Kang and Sim (Kang and Sim, 2007) developed a two-stage solvent procedure using dodecane and methanol in order to extract free astaxanthin from H. pluvialis. The culture broth was first mixed with dodecane, and than the mixture was sedimented 48 h. Later, the dodecane extract was separated from the cell debris and placed in another tank and mixed with NaOH in methanol (0.02 M) at a volume ratio of 1:1 (saponification of astaxanthin esters to free form). Later, the tank was kept in darkness at 4°C (12 h) for free astaxanthin extraction in the methanol phase. The results indicated a total recovery yield of free astaxanthin of over 85% DW.
