Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).