CN102453682A - Marine microalga and its culture method and application - Google Patents
Marine microalga and its culture method and application Download PDFInfo
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- CN102453682A CN102453682A CN201010515598XA CN201010515598A CN102453682A CN 102453682 A CN102453682 A CN 102453682A CN 201010515598X A CN201010515598X A CN 201010515598XA CN 201010515598 A CN201010515598 A CN 201010515598A CN 102453682 A CN102453682 A CN 102453682A
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- isochrysis
- ccmm5001
- isochrysis galbana
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- marine microalgae
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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Images
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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
- 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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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a marine microalga and its culture method and application. The marine microalga is isochrysis sp. CCMM5001, is preserved in the china center for type culture collection (CCTCC), and has a preservation number of CCTCC M2010138. The culture method comprises separating a single algae cell of isochrysis sp. CCMM5001, carrying out enlargement culture of the single algae cell under laboratory conditions, carrying out centrifugation in a fourth day after an isochrysis sp. CCMM5001 growth platform stage, and then drying to obtain isochrysis sp. CCMM5001 powder. The isochrysis sp. CCMM5001 powder can be utilized as a biodiesel raw material. The culture method of the marine microalga utilizes carbon dioxide in industrial exhaust gas thereby reducing greenhouse gas discharge, and realizes absorption of oxynitrides in industrial exhaust gas thereby reducing environmental pollution.
Description
Technical field
The present invention relates to marine microalgae, specifically a kind of marine microalgae and cultural method and application.
Background technology
Marine microalgae is the main provider of organism and energy in the marine ecology system; Compare with other raw material; Have the photosynthetic efficiency height, adaptive capacity to environment is strong, growth cycle is short, not limited by weather, itself can biochemical Synthetic Oil, and fat content is high; Be easy to realize advantages such as scale operation, so marine microalgae shows wide application prospect as the new source for preparing biodiesel fuel.Exploitation marine microalgae resource is utilized seawater, non-farming soil and sun power, with simple mineral matter nutritional and CO
2Change into grease, extraordinary application development prospect is arranged, become one of science and technology competition hot issue in the world.This research starts from the forties in last century, and German culturing microalgae is therefrom sought bio-oil materials as liquid fuel.Last century late nineteen seventies, USDOE has started one through National Renewable Energy Laboratory and has utilized the hydrobiont (little algae) of little algae production biofuel to plant plan, makes great efforts to gather through more than ten years to be separated to the little algae of 3000 strains, is stored in the University of Hawaii at present.The nineties, it is the project of earth research renewal technology plan that the Japan internationality trade and the Department of Industry have subsidized a key name, filters out many strains and tolerates high CO
2Concentration, high temperature, fast growth, the algae kind of high-cell density be can form, the technology platform of photosynthetic organism reactor drum and the technical scheme of little algal biomass energy development set up.Since the nineties, Israel's sand desert survey research show that all kinds of lipid acid of a kind of snowfield green alga accumulation can account for 50% of dried cell weight, wherein unsaturated fatty acids accounts for 80% of total fatty acids.U.S.'s the nineties has used modern biotechnology to develop the little ring of the little algae of transgenic oceanographic engineering algae; Lipid content surpasses 60% of dry weight under the laboratory condition; Outdoor cultivation lipid output can reach more than 40% of dry weight, and theoretical calculate is produced 1～2.5 ton of diesel oil per year for every mu.2006, US Congress's Helios (TAIYANGSHEN) plan of 400,000,000 dollars of supports from sunlight to fuel of agreeing to furnish funds for, task was to realize the industriallization of algae produce oil by 2010, and following target of producing the MMbbl bio-crude oil every day.
Domestic professors Wu Qingyu of Tsing-Hua University etc. serve as experiment algae kind with the green algate of fresh water chlorella; The Cell culture that takes the lead in technology (heterotrophic fermentation technology) is controlled supply organic and inorganic carbon, nitrogen; Obtain the heterotrophism chlorella of chlorophyll disappearance, cell variable color; Compare with the chlorella of nourishing one's nature certainly of unconverted; The crude fat content of heterotrophism frustule has improved more than 4 times, but also exists because of using the organic carbon source glucose amount to occur the problem of cost greatly, explains if improve the fat content of alga cells and just need research and propose the marine microalgae resource of cheap high oil content and more advanced technological method.Domestic biomass energy research contents mostly stresses the screening at biodiesel raw material, on the distribution of oil plant, selection, cultivation, genetic improvement and complete processing thereof and the equipment, does not relate to the marine microalgae biomass energy.
