In situ measurement of neutral lipids in Porphyridium cruentum cells using fluorescent Nile red while preventing interference from autofluorescent B-phycoerythrin

MAZZUCA SOBCZUK TANIA; IBANIEZ GONZÁLEZ, MJ; BRINDLEY ALIAS; MAZZUCA M; MOLINA GRIMA, M; CHRISTI YUSUF

Concepción

Congreso; Third Latin American Congress of Algal Biotechnology,; 2012

Universida de Concepción

A rapid and easy method was developed for monitoring the neutral lipids of the marine microalga Porphyridium cruentum by fluorescence. The fluorescent dye Nile red has been used with many green microalgae as a marker for neutral lipids (Elsey et al., 1987; Chen et al., 2011; Chen et al., 2009; Huang, 2009). This method is much easier compared to extraction of lipids from the biomass and gravimetric determination. Unlike some of the other marine algae, the red microalgae P. cruentum contains b-phycoerythrin, a fluorescent protein that emits with a high intensity in the same range of wavelengths as do neutral lipids stained with Nile red. This study defines the conditions (pH, dye concentration and biomass pretreatment) that allow for the neutral lipids content of biomass to be estimated while preventing interference from the fluorescent emission of the b-phycoerythrin. Prior to staining, the cell suspension was acidified with HCl (1.2 M) and the cells were recovered by centrifugation. The cells were then resuspended in a buffered aqueous medium (sodium acetate buffer, 260 mM, pH 5.4), stained, and measured. A linear correlation was established between the nonpolar lipids quantified by gravimetry and the fluorescence intensity of the resuspended cells. Studies were carried out with the microalgal cells grown in batches as well as in continuous cultures.