Lipids tracking in Oleaginous Yeast by flow cytometry

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.