Using flow cytometry to evaluate thermal extraction of EPS from Synechocystis sp. PCC 6803

Soluble microbial products (SMP) and extracellular polymeric substances (EPS) produced by photoautotrophic cyanobacteria become substrates for heterotrophic bacteria in photobioreactors (PBRs). Understanding the roles of SMP and EPS depends on reliable extraction and measurement methods. While SMP can be separated from biomass using filtration, EPS extraction is more challenging. Flow cytometry (FC) with the nucleic-acid (NA) stain SYTOX Green (SG) was used to evaluate EPS solubilization and cell lysis during thermal extraction of EPS from biomass of Synechocystis sp. PCC 6803. Fluorescence intensity (FI) was used to assay the binding of SG with NA, and FC made it possible to distinguish extracellular NA from intracellular NA. Thermal treatment affected the yield and accuracy of the measurement in systematic ways. For a 20-min extraction, solubilization of EPS increased and the emission FI of SG binding with extracellular NA decreased with temperature from 30 °C to 60 °C. Cell lysis and EPS denaturation occurred for temperature higher than 70 °C. High EPS-extraction efficiency without cell lysis and EPS denaturalization was achieved with thermal extraction at 60 °C for 20 min for Synechocystis PCC 6803. This work lays the foundation for using the FC + SG methodology to evaluate the effectiveness of any EPS-extraction method.