Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

Current approaches for quantification of major energy-storage forms in microalgae, including starch, protein and lipids, generally require cell cultivation to collect biomass followed by tedious and time-consuming analytical procedures. Thus, label-free, non-destructive and simultaneous quantification of such macromolecules at single-cell resolution is highly desirable in microalgal feedstock development and bioprocess control.

Here, we established a method based on single-cell Raman spectra (SCRS) that simultaneously quantifies the contents of starch, protein, triacylglycerol (TAG) and lipid unsaturation degree in individual Chlamydomonas reinhardtii cells. Measurement accuracy for the contents based on full SCRS spectrum each reached 96.86-99.24%, all significantly higher than single peak-based models. However, accuracy and reliability of measurement are dependent on the number of cells sampled, thus a formal mathematical framework was proposed and validated to rationally define "minimal sampling depth" for a given state of cellular population. Furthermore, a barcode consisting of 13 marker Raman peaks was proposed to characterize the temporal dynamics of these energy-storage products, which revealed that the average contents of starch and TAG increased, while their heterogeneity indices decreased, with those of protein being exactly the opposite. Finally, our method is widely applicable, as measurements among cells from liquid suspension culture, wet paste and frozen dried powder all exhibited excellent consistency.

When sampled at proper depth, SCRS can serve as a quantitative and generally applicable tool for characterization and screening of strains and bioprocesses based on the profile of energy-storage macromolecules and their among-cell heterogeneity.