CN106092863A - A kind of method of quick mensuration microalgae lipid content - Google Patents
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- 150000002632 lipids Chemical class 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 39
- 241000195493 Cryptophyta Species 0.000 claims abstract description 36
- KAIPKTYOBMEXRR-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole Chemical compound CCCCN1CN(C)C=C1 KAIPKTYOBMEXRR-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000000684 flow cytometry Methods 0.000 claims abstract description 5
- 239000000975 dye Substances 0.000 claims description 10
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 3
- 241000206751 Chrysophyceae Species 0.000 claims description 2
- 241000199919 Phaeophyceae Species 0.000 claims description 2
- 241000206572 Rhodophyta Species 0.000 claims description 2
- 241000206764 Xanthophyceae Species 0.000 claims description 2
- 230000003834 intracellular effect Effects 0.000 abstract description 4
- 239000002028 Biomass Substances 0.000 abstract description 2
- 238000011017 operating method Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 235000014593 oils and fats Nutrition 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 5
- 240000009108 Chlorella vulgaris Species 0.000 description 4
- 235000007089 Chlorella vulgaris Nutrition 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000003225 biodiesel Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical group C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- CMXPERZAMAQXSF-UHFFFAOYSA-M sodium;1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate;1,8-dihydroxyanthracene-9,10-dione Chemical compound [Na+].O=C1C2=CC=CC(O)=C2C(=O)C2=C1C=CC=C2O.CCCCC(CC)COC(=O)CC(S([O-])(=O)=O)C(=O)OCC(CC)CCCC CMXPERZAMAQXSF-UHFFFAOYSA-M 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 208000035126 Facies Diseases 0.000 description 1
- 235000007122 Scenedesmus obliquus Nutrition 0.000 description 1
- 244000249201 Scenedesmus obliquus Species 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
A kind of method that the invention discloses quick mensuration microalgae lipid content, belongs to biomass energy technology field.The method of the present invention is to add 50 μ g/mL 1 butyl 3 Methylimidazole. villaumites and 1 μ g/mL3 PeDPP in about 1mL algae solution, uses flow cytometry analysis after temperature bath.The method of the present invention, is not required to extract intracellular lipid and can measure composition and the content of lipid, solves existing method and needs that sample amount is big, operating procedure is many and the technical problem of time-consuming length.
Description
Technical field
A kind of method that the present invention relates to quick mensuration microalgae lipid content, belongs to biomass energy technology field.
Background technology
Biodiesel is a kind of bio-fuel obtained for raw material with plant and animal fatty acid, and it possesses and petrifaction diesel
Close performance, and there is excellent environmental protection characteristic, good fuel performance and recyclability.Owing to China's cultivated area has
Limit, it is far from being enough for the most only meeting diesel consumption by plantation xylophyta.Microalgae as photoautotrophy pattern survive micro-
Biology, living environment is various, of a great variety, and it is woody that photosynthetic efficiency, the speed of growth and oil content are significantly larger than other high oil-containings
Plant.Therefore, microalgae lipid is that the novel lipid resource with bright prospects is it is considered to be the biology of the most potential petroleum replacing
Matter resource.
Measuring the traditional method of lipid content in microalgae and be mainly gravimetric method, its principle, for first to be crushed by microalgae, is then used
Non-polar solven extracts, and finally weighs and obtains lipid content.Gravimetric method needs substantial amounts of algae solution, and operation complexity.Fluidic cell
Art is Cytobiology and molecular biology, biotechnology, monoclonal antibody technique, laser technology, photoelectron physics, fluorescence chemical, stream
The crystallization that the multidisciplinary field such as mechanics, computer technology is developed jointly, can be with fluorescent dye or fluorescein coupled antibody
As auxiliary reagent, the cell of separate state or biologic grain (cell dia is 0.2-150 μm) are carried out multiparameter, quickly,
Accurately, objective quantitative analysis.
