GRAM-SCALE PURIFICATION OF EICOSAPENTAENOIC ACID (EPA, 20 5N-3) FROM WET PHAEODACTYLUM-TRICORNUTUM UTEX-640 BIOMASS/

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalga P haeodactylum tricornutum following a three-step process: fatty acid ex traction by direct saponification of wet biomass, polyunsaturated fatt y acid (PUFA) concentration by formation of urea inclusion compounds a nd EPA isolation by preparative HPLC. Direct saponification of wet bio mass was carried out with KOH-ethanol (96% v:v) (1 h, 60 degrees C), e xtracting 91% of the EPA. PUFAs were concentrated by the urea method w ith an urea/fatty acid ratio of 4: 1 at a crystallization temperature of 28 degrees C using methanol as the urea solvent. An EPA concentrati on ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PU FA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C-18, 4.7 cm i.d. x 30 cm) and methanol- water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in the P. tricornutum bi omass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PU FA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d(-1)). Finally, a preliminary cost statement has been calculated.