CN102978114B

CN102978114B - Scenedesmus sp. and applications thereof

Info

Publication number: CN102978114B
Application number: CN201210342081.4A
Authority: CN
Inventors: 罗少敬; 邓平; 张晓楠; 吴洪; 王冰
Original assignee: ENN Science and Technology Development Co Ltd
Current assignee: ENN Science and Technology Development Co Ltd
Priority date: 2012-09-14
Filing date: 2012-09-14
Publication date: 2014-08-13
Anticipated expiration: 2032-09-14
Also published as: CN102978114A

Abstract

The present invention discloses a strain of Scenedesmus sp. and applications thereof. The Scenedesmus sp. has advantages of fast growth, rapid grease accumulation, high oil content, effective removal of nitrogen-containing compounds in water, and the like, contains a suitable part of useful pigments, and can be used for productions of biodiesel, food, health care products, feeds, aquatic product baits, drugs and cosmetics.

Description

One strain grid algae and application thereof

Technical field

The present invention relates to microorganism field, relate in particular to a kind of grid algae ENN32 (Scenedesmus sp.) and application thereof.

Background technology

Along with the progressively exhaustion of world's fossil fuel capacity, the continuous increase of CO2 emissions and global climate day by day become warm, the mankind's survival and development situation has faced unprecedented challenge.Therefore; countries in the world all can petroleum replacing product in positive searching renewable energy source; wherein, biofuel is exactly a kind of important bioenergy, but; such as the cultivation period such as corn and soybean is longer for common oil crops; take farmland more, can produce " strive grain with people, strive ground with grain " problem; thereby cause the awkward result of " solved energy dilemma, but occurred crisis in food ".

Micro-algae be a class on land, ocean is widely distributed, nutritious, growth cycle is short, photosynthetic availability high with the energy-producing lower plant of photoautotrophy, be nature origin the earliest, the biomass resource of the widest, the kind of distributing and quantity maximum.Many micro-algaes can accumulate in a large number storage property triacylglycerol (micro-algae oil) (being similar to vegetables oil) in cell under certain condition, and its high-content can reach the more than 50% of dry cell weight.Compared with high Lu Sheng oil crops, the unit surface oil yield of micro-algae is the decades of times of oilseed plant oil yield, through biological smelting can production biofuel etc. multiple biofuel, be considered to the most potential most possible biomass resource that becomes oil substitutes.

Except oil yield is very high, micro-algae is rich in proteins, fat, carbohydrate, each seed amino acid, VITAMIN, microbiotic, high unsaturated fatty acid and other various bioactivators also, therefore, there is too good DEVELOPMENT PROSPECT in fields such as protective foods, medicine and feeds, for example,, for foodstuff and pharmaceutical sector (nutritional supplement of humans and animals: VITAMIN, protein, lipid acid, polysaccharide etc.); Extract chemical industry as makeup, fine chemical product etc.; Produce biogas, fuel as the energy; For bait and feed industry (bait (CN1742570), the poultry feeds etc. of the aquatic animals such as fishes and shrimps, crustacean); In agricultural as soil redeposition, fertilizer etc.

In addition, the adaptive capacity to environment of micro-algae is very strong, also very simple to the requirement of nutrition, can under various harsh environment, grow.For example, micro-algae can absorb the organism such as the nitrogen phosphorus synthesizing amino acid, protein, phosphatide of the inorganic salt such as nitrate in sewage, nitrite, ammonium salt, phosphoric acid salt by photosynthesis, form microalgae cell, thereby the nitrogen and phosphorus content in reduction water body, therefore micro-algae can be used for removing the nitrogen phosphorus in sewage.Micro-algae also can utilize the CO in industrial gaseous waste ₂, reduce CO ₂discharge.Therefore micro-algae is being aspect industry environmental protection or also all there is good application prospect city environmental protection aspect.Also have one slightly algae can be grown in the water body of high salt, high alkali environment, can make full use of the soil that beach, saltings, desert etc. are not suitable for cultivating and carry out large scale culturing, also can utilize the non-agricultural waters such as seawater, saline-alkali water, trade effluent to cultivate, be described as " green oilfield " capable of circulation, therefore develop micro-algae cultivation and related industries thereof and be also applicable to very much especially cultivated land resource China relatively in short supply of soil.