Summary of the invention
The object of the invention is to provide a kind of marine microalgae and cultural method and application.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of marine microalgae: marine microalgae is Isochrysis galbana CCMM5001, and Isochrysis galbana CCMM5001 is preserved in Chinese typical culture collection center (CCTCC) on June 7th, 2010, and preserving number is CCTCC NO:M2010138.
(Isochrysis sp. CCMM5001) is incubated in the seawater that adds the L1 substratum, and culture temperature is 20 ± 1 ℃ with Isochrysis galbana; Light To Dark Ratio 12h: 12h, intensity of illumination 3000-4000Lux cultivated 5-7 days; The gained frustule is transferred to carries out enlarged culturing in the L1 substratum, culture temperature is 20 ± 1 ℃, Light To Dark Ratio 12h: 12h; Intensity of illumination 3000-4000Lux, treat after the plateau of Isochrysis galbana growth the 4th day centrifugal, be drying to obtain and cultivate back Isochrysis galbana algae powder.
The seawater of said interpolation L1 substratum is that interpolation L1 nutritive salt makes its salinity 30 with the equal high-temperature sterilization behind 0.45 μ m Fibrous membrane filtration of seawater.
The application of marine microalgae: said Isochrysis galbana can be used as the raw material of biofuel.
The present invention has following advantage:
Marine microalgae provided by the present invention can be used as the raw material of production biofuel; The cultivation of marine microalgae of the present invention is the carbonic acid gas that utilizes in the industrial gaseous waste; Alleviate the discharging of greenhouse gases, also can absorb the oxynitrides in the industrial gaseous waste, reduce environmental pollution; Its two, the productive rate of little algae unit surface exceeds tens of times of higher plants; Once more, little algae does not have organ differentiation such as higher plant rhizome leaf, and under conditions such as nitrogen stress, some unicellular little algae can accumulate grease in a large number, and oleaginousness can be up to 30-70%; Its three, little algae beach capable of using, saltings, desert are carried out large scale culturing, seawater capable of using, salt buck and Desert Area underground water are cultivated, therefore little algae can not strive ground with farm crop, strives water.
Description of drawings
Fig. 1 drips distribution plan for CCMM5001 fat under the Electronic Speculum of the present invention.
Embodiment
Embodiment 1
Marine microalgae is Isochrysis galbana (Isochrysis sp.CCMM5001), is preserved in Chinese typical culture collection center (CCTCC) on June 7th, 2010, and preserving number is CCTCCNO:M2010138.
Isochrysis galbana (Isochrysis sp.CCMM5001) belongs to Chrysophyta (chryspohyta) on little algae taxonomy, Chrysophyceae (chrysophyceae); Isochrysis galbana order (Iochysridales), Isochrysis galbana section (Isochysidacecae), Isochrysis galbana belongs to (Ischrysis Parke); Isochrysis galbana is exposed motor cell, and its cell shape is changeable, generally is oblong or ellipse; Juvenile cell has a slightly flat dorsal ventral side, so norma lateralis is an oblong.The long 5-6 μ of general activity cell m, wide 2-4 μ m, thick 2.2-3 μ m, sporangiocyst diameter 5-6 μ m does not have cell walls, and two flagellums are isometric, and from the cell front end, its length is 1-2 times of cell.Usually cell movement is slow, approximately with the speed rotation of per second one rotation with circle round.When cell was static, flagellum was tetanic and fall down from health, but when motion, and flagellum is towards the place ahead of cell, and carried out the motion of fluctuation formula.
The cultural method of marine microalgae chrysophyceae:
At first, and the method for utilizing kapillary to choose to leave (Chen Mingyao, biological feed is cultivated, Beijing: Chinese agriculture press, 2001,71-73; G.M.Hallegraeff, D.M.Anderson, A.D.Cembella; H.O.Enevoldsen.Manual on Harmtul Marine Microalgae.Intergovernmental Oceanographic Commission.1995; 45-50), isolate Isochrysis galbana (Isochrysis sp., single algae cell CCMM5001); In the seawater that adds the L1 substratum, cultivate, petridish is 96 orifice plates.The algal species cultivation temperature is 20 ± 1 ℃, Light To Dark Ratio 12h: 12h, intensity of illumination 3000-4000Lux.A clone clon that obtains is transferred in the larger container and cultivates after one week of cultivation, carries out enlarged culturing.
