APPLICATION OF SPECTRAL FLOW CYTOMETRY FOR CHARACTERIZATION OF ALGAL AND MICROBIAL PHENOTYPES

Abstract

Microalgae and bacteria play a crucial role in aquatic ecosystems and hold significant potential for biotechnological applications. Traditional flow cytometry (FC) faces challenges in analyzing microalgae due to interference from pigments and secondary metabolites. This study leverages spectral flow cytometry to overcome these limitations, allowing for high-resolution, label-free characterization of phenotypes of microscopic organisms by capturing full-spectrum autofluorescence signals. Focusing on one bacterial specie (Serratia marcescens) and four algae species of the Chlamydomonadales order (Chlamydomonas reinhardtii, Gonium pectorale, Chlorococcum sp., and Pandorina morum), we investigated how growth phases and cellular morphology influence spectral signatures. Our results revealed distinct subpopulations within cultures, identifiable by variations in chlorophyll autofluorescence as well as accessory pigment autofluorescence. Key findings include the correlation of accessory pigment fluorescence with growth dynamics, while chlorophyll autofluorescence combined with fluorescence of metabolic pigments distinguished viable, dying, and dead cells. Additionally, spectral variance analysis highlighted metabolic shifts during growth, demonstrating the dynamic nature of pigments in the organisms over their growth cycle. This work proposes a novel method for monitoring algal cultures in biotechnological processes using accessory pigment spectra and demonstrates the utility of spectral flow cytometry to identify and distinguish spectral phenotypes of microscopic organisms.