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An alternative high-throughput staining method for detection of neutral lipids in green microalgae for biodiesel applications

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Abstract

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO4). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure.

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  1. Phycology laboratory, Department of Botany, University of Calcutta, Kolkata, 700-019, West Bengal, India

    Gour Gopal Satpati & Ruma Pal

  2. BD Biosciences, Centre for Research in Nanoscience and Nanotechnology (CRNN), Kolkata, 700-098, West Bengal, India

    Sanjaya Kumar Mallick

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Satpati, G.G., Mallick, S.K. & Pal, R. An alternative high-throughput staining method for detection of neutral lipids in green microalgae for biodiesel applications. Biotechnol Bioproc E 20, 1044–1055 (2015). https://doi.org/10.1007/s12257-015-0281-z

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