Especially it is in the exploitation of bioenergy application aspect all to have actively developed at present both at home and abroad exploitation for micro-algae, and this is wherein in the majority with the research to chlorella.CN101460609 discloses a kind of new Chlorella (CHLORELLA) species and application CN101565675 discloses a kind of chlorella and cultural method and its application in production of biodiesel; CN101824386 discloses a chlorella, and is producing microalgae grease and fixation of C O ₂in application; CN102093955 disclose a chlorella and utilize sewage cultivate with biofuel transformation system in application; CN102229889 discloses a chlorella and cultural method thereof and at CO ₂fixing, useless water purification, the production application of the biomass such as grease, protein, pigment, starch, polysaccharide, nucleic acid.In addition, the correlative study of applying for micro-algae is also in the middle of carrying out, and CN101767893 discloses a kind of device and method that utilizes microalgae to deeply treating wastewater coupling producing biological oil.The large-scale application of micro-algae in the energy and environmental area is within sight.

Grid algae is planktonic microalgae common in fresh water, and utmost point happiness is bred in nutritious hydrostatic.Numerous species has stronger patience to organic pollutant, can be used as indicator organism in water quality assessment, and 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, is the good feeds of fish, as utilizes organic sewage oxidation pond to breed fish, and can obtain high yield.Prolific frond also can be made the feed of poultry.Not also a lot of for the exploitation of grid algae both at home and abroad at present, CN101565674 discloses strain low nutrition grid algae and the application in advanced treatment of wastewater thereof, this grid algae algae strain can be grown in low nutrient environment, therefore the nitrogen phosphorus in secondary effluent can be removed to very low level; CN102083957 discloses one and has had micro-algae of the distinctive plush copper shape of the efficient removal carbonic acid gas former mutation of grid algae and uses thereof, micro-algae of the former mutation of plush copper shape grid algae that especially carbon dioxide fixation rate is high and uses thereof; CN102250773 discloses a strain grid algae and cultural method thereof and at CO ₂fixing, useless water purification, the application in the fields such as the production of the biomass such as grease, protein, pigment, starch, polysaccharide, nucleic acid; The people such as Ji Xiang, Wang Jinrong (screening for strain scenedesmus obliquus and the optimization of growth conditions [J].Aquatic science and technology information, 2010:37 (6)) obtain that a strain oil-containing is relatively high, the strain of the good algae kind of growing way grid algae algae.

Although micro-algae has the even DEVELOPMENT PROSPECT such as medicine of the very good energy, environment protection, and relevant research work also launched gradually, current known micro-algae, especially grid algae, oleaginousness conventionally below 40%, to CO ₂the performance of the aspect such as processing power, extreme environment tolerance on also generally on the low side.Therefore need performance better, be more suitable for the grid algae of industrial application.

Summary of the invention

The object of the present invention is to provide one strong to adaptive capacity to environment, fat content is high, to CO ₂the strong grid algae that is more suitable for industrial application of processing power.

For reaching this object, the present invention by the following technical solutions:

In first aspect, the invention provides a kind of grid algae ENN32 (Scenedesmus sp.), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation date is on 07 13rd, 2012, and preserving number is CGMCC No.6346.

In grid algae ENN32 provided by the invention, the content of lipid acid in algae powder can be greater than 40% dry weight, is preferably more than 50% dry weight, is more preferably 51%～60% dry weight.

The tolerable temperature of grid algae ENN32 provided by the invention can be-10 DEG C to 44 DEG C, preferably 0 DEG C to 40 DEG C, and more preferably 10 DEG C to 35 DEG C, also more preferably 20 DEG C to 30 DEG C, most preferably 25 DEG C.

Grid algae ENN32 provided by the invention can grow at 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C, 6 DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C, 11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C, 16 DEG C, 17 DEG C, 18 DEG C, 19 DEG C, 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C.

In second aspect, the invention provides the cultural method of the grid algae ENN32 as described in first aspect, described method comprises grid algae algae strain ENN32 green cell is seeded in BG11 substratum, wherein intensity of illumination 50-500 μ mol/m ².s, preferred 100-450 μ mol/m ².s, more preferably 150-400 μ mol/m ².s, more preferably 200-350 μ mol/m also ².s, 250-300 μ mol/m most preferably ².s, pH value 6-10, preferred 7-9, more preferably 7.5-8.5, most preferably 8, temperature 10-40 DEG C, preferably 15-35 DEG C, more preferably 20-30 DEG C, most preferably 25 DEG C.

