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

Article history: Received 25 July 2016 Received in revised form 19 September 2016 Accepted 30 October 2016 Available online xxxx 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 SGwith NA, and FCmade 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 decreasedwith 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 + SGmethodology to evaluate the effectiveness of any EPS-extraction method. © 2016 Elsevier B.V. All rights reserved.