Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

Abstract

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

Microalgae degradation follow up by voltammetric electronic tongue, impedance spectroscopy and NMR spectroscopy

“…In the context of microalgae, NMR spectroscopy has been used to determine their chemical composition using diverse technical implementation to show lipophilic [55][56][57] and hydrophilic [55,57,58] compounds or the whole cells profile [55,59,60]. The usefulness of NMR spectroscopy for microalgae classification has been also demonstrated, showing the changes in the profiles of different microalgae classes [61].…”

Inhibition of DNA Methylation in Picochlorum soloecismus Alters Algae Productivity

“…Both FSC and SSC were impacted by 5AZA treatment in similar ways: initially 5AZA significantly increased both FSC ( Figure 5D, p < 0.01) and SSC ( Figure 5E, p < 0.001) until Days 8 and 10, respectively, but this effect was abrogated as the days in culture increase. In other studies of microalgae cultivation, lack of change in cell counts accompanied by an increase optical density, FSC, and SSC suggests altered cellular composition particularly of biomolecules like neutral lipids (Bono et al, 2015;Gonzalez-Esquer et al, 2019;Steadman Tyler et al, 2019). Using a BODIPY fluorescent probe (Steadman Tyler et al, 2019), we found that lipid accumulation was significantly increased after 4 days of 5AZA treatment (Figure 5F).…”

Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art

“…BODIPY 505/515 has been the fluorescent marker of choice for the evaluation of intracellular lipids in algae [166,168]. TAG content in BODIPY 505/ 515-stained C. vulgaris by single-cell fluorescence strongly correlated with the result obtained by 1 H NMR [126]. BODIPY 505/515 dissolved in DMSO at a final concentration of 1-10 μM was shown to selectively stain lipid bodies of several taxa of live algae.…”

“…This strategy was applied to quantify TAGs in living microalgae in their growth medium [125]. NMR is ideal for high-throughput calibration of flow cytometry methods aimed at the quantification of cellular TAGs in various algal species [126]. Further, semi-solid NMR techniques, such as high-resolution magic-angle spinning (HR-MAS) and intermolecular multiple-quantum coherence (iMQC), can be used to detect non-TAG lipids [126].…”

“…The latter is exacerbated in algal species with thick cell walls. This complicates the development of consistent fluorescence protocols, while dye uptake is dependent on cell viability [126]. Further, Nile Red is intensely fluorescent in organic solvents, but has a low quantum yield in water [156].…”