Label-free live microalgal starch screening via Raman flow cytometry

Microalgal carbon fixation attracts much attention as an environmentally friendly source of biomaterials. In this context, microalgal starch plays an important role because it can be converted to bioethanol and bioplastics. However, optimizing culturing conditions and generating strains with higher starch productivity is still a challenge primarily due to the lack of methods for screening starch content at a single-cell level; established starch quantification methods are time-consuming, reagent-consuming, and destructive. Here we report a high-throughput, starch-screening method based on Raman flow cytometry by measuring molecular vibrations that can be used in suspended cells in their growth medium. Our method obtains the full fingerprint spectrum of starch, providing increased specificity when compared to narrow-band Raman spectroscopy methods. Specifically, we demonstrated label-free, single-cell-resolved screening of intracellular starch in cells of microalgal species Chromochloris zofingiensis that overcame the above problems and achieved >30–100 times higher event rate than previous methods, which enabled the acquisition of >20 times more cells in a fraction of the time. We screened the single-cell starch contents of about 28,000 C. zofingiensis cells cultured under 14 different conditions (~2000 cells per condition) to adjust culture conditions for starch accumulation. Comparing the data against Lugol staining and bulk-sample enzymatic starch quantification showed the potential of Raman flow cytometry for mean starch quantification. Raman flow cytometry showed higher sensitivity than Lugol staining and linear dependence with the mean per-cell starch ratio obtained from enzymatic starch quantification. Raman flow cytometry enables single-cell label-free metabolite quantification and paves the way to accurate, cost-effective optimization of culturing conditions, with future applications in the screening of starch hyper-producing microalgae following mutagenesis or from environmental samples.