Nile Red staining of phytoplankton neutral lipids: species-specific fluorescence kinetics in various solvents.

Katariina Natunen,Timo Tamminen,Dagmar Schwenk,Heiko Rischer,Jukka Seppälä,Kristian Spilling

doi:10.1007/s10811-014-0404-5

Katariina Natunen, Timo Tamminen + Show 4 more

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https://doi.org/10.1007/s10811-014-0404-5

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Abstract

Nile Red (NR) staining potentially offers a simple method for monitoring lipid accumulation in microalgal cultivation. However, variable staining efficiencies and methods have been reported. The effect of dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol on NR penetration with four different phytoplankton species representing different taxonomical groups was studied. Treatment with the solvents enhanced the NR fluorescence of the diatom Phaeodactylum tricornutum during kinetic fluorescence measurements, but high concentrations of solvents were needed. None of the solvents improved NR staining of the green alga Chlorella pyrenoidosa and Scenedesmus obliquus, which are known to be difficult to stain due to their thick and rigid cell walls. The naked Isochrysis sp. cells stained best without solvents. The results confirm that NR staining protocol needs to be optimized for each species.

Highlights

Photosynthetic phytoplankton, referred to as microalgae, are a potential feedstock for biofuels, especially because many species produce valuable neutral lipids as storage products that can be converted to biodieselK
The objectives of this study were (i) to find out whether the Nile Red (NR) penetration into phytoplankton cells and the subsequent staining of neutral lipids could be enhanced and standardized with the use of solvents in the case of phytoplankton species that have been proven difficult to stain, (ii) to evaluate whether the improved staining method developed by Chen et al (2009) could be generalized to other algae types or (iii) whether the fluorescence kinetic parameters need to be evaluated separately for each species and growth phase, as suggested by Cooksey et al (1987)
The phytoplankton species were cultivated in 2 L polycarbonate bottles using 1.5 L of modified f/2 medium (Guillard 1975) that had been adjusted to the weight ratios of N/P=1.8 and N/Si=0.25 so that the cells would be nitrogen limited in the stationary growth phase

Summary

Introduction

Photosynthetic phytoplankton, referred to as microalgae, are a potential feedstock for biofuels, especially because many species produce valuable neutral lipids (e.g. triacylglycerols) as storage products that can be converted to biodieselK. The objectives of this study were (i) to find out whether the NR penetration into phytoplankton cells and the subsequent staining of neutral lipids could be enhanced and standardized with the use of solvents in the case of phytoplankton species that have been proven difficult to stain, (ii) to evaluate whether the improved staining method developed by Chen et al (2009) could be generalized to other algae types or (iii) whether the fluorescence kinetic parameters need to be evaluated separately for each species and growth phase, as suggested by Cooksey et al (1987). The effect of different final concentrations of DMSO (5, 10, 20 and 30 % v/v), EG (Riedel-de Haën; 1, 5, 10 and 20 % v/v) and glycerol (Normapur; 5, 10, 15 and 20 % v/v) on NR penetration into the cells was followed by kinetic fluorescence measurements.

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