The fluorescent dye being usually used in detecting intracellular oils and fats at present in flow cytometry is Nile red.The most right
After microalgae cell dyes, with flow cytomery cell at yellow spectral range passage and red color spectrum range passage
Fluorescence intensity, the content to intracellular neutral fat and polarity oils and fats is directly proportional respectively.But, the fluorescence of red color spectrum range
Intensity is easily disturbed by other auto flourescence signals in microalgae, accordingly, it would be desirable to utilize dimethyl sulfoxide as dyeing carrier, increase
Add membrane passage and enter intracellular amount to improve Nile red, thus improve fluorescence intensity, but dimethyl sulfoxide has
Volatility and toxicity, there is certain threat in healthy to operator, frustule can be produced toxic action after adding algae solution.At present
Report the colouring method of various different condition (dimethyl sulfoxide concentration, dyeing time, dyeing temperature etc.), but still generally deposit
Incomplete at tinction, fluorescence peak is overlapping and the shortcoming such as the most poor of oils and fats absolute content corresponding relation.It is another that document is reported
A kind of dyestuff for detecting oils and fats is BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-
Bora-3a, 4a-diaza-s-indacene), it has narrower emission spectrum at 515nm, can obtain separation degree higher
Peak, but also need to also exist need to use dimethyl sulfoxide, dye incomplete shortcoming, and the background fluorescence intensity of itself is excessive
Cause sensitivity relatively low, the most not yet set up the method the best with oils and fats absolute content corresponding relation.
In sum, traditional gravimetric method also exists operation length complicated, time-consuming, cannot be distinguished by neutral lipid and polar lipid
Problem, the method based on flow cytometry of report also exists that threat operator is healthy, infringement frustule, obtained
The shortcomings such as oils and fats absolute content is inaccurate.Therefore, the side of environmental protection, fast and accurately detection lipid within endothelial cells content is set up
Method, to the great significance promoting microalgae biodiesel industry.
Summary of the invention
In order to solve the problems referred to above, a kind of method that it is an object of the invention to provide quick mensuration microalgae lipid content, it
Environmentally friendly, do not damage frustule, it is only necessary to little algae solution, simple to operate, highly sensitive, result is the most accurate.
The method measuring microalgae lipid of the present invention, is to add 1-butyl-3-Methylimidazole. villaumite in microalgae algae solution to increase
Add membrane passage, and dye with 3-perylene diphenylphosphine (3-PeDPP), then use streaming thin
Born of the same parents' instrument is analyzed.
Described method is:
(1) take 5 parts or the different microalgae algae solution of the same race of more than 5 parts lipid contents, algae solution adds 1-butyl-3-methyl
Imidazoles villaumite, and dye with 3-PeDPP；
(2) FL1 (500/50) passage using flow cytometer collects the fluorescence intensity of multiple frustules, calculates the most glimmering
Light intensity MFI；
(3) relational model of actual measurement lipid content and average fluorescent strength is set up: measure in 5 parts or more than 5 parts microalgae
Lipid content C, obtains relational model (public formula (I)) with MFI matching, obtains the concrete numerical value of a, b in model:
C=a × MFI+b public affairs formula (I)
(4) in the algae solution of microalgae of the same race to be measured, add 1-butyl-3-Methylimidazole. villaumite, and dye with 3-PeDPP,
Use the condition identical with (2), under FL1 (500/50) passage, measure the average fluorescent strength MFI obtaining multiple frustule,
MFI is substituted in public formula (I), i.e. can get the lipid content of microalgae cell to be measured.
In one embodiment of the invention, 1-butyl-3-Methylimidazole. villaumite and 3-in described (1) algae solution
The concentration of PeDPP dyestuff is respectively 50 μ g/mL and 1 μ g/mL, is placed in 15min in 40 DEG C of water-baths in darkroom.
In one embodiment of the invention, described (1) is to take microalgae 1ml algae solution to cross 300 mesh sieves and be placed on sample cell
In, add 1-butyl-3-Methylimidazole. villaumite (increase membrane passage) and 3-PeDPP (dyeing).
In one embodiment of the invention, described (1) the algae density in algae solution is 105-106/ml。
In one embodiment of the invention, in described (2), flow velocity during flow cytometry analysis is 60 μ L/min.
In one embodiment of the invention, described (2) collect the fluorescence intensity of multiple frustules, refer to collect
More than 100, preferably more than 200, more than 500, more preferably more than 1000, more than 10000 or 10000
The fluorescence intensity of above frustule.
In one embodiment of the invention, the fluorescence intensity of described (2) the middle frustule collecting more than 10000.
Collecting the most of mensuration, data reliability is the strongest.
In one embodiment of the invention, described lipid content, refer to that lipid accounts for the mass percent (ratio of algae dry weight
As surveyed the total lipid content of 100g algae, content is 1%, then lipid is exactly 1g).
In one embodiment of the invention, the value of the lipid content C in mensuration microalgae in described (3), is according to weight
Mensuration mensuration obtains, and is first crushed by microalgae, then extracts with non-polar solven, finally weighs and obtain lipid content.
