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Monochloramine disinfection kinetics of Nitrosomonas europaea using propidium monoazide quantitative real-time PCR (PMA-qPCR)
Citation:
WAHMAN, D., K. A. KLEIER, AND J. G. PRESSMAN. Monochloramine disinfection kinetics of Nitrosomonas europaea using propidium monoazide quantitative real-time PCR (PMA-qPCR). Presented at 2008 AWWA WATER QUALITY AND TECHNOLOGY CONFERENCE, CINCINNATI, OH, November 16 - 20, 2008.
Impact/Purpose:
to present information
Description:
As a result of the implementation of the Stage 1 and Stage 2 Disinfectants and Disinfection Byproduct Rules, monochloramine use as a secondary disinfectant in the United States is predicted to increase to 57% of all surface and 7% of all ground water treatment systems. In addition, a recent survey suggests that 12% of drinking water utilities are contemplating a switch to monochloramine in the future. Along with the addition of monochloramine comes the risk of nitrification in the distribution system by ammonia-oxidizing bacteria (AOB). Based on utility surveys, 30 to 63% of utilities practicing chloramination for secondary disinfection experience nitrification episodes. Nitrification in drinking water distribution systems is undesirable and may result in degradation of water quality and subsequent non-compliance with existing regulations. Thus, nitrification control is a major issue in practice and likely to become increasingly important as monochloramine use increases. Based on the results of this research, we expect to gain a better understanding of the disinfection kinetics of monochloramine with AOB in chloraminated drinking water distribution systems, allowing better prevention and control of nitrification episodes in practice. Disinfection kinetic rates inherently depend on the method used to quantify viable microorganisms. Recently, researchers used LIVE/DEAD® BacLight™ (L/D), a culture-independent method based on membrane integrity, to determine monochloramine disinfection kinetics for the pure culture AOB Nitrosomonas europaea. L/D produced rates significantly lower than previous studies using the culture-dependant MPN method. Limitations with L/D include that it does not differentiate between bacteria in mixed cultures, is physically limited by the ability to count bacteria, and is subject to auto-fluorescing particles that may alter results. To address these limitations, the current research combines two DNA based methods (quantitative PCR (qPCR) and propidium monoazide (PMA)) to determine monochloramine disinfection kinetics. This PMA-qPCR method couples the ability of qPCR to quantify specific bacteria over several orders of magnitude along with PMA’s ability to selectively remove DNA from dead cells by penetrating cells with permeable membranes; therefore, the PMA-qPCR method allows specific amplification of DNA from targeted viable cells. PMA-qPCR control experiments using mixtures of viable and heat-killed N. europaea cells confirmed the ability of PMA-qPCR to detect only viable cells over several orders of magnitude. Similar experiments conducted with L/D confirmed its ability to differentiate between viable and heat-killed N. europaea. Finally, batch disinfection experiments demonstrated the applicability of PMA-qPCR to N. europaea. These research results will allow future studies to be conducted on mixed cultures in suspension and biofilm, focusing specifically on AOB present in drinking water distribution systems.