Assessment of total bacterial cells in extended aeration activated sludge plants using flow cytometry as a microbial monitoring tool

The extended aeration activated sludge (EAAS) process is one of the most applied biological processes in small towns. Here, we study the abundance and viability of total bacterial cells in two wastewater treatment plants (WWTPs) operating with an EAAS process. We use flow cytometry (FCM) combined with SYTO13 and propidium iodide (PI) dyes as a rapid, easy, reliable and accurate microbial monitoring tool. A disaggregation procedure with an ultrasonic bath was designed to detach total bacterial cells from activated sludge flocs for subsequent FCM analysis. This procedure permitted the recovery of total bacterial cells from sludge flocs without affecting bacterial viability, as indicated by bacterial strain controls. Since FCM is a multi-parameter technique, it was possible to determine total bacterial abundance and their viability in the activated sludge. As a comparative method, epifluorescence microscopy was also used to quantify total bacterial cells; both methods produced similar results. The FCM analysis revealed relative microbial stability in both the WWTPs. The total bacterial abundance quantified by FCM in the two plants studied was 1.02-6.23 x 10(11) cells L-1 with 70-72 % viability, one logarithm less than that reported in the literature for WWTPs using the conventional activated sludge process. This can be explained by the difference in the operational parameters between the conventional plant and EAAS, mainly the organic loading rate.