L1 substratum (Guillard and Hargraves; 1993.R.R.L.Guillard and P.E.Hargraves; Stichochrysis immobilis is a diatom, not a chrysophyte.Phycologia 32 (1993) pp.234-236.) comprises (1) nitrogen (NaNO
3, mother liquid concentration is 75g/L, being made into nutrient solution is 1ml NaNO
3Mother liquor: the 1L seawater of sterilizing); (2) phosphorus (NaH
2PO
4H
2The O mother liquid concentration is 5g/L, and being made into nutrient solution is 1ml NaH
2PO
4H
2O mother liquor: the 1L seawater of sterilizing); (3) micro-metals (L1 trace metal solution is made into nutrient solution and is 1ml L1 trace metal solution mother liquor: the 1L seawater of sterilizing); ((4)) VITAMINs (f/2 vitamin solution is made into nutrient solution and is 0.5ml f/2 vitamin solution mother liquor: the 1L seawater of sterilizing).
L1Medium
(Guillard?and?Hargraves?1993)
To?950mL?filtered?seawater?add：
L1Trace?Metal?Solution
(Guillard?and?Hargraves?1993)
To?950mL?dH
2O?add：
Make?final?volume?up?to?l?L?with?filtered?seawater.
f/2Vitamin?Solution
(Guillard&Ryther?1962，Guillard?1975)
To?950mL?dH
2O?add：
Secondly, carry out enlarged culturing.At volume is to cultivate above-mentioned purebred chrysophyceae 4000mL in the wide-necked bottle of 5000mL, and algae kind inoculum size is for adding 10% of L1 substratum seawater amount.Incubation time is one month.In the last week of cultivating, afterwards because the increasing of frustule quantity adopt with blood counting chamber counting, draw the growth curve of chrysophyceae with plant plankton tally counting every day.Know by the chrysophyceae growth curve of drawing, through 3 days lag phase, chrysophyceae grow into exponential phase of growth, reached plateau on the 24th day, and collected algae mud at the 4th day that gets into plateau: with nutrient solution with 3000rmin
-1, centrifugal 15min, remove supernatant after, distilled water wash deposition frond twice is once more with 3000rmin
-1, centrifugal 15min removes supernatant, collects algae mud, and vacuum lyophilization is subsequent use in-20 ℃ of preservations afterwards.
Embodiment 2
The analysis of total fat percentage composition:
Get the cryodesiccated algae powder of above-mentioned gained Isochrysis galbana take by weighing certainweight (0.5～1g), add zero(ppm) water.Be placed on-20 ℃ down freezing, then in 40 ℃ of water-baths, dissolve 5min, freezing repeatedly, dissolving 2 times, microscopically is observed broken situation (referring to Fig. 1).The dissolving back adds the 10ml volume ratio in algae liquid be 1: 2 the anhydrous diethyl ether and the mixed solvent of sherwood oil, adds the KOH deposition frustule of 5ml massfraction 10% behind the concussion mixing again, then at 4000rmin
-1Centrifugal 15min gets supernatant under the condition.60 ℃ of water-baths boil off unnecessary solvent rapidly to constant weight, and weighing gets thick fat content, calculate total fat percentage composition, (referring to table 1) with following formula.
Total fat percentage composition=(total fat/dried algae is heavy) * 100%
Table 1 chrysophyceae CCMM5001 total lipid content
Gained result of the present invention be the thick fat content of Isochrysis galbana CCMM5001 up to 45%, the highest with other types of prior art microalgae grease content, wherein at Sun Liqin (Sun Liqin, Yang Lintao; Environmental factor is to the influence of Isochrysis galbana fatty acid content and composition, food research and development, 2006,27 (5): 11-13) within the 8.5%-46.3% scope of report; Be higher than (Jiang Xiamin, Zheng Yizhou, 14 kinds of little algae total lipid contents and lipid acid composition research such as Jiang Xiamin; The hydrobiont journal, 2003,27 (3): 243-247) survey (Wang Ming such as thick fat content and Wang Ming of 4 strain chrysophyceae 25%-33%; Liu Ran, Xu Nin, Li Aifen; The fatty acid compositional analysis of the little algae of Duan Shunshan .13 kind. ecological science, 2006,25 (6): 542～544) the thick fat content of the Isochrysis galbana of surveying 19.3%.
