CN104568881B - Method for screening microalgae unicells which grow fast and are high in grease content through fluorescence microscope - Google Patents
Method for screening microalgae unicells which grow fast and are high in grease content through fluorescence microscope Download PDFInfo
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 12
- 239000013535 sea water Substances 0.000 claims description 12
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 12
- 239000007850 fluorescent dye Substances 0.000 claims description 9
- 230000003834 intracellular effect Effects 0.000 claims description 9
- 235000013619 trace mineral Nutrition 0.000 claims description 9
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- 238000000799 fluorescence microscopy Methods 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
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- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 6
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- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 6
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a biomass energy utilization technology, and aims to provide a method for screening microalgae unicells which grow fast and are high in grease content through a fluorescence microscope. The method for screening the microalgae unicells which grow fast and are high in grease content through the fluorescence microscope includes the following steps: taking a microalgae liquid for unicell separation; enlarging the cultivation to obtain a liquid containing a plurality of unicellular microalgae strains; then inoculating the unicellular microalgae liquid to 96-mesh plates for cultivation until the stable phase; screening the superior microalgae unicells with a high growth rate and dying using Nile red fluorochrome/DMSO; calculating the grease content of the microalgae unicells, so as to screen the superior microalgae unicells with a high grease content. According to the invention, superior algal strains which grow fast and are high grease content can be quickly screened from the algal cells on the 96-well plates in about 10 minutes, which greatly improves the working efficiency of screening target algal strains and reduces labor time and cost.
Description
Technical field
The present invention is with regard to biomass energy utilization technologies, grows microalgae fast and that oils and fatss are high particularly to fluorescence microscopy screening
Single celled method.
Background technology
Microalgae is rapid to Solar use efficiency high, growth and breeding, high to the adaptable, grease yield of environment.According to
The photosynthesis transformation efficiency theoretical value of report microalgae up to 10%, in unit area, produce up to Petiolus Trachycarpi oil by annual grease yield
8～24 times (Hu et al., 2008) of amount.Chisti (Chisti, 2007) founding mathematical models are calculated microalgae biology bavin
Oil is the important selection replacing petrochemical industry oil, and Cheng et al. finds that inducement of nuclear radiation can significantly improve the growth rate of chlorella
With biomass density (Cheng et al., 2013a).Khozin et al. reviews gene in diatom oil synthesis under the conditions of nitrogen stress
Expression (Khozin-Goldberg and Cohen, 2011), Mock et al. (Mock et al., 2008) find lack
Under the conditions of silicon, diatom Thalassiosira pseudonana has 75 pairs of gene inductions.But micro algae growth speed and its enrichment oil
Exist conflicting between fat, when most microalgae reach highest growth rate, in its cell fat content often than
Relatively low；And the macroscopic conditions (temperature, illumination, pH, nitrogen source, carbon source etc.) of manual control cell growth make oils and fatss in microalgae cell contain
When amount increases, the growth rate of its cell and microalgae then can substantially reduce for the transformation efficiency of solar energy.Some masters external
The germplasm innovation of high oil-producing algae strain is all classified as the top priority in whole microalgae oil-producing system engineering by the research and development institution wanting, therefore such as
How to obtain the advantage algae kind that growth rate is fast, fat content is high is key issue.
The method of traditional measurement micro algae growth speed be frustule is inoculated after the amplification culture a few days, then filtering means dehydration and
Dry the biomass density in units of measurement volume algae solution.But the method wastes time and energy relatively costly, especially for a large amount of
During thousands of algae kind mutants after screening core mutation, often it is difficult to because workload is excessively huge to complete whole screenings appoint
Business.For example by rhombus algae solution (containing frustule about 6.7 × 105Individual/mL) carry out inducement of nuclear radiation under the gamma-rays of 100Gy,
Because frustule fatality rate is about 60%, therefore produce 2.7 × 10 to I haven't seen you for ages5Individual/mL is mutated unicellular live body.Using fluorescence microscopy
The Electronic Control object stage new technique of mirror automatic shift focusing and supporting CCD camera are thin to a large amount of algaes under white light conditions
Born of the same parents carry out automatically scanning shooting, are then hopeful to filter out growth at a high speed by distinguishing that frustule color carries out quantitative data analysis
The fast frustule of speed, and the research report with regard to this respect is also fewer at present.