In one embodiment of the invention, the value of the lipid content C in mensuration microalgae in described (3), by following side
Method is carried out: weighs corresponding algae solution dried algae powder, every kind of sample about 50mg, is transferred in centrifuge tube with cover after grinding breaking cellular wall,
Adding chloroform-methanol solution that 3ml volume ratio is 1:2, whirlpool concussion 2min, add 1ml chloroform, whirlpool shakes
After 2min, 1000r/min is centrifuged, and collects supernatant；Repeat extraction 2 times, merge supernatant；Adding volume ratio is 2:2:1.8
Chloroform-methanol-water, concussion mixing after 2000r/min be centrifuged, take off layer organic facies nitrogen and dry up to constant weight.
In one embodiment of the invention, described (4) lipid content is by the mean fluorecence of FL1 (500/50) passage
Intensity calculates.
In one embodiment of the invention, the lipid content of the microalgae in described (4) refers to the percent mass of total fat
Compare content.
Microalgae of the present invention is that cell dia is at 0.2-150 μm, the do not reunite chlorella of growth, red algae, xanthophyta, silicon
Algae, Brown algae or chrysophyceae.
Described microalgae, can be chlorella, crescent moon algae, scenedesmus obliquus etc..
Beneficial effects of the present invention:
(1) little algae solution is had only to, simple to operate, highly sensitive, convenient to operation.
(2) it is not required to use the reagent of effect toxic to human body and frustule, environmental protection.
(3) cell dyeing is abundant, and the linear relationship of fluorescence intensity and lipid content can reach 0.9438.
Accompanying drawing explanation
Fig. 1: actual measurement lipid content and the relational model of average fluorescent strength.
Detailed description of the invention
Embodiment 1:
The lipid content of detection chlorella vulgaris.Take and after 1mL algae solution crosses 300 mesh sieves, add 50 μ g/mL 1-butyl-3-first
Base imidazoles villaumite and 1 μ g/mL 3-PeDPP, be placed in 15min in 40 DEG C of water-baths in darkroom, uses flow cytometer to be analyzed.
It is strong that FL1 (500/50) passage using flow cytometer collects the fluorescence of 10000 frustules in 5 parts of algae solutions respectively
Degree, its average fluorescent strength MFI is respectively 351.0608,178.81069,404.77274,270.32026 and 94.8327；
Lipid content in 5 parts of chlorellas of gravimetric detemination is respectively 32.01%, and 27.30%, 36.58%,
30.87% and 20.31%.
Matching obtains the concrete numerical value of model parameter a, b, is 0.0469 and 17.227 respectively, and linearly dependent coefficient is
0.9704。
Therefore, the lipid content computing formula of chlorella vulgaris is as follows:
C/%=0.0469 × MFI+17.227 (A)
Identical condition, under FL1 (500/50) passage, takes chlorella vulgaris to be measured, measures and obtain multiple frustule
Average fluorescent strength is 102.49228, substitutes in formula A, and the lipid content that i.e. can get microalgae cell to be measured is 22.03%.
This content, content 24.49% deviation obtained with the detection of conventional weight method is only 2.45%, illustrates that the method for the present invention is reliable
Property is strong.
Detecting other chlorella vulgaris sample by same method, the lipid content obtained is respectively 33.69%,
25.61%, 36.21%, 29.91%, 21.67, the content deviation obtained with gravimetric method is respectively 1.68%, and 1.69%,
0.37%, 0.96%, 1.36%, illustrate that this method detection accuracy is good.
The chlorella sample that lipid content is 24.49% is carried out replication 10 times, and result standard deviation is less than 2%,
Illustrate repeatable high.
Embodiment 2:
The lipid content of detection crescent moon algae.Take and after 1mL algae solution crosses 300 mesh sieves, add 50 μ g/mL 1-butyl-3-methyl miaows
Azoles villaumite and 1 μ g/mL 3-PeDPP, be placed in 15min in 40 DEG C of water-baths in darkroom.Use the FL1 (500/50) of flow cytometer
Passage collects the fluorescence intensity of 10000 frustules in 5 parts of algae solutions respectively, and its average fluorescent strength MFI is respectively 254.745,
313.7785,223.4476,298.5478 and 275.5784；
Lipid content in 5 parts of crescent moon algaes of gravimetric detemination is respectively 13.45%, and 21.37%, 11.45%,
19.87% and 18.57%
Matching obtains the concrete numerical value of model parameter a, b, is 0.1165 and-14.891 respectively, and linearly dependent coefficient is
0.9736
Therefore, the lipid content computing formula of common crescent moon algae is as follows:
C/%=0.1165 × MFI-14.891 (A)
Identical condition, under FL1 (500/50) passage, takes crescent moon algae to be measured, measures and obtain the average of multiple frustule
Fluorescence intensity is 272.9428, substitutes in formula A, and the lipid content that i.e. can get microalgae cell to be measured is 16.91%.This contains
Amount, content 17.38% deviation obtained with the detection of conventional weight method is only 0.57%, illustrates that the method for the present invention is highly reliable.
