Determination and Comparison of the Effects of Nitrogen and Sulfur Deprivations on Chlamydomonas ReinhardtII Strain CC5373-STAG
The use of microalgae in biodiesel production is of increasing interest due to the capability of enhancing the algae’s triacylglycerol (TAG) biosynthesis by the use of stress conditions to optimize microalgal TAG accumulation. Nitrogen starvation causes algal cells to accumulate starch and increase lipid content; sulfur deprivation increases H2 production and increases lipid content. Chlamydomonas reinhardtii starchless mutant cc5373-sta6, which due to an inhibited starch biosynthesis has greater accumulation of lipid bodies. To our knowledge, no studies yet have reported a combined nitrogen (N) and sulfur (S) deprivation as a stress condition for lipid production and using the starchless mutant, cc5373- sta6. We hypothesized that by removing both N and S from cc5373-sta6’s growth media, lipid bodies will over accumulate within the cell, greater than when solely using N or S deprivation. The purpose of this study was to compare the effects of nutrient deprivation conditions on cc5373-sta6 via chemical, microscopic and genetic analysis. The objectives of this study were to (1) determine the effects of nitrogen and sulfur starvation on cc5373-sta6 by conducting spectrophotometric analyses (cell density, chlorophyll content and biomass determination), (2) determine lipid accumulation via confocal microscopy and (3) identify the transcriptional expression levels of enzymes involved in lipid biosynthesis by RT-PCR. Chemical analyses for nutrient deprived cells displayed significant increase in cell density (p<0.001) and a significant decrease in chlorophyll content (p<0.001). Confocal imaging showed N and S deprived cells to accumulate large lipid bodies. More specifically, after 48 hr NS deprived cells showed significantly larger lipid body size (p<0.001). Cells subjected to N and S deprivation increased expression of ACCase after 8 hr and 24 hr (p<0.001); PEPC expression decreased after 8 hr, and increased after 24 hr (p<0.001). This study is the first to report the combinatorial effects of N and S deprivation have on lipid accumulation in cc5373-sta6. In addition, the results of this study will serve as a basis for further studies on the potential of C. reinhardtii cc-5373-sta6 for biofuel production.