In the cultural method of grid algae ENN32 of the present invention, intensity of illumination can be 50 μ mol/m ².s, 80 μ mol/m ².s, 100 μ mol/m ².s, 120 μ mol/m ².s, 150 μ mol/m ².s, 180 μ mol/m ².s, 200 μ mol/m ².s, 220 μ mol/m ².s, 250 μ mol/m ².s, 280 μ mol/m ².s, 300 μ mol/m ².s, 320 μ mol/m ².s, 350 μ mol/m ².s, 380 μ mol/m ².s, 400 μ mol/m ².s, 420 μ mol/m ².s, 450 μ mol/m ².s, 480 μ mol/m ².s or 500 μ mol/m ².s; PH value can be 6,6.5,7,7.2,7.5,7.8,8,8.2,8.5,8.8,9,9.5 or 10; Temperature can be 10,11,12,13,14,15 DEG C, 16 DEG C, 17 DEG C, 18 DEG C, 19 DEG C, 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C or 35 DEG C.

In the third aspect, grid algae ENN32 provided by the invention contains astaxanthin, β-carotene, xenthophylls, zeaxanthin and canthaxanthin.

The invention provides grid algae ENN32 as described in the first aspect application in lipid acid is produced, preferably, described lipid acid is myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid and/or behenic acid.

In fourth aspect, the invention provides grid algae ENN32 as described in the first aspect application in biofuel is produced, preferably, described biofuel is biofuel or bio-ethanol.

Aspect the 5th, the invention provides grid algae ENN32 as described in the first aspect application in environment protection, preferably described environment protection is sewage disposal or CO ₂reduce discharging, described sewage disposal comprises denitrogenation, dephosphorization, the organic matter degradation and/or heavy metal ion adsorbed of sewage.

Aspect the 6th, the invention provides grid algae ENN32 as described in the first aspect purposes in the preparation of the medicine of myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin.

Aspect the 7th, the invention provides grid algae ENN32 as described in the first aspect purposes in the food that comprises myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin, healthcare products, feed and/or fishery products bait are produced.

In eight aspect, the invention provides grid algae ENN32 as described in the first aspect application in the makeup that comprise myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin or pigment production.

In the present invention, BG11 substratum is algae media the most frequently used in this area, and its concrete component sees below.

Useful technique effect of the present invention is:

1, this algae kind fast growth, CO ₂utilization ratio is high, and cell settlement is good, is convenient to collect, and is suitable for high-density breeding, is applicable to Industry Promotion

2, this algae kind oil and fat accumulation is fast, and oleaginousness is high, is applicable to for production biofuel

3, this algae kind can effectively be removed the nitrogen in water, is suitable for wastewater treatment

4, this algae of this algae kind kind temperature tolerance range is extensive, is applicable to annual cultivation.

Brief description of the drawings

Fig. 1 is grid algae ENN32 of the present invention microscopy picture under the microscope.

Fig. 2 shows the ITS partial sequence of grid algae ENN32 of the present invention and the result that 5.8S sequence is carried out BLAST comparison in GenBank database.

Fig. 3 is the phylogenetic evolution tree that adopts the grid algae ENN32 of connected (Neighbor-joining) algorithm structure in ortho position.

Fig. 4 is the indoor cultivation growth curve of grid algae ENN32 of the present invention.

Fig. 5 is the growth curve of grid algae ENN32 of the present invention under hot conditions.

Fig. 6 is the growth curve of grid algae ENN32 of the present invention under cold condition.

Fig. 7 is the color atlas of contained pigment in grid algae ENN32 of the present invention.

Fig. 8 is the column diagram that represents the content of each pigment in grid algae ENN32 of the present invention.

Embodiment

Further illustrate technical scheme of the present invention below in conjunction with accompanying drawing and by embodiment.

Embodiment 1 screens the strain of grid algae algae

Get the soil sample of getting from Wulanchabu League of Inner Mongolia area after sterilized water dilution, add BG11 substratum to carry out enrichment culture, after 400 power microscopes are observed, approximately cell concn is 800-1200/ml, get the algae liquid of about 1ul with kapillary, being inoculated in 48 holes is equipped with in the culture plate of BG11 substratum, be to cultivate in 50umol/m2.s situation at 25 DEG C, intensity of illumination, in the time reaching certain cell concn, microscopic examination, selection only has the hole of single algae strain, and being diluted to cell concn is to pave plate after 1000-3000/ml, obtains single algae strain.

Embodiment 2 identifies algae kind and the classification on evolutionary tree thereof

The method of taking identification of morphology and Molecular Identification to combine is identified in algae strain.