Nile red dyeing proposes (Greenspan using the intracellular fat content of fluorogenic quantitative detection earliest by Greenspan
Et al., 1985), the accuracy of the method be proved in multiple biologies (Genicot et al., 2005；Huang et
Al., 2009), can replace traditional weight method that intracellular fat content is monitored.Because Nile red occurs in water quickly
Fluorescent quenching, unnecessary dyestuff need not be removed, and compares with dyestuffs such as sudan black, Nile blue-A, and Nile red can exactly will be thin
Intracellular lipid material and other repertorys make a distinction (Kranz et al., 1997), are often used in detection animal and micro-
Oils and fatss situation in biological cell.Therefore under fluorescence microscope, after being combined with lubricant component, send the characteristic of fluorescence using Nile red,
Set up the fat content that a set of utilization fluorescence microscopy measures in frustule growth course, be a kind of Rapid Screening high oil-producing algae kind
Efficiently feasible method.
Content of the invention
Present invention is primarily targeted at overcoming deficiency of the prior art, provide a kind of fluorescence microscopy screening growth fast and
The single celled method of the high microalgae of oils and fatss.For solving above-mentioned technical problem, the solution of the present invention is：
The single celled method of microalgae that fluorescence microscopy screening growth is fast and oils and fatss are high is provided, specifically includes following step：
(1) take 5 μ L microalgae liquid inoculations to carry out unicellular separation to solid plate culture medium, frustule is taken out and is inoculated into
20mL culture medium, puts into triangular flask amplification culture, obtains many unicellular algae solutions of strain；
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 8～9, in 20～30 DEG C of constant temperature, illumination
Cultivate to stable phase under the conditions of intensity 5000～7000lux, illumination 24h；96 orifice plates are placed in fluorescence microscope automatic shift pair
On burnt Electronic Control object stage, thin to the algae in hole every in 96 orifice plates under white light conditions using the supporting CCD camera of microscope
Born of the same parents carry out automatically scanning shooting, process the face to every hole frustule for the MIcrosope image by shooting for the software using MIcrosope image
Color carries out quantitative data analysis, the advantage microalgae filtering out growth rate fast (growth rate >=0.4g/L d) unicellular (by
When algae cell density is bigger, color is more deep, shows that growth rate is faster such that it is able to it is micro- to filter out the fast advantage of growth rate
Algae is unicellular)；
(3) with the Nile red fluorescent dye Nile/DMSO of 0.1g/L, screening in step (2) is obtained with fast micro- of growth rate
Algae is unicellular to be dyeed, and Nile red fluorescent dye final concentration reaches 0.1mg/L, dyeing time 7min；Dyeing frustule is put
On the object stage of fluorescence microscope, combine image processing software under EX 450-490nm wavelength and observe the glimmering of measurement frustule
Light intensity, the area of all frustules and the area of intracellular all oil droplets in the measurement visual field, the oils and fatss calculating frustule contain
Amount, thus the advantage microalgae filtering out fat content height (fat content >=40%) is unicellular；
The computational methods of fat content are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor oils and fatss in cell
Grain area summation (μm2).
In the present invention, the component of described culture medium and proportion relation are：0.15g NaHCO3、0.02g KH2PO4、0.027g
VB1、1.5×10-6g VB12、0.2g Na2SiO3·9H2O、1.0g NaNO3, 0.0005g Biotin, 1mL trace element and
980mL artificial seawater.
