Transgressive, reiterative selection by continuous buoyant density gradient centrifugation of Dunaliella salina results in enhanced lipid and starch content

Abstract

Microalgae can serve as useful feedstocks for biofuel production as they can be grown with fresh, brackish, or salt water and their lipid and starch contents can be manipulated to create customized feedstocks for different classes of biofuels. Continuous buoyant density gradient centrifugation (CBDGC) was used to perform reiterative, transgressive selection to isolate wildtype and ethyl methanesulfonate-mutagenized Dunaliella salina cells with enhanced lipid and starch production. Sixty rounds of transgressive selection resulted in the isolation of cell populations with significantly lower or higher buoyant densities. Lipid content in the low-density populations was enhanced by 1.2- to 2.9-fold in wildtype cells and 1.3- to 2.3-fold in mutagenized cells as measured by Nile Red dye staining, but the lipid content differences were not significant when quantified by liquid chromatography–tandem mass spectroscopy possibly due to the composition of the lipid pools measured by these contrasting techniques. In contrast, starch content in the high-density populations was increased by 2-fold in wild type cells and 1.4- to 1.6-fold in mutagenized cells, respectively. The observed alterations in lipid and starch contents appeared to be stable after more than 70weeks (392 cell generations). CBDGC-based selection provides a useful and accessible technological alternative to genetic engineering approaches for the customization of microbial biofuel feedstocks.