Effect of Ethanol on Triacylglycerol Synthesis in Chlorella Species Utilizing a Novel Fluorescence-Based Assay

In this study, I set out to develop a novel method of assaying acyl-CoA: diacylglycerol acyltransferase (DGAT) activity employing a fluorescently labeled substrate, and then using that protocol to determine the effects of ethanol-induced stress on DGAT activity in Chlorella species. Microsomes from Chlorella sp. were used to construct protein and time curves to characterize the method in microalgae. Microsomes from Rattus liver were used to construct a substrate concentration curve and a Lineweaver-Burk plot to characterize the method in mammalian tissue. Optimal reaction conditions were determined to be 10 ?g microsomal protein with 20 min incubation. The mammalian microsomes provided a Km of 6.26 ?M substrate concentration and a Vmax of 0.211 pmol product x min-1 x ?g protein-1. This method represents the first fluorescence based DGAT assay. Chlorella sp. cultures were exposed to 0.33%, 0.66%, and 1.00% ethanol concentrations for 3 hr, and the DGAT activity assayed under optimal conditions. Increases in DGAT activity were observed in all cultures exposed to ethanol with a maximum seen at 0.66% concentration. These results have implications towards improving biodiesel production in mass algal cultures.