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Method for screening microalgae unicells which grow fast and are high in grease content through fluorescence microscope

A single-cell and algae cell technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., to improve work efficiency and reduce labor time costs

Active Publication Date: 2015-04-29
ZHEJIANG UNIV
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  • Abstract
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Problems solved by technology

Using the new technology of electronically controlled stage with automatic focus shift of fluorescence microscope and the supporting CCD camera to automatically scan and shoot a large number of algae cells under white light conditions, and to conduct quantitative data analysis by distinguishing the color of algae cells is expected to screen out growth at a high speed. Algal cells with fast speed, but there are still relatively few research reports on this aspect

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  • Method for screening microalgae unicells which grow fast and are high in grease content through fluorescence microscope

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Experimental program
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Effect test

Embodiment 1

[0029] (1) Inoculate 5 μL Nitzki alga liquid into solid plate medium for single cell isolation, take out the algae cells and inoculate them into 20 mL medium, and put them into a Erlenmeyer flask for expansion.

[0030] The composition of described culture medium is: 0.15g NaHCO 3 , 0.02g KH 2 PO 4 , 0.027g VB 1 , 1.5×10 -6 f 12 , 0.2g Na 2 SiO 3 9H 2 O, 1.0g NaNO 3 , 0.0005g Biotin, 1mL trace elements and 980mL artificial seawater; the main components of the trace elements are: 1000mL distilled water contains 4.35g Na 2 EDTA, 7.3 mg Na 2 MoO 4 2H 2 O, 12mgCoCl 2 ·6H 2 O, 3.9g FeC 6 h 5 o 7 ·5H 2 O, 10mg CuSO 4 ·5H 2 O, 23mg ZnSO 4 , 178mg MnCl 2 4H 2 O and 600 mg H 3 BO 3 . The artificial seawater formula (salinity=3.0%) is: 1000mL distilled water contains 21.2157g NaCl, 3.407g NaCl 2 SO 4 , 0.3577g KCl, 9.3042g MgCl 2 ·6H 2 O, 1.3044g CaCl 2 , 0.0862g KBr, 0.0226g H 3 BO 3 , 0.2760g NaF and 0.0219g SrCl 2 ·6H 2 O.

[0031] (2) A 96-well pla...

Embodiment 2

[0035] (1) Take 5 μL of chlorella liquid and inoculate it into a solid plate medium for single cell isolation, take out the algae cells and inoculate them into 20 mL of medium, and put them into a Erlenmeyer flask for expansion.

[0036] The composition of described culture medium is: 0.15g NaHCO 3 , 0.02g KH 2 PO 4 , 0.027g VB 1 , 1.5×10 -6 f 12 , 0.2g Na 2 SiO 3 9H 2 O, 1.0g NaNO 3 , 0.0005g Biotin, 1mL trace elements and 980mL artificial seawater; the main components of the trace elements are: 1000mL distilled water contains 4.35g Na 2 EDTA, 7.3 mg Na 2 MoO 4 2H 2 O, 12mgCoCl 2 ·6H 2 O, 3.9g FeC 6 h 5 o 7 ·5H 2 O, 10mg CuSO 4 ·5H 2 O, 23mg ZnSO 4 , 178mg MnCl 2 4H 2 O and 600 mg H 3 BO 3 . The artificial seawater formula (salinity=3.0%) is: 1000mL distilled water contains 21.2157g NaCl, 3.407g NaCl 2 SO 4 , 0.3577g KCl, 9.3042g MgCl 2 ·6H 2 O, 1.3044g CaCl 2 , 0.0862g KBr, 0.0226g H 3 BO 3 , 0.2760g NaF and 0.0219g SrCl 2 ·6H 2 O.

[0037]...

Embodiment 3

[0041] (1) Take 5 μL of Nannochloropsis liquid and inoculate it on a solid plate medium for single cell isolation, take out the algae cells and inoculate them into 20 mL of medium, and put them into a Erlenmeyer flask for expansion.

[0042] The composition of described culture medium is: 0.15g NaHCO 3 , 0.02g KH 2 PO 4 , 0.027g VB1 , 1.5×10 -6 f 12 , 0.2g Na 2 SiO 3 9H 2 O, 1.0g NaNO 3 , 0.0005g Biotin, 1mL trace elements and 980mL artificial seawater; the main components of the trace elements are: 1000mL distilled water contains 4.35g Na 2 EDTA, 7.3 mg Na 2 MoO 4 2H 2 O, 12mgCoCl 2 ·6H 2 O, 3.9g FeC 6 h 5 o 7 ·5H 2 O, 10mg CuSO 4 ·5H 2 O, 23mg ZnSO 4 , 178mg MnCl 2 4H 2 O and 600 mg H 3 BO 3 . The artificial seawater formula (salinity=3.0%) is: 1000mL distilled water contains 21.2157g NaCl, 3.407g NaCl 2 SO 4 , 0.3577g KCl, 9.3042g MgCl 2 ·6H 2 O, 1.3044g CaCl 2 , 0.0862g KBr, 0.0226g H 3 BO 3 , 0.2760g NaF and 0.0219g SrCl 2 ·6H 2 O.

[004...

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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 [0001] The invention relates to biomass energy utilization technology, in particular to a method for screening microalgae single cells with fast growth and high oil content by fluorescence microscopy. Background technique [0002] Microalgae have high utilization efficiency of solar energy, rapid growth and reproduction, strong adaptability to the environment, and high oil production. It is reported that the theoretical value of photosynthetic conversion efficiency of microalgae can reach 10%, and the annual oil production per unit area can reach 8-24 times that of palm oil (Hu et al., 2008). Chisti (Chisti, 2007) established a mathematical model and calculated that microalgae biodiesel is an important choice to replace petrochemical oil. Cheng et al. found that nuclear radiation mutagenesis can significantly increase the growth rate and biomass density of Chlorella (Cheng et al., 2013a). Khozin et al. reviewed the expression of genes in diatom lipid synthe...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 程军岑可法周俊虎刘建忠杨卫娟张彦威黄镇宇周志军王智化
Owner ZHEJIANG UNIV
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