The applicability of 454 pyrosequencing to characterize bacterial biofilm communities from two water meters of a drinking water distribution system was assessed. Differences in bacterial diversity and composition were observed. A better understanding of the bacterial ecology of drinking water biofilms will allow for effective management of water quality in distribution systems.Drinking water distribution systems (DWDS) are interesting model systems for studies of microbial diversity and ecosystem functions in engineered environments. DWDS biofilms in particular have received much focus due to the importance of potable water delivery to end-point consumers (8). Both cultivation-based and molecular approaches have been used to reveal bacterial communities from different locations or pipe materials within a DWDS and from DWDS that differ in source water and nutrients. Studies of bacterial communities throughout DWDS have indicated that populations can differ drastically from source water to tap water (7). Activities of microbial growth and the presence of potential opportunistic pathogens have been detected in tap water, faucets, and showerheads, etc., all end points of the DWDS (8,14). Many studies attribute the survival of opportunistic pathogens in DWDS to resistance mechanisms such as cell wall permeability and biofilm formation (9, 10). The type of bacterial communities present in a DWDS and the disinfection regime applied may also be factors influencing pathogen retention in the DWDS. Nitrifying microorganisms, for example, can contribute to the depletion of monochloramine and subsequently lead to increased overall microbial growth (7).Compared to the analysis of microbial communities in source water and end-point drinking water, investigations related to the delivery process are difficult due to limited access and the high cost involved in sampling within the DWDS. To overcome these challenges, several studies have used model DWDS to mimic full-scale DWDS so as to address treatment effects on water quality. The researchers in these studies reported that episodic chlorination may accelerate the development of microbial communities with increased resistance to disinfectants (4) and that bacterial diversity can also affect disinfection efficacy and pathogen survival (1). In addition, the occurrence of bacterial community succession was observed in a model DWDS (17), which indicated the importance of longterm monitoring of bacterial biofilm development in the DWDS. These findings also demonstrated the need for the direct study of microbial communities within the DWDS, which would complement studies of the source water and end points, give a more complete view of the microbial ecology of the DWDS, and lend insights for subsequent monitoring of the DWDS.Biofilms obtained from water meters can be a good alternative to those obtained from distribution water pipes to study biofilms in DWDS because the water meters can be obtained relatively easily from private households. Moreover, the biofilms in the water meters no...

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a b s t r a c tBiological stability of drinking water implies that the concentration of bacterial cells and composition of the microbial community should not change during distribution. In this study, we used a multi-parametric approach that encompasses different aspects of microbial water quality including microbial growth potential, microbial abundance, and microbial community composition, to monitor biological stability in drinking water of the non-chlorinated distribution system of Zü rich. Drinking water was collected directly after treatment from the reservoir and in the network at several locations with varied average hydraulic retention times (6e52 h) over a period of four months, with a single repetition two years later. Total cell concentrations (TCC) measured with flow cytometry remained remarkably stable at 9.5 (AE 0.6) Â 10 4 cells/ml from water in the reservoir throughout most of the distribution network, and during the whole time period. Conventional microbial methods like heterotrophic plate counts, the concentration of adenosine tri-phosphate, total organic carbon and assimilable organic carbon remained also constant.Samples taken two years apart showed more than 80% similarity for the microbial communities analysed with denaturing gradient gel electrophoresis and 454 pyrosequencing.Only the two sampling locations with the longest water retention times were the exceptions and, so far for unknown reasons, recorded a slight but significantly higher TCC (1.3 (AE 0.1) Â 10 5 cells/ml) compared to the other locations. This small change in microbial abundance detected by flow cytometry was also clearly observed in a shift in the microbial community profiles to a higher abundance of members from the Comamonadaceae (60% vs.2% at other locations). Conventional microbial detection methods were not able to detect changes as observed with flow cytometric cell counts and microbial community analysis. Available online at www.sciencedirect.com journal home page: www.elsevier.com/loca te/watres w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 0 1 5 e3 0 2 5 0043-1354/$ e see front matter ª

Background16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platformMethodology/Principal FindingsThe following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.ConclusionsOur findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

Water utilities in parts of the U.S. control microbial regrowth in drinking water distribution systems (DWDS) by alternating postdisinfection methods between chlorination and chloramination. To examine how this strategy influences drinking water microbial communities, an urban DWDS (population Х 40,000) with groundwater as the source water was studied for approximately 2 years. Water samples were collected at five locations in the network at different seasons and analyzed for their chemical and physical characteristics and for their microbial community composition and structure by examining the 16S rRNA gene via terminal restriction fragment length polymorphism and DNA pyrosequencing technology. Nonmetric multidimension scaling and canonical correspondence analysis of microbial community profiles could explain >57% of the variation. Clustering of samples based on disinfection types (free chlorine versus combined chlorine) and sampling time was observed to correlate to the shifts in microbial communities. Sampling location and water age (<21.2 h) had no apparent effects on the microbial compositions of samples from most time points. Microbial community analysis revealed that among major core populations, Cyanobacteria, Methylobacteriaceae, Sphingomonadaceae, and Xanthomonadaceae were more abundant in chlorinated water, and Methylophilaceae, Methylococcaceae, and Pseudomonadaceae were more abundant in chloraminated water. No correlation was observed with minor populations that were detected frequently (<0.1% of total pyrosequences), which were likely present in source water and survived through the treatment process. Transient microbial populations including Flavobacteriaceae and Clostridiaceae were also observed. Overall, reversible shifts in microbial communities were especially pronounced with chloramination, suggesting stronger selection of microbial populations from chloramines than chlorine.

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Drinking water distribution systems (DWDSs) harbor the microorganisms in biofilms and suspended communities, yet the diversity and spatiotemporal distribution have been studied mainly in the suspended communities. This study examined the diversity of biofilms in an urban DWDS, its relationship with suspended communities and its dynamics. The studied DWDS in Urbana, Illinois received conventionally treated and disinfected water sourced from the groundwater. Over a 2-year span, biomass were sampled from household water meters (n = 213) and tap water (n = 20) to represent biofilm and suspended communities, respectively. A positive correlation between operational taxonomic unit (OTU) abundance and occupancy was observed. Examined under a 'core-satellite' model, the biofilm community comprised 31 core populations that encompassed 76.7% of total 16 S rRNA gene pyrosequences. The biofilm communities shared with the suspended community highly abundant and prevalent OTUs, which related to methano-/methylotrophs (i.e., Methylophilaceae and Methylococcaceae) and aerobic heterotrophs (Sphingomonadaceae and Comamonadaceae), yet differed by specific core populations and lower diversity and evenness. Multivariate tests indicated seasonality as the main contributor to community structure variation. This pattern was resilient to annual change and correlated to the cyclic fluctuations of core populations. The findings of a distinctive biofilm community assemblage and methano-/methyltrophic primary production provide critical insights for developing more targeted water quality monitoring programs and treatment strategies for groundwater-sourced drinking water systems.

Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03–14 mg kg–1 dry wt. soil) and MeHg (0.05–27 μg kg–1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB + species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.