Extraction of lipids from wet microalga Auxenochlorella protothecoides using pulsed electric field treatment and ethanol-hexane blends

Pulsed Electric Field (PEF) treatment was used as pre-treatment on the microalgae strain Auxenochlorella protothecoides (A.p.) prior to organic solvent extraction of lipids. Experiments were performed on fresh biomass from mixotrophic or autotrophic culture which both had an evaluated lipid content of 30–35% of cell dry weight. Lipid yield was determined gravimetrically and compared to the reference lipid content assessed by bead-milling and subsequent Soxhlet extraction. The biomass was concentrated at 10% w/w solids prior to PEF-treatment and further dewatered afterwards to approximately 25% w/w before extraction. PEF-treatment with an energy input of 1.5MJ per kilogram of dry matter induced electropermeabilisation of the microalgae cells detected by the increase of the conductivity of the microalgae supernatant. This greatly increased the lipid yield upon subsequent monophasic solvent extraction. A mixture of Water/Ethanol/Hexane 1:18:7.3 vol/vol/vol enabled to recover 92%, and 72%, of the evaluated lipid content of mixotrophically, and autotrophically respectively, grown A.p., after 2h of extraction. Recovery increased to 97%, and 90% respectively, after 20h of extraction. The same extraction system on untreated biomass yielded maximum 10% of lipid content. The highest yields were obtained with 80mL of solvent for 1g dry biomass but solvent volume could be reduced by a factor two in case of mixotrophically grown microalgae. However, the solvent:biomass ratio still remains high, and includes a water-miscible solvent, ethanol. Total lipid extraction was confirmed by nile red staining of residual biomass combined with fluorescence microscopy imaging and flow cytometry. Gas chromatography analyses of extracted lipids after transesterification revealed that PEF- treatment did not alter their fatty acid composition. Overall PEF-treatment shows promising features for upscaling especially in a biorefinery concept since it avoids potentially harmful temperature increase and small debris problematic for further processing.