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High-throughput screening of microalgae for use as a potential feedstock for biodiesel requires a reliable method for the rapid detection of intracellular neutral lipid content. In this study, we report a modified and improved Nile Red (NR) fluorescence staining procedure for use as a rapid and sensitive screening tool to estimate levels of intracellular neutral lipid in the picopleustonic microalgae, Nannochloropsis sp. Addition of either glycerol or dimethyl sulfoxide (DMSO) into microalgae cultures greatly enhances lipid staining efficiency and increases the fluorescence intensity of stained cells. The optimized procedure requires glycerol and DMSO at the concentration of 0.1 and 0.165 g mL −1 , respectively, for peak fluorescence in a live culture of Nannochloropsis sp. Incubation for 5 min for glycerol-NR staining and 10 min for DMSO-NR staining at room temperature, in darkness, is used for the NR concentration of 0.3 and 0.7 μg mL −1 for glycerol and DMSO, respectively. For the selection of lipid-rich cells of Nannochloropsis sp. using flow cytometric cell sorting, the glycerol-NR procedure is recommended as glycerol, unlike DMSO, does not inhibit subsequent growth of sorted cells.

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To advance the utilization of microalgae as a viable feedstock for biodiesel production, the intracellular lipid content of three strains of the marine microalgae Nannochloropsis sp. was enhanced using flow cytometry (FC) coupled with cell sorting. Total lipid content was doubled to 55% (biomass dry weight) in the sorted, daughter cells of Nannochloropsis (strain 47) after consecutive three rounds of cell sorting, and this trait was maintained for approximately 100 subsequent cell generations. In addition, daughter cells had a fatty acid profile similar to that of the parent, wild-type strain. The study demonstrates that FC coupled with cell sorting is a powerful tool for the enhancement of intracellular lipid content in microalgae exploited for biodiesel feedstock.

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