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.

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Slow sand filtration with extensive pretreatment reduces the microbial growth potential of drinking water to a minimum level at four surface water supplies in The Netherlands. The potential of these slow sand filtrates (SSFs) to promote microbial growth in warm tap water installations was assessed by measuring biofilm formation and growth ofLegionellabacteria on glass and chlorinated polyvinylchloride (CPVC) surfaces exposed to SSFs at 37 ± 2°C in a model system for up to six months. The steady-state biofilm concentration ranged from 230 to 3,980 pg ATP cm−2on glass and 1.4 (±0.3)-times-higher levels on CPVC. These concentrations correlated significantly with the assimilable organic carbon (AOC) concentrations of the warm water (8 to 24 µg acetate-C equivalents [ac-C eq] liter−1), which were raised about 2 times by mixing cold and heated (70°C) SSFs. All biofilms supported growth ofLegionella pneumophilawith maximum concentrations ranging from 6 × 102to 1.5 × 105CFU cm−2. Biofilms after ≤50 days of exposure were predominated byBetaproteobacteriales, mainlyPiscinibacter,Caldimonas,Methyloversatilis, and an unculturedRhodocyclaceaebacterium. These rapidly growing primary colonizers most likely served as prey for the host amoebae ofL. pneumophila.Alphaproteobacteria, mostlyXanthobacteraceae, e.g.,Bradyrhizobium,Pseudorhodoplanes, and other amoeba-resistant bacteria, accounted for 37.5% of the clones retrieved. A conceptual model based on a quadratic relationship between theL. pneumophilacolony count and the biofilm concentration under steady-state conditions is used to explain the variations in theLegionellaCFU pg−1ATP ratios in the biofilms.IMPORTANCEProliferation ofL. pneumophilain premise plumbing poses a public health threat. Extended water treatment using physicochemical and biofiltration processes, including slow sand filtration, at four surface water supplies in The Netherlands reduces the microbial growth potential of the treated water to a minimum level, and the distributed drinking water complies with high quality standards. However, heating of the water in warm tap water installations increases the concentration of easily assimilable organic compounds, thereby promoting biofilm formation and growth ofL. pneumophila. Prevention of biofilm formation in plumbing systems by maintenance of a disinfectant residual during distribution and/or further natural organic matter (NOM) removal is not feasible in the supplies studied. Temperature management in combination with optimized hydraulics and material selection are therefore essential to prevent growth ofL. pneumophilain premise plumbing systems. Still, reducing the concentration of biodegradable compounds in drinking water by appropriate water treatment is important for limiting theLegionellagrowth potential.

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