Detecting other crescent moon algae sample by same method, the lipid content obtained is respectively 14.78%, and 21.66%,
11.14%, 19.89%, 17.21, the content deviation obtained with gravimetric method is respectively 1.33%, and 0.29%, 0.31%,
0.02%, 1.36%, illustrate that this method detection accuracy is good.
The crescent moon algae sample that lipid content is 17.38% is carried out replication 10 times, and result standard deviation is less than 2%,
Illustrate repeatable high.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (8)
1. the method for a quick mensuration microalgae lipid, it is characterised in that described method is:
(1) take 5 parts or the different microalgae algae solution of the same race of more than 5 parts lipid contents, algae solution adds 1-butyl-3-Methylimidazole.
Villaumite, and dye with 3-perylene diphenylphosphine (3-PeDPP)；
(2) FL1 (500/50) passage using flow cytometer collects the fluorescence intensity of multiple frustules, and average fluorescent strength is
MFI；
(3) set up the relational model of actual measurement lipid content C and average fluorescent strength, obtain the concrete numerical value of a, b in model:
C=a × MFI+b public affairs formula (I)；
(4) in the algae solution of microalgae of the same race to be measured, add 1-butyl-3-Methylimidazole. villaumite, and dye with 3-PeDPP, use
The condition identical with (2), under FL1 (500/50) passage, measures the average fluorescent strength MFI obtaining multiple frustule, by MFI
Substitute in public formula (I), i.e. can get the lipid content of microalgae to be measured.
The most in accordance with the method for claim 1, it is characterised in that 1-butyl-3-Methylimidazole. villaumite in the algae solution of described (1)
It is respectively 50 μ g/mL and 1 μ g/mL with the concentration of 3-PeDPP dyestuff, darkroom is placed in 15min in 40 DEG C of water-baths.
The most in accordance with the method for claim 1, it is characterised in that in described (2), flow velocity during flow cytometry analysis is 60 μ
L/min。
The most in accordance with the method for claim 1, it is characterised in that described (2) collect the fluorescence intensity of multiple frustules, is
Refer to collect more than 100, preferably more than 200, more than 500, more preferably more than 1000, more than 10000.
The most in accordance with the method for claim 1, it is characterised in that the algae density in described algae solution is 105-106/ml。
The most in accordance with the method for claim 1, it is characterised in that described (1) is to take microalgae 1ml algae solution to cross 300 mesh sieves rearmounted
In sample cell, add 1-butyl-3-Methylimidazole. villaumite and 3-PeDPP.
The most in accordance with the method for claim 1, it is characterised in that the lipid content of the microalgae in described (4) refers to total fat
Mass percentage content.
The most in accordance with the method for claim 1, it is characterised in that described microalgae is that cell dia is not in 0.2-150 μm, reunite
Chlorella, red algae, xanthophyta, diatom, Brown algae or the chrysophyceae of growth.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109797105A (en) * | 2019-02-22 | 2019-05-24 | 中国科学院水生生物研究所 | A kind of screening technique of microalgae cell wall mutant |
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2016
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| CN103163113A (en) * | 2013-03-25 | 2013-06-19 | 哈尔滨工业大学 | Method for detecting content of microalgal oil by ultrasound-assisted fluorescent staining |
| CN103555587A (en) * | 2013-11-07 | 2014-02-05 | 三亚深海科学与工程研究所 | Method of screening high grease algae from natural water body |
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Cited By (2)
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| CN109797105A (en) * | 2019-02-22 | 2019-05-24 | 中国科学院水生生物研究所 | A kind of screening technique of microalgae cell wall mutant |
| CN109797105B (en) * | 2019-02-22 | 2021-04-13 | 中国科学院水生生物研究所 | Screening method of microalgae cell wall mutant |
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