Key role of fluorescence quantum yield in Nile Red staining method for determining intracellular lipids in yeast strains

Abstract

Background Microbial lipids are found to be an interesting green alternative to expand available oil sources for the chemical industry. Yeasts are considered a promising platform for sustainable lipid production. Remarkably, some oleaginous yeasts have even shown the ability to grow and accumulate lipids using unusual carbon sources derived from organic wastes, such as volatile fatty acids. Recent research efforts have been focused on developing rapid and accurate fluorometric methods for the quantification of intracellular yeast lipids. Nevertheless, the current methods are often tedious and/or exhibit low reproducibility. Results This work evaluated the reliability of different fluorescence measurements (fluorescence intensity, total area and fluorescence quantum yield) using Nile Red as lipid dye in two yeast strains (Yarrowia lipolytica ACA-DC 50109 and Cutaneotrichosporon curvatum NRRL-Y-1511). Different standard curves were obtained for each yeast specie. Fermentation tests were carried with 6-month difference to evaluate the effect of the fluorometer lamp lifetime on lipid quantification. Conclusions Fluorescence quantum yield presented the most consistent measurements along time and the closer estimations when compared with lipids obtained by conventional methods (extraction and gravimetrical determination). The need of using fluorescence quantum yield to estimate intracellular lipids, which is not the common trend in studies focused on microbial lipid production, was stressed. The information here provided will surely enable more accurate results comparison.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform

“…Due to the lack of standard curves for the lipid determination by fluorescence for all the tested strains, lipid content was determined gravimetrically as detailed in Morales-Palomo et al 48 . Briefly, cell biomass (0.4 g/L) was centrifuged at 5000 rpm for 10 min at 4 °C (Heraeus, Megafuge, Thermo Scientific) and dried at 105 °C overnight.…”

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform

“…Lipid content was fluorometrically determined with Nile Red (9-diethylamino-5 H-benzo[ a ]phenoxazine-5-one, Acros Organics) as described by Morales-Palomo and colleagues (2022) [ 24 ]. Fluorescence curves in each time point were converted to quantum yield to accurately determine lipid content.…”

Phosphate limitation as crucial factor to enhance yeast lipid production from short‐chain fatty acids

“…Lipid content was calculated by fluorimetrical means using the optimized protocol described in Morales‐Palomo, Liras, et al, 2022 . The data obtained from the area of each fluorescence curve were converted to quantum yield.…”