The Enrichment,Isolation And Characterization Of Lipid Droplets From Oleaginous Microalgae

Oleaginous microalgae is important feedstock for biodiesel production.Research efforts that explore mechanisms of lipid accumulation and metabolism pathway of microalgae are needed to improve the lipid content of cells.Lipid droplets(LDs)are the cell organelle of lipid storage and metabolism.A deep explore to them will probably be helpful to the improvement of lipid accumulation in microalgal cells.Selecting Botryococcus braunii and Platymanas subcordiformis as the research objects,this paper explored the Nile red detection method to determine the lipid content of cells and the method of lipid accumulation in cells.Meanwhile this paper explored the lipid droplets isolation protocol suitable for the two samples,and subsequently characterized the isolated LDs(size,lipid composition,protein composition and so on).By optimizing the pretreatment and staining conditions,the Nile red detection method was improved as follows: treating algal cells by ultrasonication for 180 s,centrifugation to remove the supernatant,suspending the cells with 8% dimethylsulfoxide to OD750 of 1.6,adding Nile red(final concentration 1.2 ?g/mL),incubating the staining system at 40? for 10 min.Then the sample was measured by fluorescence spectrometer excited at 520 nm.The interaction between Nile red and microalgal cells,and the accuracy of fluorescence intensity was enhanced after optimization.Meanwhile,the staining conditions of Platymanas subcordiformis was explored as similar to Botryococcus braunii.The biomass of microalgae was increased by improving culture apparatus and optimizing culture medium,while the lipid content of cells was increased with high irradiance illumination.Afterwards,intact LDs were isolated from the two samples successfully.Afterwards this paper characterized the isolated LDs and reached the following conclusion:(a)The size of LDs from Botryococcus braunii and Platymanas subcordiformis were about 1.1 ?m and 0.6 ?m respectively;(b)Triacylglycerols(TAG)was the main lipid of LDs,while a small quantity of diacylglycerol and sterol ester may existed in the LDs of Botryococcus braunii;(c)No homologous LD-specific proteins with other plant were found in the LDs of two samples.At last,proteins extracted from the LDs were further separated by two-dimensional electrophoresis,which laid a basis on the identification of novel LD proteins.