Drinking water distribution systems (DWDSs) are dark, wet environments that provide opportunity for microbial biofilm development and long-term occupancy of infrastructure surfaces. Historically, identifications of microorganisms in natural environments have relied on culture-based techniques, which can underestimate the diversity of microbial assemblages. Consequently, information regarding the microbial inhabitants of DWDSs was previously limited to those that were cultivated and observed in the laboratory. Recent technological developments in molecular biology now afford the capability to directly identify organisms in the environment based on their ribosomal RNA (rRNA) gene sequences, thus circumventing the need to isolate and culture the microbe in the laboratory setting.;In this study, I used rRNA sequences to identify microorganisms and investigate microbiological variation within and between samples collected from two DWDSs spatially and over time. Despite differences in the age, pipe composition, source water, and disinfectant residual type, the two DWDSs were shown to be broadly similar to each other in phylogenetic composition. For example, at the phylum level, members of the Proteobacteria, Actinobacteria, and Cyanobacteria dominated both systems. However, the DWDS using chloramine residual consistently contained more taxa, and therefore greater diversity, than the DWDS with chlorine residual. My findings further indicated the rather remarkable stability of the microbial constituents over space and time, with some variability of the abundances of microbial populations among different sample sites within each system.;To expand the results and assess the microbiological similarity of additional DWDS ecosystems, I investigated six other DWDSs from separate geographical locations. All DWDSs that utilized physical water treatment processes accompanied by chemical (chlorine or chloramine) disinfection demonstrated remarkable similarity in microbiological composition. By contrast, samples from a DWDS exempt from physical processing and chemical disinfection were highly dissimilar to samples from all other DWDSs investigated. Notably, Mycobacterium spp. were identified in all disinfected DWDSs, and were notably absent in all non-chlorinated samples. This study provides the first multi-year, longitudinal, molecular microbiological examination of two DWDSs and includes a broader geographical representation of systems for comparison. These data provide a significant foundation for describing the resident microbiology of DWDSs from a culture-independent, molecular perspective.
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