Aims: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. Methods and Results: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight TM kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight TM kit and CFDA assay by 2AE89 and 2AE81 log respectively. Bacterial diversity was also reduced from >20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. Conclusions: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. Significance and Impact of the Study: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.
Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA genedirected PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 ؋ 10 5 bacteria ml ؊1 . The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 ؋ 10 3 active AOB ml ؊1 were detected in the membraneintact fraction, and 1.40 ؋ 10 4 active AOB ml ؊1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml ؊1 detected using a routine liquid most-probablenumber assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.For extended drinking water systems, chloramine (in particular, monochloramine) is often the preferred disinfectant. Chloramine is less reactive than free chlorine, maintains an extended disinfection residual (45), and produces lower concentrations of disinfection by-products, such as trihalomethanes and haloacetic acids (5,23,28). Despite these advantages, the use of chloramine can introduce ammonia into a distribution system, both as excess ammonia from chloramine formation and as released ammonia from chloramine decay. These factors may lead to biological instability in such systems due to biological nitrification by nitrifying bacteria, such as the chemolithotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) (34,35).Nitrification episodes in chloraminated distribution systems are common, and there is evidence that 63% of medium and large utilities in the United States have experienced episodes of nitrification (45). Cunliffe (6) studied five chloraminated distribution systems in South Australia and found that 64...
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