The development and use of real-time PCR for the quantification of nitrifiers in activated sludge

Chemical analytical data has long been used to monitor the performance of activated sludge plants even though the process relies on the performance of microorganisms. It is now evident that a rapid and reliable quantitative method is required, to be able to monitor the organisms responsible for nutrient transformation and their activities, allowing avenues for more efficient nutrient removal. The development of real-time or quantitative polymerase chain reaction (PCR) also known as TaqMan® or 5′-nuclease assay has allowed the rapid, quantitative analysis of DNA templates, eliminating some of the variability traditionally associated with other quantitative techniques. In this study analysis of Nitrospira spp., one of the key organisms in nitrite oxidation in wastewater treatment, was used to validate real-time PCR for the their quantification in activated sludge. A probe and primer set, targeting the 16S rRNA gene of Nitrospira spp. was designed according to the constraints of the TaqMan® specifications. Samples used to evaluate the method included DNA from the sludge from full-scale wastewater treatment plants and laboratory scale systems. The reproducibility, quantitative efficiency and specificity were assessed in the evaluation. It was concluded that the method is sensitive and reproducible but has some constraints on the quantitative efficiency. A survey of full-scale systems for Nitrospira spp. was carried out and the results are presented here.