Abstract(s) :

(Anglais) Alpo project aims at using carbohydrates and lipids from microalgae to produce biosourced polymer. It is sought to add value to other compounds by fermentation with oleaginous yeasts, as they are able to metabolize different sources of carbon (e.g., monosaccharides, disaccharides, glycerol) into various kinds of lipids1, including polyunsaturated fatty acids, that are used as building blocks for polymers synthesis. Yeasts lipids extraction and quantification is a fastidious and time-consuming process involving several steps with the use of non-environmentally friendly solvent such as chloroform / methanol or hexane. Furthermore, the cost of substrate and the poor growth on it limited the quantity of biomass available for FAMEs quantification by GC-FID. An alternative method to assess yeasts lipids production during fermentation consists in dyeing intracellular lipids with a fluorochrome. The most popular lipid dye is Nile red, that binds non-specifically neutral lipids. The BODIPY 493/503 dye is more specific to neutral lipid than Nile red 2,3. In this study, four oleaginous yeast strains (two strains of Yarrowia lipolytica, one Lipomyces starkeyi and one Meyerozyma guillermondii) were compared for their ability to accumulate lipids during growth. Intracellular lipids accumulation was monitored during yeasts growth, both by Folch protocol and flow cytometry after cell dyeing with BODIPY 493/503. Good correlations between fluorescence intensity with both total lipid accumulation and polyunsaturated fatty acid concentration were found. GC analysis show that Yarrowia lipolytica accumulated more lipids but Lipomyces starkeyi presented the highest production of α-linolenic acid and linoleic acid, a long chain polyunsatured acid of interest for the chemical industry. Yeast marking during fermentation with BODIPY 493/503 associated with flow cytometry is thus a very promising technique to measure and compare the ability of yeasts to synthesize and accumulate lipids. This method makes easier the screening on high cost substrate such as microalgae. Indeed, one can by-pass lipid extraction from biomass to monitor lipid production and perform tests with few substrate in flask.