CN103184156B

CN103184156B - Scenedesmus algal strain and its use

Info

Publication number: CN103184156B
Application number: CN201110448718.3A
Authority: CN
Inventors: 邓平; 罗少敬; 陈传红; 吴洪; 马建源
Original assignee: ENN Science and Technology Development Co Ltd
Current assignee: ENN Science and Technology Development Co Ltd
Priority date: 2011-12-29
Filing date: 2011-12-29
Publication date: 2014-08-20
Anticipated expiration: 2031-12-29
Also published as: CN103184156A

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 the 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.The problems such as environmental pollution are day by day serious.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 subject to people with the research of the micro-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.Micro-algae photosynthetic efficiency is high, absorbs a large amount of CO in process of growth ₂, can effectively reduce CO ₂discharge.There is algae slightly can utilize the moiety in waste stream, convert it into the biological substance of self, therefore can be effectively used to the processing of trade effluent, waste material.

Grid algae is planktonic algae common in fresh water, and utmost point happiness is bred in nutritious hydrostatic.Wherein numerous species has stronger patience to organic pollutant.Grid algae has certain effect in self purification of water body and sewage purification, is the dominant species in organic sewage oxidation pond biophase.It can be attached in water on organism fragment and other waterplant bodies with bacterium simultaneously, forms gelatinous layer, adsorb organic compound.When grid algae carries out photosynthesis, produce oxygen for the needs of bacterial degradation organic matter on the one hand, also can directly utilize on the other hand organic matter as Carbon and nitrogen sources, organism in water is degraded rapidly, thus purifying water body.Grid frustule contains rich in protein and VITAMIN, and grid algae propagation is fast, is of high nutritive value, and can be used as artificial a large amount of material of cultivating.In part grid frustule, abundant fatty acid can be accumulated, by manual operation, biofuel can be converted into.

Application number is to be separated to the new chlorella of a strain in 200780020794.6 patent, and can Application Areas to multiple fields, comprise the processing of discarded carbonic acid gas in environment or industrial production, production and the wastewater treatment of bioenergy.

Application number is to utilize self-separation grid algae in Treating Municipal Sewage in 201010111834 patent, and coupling produce the device of diesel oil can be for the production of micro-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, obtain the strain of a kind of grid algae algae, and the inventor is surprised to find, this grid algae algae strain can for example, be grown in wide temperature range (9-43 DEG C), fast growth and be easy to collect, and can effectively produce the material such as lipid acid, carotenoid, there are the potentiality in application such as sewage disposal, production of biodiesel and feed or food service industrys.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's common micro-organisms center.

Described grid algae algae strain ENN2201A (Desmodesmus sp.), its nucleotide sequence comprises the nucleotide sequence shown in 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 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 Deyang City Zhongjiang County, adds the BG11 substratum of 1/3 volume, is 40 μ mol/m at 25 DEG C, intensity of illumination ².s under condition, cultivate about 5 days, get this water sample in micro-Microscopic observation, judge the main species that exist in 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 DEG C, intensity of illumination ².s under the condition of left and right, cultivate about 10 days, can see that single algae falls to growing, fall to being inoculated in BG11 substratum with transfering loop or aseptic toothpick picking list algae and cultivate.Repeat this process, until obtain the algae strain of pure culture, in the present invention, its numbering is called to " ENN2201A ".

In embodiment 1, provide concrete separation screening process.

In embodiment 2, from form level and molecular level, the algae strain obtaining is carried out to classification qualification, and analyzed and compare.According to the result of identification of morphology and Molecular Identification, determine that ENN2201A is Shan Zao section (Scenedesmaceae), chain band Trentepohlia (Desmodesmus).

Another aspect of the present invention relates to the cultural method of described grid algae algae strain, and it comprises any one in following (1)-(4) or multinomial:

(1) described substratum is algae substratum, for example BG11, BBM, CT, SE, HB111, HB 119 or MA;

(2) culture temperature is 9-43 DEG C; Be preferably 15-40 DEG C; More preferably 25-30 DEG C;

(3) pH is 7-9;

(4) illumination is 200-1500 μ mol/m ²/ s.

About culture temperature, best culture temperature is 25-30 DEG C, is equaling higher than 9 DEG C, but also can grow while being less than 15 DEG C, and the speed of growth can be subject to certain impact.Higher than 40 DEG C and be less than 43 DEG C, continue can grow under 2-4h, see embodiment 8, but the speed of growth can be influenced.

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 processing 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 effectively purify waste water 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; Or 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.