In the present invention, the component of described trace element and proportion relation are：4.35g is contained in 1000mL distilled water
Na2EDTA、7.3mg Na2MoO4·2H2O、12mgCoCl2·6H2O、3.9g FeC6H5O7·5H2O、10mg CuSO4·5H2O、
23mg ZnSO4、178mg MnCl2·4H2O and 600mg H3BO3.
In the present invention, the component of described artificial seawater and proportion relation are (salinity=3.0%)：Contain in 1000mL distilled water
There are 21.2157g NaCl, 3.407g Na2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、1.3044g CaCl2、
0.0862g KBr、0.0226g H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
In the present invention, the algae kind in described microalgae liquid be following any one：The sky obtaining is screened from natural environment
Algae kind mutant or the transgenic algae kind obtaining through improvement of genes that so algae kind, physics and chemistry behavior obtain.
Compared with prior art, the invention has the beneficial effects as follows：
Using Electronic Control object stage and the Nile red staining technique of the focusing of fluorescence microscope automatic shift, in white light conditions
The color data of lower quantitative analyses frustule filters out the fast frustule of growth rate, then the fluorescence according to frustule and oil droplet
Ionization meter calculates fat content such that it is able to go out growth rate in 10 minutes about the frustule Rapid Screenings to 96 orifice plates
Hurry up (>=0.4g/L d) and fat content height (>=40%) advantage algae strain, substantially increase screening target algae strain work effect
Rate, decreases cost in labor time.
Brief description
Fig. 1 is the process chart of the present invention.
Specific embodiment
With specific embodiment, the present invention is described in further detail below in conjunction with the accompanying drawings：
A kind of single celled method of microalgae that fluorescence microscopy screening growth is fast and oils and fatss are high as shown in Figure 1, specifically includes down
State step：
(1) 5 μ L microalgae liquid inoculation to solid plate culture medium are taken to carry out unicellular separation, the algae kind of described microalgae liquid
Including screening the natural algae kind obtaining from natural environment, algae kind mutant that physics and chemistry behavior obtains, through improvement of genes
The transgenic algae kind obtaining.Frustule is taken out and is inoculated into 20mL culture medium, put into triangular flask amplification culture.
The component of described culture medium and proportion relation are：0.15g NaHCO3、0.02g KH2PO4、0.027g VB1、1.5
×10-6g VB12、0.2g Na2SiO3·9H2O、1.0g NaNO3, 0.0005g Biotin, 1mL trace element and 980mL artificial
Sea water.Described trace element component and proportion relation are：4.35g Na is contained in 1000mL distilled water2EDTA、7.3mg
Na2MoO4·2H2O、12mgCoCl2·6H2O、3.9g FeC6H5O7·5H2O、10mg CuSO4·5H2O、23mg ZnSO4、
178mg MnCl2·4H2O and 600mg H3BO3.Described artificial seawater formula (salinity=3.0%) is：In 1000mL distilled water
Containing 21.2157g NaCl, 3.407g Na2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、1.3044g CaCl2、
0.0862g KBr、0.0226g H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 8～9, in 20～30 DEG C of constant temperature, illumination
Cultivate to stable phase under the conditions of intensity 5000～7000lux, illumination 24h；96 orifice plates are placed in fluorescence microscope automatic shift pair
On burnt Electronic Control object stage, thin to the algae in hole every in 96 orifice plates under white light conditions using the supporting CCD camera of microscope
Born of the same parents carry out automatically scanning shooting, carry out quantitative data using MIcrosope image process software to the color of every hole frustule and divide
Analysis.Because when algae cell density is bigger, color is more deep, show that growth rate is faster such that it is able to filter out growth rate fast (>=
0.4g/L d) advantage microalgae unicellular；
(3) with the Nile red fluorescent dye Nile/DMSO of 0.1g/L, the fast microalgae of growth rate is obtained to screening unicellular
Dyeed, Nile red fluorescent dye final concentration reaches 0.1mg/L, dyeing time 7min；Dyeing frustule is placed in fluorescence show
On the object stage of micro mirror, combine, under EX 450-490nm wavelength, the fluorescence intensity that image processing software observes measurement frustule,
The area of all frustules and the area of intracellular all oil droplets in the measurement visual field, calculate the fat content of frustule, thus
The advantage microalgae filtering out fat content height (>=40%) is unicellular；
The computational methods of fat content are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor oils and fatss in cell
Grain area summation (μm2).
