Innovative screening approach for the identification of triacylglycerol accumulating oleaginous strains

Currently, triacylglycerides (TAG) accumulation in the form of lipid droplets (LDs) in oleaginous microorganisms is of immense importance due to their ability to get transesterified into value-added products in the form of biodiesel. Hence, in order to search for oleaginous microorganisms having high lipid content among a wide range of samples from different niches, there is a compulsive need to develop simple, reliable and rapid methods for screening of TAG accumulating strains. Conventional methods require multistep processes for the isolation, cultivation, extraction and estimation of lipids to identify oleagenic strains. To overcome these challenges, we are proposing an easy, live cell imaging technique for the estimation of lipids via visualization of TAG accumulation in probable strains at the single cell level that gives real-time monitoring of intracellular lipid accumulation in yeasts. In this screening technique, only 100 mu l of specific neutral lipid accumulating medium was used to grow the isolated culture in the microtiter plate. The harvested cells were stained with LipidTOX (TM) Green and visualized by a LED based digital inverted fluorescence microscope. Among 446 yeast colonies screened, maximum lipid producing yeast strains Rhodosporidium kratochvilovae HIMPAI and Rhodotorula minuta, having supersized lipid body of 5.05 +/- 0.87 mu m and 4.46 +/- 0.61 mu m, respectively, were identified as potential candidates for biodiesel production. To the best of our knowledge, this is the first report of using LipidTOX (TM) Green for the staining of lipid droplets present in yeast cells as per the literature. (C) 2018 Elsevier Ltd. All rights reserved.