Of the present inventionly relate in one aspect to again described grid algae algae strain in the purposes of preparing in feed; Particularly, described feed is that feed can all or part ofly be made up of grid algae algae of the present invention strain described in fishery products feed, animal and fowl fodder or feed for pet.Grid algae algae of the present invention strain can be separately as feed, also can be used as additive and prepares feed.Can directly use frond, i.e. algae powder, adds in feed, also can after broken wall, add.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 Chang, Wu Weixiong, fodder additives manufacturing technology, the 2011-02 of scientific and technical literature press, etc.

Of the present inventionly relate in one aspect to again described grid algae algae strain in the purposes of preparing in carotenoid; Particularly, described carotenoid is selected from any one or more in zeaxanthin, canthaxanthin and β-carotene.

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 processed or purified waste water or the method for waste gas, comprises the step that uses 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 again a kind of method of preparing biofuel and/or grease, comprise the step that uses grid algae algae of the present invention strain.

Of the present inventionly relate in one aspect to again a kind of method of preparing feed, comprise the step that uses grid algae algae of the present invention strain.

Of the present inventionly relate in one aspect to again a kind of method of preparing carotenoid, comprise the step that uses grid algae algae of the present invention strain; Particularly, described carotenoid is selected from any one or more in zeaxanthin, canthaxanthin and β-carotene.

The beneficial effect of the invention

1) this algae kind suitable temperature range is wide, and cultivation also has good performance at low temperatures, can effectively increase annual cultivation number of days.

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.

Brief description of the drawings

Fig. 1: form under ENN2201A microscope.Line segment in figure A and B represents 10 microns.

Fig. 2: ENN2201A algae 18S sequence B last result.

Fig. 3: ENN2201A phylogenetic analysis (18S).

Fig. 4: ENN2201A algae ITS sequence B last result.

Fig. 5: ENN2201A phylogenetic analysis (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.Note: when inoculation, three algae strain starting point concentrations are respectively 1: 0.914,2: 0.812,1.288, and gap is not very large, can not have great especially impact to subsequent growth.

Fig. 9: the ENN2201A algae kind that is in logarithmic phase is inoculated in the plate-type reactor of 50 × 3 × 50cm, with BG11 culture medium culturing.And, inoculate the two strains grid algae algae strain faster of growing with the same terms and compare.

Each substances content in carotenoid when Figure 10: ENN2201A growth 16 days.Account for dry weight ratio: left for zeaxanthin, in for canthaxanthin, right be β-carotene.

The growing state of Figure 11: ENN2201A in tap water and waste water.

Biomaterial preservation explanation:

Grid algae algae strain ENN2201A (Classification And Nomenclature of suggestion is Desmodesmus sp.), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on August 17th, 2011, preserving number is CGMCC No.5150, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.

Embodiment

Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples are only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " 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: screening produce oil grid algae algae strain

The triumphant rivers water sampling in Deyang City Zhongjiang County, adds the BG11 substratum of 1/3 volume, is 40 μ mol/m at 25 DEG C, intensity of illumination ².s under condition, cultivate about 5 days, get this water sample in micro-Microscopic observation, judge the main species that exist in 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 DEG C, intensity of illumination ².s under the condition of left and right, cultivate about 10 days, can see that single algae falls to growing, fall to being inoculated in BG11 substratum with transfering loop or aseptic toothpick picking list algae and cultivate.Repeat this process, until obtain the algae strain of pure culture.The pure lines algae liquid growing is cultivated respectively in triangular flask, column reactor, and results of regular determination dry weight and fat content, therefrom select fast growth, algae strain that fat content is high.By this method, obtain an algae strain called after ENN2201A.

The present invention's BG11 substratum used all as shown in Table 1 below.

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 1000 ml deionized waters) is as follows:

embodiment 2: identification of morphology

The above-mentioned algae strain ENN 2201A separating is observed under 1000 power microscopes, the results are shown in Figure 1.Frond is unicellular or sizing colony form exists, and unicellular spherical in shape or avette, sizing colony is made up of 2-4 cell, colony's cell straight line arranged side by side becomes row, the upper and lower two ends of each cell are extensively round, and cell walls is level and smooth, and the two ends up and down of the 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.Be initially identified as 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; Again to the phenol that adds 600 μ l in reaction system: chloroform (volume ratio 1: 1) extracting, 14, the centrifugal 15min of 000rpm, get supernatant, add 0.6 times of volume isopropanol precipitating of supernatant, after 75% washing with alcohol, be dissolved in 100 μ l sterilizing distilled waters its concentration of UV spectrophotometer measuring and purity.