The following examples can make this professional professional and technical personnel that the present invention is more fully understood, but not with any side
Formula limits the present invention.
Embodiment 1
(1) take 5 μ L rhombus algae solution bodies to be seeded to solid plate culture medium and carry out unicellular separation, frustule is taken out inoculation
To 20mL culture medium, put into triangular flask amplification culture.
The consisting of of described culture medium：0.15g NaHCO3、0.02g KH2PO4、0.027g VB1、1.5×10-6g VB12、
0.2g Na2SiO3·9H2O、1.0g NaNO3, 0.0005g Biotin, 1mL trace element and 980mL artificial seawater；Described micro-
Secondary element main component is：4.35g Na is contained in 1000mL distilled water2EDTA、7.3mg Na2MoO4·2H2O、
12mgCoCl2·6H2O、3.9g FeC6H5O7·5H2O、10mg CuSO4·5H2O、23mg ZnSO4、178mg MnCl2·4H2O
With 600mg H3BO3.Described artificial seawater formula (salinity=3.0%) is：In 1000mL distilled water contain 21.2157g NaCl,
3.407g Na2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、1.3044g CaCl2、0.0862g KBr、0.0226g
H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 8, in 20 DEG C of constant temperature, intensity of illumination
Cultivate to stable phase under the conditions of 5000lux, illumination 24h.96 orifice plates are placed in the electronics control of fluorescence microscope automatic shift focusing
On object stage processed, under white light conditions the frustule in hole every in 96 orifice plates is carried out automatically using the supporting CCD camera of microscope
Scanning shoot, processes software using MIcrosope image and carries out quantitative data analysis to the color of every hole frustule.Because algae is thin
When born of the same parents' density is bigger, color is more deep, shows that growth rate is faster such that it is able to filter out growth rate fast (0.42g/L d)
Advantage microalgae is unicellular.
(3) Nile red fluorescent dye (0.1g/L, Nile/DMSO) is used to obtain the fast microalgae of growth rate to screening unicellular
Dyeed (Nile red final concentration 0.1mg/L, dyeing time 7min), dyeing frustule is placed in the object stage of fluorescence microscope
On, combine the fluorescence intensity that image processing software observes measurement frustule, institute in the measurement visual field under EX 450-490nm wavelength
There are the area of frustule and the area of intracellular all oil droplets, calculating the fat content of frustule, thus filter out oils and fatss containing
The advantage microalgae of amount high (47%) is unicellular.Fat content computational methods are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor oils and fatss in cell
Grain area summation (μm2).
Embodiment 2
(1) take 5 μ L chlorella liquid inoculations to carry out unicellular separation to solid plate culture medium, frustule is taken out inoculation
To 20mL culture medium, put into triangular flask amplification culture.
