CN108588171A - A method of screening high yield biodiesel microalgae using Nile red - Google Patents
A method of screening high yield biodiesel microalgae using Nile red Download PDFInfo
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- CN108588171A CN108588171A CN201810566163.4A CN201810566163A CN108588171A CN 108588171 A CN108588171 A CN 108588171A CN 201810566163 A CN201810566163 A CN 201810566163A CN 108588171 A CN108588171 A CN 108588171A
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- microalgae
- nile red
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- algae solution
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/405—Assays involving biological materials from specific organisms or of a specific nature from algae
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Abstract
Disclosed by the invention to belong to microalgae screening technique field, specially a kind of method for screening high yield biodiesel microalgae using Nile red, this is included the following steps using the method that Nile red screens high yield biodiesel microalgae：S1：By known OD680nmLogarithmic phase latter stage microalgae algae solution be added 96 hole black ELISA Plates in multi-function microplate reader read algae solution initial fluorescence value X0；S2：Nile red dye is configured to Nile red dye liquor with acetone, Nile red dye liquor is added in the microalgae algae solution in step S1, mixing surveys its fluorescence intensity X after being incubated in the dark；S3：According to formula Y=(X X0)/OD680Calculate opposite fat content, OD in formula680For a kind of method for screening high yield biodiesel microalgae using Nile red that the absorbance of algae solution under 680nm, the invention propose, the screening efficiency of high yield biodiesel microalgae is improved, screening technique is simplified, breakneck acceleration is fast, easy to operation, and screening cost is cheap.
Description
Technical field
It is specially a kind of to screen high yield biodiesel microalgae using Nile red the present invention relates to microalgae screening technique field
Method.
Background technology
With the development of economy, requirement of the mankind to the energy is higher and higher, and microalgae is considered as preparing biodiesel fuel
Desirable feedstock.Algae has many advantages, such as that photosynthetic efficiency height, zero net carbon value, easy culture, growth cycle is short, petroleum hydrocarbon content is high, and
Ground is not striven with agriculture, grain is not striven with people, global energy distribution is not influenced, is used as bioenergy, has caused national governments and scholar
Highest attention.Microalgae prepare biodiesel research technique include the screening of oil-producing microalgae, microalgae culture system, microalgae receipts
Collect the processes such as separation, the extraction of algae oil, microalgae grease preparing biological diesel oil.The measurement of wherein microalgae grease content is research and opens
The key link for sending out oil-producing microalgae high.Traditional weight method come measure algae content needs frustule amount is big and processing procedure is multiple
It is miscellaneous cumbersome, it is bound to expend a large amount of space and time, measurement data is inaccurate, and screening efficiency is low.For this purpose, it is proposed that a kind of
The method for screening high yield biodiesel microalgae using Nile red.
Invention content
The purpose of the present invention is to provide a kind of methods for screening high yield biodiesel microalgae using Nile red, in solution
Stating traditional weight method for being proposed in background technology, the frustule amount of algae content needs is big and complex disposal process is numerous to measure
It is trivial, it is bound to expend a large amount of space and time, measurement data is inaccurate, the low problem of screening efficiency.
To achieve the above object, the present invention provides the following technical solutions：It is a kind of to screen high yield biodiesel using Nile red
The method of microalgae, this is included the following steps using the method that Nile red screens high yield biodiesel microalgae：
S1：By known OD680nmThe microalgae algae solution in logarithmic phase latter stage be added in 96 hole black ELISA Plates with multifunctional enzyme mark
Instrument reads the initial fluorescence value X of algae solution0；
S2：Nile red dye is configured to Nile red dye liquor with acetone, the microalgae in step S1 is added in Nile red dye liquor
In algae solution, mixing surveys its fluorescence intensity X after being incubated in the dark；
S3：According to formula Y=(X-X0)/OD680Calculate opposite fat content, OD in formula680For the extinction of algae solution under 680nm
Degree.
Preferably, a concentration of 0.025mg/ml-0.4mg/ml of the Nile red dye liquor.
Preferably, the dyeing time of the Nile red dye liquor is 5min-25min.
Preferably, 25 DEG C -45 DEG C of the dyeing temperature of the Nile red dye liquor.
Preferably, the microalgae algae solution OD680nmExtension rate be 0.1-0.8.
Compared with prior art, the beneficial effects of the invention are as follows：The a kind of of invention proposition screens high yield using Nile red
The method of biodiesel microalgae improves the screening efficiency of high yield biodiesel microalgae, simplifies screening technique, breakneck acceleration
Soon, easy to operation, screening cost is cheap.
Description of the drawings
Fig. 1 is the line chart that Nile red dye concentration of the present invention influences coloration result；
Fig. 2 is the line chart of influence of the dyeing time of the present invention to coloration result；
Fig. 3 is the line chart of influence of the dyeing temperature of the present invention to coloration result；
Fig. 4 is the line chart of influence of the algae solution concentration of the present invention to coloration result.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The present invention provides a kind of technical solution：A method of screening high yield biodiesel microalgae, the profit using Nile red
Included the following steps with the method that Nile red screens high yield biodiesel microalgae：
S1：By known OD680nmThe microalgae algae solution in logarithmic phase latter stage be added in 96 hole black ELISA Plates with multifunctional enzyme mark
Instrument reads the initial fluorescence value X of algae solution0；
S2：Nile red dye is configured to Nile red dye liquor with acetone, the microalgae in step S1 is added in Nile red dye liquor
In algae solution, mixing surveys its fluorescence intensity X after being incubated in the dark；
S3：According to formula Y=(X-X0)/OD680Calculate opposite fat content, OD in formula680For the extinction of algae solution under 680nm
Degree.