Adopt eukaryote 18S amplification universal primer (primer is synthetic synthetic by Shanghai Sheng Gong bio-engineering corporation) micro-algae genome 18S gene fragment that increases, 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 DEG C of denaturation 5min, and then 94 DEG C of sex change 50s, 56 DEG C of annealing 50s, 72 DEG C are extended 1min 30s, react 30 circulations, and last 72 DEG C are extended 10min.1.0% agarose gel electrophoresis detects its amplified production.Obtain the 18S sequence of ENN2201A.Below listed 18S sequence is the gene order that order-checking obtains, as shown in SEQ ID NO:3 below:

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 cloned

The 18S sequence login GenBank database of the ENN2201A that amplification is obtained carries out BLAST comparison, and two sequences that result demonstration and its matching degree and fraction of coverage are the highest are does not identify sequence.Come the 3rd be No. ACCESSION Scenedesmuscommunis gene for 18S small subunit rRNA sequence for X73994.1, 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 for No. ACCESSION the Scenedesmus abundans gene for 18S small subunit rRNA sequence of X73995.1, 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. phylogenetic analysis

Utilize the BLAST instrument in ncbi database to carry out sequence similarity analysis to the 18S partial sequence of algae strain ENN 2201A.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/) carry out sequence homology evolution comparison, form a multiple sequence coupling permutation matrix.Then, use Mega4.0 software (Tamura K etc., 2007), adopt connected (Neighbor-joining) algorithm in ortho position, the data set bootstraps value of bootstrapping is that 1000 constructing systems are grown evolutionary trees.

According to 18S sequence B last result, choose 6 strains wherein, 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 again Chlamydomonas reinhardtii Chlamydomonas reinhardtii, the 18S sequence of two chlorella Chlorella vulgaris strain CCAP 211/113 and Chlorella vulgaris strain KMMCC FC-16, common structure evolutionary tree (Fig. 3).

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).Apart from other kinds, as chlamydomonas (chlamydomonas), chlorella (chlorella) is equidistant far away.

embodiment 4: Molecular Identification 2 (ITS)

1. genome extracts

ITS sequence amplification adopts eukaryote ITS amplification universal primer (primer is synthetic by Shanghai Sheng Gong bio-engineering corporation), 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 DEG C of sex change 5min, then 94 DEG C of 40s, 56 DEG C of 40s, 30 circulations of 72 DEG C of 2min, last 72 DEG C of 10min.1.0% agarose gel electrophoresis detects its amplified production, and pcr amplification obtains about 692bp segment.The SEQ ID NO:6 obtaining is the ITS partial sequence of ENN 2201A.Sequence is as shown in SEQ ID NO:6:

TGGGTTGGAAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA

TCATTGAATATGCAAACCACAACACGCACTCTTTATTTGTGTTCGACGTTAGGTCAACA

CGCGCAAGCGTGTGGCCTACTAACCTACACACCATTGACCAACCATTTATCAAACCAAA

CTCTGAAGCTTTGGCTGCCGTTAACCGGCAGTTCTAACAAAGAACAACTCTCAACAACG

GATATCTTGGCTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGTGAATT

GCAGAATTCCGTGAACCATCGAATCTTTGAACGCATATTGCGCTCGACTCCTCGGAGAA

GAGCATGTCTGCCTCAGCGTCGGTTTACACCCTCACCCCTCTTCCTTAACAGGAGGCGC

CTGTCGTGCTTGCTTAAGCCGGCAGCAGGGGTGGATCTGGCTCTCCCAATCGGATTCAC

TCTGGTTGGGTTGGCTGAAGCACAGAGGCTTAAACTGGGACCCAATTCGGGCTCAACTG

GATAGGTAGCAACACCCTCGGGTGCCTACACGAAGTTGTGTCTGAGGACCTGGTTAGGA

GCCAAGCAGGAAACGCGTCTCTGGCGCGTACTCTGTATTCGACCTGAGCTCAGGCAAGG

CTACCCGCTGAACTTAAGCATATCAATAAGCGGGAGGAACCCCTTTT (SEQ ID NO：6)

2. nucleotide sequence (SEQ ID NO:6) the homology search cloned:

The ITS partial sequence and the 5.8S sequence login GenBank database that obtain E are carried out to BLAST comparison, result shows the internal transcribed spacer 1 with No. ACCESSION desmodesmus armatus var. that is DQ414547.1,5.8S ribosomal RNA gene and internal transcribed spacer 2 sequence have higher similarity and reach 97%, and fraction of coverage is also up to 96% (Fig. 4).

