The present work is the first to examine temporal variability of bacterial diversity in a free-chlorinated drinking water distribution system over multiple years. The authors examined seasonal and monthly bacterial diversity by using three different molecular methods. 16S rRNA gene clone library analysis identified the bacteria in Pittsburgh seasonal water samples, collected in fall 2006, and winter, spring, and summer 2007 and an event sample collected following a major pipe break, as primarily members of the alpha-, beta-, and gammaproteobacteria classes, which agrees with previous studies of smaller numbers of bulk water samples over shorter durations. However, in this study, seasonal shifts in relative populations were observed with decreased alphaproteobacteria and increased betaproteobacteria diversity in the winter sample compared to the other seasonal samples, which may be caused by changes in chlorine dosing. Further, in 11 monthly samples collected at the same location from September 2008 to August 2009, quantitative polymerase chain reaction (qPCR) results indicated that alpha- and betaproteobacteria predominated over the year, except in May and October, when changes in chlorine dosing as a result of temperature shifts may have influenced bacterial diversity. Denaturing gradient gel electrophoresis (DGGE) cluster analyses suggested that the monthly samples cluster seasonally with greater than 85% similarity between March and April, June and July, and December and January. These results demonstrate the consistent presence of alpha-, beta-, and gammaproteobacteria in drinking water distribution systems across seasons during routine operational conditions and suggest that changes in bacterial class distributions may be useful indicators of system disruptions.
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