The consisting of of described culture medium：0.15g NaHCO3、0.02g KH2PO4、0.027g VB1、1.5×10-6g VB12、
0.2g Na2SiO3·9H2O、1.0g NaNO3, 0.0005g Biotin, 1mL trace element and 980mL artificial seawater；Described micro-
Secondary element main component is：4.35g Na is contained in 1000mL distilled water2EDTA、7.3mg Na2MoO4·2H2O、
12mgCoCl2·6H2O、3.9g FeC6H5O7·5H2O、10mg CuSO4·5H2O、23mg ZnSO4、178mg MnCl2·4H2O
With 600mg H3BO3.Described artificial seawater formula (salinity=3.0%) is：In 1000mL distilled water contain 21.2157g NaCl,
3.407g Na2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、1.3044g CaCl2、0.0862g KBr、0.0226g
H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 8.5, in 25 DEG C of constant temperature, intensity of illumination
Cultivate to stable phase under the conditions of 6000lux, illumination 24h.96 orifice plates are placed in the electronics control of fluorescence microscope automatic shift focusing
On object stage processed, under white light conditions the frustule in hole every in 96 orifice plates is carried out automatically using the supporting CCD camera of microscope
Scanning shoot, processes software using MIcrosope image and carries out quantitative data analysis to the color of every hole frustule.Because algae is thin
When born of the same parents' density is bigger, color is more deep, shows that growth rate is faster such that it is able to filter out growth rate fast (0.84g/L d)
Advantage microalgae is unicellular.
(3) Nile red fluorescent dye (0.1g/L, Nile/DMSO) is used to obtain the fast microalgae of growth rate to screening unicellular
Dyeed (Nile red final concentration 0.1mg/L, dyeing time 7min), dyeing frustule is placed in the object stage of fluorescence microscope
On, combine the fluorescence intensity that image processing software observes measurement frustule, institute in the measurement visual field under EX 450-490nm wavelength
There are the area of frustule and the area of intracellular all oil droplets, calculating the fat content of frustule, thus filter out oils and fatss containing
The advantage microalgae of amount high (58%) is unicellular.Fat content computational methods are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor oils and fatss in cell
Grain area summation (μm2).
Embodiment 3
(1) take 5 μ L micro- plan ball algae solution body to be seeded to solid plate culture medium and carry out unicellular separation, frustule is taken out and connects
Plant and arrive 20mL culture medium, put into triangular flask amplification culture.
The consisting of of described culture medium：0.15g NaHCO3、0.02g KH2PO4、0.027g VB1、1.5×10-6g VB12、
0.2g Na2SiO3·9H2O、1.0g NaNO3, 0.0005g Biotin, 1mL trace element and 980mL artificial seawater；Described micro-
Secondary element main component is：4.35g Na is contained in 1000mL distilled water2EDTA、7.3mg Na2MoO4·2H2O、
12mgCoCl2·6H2O、3.9g FeC6H5O7·5H2O、10mg CuSO4·5H2O、23mg ZnSO4、178mg MnCl2·4H2O
With 600mg H3BO3.Described artificial seawater formula (salinity=3.0%) is：In 1000mL distilled water contain 21.2157g NaCl,
3.407g Na2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、1.3044g CaCl2、0.0862g KBr、0.0226g
H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 9, in 30 DEG C of constant temperature, intensity of illumination
Cultivate to stable phase under the conditions of 7000lux, illumination 24h.96 orifice plates are placed in the electronics control of fluorescence microscope automatic shift focusing
On object stage processed, under white light conditions the frustule in hole every in 96 orifice plates is carried out automatically using the supporting CCD camera of microscope
Scanning shoot, processes software using MIcrosope image and carries out quantitative data analysis to the color of every hole frustule.Because algae is thin
When born of the same parents' density is bigger, color is more deep, shows that growth rate is faster such that it is able to filter out growth rate fast (0.56g/L d)
Advantage microalgae is unicellular.
(3) Nile red fluorescent dye (0.1g/L, Nile/DMSO) is used to obtain the fast microalgae of growth rate to screening unicellular
Dyeed (Nile red final concentration 0.1mg/L, dyeing time 7min), dyeing frustule is placed in the object stage of fluorescence microscope
On, combine the fluorescence intensity that image processing software observes measurement frustule, institute in the measurement visual field under EX 450-490nm wavelength
There are the area of frustule and the area of intracellular all oil droplets, calculating the fat content of frustule, thus filter out oils and fatss containing
The advantage microalgae of amount high (52%) is unicellular.Fat content computational methods are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor oils and fatss in cell
Grain area summation (μm2).
