Measuring lipid content as % dry weight in C. reinhardtii
Sabine Waffenschmidt1, Lilija Brant1, and Ursula Goodenough2
1) Dept. Biochemistry, University of Cologne, Cologne 4750674 Germany
2) Dept. Biology, Washington University, St. Louis MO 63130
We recently characterized the lipid body (LB) production profiles of the cw15 and cw15 sta6 mutant strains of C, reinhardtii following nitrogen starvation in 20 mM acetate (Wang et al., Eukaryotic Cell 8: 1856-1868, 2009), where we expressed LB yield on a per cell basis. In the algal biodiesel field, it is customary to express yield on a dry-weight basis, either as % total lipid or % fatty acids (e.g. Hu et al., Plant J. 54: 621, 2009). We have therefore developed rigorous protocols for total lipid extraction and dry-weight determination in C. reinhardtii to allow its lipid-biosynthetic potential to be compared between strains and with other organisms. For lipid extraction, we add 70 ml hot isopropanol (~80°C) to 4x109 cells and agitate 1-2 min in a hot Waring Blender. The hot homogenate is glass-filtered with suction; the filter cake is next blended with 70 ml chloroform:isopropanol (1:1), filtered, and then blended with 70 ml chloroform and filtered. The combined filtrates are concentrated in high vacuum overnight. For dry-weight determination, 4x109 cells are pelleted, resuspended in glass-distilled water, lyophilized, and the lyophilate further dried in an oven (105°C) until a constant weight is reached. All procedures are done in glass or metal vessels. Applying these protocols to the cw15 sta6 mutant, we obtained values of 33.2% lipid at the end of log-phase mixotrophic growth, and 65.3% after 30 hr N-starvation in 20 mM acetate. These percentages compare favorably with those reported for other microalgae (Hu et al. op cit). Data for other strains and growth conditions will be presented.
e-mail address of presenting author: waff@Uni-Koeln.DE