Optimization of One-Step In Situ Transesterification Method for Accurate Quantification of EPA in Nannochloropsis gaditana

Abstract

Microalgae are a valuable source of lipid feedstocks for biodiesel and valuable omega-3 fatty acids. Nannochloropsis gaditana has emerged as a promising producer of eicosapentaenoic acid (EPA) due to its fast growth rate and high EPA content. In the present study, the fatty acid profile of Nannochloropsis gaditana was found to be naturally high in EPA and devoid of docosahexaenoic acid (DHA), thereby providing an opportunity to maximize the efficacy of EPA production. Using an optimized one-step in situ transesterification method (methanol:biomass = 90 mL/g; HCl 5% by vol.; 70 • C; 1.5 h), the maximum fatty acid methyl ester (FAME) yield of Nannochloropsis gaditana cultivated under rich condition was quantified as 10.04% ± 0.08% by weight with EPA-yields as high as 4.02% ± 0.17% based on dry biomass. The total FAME and EPA yields were 1.58-and 1.23-fold higher separately than that obtained using conventional two-step method (solvent system: methanol and chloroform). This one-step in situ method provides a fast and simple method to measure fatty acid methyl ester (FAME) yields and could serve as a promising method to generate eicosapentaenoic acid methyl ester from microalgae.

Microalgae Characterization for Consolidated and New Application in Human Food, Animal Feed and Nutraceuticals

“…The lipid content was extracted from about 120 mg of each lyophilized microalgae using a mixture of chloroform:methanol:distilled water with a ratio of 1:2:0.75 (3.75 mL) in continuous stirring for 1 h using the Bligh and Dyer method as modified by Tang et al [ 38 ]. After extraction, 1 mL of chloroform and 0.5 mL of double-distilled water were added.…”

Oil extraction and derivatization method: a review

“…Furthermore, the best method for one species was not the same for the others, which points to a matrix effect and that the method used for the analysis of the fatty acid content of different organisms must be carefully selected. Tang et al, 125 performs an optimization of the method for the determination of fatty acids in Nannochloro psisgaditana. Total yields of methylated fatty acids and EPA were 1.58 and 1.23 times higher separately than those obtained by the conventional two-step method (solvent system: methanol and chloroform).…”

“…[145][146][147] This method presents some complications, such as: the addition of acetyl chloride generates an exothermic reaction that sometimes results in sample loss and possible injury to the analyst; adding acetyl chloride slowly and with stirring is difficult for large numbers of samples; in addition, certain polyunsaturated fatty acids are not very stable at 100°C during the transesterification process; and the generation of molecular species that can contribute to fatty acids degradation. 148 This method has also been used by Guil-Guerrero et al, 143 and Masoodet al 147 Tang et al, 125 optimizes a transesterification process in situ for the quantification of EPA in Nannochloropsis gaditana. This method was previously described by Laurens et al, 149 and it provides advantages over the conventional two-stage method, such as speed, which is simple and reliable.…”

Supercritical Fluid Extraction of Lutein from Scenedesmus almeriensis

“…The lipid content was extracted from about 120 mg of each lyophilized microalgae using a mixture of chloroform:methanol:distilled water with a ratio of 1:2:0.75 (3.75 mL) in continuous stirring for 1 h using the Bligh and Dyer [40] method, as modified by Tang et al [41]. After extraction, 1 mL of chloroform and 0.5 mL of double-distilled water were added.…”