| Microalgae that can accumulate lipid accounting for more than 20% of their biomass weight are called oleaginous microalgae.They are becoming increasingly attractive as raw materials for biodiesel production since they have several advantages,including high capacity of carbon fixation,high growth rate,not competing with arable land etc.Dunaliella tertiolecta was the subject of this research,enhancement of the lipid content of which was focused.First,the application of BODIPYstaining method for the dertermination of lipid content in D.tertiolecta were explored.Second,in search for effective chemical compounds for improving the lipid content or biomass of D.tertiolecta,screening in nutrients(glycerol,glycine,fructose and sodium acetate),auxins(gibberellin and inositol)and chemical trrigers(azelaic acid,nicotinic acid,triethylamine,sodium azide and sodium diacetate)were conducted,during which the effects of those compounds on cell growth,pigment content,lipid accumulation and cell morphology of D.tertiolecta were also investigated.To further improved the lipid yield of D.tertiolecta,two phase cultivation was adopted in the optimization experiment of glycerol-triethlylamien and sodium azide-salinaty.The effect of triethylamine,a compround proved to be effective in improving the lipid yield and lipid content of D.tertiolecta,on gene expression of lipid biosynthesis related enzymes were investigated.Fatty acid profile changes were analyzed by GC-MS.The transcript level of glycerol-3-phophate dehydrogenase(GPDH)response to the treatment of triethylamine was analyzed by qRT-PCR.The result of optimazition on BODIPY staining conditions showed that the best stain effect can be obtained when cell concentration,BODIPY concentration,staining temperature and staining time were 104-106 cells/mL,0.496 ?g/mL,35 oC and 10 min,respectively.Comparing the results of lipid content measured by BODIPY staining method and traditonal gravity weighing method,a high correlation was found,manifesting in regression equation as y=0.115x+0.170,R2=0.978.Results from single factor experiment screening of chemical compounds showed that glycerol can dramatially enhance cell growth of D.tertiolecta during exponential phase,however lipid yield was reduced due to the increased biomass.Triethylamine was inhibitive to the cell growth of D.tertiolecta,but can increase the lipid yiled andlipid content per cell up to 20% and 80% at 100 ppm,comparing to the control.Sodium azide can improve the lipid content of D.tertiolecta without significantly affacting the biomass productivity.When added 50 ?M,increase of 10.4% and 21.7%in lipid yiled and lipid content per cell respectively were obtained in sodium azide groups comparing to the control.No significant changes on lipid production in D.tertiolecta were observed with the treatment of other chemical compounds mentioned aboved.In the two phase cultivation of glycerol-triethylamine treated D.tertiolecta,significant increase of biomass productivity was obtained due to the glycerol treatment in phase one.Furthermore,inhibitive effect of trithylamine on cell growth was also alleviated by glycerol during phase two.When glycerol addition was 1 g/L and triethylamien 100 ppm,the highest lipid yield was obtained,26.7% higher than that of the control group.The result of two phase cultivation treated with different concentrations of sodium azide and salinity showed that biomass was slightly lower than the control in sodium azide groups.In the second phase of cultivation,synergistic effect concerning the enhancement of lipid accumulation in D.tertiolecta was observed in sodium azide-salinity groups.The highest lipid yiled and lipid content per cell were obtained when sodium azide concentration was 50 ?M and the salinity was 2.5 M,increasing 10% and 70.5% respectively compared to the control.Analysis on the fatty acid profile of D.tertiolecta showed that triethylamine treatment can significantly increase the content of C16 while decrease the content of C18.RT-PCR resuted showed that gene expression of the two isodynamic enzymes of GPDH,mGPDH and cGPDH,were both up-regulated under the treatment of triethylaime.MGPDH was up-regulated the most at 100 ppm while cGPDH up-regulated the most at 150 ppm.In conclusion,BODIPY staining method for lipid content determination can be applied in D.tertiolecta.Single factor experiment for effective chemical compounds screening is an practical way for lipid content enhancing explorations.Two phase cultivation are beneficial for improving lipid yield in microalgae.Fatty acid profile was also affacted by triethylamine.Triethylamine treatment can up-regulate the gene expression of GDPH,resulting in the alteration of lipid metabolism in D.tertiolecta. |
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