Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture

•Heating at 55℃ and 30 min was reliable for total EPS extraction from MBSC.•The optimum condition was applicable for mixture with a wide range of M:B mass ratios.•AFCS enabled accurate seperation of microalgae and bacteria in the MBSC.•AFCS-heating accurately differentiated EPS from microalgae versus bacteria with low EPS loss.•The EPS content of bacteria was about 4.7-fold greater than the EPS content of microalgae. Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (