Investigation of Neutral Lipid Production in Desmodesmus armatus and Synechocystis sp PCC6803

Microalgae has been introduced in the last decades to be an optimum source for biofuel production that can be utilised efficiently to replace the conventional fossil fuels as a source of energy. In our study, a local strain of green microalgae was isolated from the Weston Park pond (Sheffield, UK) and identified, using different molecular markers including 18S rDNA, ITS1, ITS2, and 5.8S rDNA, as Desmosdesmus armatus. The neutral lipid content (as the basis of renewable biodiesel production) of both D. armatus and the model cyanobacterium Synechocystis sp. was measured using the lipophilic fluorescent dye Nile Red. The two strains were grown in normal BG11 medium and under stress conditions including high salinity (0.2, 0.4 M NaCl), different concentrations of sodium nitrate in the BG11 media (10% NaNO3, N-free), and different sources of nitrogen (NH4Cl, urea). The results showed that N-free BG11 medium was the best stress conditions in both strains for inducing a significant (p<0.05) increase in neutral lipid content.
Further work concentrated on D. armatus and the Fatty Acid Methyl Esters (FAME) conversion yield was examined using a direct transesterification method and the composition of fatty acids was investigated using GC-MS. Desmodesmus armatus grown in N-free BG11 medium showed the highest yield and the contents of C16 and C18 fatty acids (useful for biodiesel production) increased significantly under this stress condition. Further analysis of D. armatus lipid content was conducted using 1H NMR and the results confirmed that the fatty acid content is much higher in the N free grown cells.
The final set of experiments focused on random mutation of D. armatus cells using Ultra Violet light (254 nm) to generate new strains with high neutral lipid content. The high lipid containing cells were isolated using Nile Red staining and automated fluorescence assisted cell sorting (FACS) flow cytometry technique. A mutant strain was isolated with 5 times greater yield of neutral lipid than the wild type strain based on the median of the Nile Red fluorescence of the wild type and the mutant cells. This significant increase in the lipid yield using UV-induced random mutation, Nile Red staining and FACS has
paved the way for further investigation of the molecular and genetic approaches to identify the key genes that control neutral lipid biosynthesis in microalga