The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments inside loose deposits structures for bacteria to grow. Moreover, loose deposits protect the associated bacteria from disinfectants, and due to their mobility, the associated bacteria reach taps easily.

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The objective of this study was to evaluate the application of flow cytometry total cell counts (TCCs) as a parameter to assess microbial growth in drinking water distribution systems and to determine the relationships between different parameters describing the biostability of treated water. A one-year sampling program was carried out in two distribution systems in The Netherlands. Results demonstrated that, in both systems, the biomass differences measured by ATP were not significant. TCC differences were also not significant in treatment plant 1, but decreased slightly in treatment plant 2. TCC values were found to be higher at temperatures above 15°C than at temperatures below 15°C. The correlation study of parameters describing biostability found no relationship among TCC, heterotrophic plate counts, and Aeromonas. Also no relationship was found between TCC and ATP. Some correlation was found between the subgroup of high nucleic acid content bacteria and ATP (R 2 = 0.63). Overall, the results demonstrated that TCC is a valuable parameter to assess the drinking water biological quality and regrowth; it can directly and sensitively quantify biomass, detect small changes, and can be used to determine the subgroup of active HNA bacteria that are related to ATP.

This study assessed the characteristics of and changes in the suspended particles and the associated bacteria in an unchlorinated drinking water distribution system and its reservoirs with different water sources. The results show that particle-associated bacteria (PAB) were present at a level of 0.8–4.5 × 103 cells ml−1 with a biological activity of 0.01–0.04 ng l−1 ATP. Different PAB communities in the waters produced from different sources were revealed by a 16S rRNA-based pyrosequencing analysis. The quantified biomass underestimation due to the multiple cells attached per particle was ≥ 85%. The distribution of the biologically stable water increased the number of cells per particle (from 48 to 90) but had minor effects on the PAB community. Significant changes were observed at the mixing reservoir. Our results show the characteristics of and changes in suspended PAB during distribution, and highlight the significance of suspended PAB in the distribution system, because suspended PAB can lead to a considerable underestimation of biomass, and because they exist as biofilm, which has a greater mobility than pipe-wall biofilm and therefore presents a greater risk, given the higher probability that it will reach the customers’ taps and be ingested.