Micro-Raman spectroscopy of the light-harvesting pigments in Chlamydomonas reinhardtii under salinity stress.

Microalgae are a rich source of carotenoids with enhanced yields during biotic or abiotic stresses, which often impose survival challenges on the cells. Using a non-invasive pigment profiling approach with micro-Raman spectroscopy, we have analyzed the effect of salinity stress on carotenoids in autotrophic Chlamydomonas reinhardtii. Raman spectral analysis of ν(C = C) mode indicates an increase in the carotenoids with lower conjugation length (lutein and zeaxanthin) compared to β-carotene, as the function of culture age and salinity stress, but especially when salinity stress was imposed in two-stage mode (stress imposed on 2nd day, D2_100, and 4th day, D4_100, during exponential phase). Population-scale heterogeneities in carotenoid Raman mode peak center, quantified with heterogeneity index (HI), were highest during the stationary phase of the cultures and under salinity stress. Although the Raman signal was obtained from a randomly selected small focal volume in the cell, a decrease in chlorophyll Raman mode intensities with age and salinity stress was well corroborated by single-cell population fraction measurements by microscopy. Raman intensity fluctuations (I) were high for both chlorophyll and carotenoid modes under salinity stress, which can arise due to variations in chlorophyll/carotenoid content and composition, or conformational changes in the pigments in C. reinhardtii cells. Interestingly, in all growth conditions, chlorophyll a Raman mode intensity was found to show a high correlation to that of β-carotene, pointing out a high degree of cooperativity in the light-harvesting complex pigments even during salinity stress. Thus, we demonstrate the usefulness of non-invasive pigment profiling with micro-Raman spectroscopy for developing an optimization for salinity stress conditions for high biomass yield and proper harvest time to obtain carotenoids with desired chemical composition.