Finally it should be noted that listed above be only the present invention specific embodiment.It is clear that the invention is not restricted to
Above example, can also have many variations.Those of ordinary skill in the art directly can lead from present disclosure
The all deformation going out or associating, are all considered as protection scope of the present invention.
Claims (5)
1. the single celled method of microalgae that fluorescence microscopy screening growth is fast and oils and fatss are high is it is characterised in that specifically include following steps
Suddenly：
(1) take 5 μ L microalgae liquid inoculations to carry out unicellular separation to solid plate culture medium, frustule is taken out and is inoculated into 20mL
Culture medium, puts into triangular flask amplification culture, obtains many unicellular algae solutions of strain；
(2) inoculate the unicellular algae solution of 300 μ L with 96 orifice plates, initial inoculation pH is adjusted to 8～9, in 20～30 DEG C of constant temperature, intensity of illumination
Cultivate to stable phase under the conditions of 5000～7000lux, illumination 24h；96 orifice plates are placed in the focusing of fluorescence microscope automatic shift
On Electronic Control object stage, under white light conditions the frustule in hole every in 96 orifice plates is entered using the supporting CCD camera of microscope
Row automatically scanning shoots, and processes software using MIcrosope image and by the MIcrosope image shooting, the color of every hole frustule is entered
Row quantitative data is analyzed, and filters out the fast advantage microalgae of growth rate unicellular；
(3) with the Nile red fluorescent dye Nile/DMSO of 0.1g/L, the fast microalgae list of growth rate is obtained to screening in step (2)
Cell is dyeed, and Nile red fluorescent dye final concentration reaches 0.1mg/L, dyeing time 7min；Dyeing frustule is placed in glimmering
On the object stage of light microscope, the fluorescence combining image processing software observation measurement frustule under EX 450-490nm wavelength is strong
Degree, the area of all frustules and the area of intracellular all oil droplets in the measurement visual field, calculate the fat content of frustule, from
And it is unicellular to filter out the high advantage microalgae of fat content；
The computational methods of fat content are：
Fat contentWherein ACArea summation for frustule (μm2), ALFor fat particles in cell
Area summation (μm2).
2. method according to claim 1 is it is characterised in that the component of described culture medium and proportion relation are：0.15g
NaHCO3、0.02g KH2PO4、0.027g VB1、1.5×10-6g VB12、0.2g Na2SiO3·9H2O、1.0g NaNO3、
0.0005g Biotin, 1mL trace element and 980mL artificial seawater.
3. method according to claim 2 is it is characterised in that the component of described trace element and proportion relation are：
4.35g Na is contained in 1000mL distilled water2EDTA、7.3mg Na2MoO4·2H2O、12mgCoCl2·6H2O、3.9g
FeC6H5O7·5H2O、10mg CuSO4·5H2O、23mg ZnSO4、178mg MnCl2·4H2O and 600mg H3BO3.
4. method according to claim 2 is it is characterised in that the component of described artificial seawater and proportion relation are：
21.2157g NaCl, 3.407g Na is contained in 1000mL distilled water2SO4、0.3577g KCl、9.3042g MgCl2·6H2O、
1.3044g CaCl2、0.0862g KBr、0.0226g H3BO3, 0.2760g NaF and 0.0219g SrCl2·6H2O.
5. method according to claim 1 it is characterised in that the algae kind in described microalgae liquid be following any one：
Screen the natural algae kind obtaining from natural environment, algae kind mutant that physics and chemistry behavior obtains or obtain through improvement of genes
Transgenic algae kind.
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| CN102565012A (en) * | 2010-12-24 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Method for detecting oil content of microalgae |
| CN102943048A (en) * | 2012-11-30 | 2013-02-27 | 北京联合大学生物化学工程学院 | High-oil-content cyclotella menhiniana mutant strain, as well as screening and culturing method thereof |
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| CN102565012A (en) * | 2010-12-24 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Method for detecting oil content of microalgae |
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