Bacteriology of drinking water distribution systems: an integral and multidimensional review

A drinking water distribution system (DWDS) is the final and essential step to supply safe and high-quality drinking water to customers. Biological processes, such as biofilm formation and detachment, microbial growth in bulk water, and the formation of loose deposits, may occur. These processes will lead to deterioration of the water quality during distribution. In extreme conditions, pathogens and opportunistic pathogens may proliferate and pose a health risk to consumers. It is, therefore, necessary to understand the bacteriology of DWDSs to develop effective strategies that can ensure the water quality at consumers' taps. The bacteriology of DWDSs, both the quantitative growth and the qualitative bacterial community, has attracted considerable research attention. However, the researchers have focused mainly on the pipe wall biofilm. In this review, DWDS bacteriology has been reviewed multidimensionally, including both the bacterial quantification and identification. For the first time, the available literature was reviewed with an emphasis on the subdivision of DWDS into four phases: bulk water, suspended solids, loose deposits, and pipe wall biofilm. Special concentration has been given to potential contribution of particulate matter: suspended particles and loose deposits. Two highlighted questions were reviewed and discussed: (1) where does most of the growth occur? And (2) what is the contribution of particle-associated bacteria to DWDS bacteriology and ecology? At the end of this review, recommendations were given based on the conclusion of this review to better understand the integral DWDS bacteriology.     

Diversity of bacteria and mycobacteria in biofilms of two urban drinking water distribution systems

In this study, to give insight into the bacterial diversity of biofilms from full-scale drinking water distribution systems (DWDSs), the bacterial community compositions of biofilms from two urban DWDSs (Guangzhou and Beijing, China) were determined using a 16S rRNA gene library technique. Meanwhile, the occurrence and diversity of mycobacteria were also analyzed by a Mycobacterium -specific hsp gene assay. The biofilms from the full-scale DWDSs have complex bacterial populations. Proteobacteria was the common and predominant group in all biofilm samples, in agreement with previous reports. The community structures of bacteria at the three sites in Guangzhou DWDS were significantly different, despite the similar physicochemical properties of portable water. Some abundant and peculiar bacterial phylotypes were noteworthy, including Methylophilus , Massilia, and Planomicrobium , members of which are rarely found in DWDSs and their roles in DWDS biofilms are still unclear. The diversity of Mycobacterium species in biofilm samples was rather low. Mycobacterium arupense and Mycobacterium gordonae were the primary Mycobacterium species in Guangzhou and Beijing biofilms, respectively, indicating that M. arupense may be more resistant to chloride than M. gordonae.     

Core-satellite populations and seasonality of water meter biofilms in a metropolitan drinking water distribution system

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.     

Microbial Community Dynamics of an Urban Drinking Water Distribution System Subjected to Phases of Chloramination and Chlorination Treatments

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 ≅ 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.     

Phylogenetic diversity of microbial communities in real drinking water distribution systems

Microbial regrowth in drinking water distribution systems (DWDS) is a major concern in the water supply industry. Detailed knowledge of the microbial community in DWDS will be of great importance for assessing the microbiological risks of drinking water. The spatial heterogeneity of microbial community structures in the bulk waters of a large real DWDS was investigated using 16S rRNA clone library analysis. The results indicate that high residual chlorine in drinking water could not control microbial regrowth in DWDS. The bacterial communities in the bulk waters were spatially heterogenic, mainly composed of Alphaproteobacteria and Betaproteobacteria (or Cyanobacteria). Microorganisms from the genera Acinetobacter, Sphingomonas and Gemella were detected, implying there is microbiological risk from drinking water. This work provides new insight into microbial ecology in DWDS.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

Terrestrial Runoff Controls the Bacterial Community Composition of Biofilms along a Water Quality Gradient in the Great Barrier Reef

16S rRNA gene molecular analysis elucidated the spatiotemporal distribution of bacterial biofilm communities along a water quality gradient. Multivariate statistics indicated that terrestrial runoff, in particular dissolved organic carbon and chlorophyll a concentrations, induced shifts of specific bacterial communities between locations and seasons, suggesting microbial biofilms could be suitable bioindicators for water quality.     

Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities

Next‐generation sequencing has dramatically changed the landscape of microbial ecology, large‐scale and in‐depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon‐specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine‐tuning experimental and computational parameters used to characterize natural communities.     

Variations of bacterial 16S rDNA phylotypes prior to and after chlorination for drinking water production from two surface water treatment plants

We examined the variations of bacterial populations in treated drinking water prior to and after the final chlorine disinfection step at two different surface water treatment plants. For this purpose, the bacterial communities present in treated water were sampled after granular activated carbon (GAC) filtration and chlorine disinfection from two drinking water treatment plants supplying the city of Paris (France). Samples were analyzed after genomic DNA extraction, polymerase chain reaction (PCR) amplification, cloning, and sequencing of a number of 16S ribosomal RNA (rRNA) genes. The 16S rDNA sequences were clustered into operational taxonomic units (OTUs) and the OTU abundance patterns were obtained for each sample. The observed differences suggest that the chlorine disinfection step markedly affects the bacterial community structure and composition present in GAC water. Members of the Alphaproteobacteria and Betaproteobacteria were found to be predominant in the GAC water samples after phylogenetic analyses of the OTUs. Following the chlorine disinfection step, numerous changes were observed, including decreased representation of Proteobacteria phylotypes. Our results indicate that the use of molecular methods to investigate changes in the abundance of certain bacterial groups following chlorine-based disinfection will aid in further understanding the bacterial ecology of drinking water treatment plants (DWTPs), particularly the disinfection step, as it constitutes the final barrier before drinking water distribution to the consumer’s tap.     

Temporal Variations in the Abundance and Composition of Biofilm Communities Colonizing Drinking Water Distribution Pipes

Pipes that transport drinking water through municipal drinking water distribution systems (DWDS) are challenging habitats for microorganisms. Distribution networks are dark, oligotrophic and contain disinfectants; yet microbes frequently form biofilms attached to interior surfaces of DWDS pipes. Relatively little is known about the species composition and ecology of these biofilms due to challenges associated with sample acquisition from actual DWDS. We report the analysis of biofilms from five pipe samples collected from the same region of a DWDS in Florida, USA, over an 18 month period between February 2011 and August 2012. The bacterial abundance and composition of biofilm communities within the pipes were analyzed by heterotrophic plate counts and tag pyrosequencing of 16S rRNA genes, respectively. Bacterial numbers varied significantly based on sampling date and were positively correlated with water temperature and the concentration of nitrate. However, there was no significant relationship between the concentration of disinfectant in the drinking water (monochloramine) and the abundance of bacteria within the biofilms. Pyrosequencing analysis identified a total of 677 operational taxonomic units (OTUs) (3% distance) within the biofilms but indicated that community diversity was low and varied between sampling dates. Biofilms were dominated by a few taxa, specifically Methylomonas, Acinetobacter, Mycobacterium, and Xanthomonadaceae, and the dominant taxa within the biofilms varied dramatically between sampling times. The drinking water characteristics most strongly correlated with bacterial community composition were concentrations of nitrate, ammonium, total chlorine and monochloramine, as well as alkalinity and hardness. Biofilms from the sampling date with the highest nitrate concentration were the most abundant and diverse and were dominated by Acinetobacter.     

RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities.     