1. shape qualification (seeing Fig. 1): the above-mentioned algae strain ENN32 separating is observed at 1000 power microscopes, and frond is that unicellular form exists, and cell walls is level and smooth, unicellular ovalize.Cell major diameter 8-10 μ m, minor axis 6-8 μ m.Chromatoplast Zhousheng, sheet, one piece of tool pyrenoids, vegetative propagation produces autospore.Retrieval " CHINESE FRESHWATER algae-system, classification and ecology ", finds that it is Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlorococcale (Chlorococcales), Shan Zao section (Scenedesmaceae), Scenedesmus (Scenedesmus) that this algae is classified identical in form.

2. Molecular Identification:

A, from the ENN32 increase its ITS and the 18S partial nucleotide sequence that separate

Extract the ENN32 algae genomic dna of cultivating 4 days by CTAB method.Get a certain amount of cell, after milled processed, add the addition of C TAB damping fluid, homogenate, add isopyknic phenol: chloroform extracting, equal-volume isopropanol precipitating, is dissolved in certain volume sterilizing distilled water its concentration of UV spectrophotometer measuring and purity after 75% washing with alcohol.

ITS sequence amplification adopts eukaryote ITS amplification universal primer (primer is synthetic synthetic by Shanghai Sheng Gong bio-engineering corporation).

Primer 15 ' GGAAGTAAAAGTCGTAACAAGG 3 ' (SEQ ID NO:1)

Primer 25 ' GCATATCAATAAGCGGAGGA 3 ' (SEQ ID NO:2)

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 750bp segment.Sequence following (underscore part is primer sequence):

GGAAGTAAAAGTCGTAACAAGGGTTTTCCCCCCGCTTATTGATATGCTTAA

GTTCAGCGGGTAGCCTTGCCTGAGCTCAGGTCGAAAGTTTAGACATACCAT

AGGTATGTTCCTGCTTGGCTCCTAGCAAGATCCACAAGCAACAACTTCGTG

TAGTCGGCAGAAGCCGGTGCTACCTATCCAGTTGAAGCCCATACGGGTCC

TTGCTTAAGCCTCTGCACTTCAGCCAACCCAATCCGCAGTAAAGCAGATTG

GGGAAGCCAGATCCACCCCAATGGCCAGCTAGAGAAGCCGACACACCCA

ACAAGTTGGGAGGGGTGAGGGTGTAAACCGACGCTGAGGCAGACATGCT

CTTGCCCGAAGGCTCGAGCGCAATATGCGTTCAAAGATTCGATGGTTCACG

GAATTCTGCAATTCACACTACGTATCGCATTTCGCTGCGTTCTTCATCGTTG

CGAGAGCCAAGATATCCGTTGTTGAGAGTTGTCTTTGGTTAAGATTGCCAG

TTACTAGCAATCACAACTTCAGAGTTTAGTTTTAACAGTGGTTAGCACTGG

TGTATAGTCATGCCAAGGCGCCACTGAGCAGGCAGCAATGCCCACCCAGC

TGTGAGATGCGAGCGGGTTTTAAGCCGAACGCCCCACTGGCAAGCCAAGG

CACGGAACGTTGAGGTTCACATTGTGGTTTTAATAATTCAATGATCCCTCCG

CAGGTTCACCTACGGAGACCTTGTTTACGACTTTTACTTTCCAT TCCTCCGC

TTATTGATATGC(SEQ ID NO:3)。

B, the nucleotide sequence homology of cloning search

The ITS partial sequence of the ENN32 of acquisition and 5.8S sequence login GenBank database are carried out to BLAST comparison, result shows that having higher similarity with No. ACCESSION 18S ribosomal RNA gene and internal transcribed spacer 2sequence that is the Scenedesmus sp.Isolate pic 6/16T-1W of DQ417571.1 reaches 91%, fraction of coverage 92% (Fig. 2).

C, makes evolutionary tree according to above-mentioned sequence, sees Fig. 3.

Utilize the BLAST instrument in ncbi database to carry out sequence similarity analysis to the ITS partial sequence of algae strain ENN32.The ITS partial sequence of selected part homologous sequence and this algae strain utilizes clustaLx software package to carry out sequence homology evolution comparison, forms a multiple sequence coupling permutation matrix.Then, use Mega4.0 software, adopt connected (Neighbor-joining) algorithm in ortho position, the data set bootstraps value of bootstrapping is that 1000 constructing systems are grown evolutionary trees (seeing Fig. 3).