3. phylogenetic analysis

The results are shown in Figure 5.

Utilize the BLAST instrument in ncbi database to carry out sequence similarity analysis to the ITS partial sequence of algae strain ENN 2201A.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/) carry out sequence homology evolution comparison, form a multiple sequence coupling permutation matrix.Then, use Mega4.0 software (Tamura K etc., 2007), adopt connected (Neighbor-joining) algorithm in ortho position, the data set bootstraps value of bootstrapping is that 1000 constructing systems are grown evolutionary trees.

The sibship of ENN2201A and desmodesmus armatus var. is nearer as can be seen from Figure 5, is positioned in same branch.Illustrate that ENN2201A should belong to chain band Trentepohlia.

According to the result of identification of morphology and Molecular Identification (18S and ITS), limiting ENN 2201A is Shan Zao section (Scenedesmaceae), and chain band Trentepohlia (Desmodesmus), is initially identified as Desmodesmus sp..

the triangular flask cultivation of embodiment 5:ENN 2201A algae

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, is placed on shaking table and cultivates.Initial inoculation OD750 is 0.086 left and right; Intensity of illumination approximately 35 μ molm-2s-1, temperature is 25+1 DEG C, rotating speed 100rpm/min, light source is fluorescent lamp.Its OD680 of periodic measurement and 750 changing conditions.The results are shown in Figure 6.

As seen from Figure 6, ENN2201A well-grown, compares with bibliographical information (Ji Xiang etc., a screening for strain scenedesmus obliquus and the optimization of growth conditions, aquatic science and technology information, 2010,37 (6), 265-268), has 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 is in logarithmic phase is seeded in by 20% inoculative proportion in the BG11 substratum (formula is in table 1) preparing, uses the column reactor of 40mm internal diameter, 600mm length to carry out both culturing microalgae.In culturing process, intensity of illumination is controlled at 150-600 μ mol/m ².s in scope.In incubation period, by pass into the mixed gas (carbonic acid gas that is 1.5-2% containing volume fraction) of air and carbonic acid gas in nutrient solution, to provide the pH value of carbon source regulation culture base between 7-9.Temperature adjusting is within the scope of 25-35 DEG C.In 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 obtains algae mud by the method for centrifugal or natural subsidence, and algae mud is being carried out to vacuum lyophilization.Select other algae strains of grid algae (numbering is respectively No. 1, No. 2) that this laboratory self-separation obtains together to evaluate, to compare its growing state.Three Duplicate Samples of each algae strain inoculation, to verify its collimation.

After being dried, prepare to measure its oil component content.

2) lipid acid extracts:

In the phial that to get volume that 50mg or 100mg freeze-dried algae powder be placed on tool Telfnon bottle screw cap be 15-20ml, place again a little magnetic bar, add 2-4ml 10%DMSO-Methanol solution, 40 DEG C of sand baths (beaker of containing sand is placed on constant-temperature heating magnetic stirring apparatus) 5 minutes; Then stir extracting 30 minutes at 4 DEG C of lower magnetic forces, 3500 leave the heart, shift supernatant liquor in another bottle.4 DEG C of lower magnetic forces of ether, normal hexane 4-8ml that remaining algae-residue adds 1: 1 again stir extracting 1 hour, and 3500 leave the heart, shift supernatant liquor in an above-mentioned bottle.Said process can be repeated until algae-residue bleaches.In above-mentioned merging extract, adding pure water to make four (water, DMSO-Methanol, ether, normal hexane) volume ratio is 1: 1: 1: 1, concussion phase-splitting, pipetting organic phase transfers in another phial, in stink cupboard, blow to becoming concentrated solution with nitrogen, then transfer in the 1.5ml plastic centrifuge tube of weighing in advance, then dry up to constant weight with nitrogen.

