An efficient modification of the Bligh and Dyer method, given below, was proposed for the extraction of lipids from unicellular organisms. Several parameters were optimized to improve the fatty acid recovery (Lewis T et al., J Microbiol Meth 2000, 43, 107). Thus, it was shown that the total amount of recovered fatty acids increased by about 30% by adding solvents to the biomass in order of increasing, as opposed to decreasing, polarity.
Cells were harvested by centrifugation at high speed for 15 min. The supernatant was discarded, the cell pellet re-suspended in 100 ml 1.0% NaCl (w/v), and re-centrifuged. The second supernatant was discarded and the cell pellet frozen overnight at -30°C. Frozen biomass was freeze dried for 15 h and subsequently stored in a sealed glass container at -30°C.
To freeze-dried cells (about 100 mg) to which a total of 114 ml solvents were added in the sequence: chloroform, methanol, water to achieve a final chloroform/methanol/water ratio of 1/2/0.8 (v/v/v). Samples were shaken for 15 s immediately following the addition of each solvent, and allowed to stand for about 18 h, with occasional shaking by hand.
Phase separation of the biomass-solvent mixtures in the separatory funnels was achieved by adding chloroform and water to obtain a final chloroform/methanol/water ratio of 1/1/0.9 (v/v/v). A known portion of each total lipid extract recovered from the lower chloroform phase was used for further analysis.
Comparing various extraction procedures, it has been shown that a modified (miniaturized) Bligh and Dyer extraction technique was the most efficient with an oleaginous bacteria Thraustochytrium sp (Burja AM et al., J Agric Food Chem 2007, 55, 4795). If only fatty acid determination is required, a direct saponification using KOH in ethanol was almost as efficient as the previous one.
An hexane-isopropanol solvent system was found to be effective for the bacterium Pseudomonas atlantica (Guckert J B et al., J Microbiol Methods 1988, 8, 131), this system gaving a lower recovery of lipids in the green alga Chlorella sp. (Guckert J B et al., J Microbiol Methods 1988, 8, 139).
At work using a yeast, Yarrowia lipolytica, has reported that bio-based solvents could be an alternative to petrochemical solvents, such as hexane. Some differences were noted between experimental and theoretical studies (Breil C et al., Molecules 2016, 21, 196). Ethyl acetate and methyltetrahydrofuran are the best candidate solvents to extract all of the lipids of Yarrowia lipolytica (triglycerides, diglycerides, free fatty acids and phospholipids) and are derived from renewable resources.