Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

UV Mutagenesis as a Strategy to Enhance Growth and Lipid Productivity of Chlorella sp. 042

Microalgae appeared to be an alternative feedstock for renewable biodiesel production due to their capability to accumulate considerable amounts of lipids. In this study, mutagenesis using UVC light with different periods was applied to Chlorella sp. 042 to produce a microalgae strain with high lipid productivity of 45, 60, and 75 min. The Nile red fluorescence method was conducted to select a Chlorella sp. mutant with high neutral lipid and generated one mutant from every UV mutation period, M45-06, M60-02, and M75-21. All of the mutants have higher growth rates than the wild type. Chlorella sp. 042 M60-02 achieved the highest lipid productivity, with 34 mg L-1 day-1. Furthermore, as other major biochemical components, carbohydrate and protein contents were determined. Our results showed that all the mutants enhance their carbohydrate and protein contents compared to the wild type. However, mutations for more than 60 min do not intensely change the protein content of mutant microalgae. Gas chromatography-mass spectrophotometry analysis revealed that M60-02 mutant has similar FAME profiles with the wild type, which contain palmitic acid (C16:0), stearic acid (C 18:0), oleic acid (C18:1), and linoleic acid (C18:2). These results demonstrate that the UV mutation of Chlorella sp. 042 for 60 min is suitable as a source of biodiesel production.

Quantification of microplastics by count, size and morphology in beverage containers using Nile Red and ImageJ

Abundant evidence of microplastics (MP) found in the environment, and its toxicity effect in animals calls for human-related research. However, well-established quantitative controlled studies on the potential route of human exposure to MP are still sparse. MP count, size and morphology in 15 polylactic acid (PLA)-lined plastic cups and 15 PLA-lined paper cups were examined using Nile Red fluorescence tagging, microscopic photography, and morphology assessment and quantification based on ImageJ. In the plastic cups, the count and area of MP fibers were found to be significantly higher compared with blanks (p < 0.05), but not MP particles or total MP. In paper cups, count or area was not significantly different in terms of MP particle, MP fibers or total MP. No interesting trend was observed in the distribution regarding the size of MP particles or fibers. These results indicate that selected paper cups and plastic cups could be considered as safe beverage containers, but further research on the toxicological effects of MPs in different morphologies released from plastic cups on human health is needed.

Dataset of Nile Red Fluorescence Readings with Different Yeast Strains, Solvents, and Incubation Times

We used Nile red to estimate lipid content in oleaginous yeasts using a high-throughput approach. We measured the fluorescence intensity of Nile red using different solvents, yeast strains, and incubation times in optimized excitation/emission wavelengths. The data show the relative fluorescence units (RFU) for Nile red excitation, using 1× PBS, 1× PBS and 5% v/v isopropyl alcohol, 50% v/v glycerol, culture medium A-gly broth, and A-gly broth supplemented with 5% v/v DMSO. In addition, we showed the RFU for the Nile red dye for different oleaginous and non-oleaginous yeast strains, such as Meyerozyma guilliermondii BI281A, Yarrowia lipolytica QU21 and Saccharomyces cerevisiae MRC164. Other measurements of lipid accumulation kinetics were shown for the above and additional yeast strains. These datasets provide the guidelines to obtain the optimal solvent system and the minimal interaction time for the Nile red dye to enter in the cells and obtain a stable readout.

Quantification of micropolarity and microviscosity of aggregation and salt-induced gelation of sodium deoxycholate (NaDC) using Nile red fluorescence

Nile red fluorescence properties can be used for the estimation of micropolarity and microviscosity of the gel medium.