3) fatty acid analysis:

After method is extracted above, with 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 DEG C, detector temperature: 280 DEG C, detector type: Agilent FID, chromatographic column: Agilent DB-5 capillary chromatographic column (30m × 0.25mm, 0.25 μ m), splitting ratio: 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, ENN2201A fast growth, high level cadre Beijing South Maxpower Technology Co. Ltd reaches 11.552g/L.No. 1 Zao Zhu high level cadre Beijing South Maxpower Technology Co. Ltd reaches 10.266g/L, and No. 2 Zao Zhu high level cadre Beijing South Maxpower Technology Co. Ltd reaches 7.869g/L.From result, ENN2201A has the speed of growth faster compared with other two strains grid algaes.Oil length three approaches, and high energy reaches 54% left and right.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

under embodiment 7:ENN 2201A low temperature environment cultivation and with other grid algae comparisons (1)

The ENN2201A algae kind that is in logarithmic phase is inoculated in the plate-type reactor of 50 × 3 × 50cm to BG11 (formula is in 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 variation of ambient temperature scope mainly between-7 to 10 DEG C, algae liquid temp variation range is mainly between 9-15 DEG C.Daylight variation range is mainly at 200-1500 μ mol/m ²between/s.

Growth velocity calculation formula: v=(m1-m2)/(t1-t2) m: algae liquid dry weight, t: incubation time.

As seen from Figure 8, under low temperature environment, ENN2201A growth better, compared with other two strains algae strains, still has obvious growth vigor.With growth velocity comparison, ENN2201A improves 35.5% compared with No. 1 algae, improves 12.5% compared with No. 2 algaes.In conjunction with Fig. 7, Fig. 8 result, under No. 2 algae strain normal temperature, growth is slower than No. 1, at low temperatures faster than No. 1 algae strain.ENN2201A is under two kinds of conditions, and the speed of growth is all better than this two strains algae strain.

under embodiment 8:ENN 2201A low temperature environment cultivation and with other grid algae comparisons (2)

The ENN2201A algae kind that is in logarithmic phase is inoculated in the plate-type reactor of 50 × 3 × 50cm to BG11 (formula is in 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 lower Fig. 9.

This experiment variation of ambient temperature on daytime scope is mainly 4-20 DEG C, and algae liquid temp variation range is mainly between 10-25 DEG C.Daylight variation range is mainly at 200-1500 μ mol/m ²between/s.

As seen from Figure 9, under low temperature environment, ENN2201A growth better, compared with other two strains algae strains, still has obvious growth vigor.With growth velocity comparison, ENN2201A exceeds 49.3% compared with No. 1 algae, exceeds 24% compared with No. 2 algaes.

the settleability research of embodiment 9:ENN 2201A

The ENN2201A cultivating in embodiment 5 20 days is left standstill to 8 hours, and before leaving standstill, measuring dry weight is 4.7g/L, leaves standstill after 8 hours, and in the sampling of reactor middle part, measurement dry weight is 0.3g/L, and rate of descent reaches 93.6%.Illustrate that ENN2201A effect of settling is better, be beneficial to collection, be conducive to apply in industrialization.

embodiment 10:ENN 2201A algae carotene carotene content is measured

Carotenoid is natural pigment necessary in animal body and self cannot be synthetic.Existence, breeding as fodder additives to animal have good promoter action.Green logarithmic phase frustule ENN2201A is inoculated in the tubular reactor of 4cm optical path, and centrifugal collection frustule after continuous illumination in 24 hours cultivation 16d, measures the carotenoid content in cell.

1, the extraction of carotenoid and saponification:

(1) accurately take 100mg algae powder, be placed in 15ml phial with cover, add 2-4mLDMSO, put into the ultrasonic 15min of ultrasonic washing instrument of 20 DEG C.

(2) take out bottle, add the extract (methylene dichloride: methyl alcohol=V25: V75) of 2-4mL, put into the ultrasonic 15min of ultrasonic washing instrument of 20 DEG C.

(3) take out bottle, 4 DEG C of centrifugal 5min of 3500rpm, transfer to supernatant liquor in long Glass tubing.

(4) remaining algae-residue adds the extract (methylene dichloride: methyl alcohol=V25: V75) of 4ml, puts into the ultrasonic 15min of ultrasonic washing instrument of 20 DEG C.

(5) take out bottle, 4 DEG C of centrifugal 5min of 3500rpm, transfer to supernatant liquor in above-mentioned long Glass tubing.

(6) repeating step 3,4 until supernatant liquor and algae-residue bleach.

(7), in the long Glass tubing of above-mentioned united extraction liquid, add the 0.107MNaOH-CH of 1/6 volume ₃oH solution, mixes, and rushes nitrogen, sealing, lucifuge saponification 12h in 4 DEG C of refrigerators.

(8) in above-mentioned saponification liquor, add 3-5ml sherwood oil, vibration mixes, after be incorporated in the isopyknic distilled water of saponification liquor, after stratification, draw supernatant liquid, transfer in phial.

(9) remaining liq continues to add sherwood oil extracting, until the whole extractings of pigment are complete.

