Sensitive and Accurate Differentiation of Extracellular Polymeric Substance Hydrolysis Using Flow Cytometry during Biofilm Extracellular Polymeric Substance Extraction

Abstract

Extracellular polymeric substances (EPS) affect the formation, properties, and function of biofilms. Understanding the roles of EPS in biofilm-related processes depends on reliable extraction and evaluation methods. However, differentiating true EPS from intracellular organic polymers released by cell lysis has proven to be difficult during EPS extraction. In this study, thermal and cation exchange resin (CER) treatments were used to extract biofilm EPS. SYTOX Green (SG) dye was used to stain nucleic acid (NA), and the enhanced fluorescence signal (FS) from the NA-SG complex was sensitively recorded by flow cytometry (FC). Results showed that thermal extraction could achieve effective EPS release, but a higher temperature and longer time caused a significant cell activity decline, protein denaturation, and cell lysis. In contrast, CER extraction had no effects on cell activity and organics denaturation, and it only caused minor cell lysis at a high CER dose. Combining all the results indicates that CER extraction using 70 g-CER/g-VSS for 2 h provided reliable and efficient extraction of biofilm EPS: high EPS extraction efficiency with minimal EPS denaturation, cell lysis, and loss of cell activity. FC combined with SG staining should be applicable for evaluating the effectiveness of EPS extraction from any microbial aggregates.

Mass Flow and Metabolic Pathway of Nonaeration Greywater Treatment in an Oxygenic Microalgal–Bacterial Biofilm

“…In a microalgal–bacterial symbiotic biofilm, extracellular polymeric substances (EPS) play important roles in biosorption and biotransformation. , EPS’s multiple functional groups, including –COOH, –OH, –NH, –CONH, and –NH 3 + , enable the complexation of linear alkylbenzenesulfonates (LAS) and various species of nitrogen. ,− For the biological transformations of C and N, metabolic pathways involve uptake, dissimilation, assimilation, and mineralization steps catalyzed by multiple functional enzymes that are associated with the bacteria. ,,, According to a published stoichiometric analysis, 49% of C was removed by bacterial assimilation when acetate was the input organic, while microalgae and bacteria contributed to 25 and 59% of N removal, respectively . However, the mass flows of C and N in the symbiotic biofilms were not verified experimentally.…”