Characterization of nutrient-removing microbial communities in two full-scale WWTP systems using a new qPCR approach.

Biological wastewater treatment processes involve very complex microbial communities. Culture-independent molecular methods are feasible tools used to analyze and control the structure of different microbial communities, such as bacterial communities that remove nutrients. Here, we used the gBlocks gene fragments method, a new real-time PCR approach for the development of DNA standards, to quantify total bacterial cells, AOB, NOB, and Archaeal genes at two different WWTPs. PAOs were also quantified using the FISH technique. Our findings highlight a significant improvement in real-time PCR detection for the microorganisms studied. The qPCR and FISH technique applied allowed characterization of the microbial composition of two WWTPs operated as a conventional WWTP and a biological nutrient-removal WWTP. The results revealed a significant difference in the microbial profiles of the WWTPs, with a higher abundance of nitrifying bacterial communities and PAOs in the nutrient removal plant, which were in accordance with operational performance.

Tracking and quantification of nitrifying bacteria in biofilm and mixed liquor of a partial nitrification MBBR pilot plant using fluorescence in situ hybridization.

A moving bead biofilm reactor (MBBR) pilot plant was implemented as a partial nitrification process for pre-treatment of ammonium-rich liquors (676 ± 195 mg L(-1)), and studied for 479 days under variations in hydraulic retention time. The main purpose of this work, was the study of dynamics abundance of total bacteria and single-cells nitrifying bacteria belonging to ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in biofilms and mixed liquor of the plant. The microbial monitoring was successfully achieved using fluorescence in situ hybridization combined with flocs disaggregation protocol as a useful microbial monitoring tool. A partial nitrification process with a N-NH4(+) removal rate of about 38.6 ± 14.8% was successfully achieved at 211 days after start-up, with a clear dominance of AOB, which accounted for 11.3 ± 17.0% of total bacterial cells compared with only 2.1 ± 4.0% of NOB. The effluent obtained was subsequently supplied to an Anammox reactor for complete ammonium treatment.

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 × 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.