Simultaneous Detection of Multiple Carotenoid Pigments in Algae by Raman Spectroscopy and Classical Least Squares (CLS) Analysis

Synthetic carotenoid pigments derived from petrochemicals are used for feed industry. However, due to health safety concerns these synthetic forms cannot be used for direct human consumption. Algae cells naturally produce multiple carotenoid pigments, including astaxanthin and lutein that have antioxidants, antitumor and antiviral properties. Hence, these natural carotenoids have high market value as nutraceutical supplements, and also as natural food coloring and cosmetics ingredient. Cost-efficient extraction and purification of these high value carotenoids from algae is challenging as the pigment profiles of algae cells is complex and dynamic. Cells contain a mixture of multiple pigments dissolved in lipids and the composition varies with growth stages, changes in environment, and nutrient availability. To be commercially beneficial, harvesting and extraction of algal cultures should be done at an optimal time point, when both biomass and the desired high-value carotenoid accumulation are at its peak. Pigment composition and quantity are conventionally determined by chromatography and UV/ IR spectroscopy. These methods although can provide high accuracy level, are dependent on multi-step process of pigment extraction, needs large volume of algal biomass, expensive chemicals, laboratory equipments, and hours of skilled manpower. In this paper, we present a high-throughput method of in situ pigment profiling by Raman spectroscopy. Pigment spectra could be obtained under 5 min from live algae cells, without any extraction, drying or grinding. Identification and relative quantification of 3-6 carotenoid pigments simultaneously from the spectra was possible using CLS analysis method. The method was found to be sensitive, versatile and robust by statistical analysis. This approach can be used for pigment profiling and screening of live algal cell cultures with population variability for real-time assessment of their health and productivity. It additionally has the potential to be used for live pond monitoring using portable Raman spectrometers.