Directed evolution of Chlorella sp. HS2 towards enhanced lipid accumulation by ethyl methanesulfonate mutagenesis in conjunction with fluorescence-activated cell sorting based screening

Schematic illustration of the present study. A directed evolution approach by EMS mutagenesis in conjunction with FACS-based selection was used to enhance the lipid productivity of Chlorella sp. HS2. [Display omitted] •EMS mutagenesis with FACS was used for strain improvement of Chlorella sp. HS2.•SMS showed high lipid content of 35.6% and lipid productivity of 248.7 mg L-1 d-1.•Cells that underwent mutagenesis had higher content of SFA (43.1%) and MUFA (33.6%).•Biodiesel fuel properties of SMS (TE+F) were compliant with US and EU standards. The present study aimed to improve the lipid content and lipid productivity of Chlorella sp. HS2 by exposing them to ethyl methanesulfonate (EMS) to generate a mutant and followed with fluorescent assorted cell sorting (FACS) for the selection of mutant cells having desirable phenotypes. The microalgae were subjected under 100 μ mol mL−1 concentration of EMS for 30 min, followed by intracellular lipid staining with boron-dipyrromethene (BODIPY) fluorophore for FACS based selection. The biomass growth, lipid content, lipid productivity, and fatty acid composition of the selected mutagenized strain (SMS, TE+F) of Chlorella sp. HS2 were compared against those of FACS selected strain without mutagenesis (FSS, TF) and parent wild strain (PWS, TC), under two different carbon supplementation schemes (only CO2 supply and optimized carbon supply). The highest lipid content and productivity of 35.6% and 248.7 mg L-1 d-1, respectively were observed in the SMS grown under optimized carbon supplementation (1% CO2 (v/v) and 0.5 g L-1 NaHCO3), which was a substantial improvement over those of FSS (26.6% and 193.4 mg L-1 d-1) and PWS (24.7% and 153.6 mg L-1 d-1). The fatty acid methyl ester (FAME) profile of the lipids derived from SMS showed a marked increase in the proportion of saturated fatty acids (SFA) and mono-unsaturated fatty acids (MUFA) (76.7%) over those of FSS (70.2%) and PWS (69.6%). Lastly, fuel properties were found to be appropriate for biodiesel production, as they corroborate with international fuel standards. These results indicate that directed evolution using EMS mutagenesis with FACS is a powerful tool to achieve microalgal strain improvement for biofuel production.