"Bligh and Dyer" and Folch Methods for Solid-Liquid-Liquid Extraction of Lipids from Microorganisms. Comprehension of Solvatation Mechanisms and towards Substitution with Alternative Solvents

Abstract

Bligh and Dyer (B & D) or Folch procedures for the extraction and separation of lipids from microorganisms and biological tissues using chloroform/methanol/water have been used tens of thousands of times and are "gold standards" for the analysis of extracted lipids. Based on the Conductor-like Screening MOdel for realistic Solvatation (COSMO-RS), we select ethanol and ethyl acetate as being potentially suitable for the substitution of methanol and chloroform. We confirm this by performing solid-liquid extraction of yeast (Yarrowia lipolytica IFP29) and subsequent liquid-liquid partition-the two steps of routine extraction. For this purpose, we consider similar points in the ternary phase diagrams of water/methanol/chloroform and water/ethanol/ethyl acetate, both in the monophasic mixtures and in the liquid-liquid miscibility gap. Based on high performance thin-layer chromatography (HPTLC) to obtain the distribution of lipids classes, and gas chromatography coupled with a flame ionisation detector (GC/FID) to obtain fatty acid profiles, this greener solvents pair is found to be almost as effective as the classic methanol-chloroform couple in terms of efficiency and selectivity of lipids and non-lipid material. Moreover, using these bio-sourced solvents as an alternative system is shown to be as effective as the classical system in terms of the yield of lipids extracted from microorganism tissues, independently of their apparent hydrophilicity.

Keywords: Bligh and Dyer; Folch; bio-sourced solvent; lipids; yeast.

Figure 1

Initial test of solubilities in pure solvents. Relative distribution (weight) of different extracted compounds (lipids, proteins and sugars) in the pure solvents.

Figure 2

Extraction yields (g for 100 g of dry matter (DM)): Results of different extractions realized with the chloroform–methanol–water system and with the ethyl acetate–ethanol–water system, given by the gravimetric method and gas chromatography compared to the Bligh and Dyer reference method (1959).

Figure 3

High performance thin-layer chromatography (HPTLC) plates of classical and alternative systems; validation of methods by the presence of lipids in organic phases. (PI: phosphatidylinositol, PE: phosphatidylethanolamine, PC: phosphatitylcholine, Lyso: lysophosphatidylcholine, FFA: Free Fatty Acids, DAG: diacylglycerol, TAG: Triacylglycerol, MAG: monoacylglycerol).

Figure 4

Relative profiles of fatty acids and lipid classes given by gas chromatography and HPTLC. The aim was to determine whether there was selectivity of different lipid classes (PI: phosphatydilinositol, PE: phosphatidylchoine, PC: phosphatitylcholine, FFA: Free Fatty Acids, DAG: diacylglycerol, TAG: Triacylglycerol) and fatty acids (C16: palmitic acid, C16:1: hexadecanoic acid, C18: stearic acid, C18:1n9: oleic acid, C18:2n6: linoleic acid, C18:3n6 : linolenic acid) between the different extraction compositions of points 1 to 13 in a classical diagram and A to N in an alternative diagram.

Figure 5

Extraction yields of proteins in both systems. Yields obtained by UV spectrometry in aqueous and organic phases. Points 1 to 13 are in the diagram with the classical solvents and points A to N are in the diagram with the green alternative solvents. The orange part is the organic phase and the blue part is the aqueous phase.

Figure 6

Extraction yields of glucose in both systems. Yields obtained by UV spectrometry in aqueous and organic phases.

Figure 7

Ternary systems of classical Bligh and Dyer (B &D) (Methanol/Chloroform/Water) and Greener Bligh and Dyer (ethanol–ethyl acetate–water). The black curve separates the monophasic region (above) from the diphasic region (below).