Autofluorescence-based High-throughput Isolation of Nonbleaching Strains Under Nitrogen-depletion

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1-4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching strains in which phycocyanin was not degraded under N-depletion, autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.