Embodiment 3
The analysis of chrysophyceae fatty acid component
With adding 5mL 0.4molL in the embodiment 2 dry above-mentioned thick fat samples
-1KOH-CH
3OH solution, 60 ℃ of heating in water bath saponification 2h are until the oil droplet completely dissolve.After the cooling, move in the centrifuge tube, (9: 1=commodity concentrated hydrochloric acid: zero(ppm) water) regulate pH to 1-2, add 1mL 5%NaCl solution again, then using the 6ml volume ratio is 1: 4 CHCl to add hydrochloric acid
3-C
6H
14Organic phase is got in the mixing solutions extraction.Adding 5mL massfraction is 14% BF in organic phase
3-CH
360 ℃ of esterification 1h of OH solution.Nitrogen dries up, and 2mL normal hexane constant volume is got 1mL solution in last appearance bottle, carries out GC/MS and detects (referring to table 2).Laboratory apparatus is Agilent 7890N gas chromatograph-975C GC-MS (GC/MS).Chromatographic column is: HP-5MS, and (30m * 0.25mm 20 * 0.25um); Gc operational condition: 250 ℃ of vaporizer temperature; 280 ℃ of transmission line temperature; The chromatographic column heating schedule: 150 ℃ of initial temperature rise to 280 ℃ with 10 ℃ of min-1, and keep 5min; Input mode: split stream sampling, splitting ratio are 100: 1; Sample size: 1 μ L.Retrieval NIST, the mass spectrum picture library is confirmed lipid acid kind in the sample, the method for definite employing area normalization of each lipid acid relative content is calculated.
Said KOH-CH
3OH is that KOH is dissolved in CH
3Among the OH, KOH is at CH
3Molar density among the OH is 0.4molL
-1Said BF
3-CH
3OH is BF
3Be dissolved in CH
3In the OH solution, its volume(tric)fraction is 14%.
The main lipid acid of table 2 CCMM5001 chrysophyceae algae strain is formed
Gained result of the present invention has comprised C basically for CCMM5001
14:0, C
16:0, C
18:1, C
18:4And C
22:5In lipid acid, and content is all higher, C
16And C
18Be content up to 85.27%, C wherein
16:0, C
16:1, C
18:1Content abundant, meet the requirement for preparing biofuel from molecular structure analysis.The grease composition of most of little algaes is mainly triglyceride level (>=80%) and C
14-C
22Longer chain fatty acid (Metting F B, Biodiversity and application of microalgae, J Inorg Biochem, 1996,17 (5-6): 477-489), wherein lipid acid is with C
16With C
18Be that lipid acid is main (Guil-Guerrero etc., 2004), PUFAs is the main ingredient of unsaturated fatty acids; Palmitinic acid is main ingredient (the Harwood J L of sfas; Guschina I A, The versatility of algae and their lipid metabolism, Biochimie; 2009,91:679-684.).
Claims (4)
1. marine microalgae, it is characterized in that: marine microalgae is Isochrysis galbana (Isochrysis sp.CCMM5001), is preserved in Chinese typical culture collection center (CCTCC) on June 7th, 2010, and preserving number is CCTCC NO:M2010138.
2. cultural method by the said marine microalgae of claim 1, it is characterized in that: (Isochrysis sp. CCMM5001) is incubated in the seawater that adds the L1 substratum with Isochrysis galbana; Culture temperature is 20 ± 1 ℃, Light To Dark Ratio 12h: 12h, intensity of illumination 3000-4000Lux; Cultivated 5-7 days; The gained frustule is transferred to carries out enlarged culturing in the L1 substratum, culture temperature is 20 ± 1 ℃, Light To Dark Ratio 12h: 12h; Intensity of illumination 3000-4000Lux, treat after the plateau of Isochrysis galbana growth the 4th day centrifugal, be drying to obtain and cultivate back Isochrysis galbana algae powder.
3. by the cultural method of claim 1 or 2 described marine microalgaes, it is characterized in that: the seawater of said interpolation L1 substratum is that interpolation L1 nutritive salt makes its salinity 30 with the equal high-temperature sterilization behind 0.45 μ m Fibrous membrane filtration of seawater.
4. application by the described marine microalgae of claim 1, it is characterized in that: said Isochrysis galbana can be used as the raw material of biofuel.
Priority Applications (1)
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| CN103966100A (en) * | 2014-05-26 | 2014-08-06 | 临沂大学 | Culture medium and culture method for culturing isochrysis galbana and heterogloea sp. by use of slaughtering plant wastewater |
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