Mechanism of Nutrient Deprivation Induced Triacylglyceride Accumulation in Alga Indicated by Fluorescence Hyperspectral Imaging

Abstract

Microalgae have been identified as a promising renewable feedstock for production of lipids for feeds and fuels. For a wide variety of algal species, depletion of major nutrients from the growth medium results in dramatically increased biosynthesis of triacylglycerides, in some cases exceeding 70% of the dry weight of the biomass. In recent work, we explored this process in a number of promising algal production strains (Chlorophyceae and Eustigmophyceae) using hyperspectral confocal fluorescence imaging with multivariate spectral unmixing. By coupling intensity modulation of the excitation laser and attenuation of the dominant chlorophyll fluorescence emission, we identified a distinct spectral component composed of chlorophyll fluorescence and resonance Raman emission for lipid-solubilized carotenoids allowing label-free projections of lipid bodies in addition to plastids. Furthermore, cells exposed to the lipophilic dye, Nile Red, exhibited distinct spectral components corresponding to the polar and neutral lipid fractions. Combining these capabilities for analysis of nutrient deprivation time courses revealed increases in the lipid fraction of the cellular volume of ∼500%, as well as a correlated decrease in the plastid fraction of the total cellular volume. Additionally, diffraction limited tubules connecting the lipids bodies to the plastids and the presence of unbound chlorophyll in the lipid bodies provided evidence for direct shuttling of lipids from the chloroplast to the lipid bodies, in opposition to the expected ER to lipid body transfer dictated by the Kennedy pathway. The results suggest that nutrient deprivation-based lipid accumulation in these species is dominated by plastid membrane recycling, independent of acyl-CoA. Finally, an additional spectral component exhibiting high spectral overlap with Nile Red and varying inversely with the chlorophyll emission was detected during senescence, potentially resulting in false positives for lipid enrichment in low spectral resolution assays.