Wherein, a concentration of 0.025mg/ml-0.4mg/ml of the Nile red dye liquor, when the dyeing of the Nile red dye liquor
Between be 5min-25min, 25 DEG C -45 DEG C of the dyeing temperature of the Nile red dye liquor, the microalgae algae solution OD680nmExtension rate
For 0.1-0.8.
Nile red fluorescence colour condition optimizing
Referring to Fig. 1, the concentration of Nile red dye liquor is in the range of 0.025ug/ml to 0.2ug/ml, six plants of algaes of selection
The tendency of the relative intensity of fluorescence of kind is roughly the same, and most of in 0.05ug/ml or 0.1ug/ml, relative intensity of fluorescence reaches
To maximum, it is contemplated that the relative intensity of fluorescence of 0.05ug/ml and 0.1ug/ml is not much different, and saves dyestuff and determine to choose
Dye strengths of the 0.05ug/ml as follow-up high-volume screening.
Referring to Fig. 2, the dyeing time of Nile red dye liquor is basic for the tendency of the relative intensity of fluorescence of six kinds of frustules
Unanimously, relative intensity of fluorescence has raised trend in 5min to 10min, but more than the situation slowly declined is presented after 10min, because
And it determines to choose dyeing times of the 10min as follow-up high-volume screening.
Referring to Fig. 3, in the range of 25-45 DEG C, the dyeing temperature of Nile red dye liquor is opposite for six kinds of frustules
The influence of fluorescence intensity is not highly significant, and tendency is also roughly the same, chooses 35 DEG C as the dyeing temperature subsequently in high volume screened
Degree.
Referring to Fig. 4, influence of the cell density of six kinds of microalgaes to coloration result, in algae solution concentration in OD680nmIt is in
In the range of 0.1-0.4, there are good linear relationship (each R with the concentration of its algae solution for the relative intensity of fluorescence of each seeds algae strain2
It can reach 0.99 or more).
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of method for screening high yield biodiesel microalgae using Nile red, it is characterised in that：This is screened high using Nile red
The method of generation diesel oil microalgae includes the following steps：
S1：By known OD680nmLogarithmic phase latter stage microalgae algae solution be added 96 hole black ELISA Plates in read with multi-function microplate reader
Take the initial fluorescence value X of algae solution0；
S2：Nile red dye is configured to Nile red dye liquor with acetone, the microalgae algae solution in step S1 is added in Nile red dye liquor
In, mixing surveys its fluorescence intensity X after being incubated in the dark；
S3：According to formula Y=(X-X0)/OD680Calculate opposite fat content, OD in formula680For the absorbance of algae solution under 680nm.
2. a kind of method for screening high yield biodiesel microalgae using Nile red according to claim 1, it is characterised in that：
A concentration of 0.025mg/ml-0.4mg/ml of the Nile red dye liquor.
3. a kind of method for screening high yield biodiesel microalgae using Nile red according to claim 1, it is characterised in that：
The dyeing time of the Nile red dye liquor is 5min-25min.
4. a kind of method for screening high yield biodiesel microalgae using Nile red according to claim 1, it is characterised in that：
25 DEG C -45 DEG C of the dyeing temperature of the Nile red dye liquor.
5. a kind of method for screening high yield biodiesel microalgae using Nile red according to claim 1, it is characterised in that：
The microalgae algae solution OD680nmExtension rate be 0.1-0.8.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810566163.4A CN108588171A (en) | 2018-06-04 | 2018-06-04 | A method of screening high yield biodiesel microalgae using Nile red |
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| CN201810566163.4A CN108588171A (en) | 2018-06-04 | 2018-06-04 | A method of screening high yield biodiesel microalgae using Nile red |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090298159A1 (en) * | 2008-05-27 | 2009-12-03 | Tsinghua University | Method for producing biodiesel from an alga |
| CN102033059A (en) * | 2010-11-29 | 2011-04-27 | 哈尔滨工业大学 | Method for detecting microalgae oil |
| CN103808697A (en) * | 2012-11-08 | 2014-05-21 | 中国科学院大连化学物理研究所 | Method using BODIPY (4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene) fluorescent dye for determination of fat content in microalgae |
-
2018
- 2018-06-04 CN CN201810566163.4A patent/CN108588171A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090298159A1 (en) * | 2008-05-27 | 2009-12-03 | Tsinghua University | Method for producing biodiesel from an alga |
| CN102033059A (en) * | 2010-11-29 | 2011-04-27 | 哈尔滨工业大学 | Method for detecting microalgae oil |
| CN103808697A (en) * | 2012-11-08 | 2014-05-21 | 中国科学院大连化学物理研究所 | Method using BODIPY (4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene) fluorescent dye for determination of fat content in microalgae |
Non-Patent Citations (2)
| Title |
|---|
| JUDITH RUMIN ET AL.: ""The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae"", 《BIOTECHNOLOGY FO BIOFUELS》 * |
| 王金娜等: ""产油微藻的筛选及中性脂动态积累过程的检测"", 《生物物理学报》 * |
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Application publication date: 20180928 |