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.
Highlights
The development of rapid, accurate methods to measure the lipid content of algae cells is important to the success of biodiesel production from this promising biomass resource [1, 2]
Flow cytometry experiments revealed that Chlorella vulgaris cells cultured in nitrogen-limited medium to induce lipid accumulation [2, 17] exhibited greater BODIPY fluorescence per cell than cells cultured in nitrogen-replete medium (Fig 1)
As we have shown that the mean BODIPY fluorescence of labeled cells in a culture measured via flow cytometry is strongly correlated with TAG content per cell by Nuclear magnetic resonance (NMR), we can hypothesize that the distribution of measured BODIPY fluorescence for cells within a culture indicates the distribution of lipid content for those cells
Summary
The development of rapid, accurate methods to measure the lipid content of algae cells is important to the success of biodiesel production from this promising biomass resource [1, 2]. Because TAG biosynthesis is enhanced when algae are subjected to stresses that frequently inhibit cell growth, algal lipid content can vary widely with growth conditions and over time [1, 2]. This variability necessitates timeseries measurement of lipid content for different growth conditions in order to improve cultivation protocols and monitor changes in lipid content during industrial production [1, 4]. Single-cell lipid measurement facilitates sorting of cells with high lipid content for the development of more productive algae strains [7] and fundamental investigation of the dynamics of algal lipid accumulation [6], yielding knowledge that will enable genetic engineering of improved strains [8, 9]
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