Abstract
Yarrowia lipolytica, a non-conventional and strictly aerobic yeast, has been extensively studied due to its ability in producing substances attractive for the industry, such as organic acids, proteins, lipases and lipids. Oleaginous microorganism refers to a group that can present a lipid content higher than 20% of its cell dry weight. Y. lipolytica is described by literature as an oleaginous yeast because the presence of a large amount of oil into the cell. Oil is stored usually in a special organelle that is designated by different terms in literature, such as lipid particle, oil body, lipid droplet (LD) or lipid body (LB). The structure comprehends neutral lipids, mainly triacylglycerol and steryl esters, forming a hydrophobic core encompassed by a phospholipid monolayer with some proteins. There are several methods available to determinate intracellular oil; however all of them demand a long time to provide data. Flow cytometry is a technique that provides data almost in real time (at line). Therefore it is possible to evaluate the biological system in a qualitative and quantitative way and faster than the traditional methods. The yeast strain Yarrowia lipolytica IMUFRJ 50682 is currently being used in our laboratory as a model to investigate the production of a plurality of desired substances, lipase, citric acid and the like. In the present work, this yeast was cultivated on three different media in flasks. Medium 1: complex mineral medium plus pure glycerol; Medium 2: simple mineral medium plus crude glycerin and; Medium 3 simple mineral medium plus glucose. Lipid accumulation was monitored by flow cytometry associated with the fluorescent stain Nile Red (9-diethylamina-5H-benzo[a]phenoxazine-5-one). By using this technique, it was able to determinate cell total lipid and distinguish polar lipids and neutral lipids into the cell, the last ones related with stored oil particle. Although three media used showed differences in oil accumulation, their growth profiles were very similar. A significant increment in total lipid content was detected in Medium 1 and 3, using pure glycerol and glucose, respectively. The results obtained demonstrate flow cytometry as a successful technique to monitor lipid storage in cultures of Y. lipolytica.
