Better understanding of the activated sludge process combining fluorescence-based methods and flow cytometry: A case study.

This study aims to demonstrate the validity of fluorescence-based methods, together with flow cytometry, as a complementary tool to conventional physicochemical analyses carried out in wastewater treatment plants (WWTPs), for the control of the currently largely unknown activated sludge process. Staining with SYTO 9, propidium iodide and 5-(and 6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-HDFFDA) was used for cell viability and oxidative stress monitoring of the bacterial population forming the activated sludge of a WWTP. Throughout the period of research, several unstable periods were detected, where the non-viable bacteria exceeded the 75% of the total bacterial population in the activated sludge, but only in one case the cells with oxidative stress grew to 9%, exceeding the typical values of 2%-5% of this plant. These periods coincided in two cases with high values of total suspended solids (SST) and chemical oxygen demand (COD) in the effluent, and with an excess of ammonia in other case. A correlation between flow cytometric and physicochemical data was found, which enabled to clarify the possible origin of each case of instability in the biological system. This experience supports the application of bacterial fluorescence staining, together with flow cytometric analysis, as a simple, rapid and reliable tool for the control and better understanding of the bacteria dynamics in a biological wastewater treatment process.