(625j) Cytomic Analysis and Fatty Acids Profiling of the Snow Algae Chlamydomonas Nivalis by NaCl Stress Using Laser Scanning Confocal Microscope, Flow Cytometry and GC-MS

The snow algae Chlamydomonas nivalis, a typical microalgal species discovered on snow field at Antarctic and similar extreme environments on earth, can adapt to very low temperature, high light intensity and strong UV radiation for survival by forming aplanospore rich in astaxanthin, a brown red pigment with super anti-oxidative effect. C. nivalis is realized not only a model species to investigate the mechanism of cell response and adaptation to stress conditions, but also a potential alternative bioresource for astaxanthin production by microalgal biotechnology. The response and resistance of microalgae to stress environment depend on a series of essential regulation of metabolic pathway, especially on lipid biosynthesis and cell wall structure. However, current research of C. nivalis mostly focuses on the classification, life cycle, modes of reproduction, regional distribution and cell ultra-structure. Cytomic analysis and fatty acid profiling of C. nivalis in resistant physiology has paid little attention.

In the present work, the 6-day culture of C. nivalis UTEX LB 2824 in Bold 1NV medium under autotrophic growth was treated by NaCl with final concentrations of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50% for 1, 2, 3, 5, 7, 11, 15, 24 or 48 h, respectively. The content of neutral and polar lipid referred as fluorescent intensity in stressed cells after stained with specific dye Nile Red was quantified by cytomic analysis based on laser scanning confocal microscope (LSCM, Ex: 543 nm; scan: 560-615 nm) and flow cytometry (FCM, Ex: 488 nm, FL1 at 530 nm, FL2 at 575 nm and SSC signal). After extraction and derivation of triacylglycerol (TG) and free fatty acids (FFA) from stressed cells, the profiles of FFA and FA in TG were analyzed by GC-MS. In general, the content of neutral and polar lipid showed the similar trends with total lipid in cells. The fluorescent intensities of lipids constantly increased to the maximum during 7 h, then dropped to a stable level within 24 to 48 h. The trend was not rigorous within all NaCl concentrations, but the highest increase of 211% for total lipid at 0.75% NaCl for 7 h, 68 times increase for neutral lipid at 1.00% NaCl for 7 h and 10 times increase for polar lipid at 1.25% NaCl for 5 h were observed, respectively. The cell size in all groups of NaCl stress enlarged quickly during 3 to 5 h, then stopped to become large. Big changes of composition and content of several FFA (C12:0, C16:0, C16:3, C18:0, C18:1 and C18:3) were observed with variation for 4.61, 3.70, 4.37, 2.68, 2.64, 3.14 times, respectively. C16:0, C18:0, C18:1 and C18:3 in TG also varied much for 2.67, 2.53, 4.91, 2.42 times, respectively. The results indicated C. nivalis could synthesize, accumulate lipid and change fatty acids profile very quickly during several hours by stress induction. The content of C16:0, C18:0, C18:1 and C18:3 fatty acids could change the degree of lipid unsaturation (DLU) and play an important role on regulating the membrane fluidity, which result an enhancement of cell response and resistance to NaCl stress. This is the first report to demonstrate the lipid changes corresponding to NaCl stress by “omics” technology, and figure out the regulation mechanism of lipid biosynthesis by NaCl induction in C. nivalis.