(10) above-mentioned phial is dried up in nitrogen, rear with moving phase dissolving, through the organic membrane filtration of syringe-type of 0.22 μ m, carry out HPLC and detect analysis.

2, carotenoid detects:

After method is extracted above, (detector: UV/VIS, detects wavelength: 450nm to use Agilent1100 high performance liquid chromatograph; Chromatographic column: 4.6mm × 250mm kromasilC18; Column temperature: 25 DEG C; Flow velocity: 0.8mL/min; Moving phase: methyl alcohol: acetonitrile=50: 50; Analytical procedure: marker method; Sample size: l), in its carotenoid, each substances content is as Figure 10 for 20 μ.

This detects algae powder is the result obtaining under common cultivation, carotenoid is not carried out to specific aim inducing culture.

the cultivation of embodiment 11:ENN 2201A waste water and gas

The ENN2201A algae kind that is in logarithmic phase is seeded in 50 × 10 × 50cm plate-type reactor, two experimental group are set: first group of use tap water prepared BG11 substratum according to table 1 and cultivated, second group uses 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 cultivates.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

From table 3, the waste water various ion concentration of chemical plant emission is far above the theoretical value of BG11 substratum various ion concentration.Illustrate, this waste water of chemical plant emission is enough to meet micro algae growth nutritional need completely, after interpolation nitrogen phosphorus, can use.

Utilize natural condition to carry out grown cultures, between incubation period, natural condition are as follows: culture cycle is 10 days, and day temperature variation range is mainly between 23-43 DEG C.The illumination variation scope sunny side of 9:00-17:00 is mainly at 200-1300 μ mol/m ²between/s.Pass into the waste gas (content is in table 4) of 2-5% daytime, carbon source to be provided and to regulate and control pH value between 7-9.In culturing process, be interrupted sampling and measuring dry weight (the results are shown in Figure 11).

As seen from Figure 11, the strain of ENN2201A algae can normal growth in tap water and trade effluent.The algae strain that proves present patent application protection can be used industrial gaseous waste waste water to cultivate, can be obviously cost-saving, be conducive to industrialization actual production.And in chemical plant wastewater component, part concentration of component is far above medium standard component.Illustrate that this algae strain adapts to component concentration ranges wide, applied range.

Table 4: chemical plant emission waste gas component

Note: butt generally refers to the character such as the humidity, specific heat, specific volume, enthalpy of the wet air that represents taking the dry air of unit mass or dry gas as benchmark or humid gas.Represent the moisture in wet solid during taking unit mass anhydrous solid as benchmark, also referred to as butt.

In experiment, in waste water, only added nitrogen, phosphorus cultivates, algal grown must absorb other inorganic salt and CO ₂, this can only be from waste water, thereby waste water is had to 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 amendments 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.

Claims

1. a grid algae algae strain ENN2201A, is Desmodesmus sp., and 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's common micro-organisms center.

2. grid algae algae strain ENN2201A according to claim 1, its nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:3.

3. grid algae algae strain ENN2201A according to claim 1 and 2, its nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:6.

4. the cultural method of grid algae algae claimed in claim 1 strain, substratum is algae substratum, and culture temperature is 9-43 DEG C, and pH is 7-9, and illumination is 200-1500 μ mol/m ²/ s.

5. cultural method according to claim 4, is characterized in that, described algae substratum is BG11, BBM, CT, SE, HB111, HB119 or MA.

6. according to the cultural method described in claim 4 or 5, it is characterized in that, described culture temperature is 15-40 DEG C.

7. cultural method according to claim 6, is characterized in that, described culture temperature is 25-30 DEG C.

Grid algae algae claimed in claim 1 strain dispose of sewage and/or waste gas in purposes.

9. purposes according to claim 8, is characterized in that, described in be treated to purification.

10. purposes according to claim 8 or claim 9, is characterized in that, described sewage is trade effluent or city domestic sewage, and described waste gas is industrial gaseous waste.

The purposes of 11. grid algae algae claimed in claim 1 strains in preparation biofuel and/or grease.

12. grid algae algae claimed in claim 1 strains are in the purposes of preparing in feed.

13. purposes according to claim 12, is characterized in that, described feed is fishery products feed, animal and fowl fodder or feed for pet.

14. grid algae algae claimed in claim 1 strains are in the purposes of preparing in carotenoid.

15. purposes according to claim 14, is characterized in that, described carotenoid is selected from least one in zeaxanthin, canthaxanthin and β-carotene.