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.

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.Free-living protozoa are ubiquitous in natural freshwater environments (7,38,51,71) but also proliferate in engineered water systems, including water treatment systems (3,47,70), distribution systems (6, 75), and tap water installations inside buildings (54, 69). Concentrations of protozoa, determined using cultivation methods and microscopy, range from Ͻ1 to 10 4 cells liter Ϫ1 in treated water (3,47,70,75) and from Ͻ1 to 7 ϫ 10 5 cells liter Ϫ1 in distribution systems (6,61,64,75). Genera of free-living protozoa commonly observed in these systems and in tap water installations include Acanthamoeba,

The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration.

Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila. The biofilm on glass (average steady-state concentration, 23 Ϯ 9 pg ATP cm Ϫ2 ) exposed to treated aerobic groundwater (0.3 mg C liter Ϫ1 ; 1 g assimilable organic carbon [AOC] liter Ϫ1 ) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 Ϯ 9 pg ATP cm Ϫ2 ) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm Ϫ2 in the biofilms on glass (1,055 Ϯ 225 pg ATP cm Ϫ2 ) and CPVC (2,755 Ϯ 460 pg ATP cm Ϫ2 ) exposed to treated anaerobic groundwater (7.9 mg C liter Ϫ1 ; 10 g AOC liter Ϫ1 ). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis. This amoeba was rarely detected at biofilm concentrations of Ͻ100 pg ATP cm Ϫ2 . A threshold concentration of approximately 50 pg ATP cm Ϫ2 (TCC ϭ 1 ϫ 10 6 to 2 ϫ 10 6 cells cm Ϫ2 ) was derived for growth of L. pneumophila in biofilms.IMPORTANCE Legionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter Ϫ1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila. Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter Ϫ1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

Fifty-seven per cent of all water supply systems in the Netherlands are controlled by model predictive flow control; the other 43% are controlled by conventional level-based flow control. The differences between conventional level-based flow control and model predictive control were investigated in experiments at five full-scale water supply systems in the first half of 2011. Quality parameters of the drinking water and energy consumption of the treatment and distribution processes were measured and analysed. The experiments showed that the turbidity values are 12-28% lower, and particle volume values 12-42% lower for the systems which are controlled by model predictive flow control. The overall energy consumption of water supply systems controlled by predictive flow control is 1.0-5.3% lower than conventional level-based flow controlled systems, and the overall energy costs are 1.7-7.4% lower.

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The objective of our study was to explore microbiological parameters that are suitable as indicators for regrowth in distribution systems that receive unchlorinated drinking water in the Netherlands. Treated water and distributed water at two locations in the distribution system of 28 treatment plants were analyzed for a range of biological parameters. The results demonstrated that Aeromonas, HPCs, and Mycobacterium were significantly (more often) higher and ATP was significantly (more often) lower in the distributed water than in the treated water, whereas cell numbers remained stable during distribution. Average fungal 18S rRNA gene copies did not differ significantly between the treated and the distributed water, but fungal numbers were significantly more often higher in the distributed water than in the corresponding treated water. These results demonstrate that the distribution system has a clear effect on HPCs, Aeromonas, Mycobacterium, ATP and, to a lesser extent, on fungi in drinking water, but not on cell numbers. Furthermore, we noticed that ATP in the treated water is a better predictor for the regrowth level in the distribution system than total, membrane-intact or HNA cell numbers. In contrast to previous studies, we conclude that ATP and cell numbers seem to be poor indicators for regrowth in the drinking water environment, whereas HPCs, Aeromonas, Mycobacterium and, to a lesser extent, fungi appear to be more reliable indicators to monitor regrowth.