According to the result of form and ITS blast, restriction ENN32 is Scenedesmus (Scenedesmus).Contriver is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC is called for short in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) by described algae strain on July 13rd, 2012, and its preserving number is CGMCC No.6346.

Indoor culture and the oil and fat accumulation of embodiment 3ENN32 grid algae

The grid algae algae strain ENN 32 green swarm cells that are in logarithmic phase are seeded in the BG11 substratum (formula is in table 1) preparing, make cell density reach OD ₇₅₀between 0.2-0.8.Culturing process intensity of illumination is controlled at 50-500umol/m ².s, in incubation period, by pass into the carbonic acid gas of 1.5-2% and the mixed gas of air in nutrient solution, the pH value of substratum is adjusted between 7-9, temperature adjusting is within the scope of 15-35 DEG C.The column reactor that the reactor that cultivation is used is 40mm internal diameter, length 600mm.In culturing process, timing sampling is measured dry weight, the results are shown in Figure 4.Cultivation proceeds to the 16th day, when algae liquid its colour changed into yellow, 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.

Table 1BG11 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
A ₅Trace element *	1ml

* A ₅the moiety of trace element

Be added in 1000ml deionized water

H ₃BO ₃	2.86g
		MnCl ₂·H ₂O	1.81g
ZnSO ₄·7H ₂O	0.222g
		CuSO ₄·5H ₂O	0.079g
Na ₂MoO ₄·2H ₂O	0.390g
		Co(NO ₃) ₂·6H ₂O	0.0494g

After being dried, measure its oil component content as following table 2, analytical procedure is as follows:

1) 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.Remaining algae-residue adds 4 DEG C of lower magnetic forces of ether, normal hexane 4-8ml of 1:1 to stir extracting 1 hour again, 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.

2) 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: FID, chromatographic column: DB-5 capillary chromatographic column (30m × 0.25mm, 0.25 μ m), splitting ratio: 4:1.Analytical procedure: marker method (gas-chromatography is done carrier gas with nitrogen, is equivalent to the moving phase of liquid chromatography).

The total fat component of the sample table of table 2 gas Chromatographic Determination

The lipid acid of multiple ENN 32 algae strains extracts and analyzes experimental result and shows, in ENN 32 algae strains, the content of lipid acid is 51%～60% dry weight.

embodiment 4ENN 32 high temperature tolerances

The grid algae algae strain ENN 32 green swarm cells that are in logarithmic phase are seeded in the BG11 substratum (formula is in table 1) preparing, make cell density reach OD ₇₅₀between 0.2-0.8.In incubation period, by pass into the carbonic acid gas of 1.5-2% and the mixed gas of air in nutrient solution, the pH value of substratum is adjusted between 7-9, cultivating the reactor using is 50*50*5cm plate-type reactor.Inoculate other two kinds of green algas as a control group with above-mentioned the same terms.Indoor control high-temperature cultivation condition (40 DEG C of left and right maintain 8-10h, and all the other time normal temperature are cultivated) cultivates.In culturing process, timing sampling is measured dry weight, the results are shown in Figure 5.

embodiment 5ENN32 cold tolerance

The grid algae algae strain ENN 32 green swarm cells that are in logarithmic phase are seeded in the BG11 substratum (formula is in table 1) preparing, and making cell density reach OD750 is between 0.2-0.8.In incubation period, by pass into the carbonic acid gas of 1.5-2% and the mixed gas of air in nutrient solution, the pH value of substratum is adjusted between 7-9.Cultivating the reactor using is 50*50*5cm plate-type reactor.At sunlight booth, normal temperature group is set and cultivates, temperature is maintained at 25 DEG C of left and right.Utilize outdoor winter low temperature environment that low temperature group is set and cultivate, daytime 10 DEG C of temperature on average, the lowest temperature is below 0 DEG C.In culturing process, timing sampling is measured dry weight, the results are shown in Figure 6.

The omnidistance difference of the normal temperature group of ENN22 and low temperature group dry weight is all little, and final 15 days time, dry weight reaches 3.5g/L left and right, and 8-12h high temperature does not almost affect the growth of ENN22.But 40 DEG C of high temperature are for other algae strains, are possible cause dead temperature.

