gas is industrial gaseous waste.
10. a method for preparing biofuel and/or grease comprises that right to use requires the step of 1 described grid algae algae strain.
11. a method for preparing feed comprises that right to use requires the step of 1 described grid algae algae strain.
12. a method for preparing carotenoid comprises that right to use requires the step of 1 described grid algae algae strain; Particularly, described carotenoid is selected from any one or more in zeaxanthin, canthaxanthin and the β-Hu Luobusu.
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CN105713950A (en) * 2014-12-05 2016-06-29 中国石油化工股份有限公司 Method for producing microalgal oil by using flue gas
CN105713935A (en) * 2014-12-05 2016-06-29 中国石油化工股份有限公司 Method for producing lipid through mixed culture of microalgae
CN105648023B (en) * 2014-12-05 2019-01-25 中国石油化工股份有限公司 A kind of method that microalgae mixed culture prepares grease
CN105713950B (en) * 2014-12-05 2019-06-11 中国石油化工股份有限公司 A method of microalgae grease is produced using flue gas
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CN107365708A (en) * 2016-05-12 2017-11-21 财团法人食品工业发展研究所 Grid algae (DESMODESMUS SP.) and its application on Synthetic Oil and raw matter fuel
TWI640626B (en) * 2016-05-12 2018-11-11 財團法人食品工業發展研究所 Desmodesmus sp. and its applications in the synthesis of oils and biofuels
CN115161201A (en) * 2022-05-26 2022-10-11 珠海元育生物科技有限公司 Scenedesmus strain and culture method and application thereof
CN115161201B (en) * 2022-05-26 2024-03-19 珠海元育生物科技有限公司 Sclerophyllum strain and culture method and application thereof

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