| With the rapid development of global economy,the shortage of fossil energy and global warming have attracted more attention.Biodiesel as a renewable new energy is expected to gradually replace the traditional fossil energy.Not only because of high growth rate and high oil content in microalga,but also non-competition with the food demand of human being,so microalga are potential materials for biodiesel.How to improve the yield of biodiesel is a major bottleneck limiting the industrialization of biodiesel production.In this study,we use Nannochloropsis gaditana 527 as material,to induce and screen higher fatty acid production strains.The main results are as follows:1.In this study,a new method was developed for the rapid detection of the oil content in the cells by the method of Nile-red fluorescence staining.Under the condition of nitrogen deficiency the Nannochloropsis cells were scanned with excitation spectra and emission spectra.We systematically obtained the optimal excitation wavelength and emission wavelength were 515 nm and 570 nm.And then the influence factors of Nile red staining were optimized to get that the optimum concentration of DMSO was 5%,the final Nile red concentration was 1 mg/L,staining time was 6 min.The lipid content measured by gravimetric method and Triolein standard was correlated with the fluorescence intensity of Nile red staining.The results showed that the Nile red fluorescence staining method could be used to detect the intracellular lipid content quickly and accurately,and there was a positive correlation between oil content and fluorescence intensity,the correlation coefficient R~2 was 0.9973.The lower limit of lipid detection was 2 ?g,which greatly reduced the amount of cells required for the determination of oil content.2.The combination method of ultraviolet mutation and DCMU to screen higher lipid content mutants were applied to Nannochloropsis,more than 50 strains were screened by Nile red staining and among them mutants of S10 and P16 were two of the highest.Comparing the lipid content and fatty acid composition of wild-type and mutant strains under different nutrient conditions,we found that under normal culture condition the oil content of wild-type and mutant strains S10 and P16 were 15.12%,24.35% and 22.55%,and the cell density were 46.57×106 cells/mL,42.5×106 cells/mL and 43.23×106 cells/mL.The lipid productivity of mutant strains S10 and P16 reached 18.37 g·m-3·d-1 and 16.90 g·m-3·d-1 respectively,63% and 50% higher than the wild-type strain which was 11.27 g·m-3·d-1.The fatty acid composition of mutant S10 and P16 was different from wild-type strain,and the proportion of long chain fatty acid C20:5n3 was significantly lower than the wild-type strain,which decreased by 39.18% and 8.19%,respectively.While under the condition of nitrogen deficiency,the lipid content of wild-type and mutant strains S10 and P16 were 36.81%,51.59% and 41.81%,and the cell density were 19.44×106 cells/mL,22.69×106 cells/mL and 20.25×106 cells/mL,which had significant differences in the growth and lipid content.And the lipid content of mutant strains S10 and P16 were 40.15% and 13.58% higher than the wild-type,respectively.Compared with the wild-type,the fatty acid C20:4n6 of mutant strains S10 and P16 decreased by 51.50% and 32.84%,C20:5n3 decreased by 62.40% and 69.97%,respectively.Under the condition of nitrogen deficiency the lipid content of wild-type and mutant strains S10 and P16 were 24.85%,26.93% and 25.57%,and the cell density were 36.07×106 cells/mL,38.87×106 cells/mL and 38.26×106 cells/mL,there was significant differences in the growth and lipid content.Results showed that the mutants hardly involves changing phosphorus metabolism.However,compared with the wild-type,the fatty acid C20:4n6 of mutant strains S10 and P16 increased by 41.17% and 79.17%,C20:5n3 increased by 54.92% and 44.49%,respectively.3.When glucose was introduced as carbon source,the mutant strain S10 and P16 showed the same tendency of biomass production and lipid content as the wild-type strain,and the lipid content of mutant strain S10 and P16 were about 50% and 25% higher than that of wild-type strain,respectively.But the fatty acid composition was different in mutant strain S10 and P16 compared with the wild-type strain.C16:0 content of S10 was lower than its control group,but C18:1 and C18:2n6 were higher.C20:4n6 and C20:5n3 of P16 were slightly lower than the control group,and C14:0 and C16:0 were unchanged,but C16:1 was higher.4.When glycerol was applied as carbon source,S10 and P16 performed the same tendency in biomass productivity and lipid content as the wild-type strain,and the lipid content in both S10 and P16 were increased.Compared with the wild-type strain,the biomass of S10 decreased but the lipid content was 40% higher,while the biomass of P16 was almost unchanged but lipid content was 30% higher.The fatty acid C16:0 of S10 decreased and the fatty acid of P16 was almost unchanged.5.The gene expression of the key enzyme LACS during the synethesis of long-chain fatty acid were comparied in both wild-type and mutants.RT-qPCR results showed that compared to the wild-type strain,the expression level of LACS gene of mutant strains S10 and P16 were 1.43 times and 1.46 times higher,and the lipid productivity were 63% and 50% higher,and the fatty acid EPA were 28.35% and 18.88% higher.The relationship between the expression of LACS gene and the lipid content was analyzed by sequencing the wild-type and mutant strains.The results showed that there was a positive correlation between the expression of LACS gene and lipid content. |
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