Embodiment 6ENN 32 is for nitric wastewater processing

Utilize plant effluent cultivating microalgae algae strain ENN 22, process after 5 days, the concentration of N becomes 0.009g/L from 0.21g/L, and clearance is 95.7%.Prove that algae strain ENN 22 can be for nitrogenous wastewater treatment.

Embodiment 7ENN 32 pigment contents are measured

1, the extraction of pigment in frustule: get the frustule of certain centrifugal collection, soak and extract for several times with methyl alcohol, merge methanol extract liquid, remove methyl alcohol with rotary vacuum evaporator, enriched material is used CCl again ₄dissolve, to be measured.

2, the preparation of pigment standardized solution: take a certain amount of astaxanthin, xenthophylls, zeaxanthin, canthaxanthin, β-carotene standard substance, with the standard operation liquid of methyl alcohol preparation different mass concentration.

3, upper liquid chromatography, color atlas (seeing Fig. 7) and the typical curve of drafting pigment

4, by external standard method, with pigment standard substance, quantitative Analysis goes out the content of pigment, in 1g ENN32 algae powder, astaxanthin-containing 0.15～0.65mg, xenthophylls 0.20～0.80mg, zeaxanthin 0.61～1.5mg, canthaxanthin 0.16～0.63mg, Β-carotene 1.73～5mg. (seeing Fig. 8)

Astaxanthin chemical name is 3,3 '-dihydroxyl-4, and 4 '-diketo-β, β '-carotene, can be with protein bound blue or green, blue in vivo.There is anti-oxidant, anti-ageing, antitumor, prevention cardiovascular and cerebrovascular diseases effect;

Xenthophylls has another name called " Lutein ", is the main pigment that forms human eye retina macular region, has protection eyes, delays eyes aging and prevent pathology and antioxygenation;

Zeaxanthin is the isomer of xenthophylls, has the effect of particularly important to maintaining eye health;

Canthaxanthin (food additive international numbering: INS161g) mainly, as animal feedstuff additive, also can be used for the foods such as jam, candy, syrup, sauce, carbonated beverage, as tinting material.

Β-carotene is a kind of antioxidant, there is detoxification, to safeguard the indispensable nutrient substance of HUMAN HEALTH, anticancer, preventing cardiovascular disease, cataract and anti-oxidant on have significant function, and and then prevent the aging and old and feeble multiple degenerative disorders causing.

Applicant's statement, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not mean that the present invention must rely on above-mentioned detailed features and method could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention, and the selections of the equivalence replacement to the selected condition of the present invention and the increase of accessory, concrete mode etc., within all dropping on protection scope of the present invention and open scope.

Claims

1. a grid algae ENN32 (Scenedesmus sp.), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and preservation date is on 07 13rd, 2012, and deposit number is CGMCC No.6346.

2. the cultural method of grid algae ENN32 as claimed in claim 1, described method comprises grid algae algae strain ENN32 green cell is seeded in BG11 substratum, wherein intensity of illumination 50-500 μ mol/m ².s, pH value 7-9, temperature 15-35 DEG C.

3. the cultural method of grid algae ENN32 according to claim 2, is characterized in that, described intensity of illumination is 100-450 μ mol/m ².s, pH value 7.5-8.5, temperature is 20-30 DEG C.

4. the cultural method of grid algae ENN32 according to claim 3, is characterized in that, described intensity of illumination is 150-400 μ mol/m ².s, pH value 8, temperature is 25 DEG C.

5. the cultural method of grid algae ENN32 according to claim 4, is characterized in that, described intensity of illumination is 200-350 μ mol/m ².s.

6. the application of the grid algae ENN32 as described in any one in claim 1 to 5 in lipid acid is produced, it is characterized in that, described lipid acid is myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid and/or behenic acid.

7. the application of the grid algae ENN32 as described in any one in claim 1 to 5 in biofuel is produced, is characterized in that, described biofuel is biofuel or bio-ethanol.

8. the application of the grid algae ENN32 as described in any one in claim 1 to 5 in environment protection, is characterized in that, described environment protection is sewage disposal or CO ₂reduce discharging, described sewage disposal comprises denitrogenation, dephosphorization, the organic matter degradation and/or heavy metal ion adsorbed of sewage.

9. the application in the medicine that the grid algae ENN32 as described in any one in claim 1 to 5 comprises myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin in preparation.

10. the purposes of the grid algae ENN32 as described in any one in claim 1 to 5 in the food that comprises myristic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, octadecadienoic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin, healthcare products, feed, fishery products bait, makeup and/or pigment production.