An ignored and potential source of taste and odor (T&O) issues—biofilms in drinking water distribution system (DWDS)

It is important for water utilities to provide esthetically acceptable drinking water to the public, because our consumers always initially judge the quality of the tap water by its color, taste, and odor (T&O). Microorganisms in drinking water contribute largely to T&O production and drinking water distribution systems (DWDS) are known to harbor biofilms and microorganisms in bulk water, even in the presence of a disinfectant. These microbes include T&O-causing bacteria, fungi, and algae, which may lead to unwanted effects on the organoleptic quality of distributed water. Importantly, the understanding of types of these microbes and their T&O compound-producing mechanisms is needed to prevent T&O formation during drinking water distribution. Additionally, new disinfection strategies and operation methods of DWDS are also needed for better control of T&O problems in drinking water. This review covers: (1) the microbial species which can produce T&O compounds in DWDS; (2) typical T&O compounds in DWDS and their formation mechanisms by microorganisms; (3) several common factors in DWDS which can influence the growth and T&O generation of microbes; and (4) several strategies to control biofilm and T&O compound formation in DWDS. At the end of this review, recommendations were given based on the conclusion of this review.

     

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set.     

Metagenomic profiling of fresh water lakes at different altitudes in Pakistan

Freshwater lakes play a crucial role in sustainability of ecosystem in the favour of communities flourishing around it. In this study, differential metagenomic evaluation of three freshwater ecosystems in Pakistan was carried out. These included Lakes Keenjhar, Rawal and Saif-ul-Muluk situated at the altitudes 21, 527, 3224 m respectively. Former two lakes serve as water reservoirs for three major cities of Pakistan, while the high-altitude Lake Saif-ul-Muluk is located in a pristine environment at the meeting point of Himalayan and Karakoram ranges. Next generation sequencing (NGS) of metagenomic DNA of microbial communities was carried out using surface water samples from these lakes by Illumina Hi-Seq 2500 sequencing technology. Bioinformatics analysis identified Proteobacteria as the most dominant phylum (58–79%) followed by Planctomycetes (34%), Cyanobacteria (12%) and Bacteroidetes (15%). Lake Saif-ul-Muluk contained highest abundance of bacterial genera including Vibrio, Bordetella, Pseudomonas, Burkholderia and Escherichia; while Microcystis were found to be abundant in Lakes Keenjhar and Rawal. Non-parametric multi-dimensional scaling (NMDS) principle component analysis on genus level revealed that these ecosystems were divergent from each other and from metagenomes of lakes in the Americas. Characterization of virulence factors and antibiotics resistant genes in metagenomic contigs were also identified. Chemical profiling of Lakes Keenjhar and Rawal showed higher concentration of phosphorus which indicated eutrophication in these lakes might be due to Microcystis infestation.     

Selective Extraction of Bacterial DNA from the Surfaces of Macroalgae

A novel method has been developed for the selective extraction of DNA from surface-associated bacterial communities from the two model marine benthic algae Ulva australis and Delisea pulchra. The extracted DNA had no detectable contamination with host DNA, was recovered in high yield and quality, and was representative of the bacterial community on the algal surfaces. The DNA is suitable for a variety of subsequent applications, including the construction of large-insert clone libraries and metagenomic sequencing.     

Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon results and provides functional insights

Prevailing 16S rRNA gene-amplicon methods for characterizing the bacterial microbiome of wildlife are economical, but result in coarse taxonomic classifications, are subject to primer and 16S copy number biases, and do not allow for direct estimation of microbiome functional potential. While deep shotgun metagenomic sequencing can overcome many of these limitations, it is prohibitively expensive for large sample sets. Here we evaluated the ability of shallow shotgun metagenomic sequencing to characterize taxonomic and functional patterns in the faecal microbiome of a model population of feral horses (Sable Island, Canada). Since 2007, this unmanaged population has been the subject of an individual-based, long-term ecological study. Using deep shotgun metagenomic sequencing, we determined the sequencing depth required to accurately characterize the horse microbiome. In comparing conventional vs. high-throughput shotgun metagenomic library preparation techniques, we validate the use of more cost-effective laboratory methods. Finally, we characterize similarities between 16S amplicon and shallow shotgun characterization of the microbiome, and demonstrate that the latter recapitulates biological patterns first described in a published amplicon data set. Unlike for amplicon data, we further demonstrate how shallow shotgun metagenomic data provide useful insights regarding microbiome functional potential which support previously hypothesized diet effects in this study system.     

Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities

Producing cellulosic biofuels from plant material has recently emerged as a key US Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.     

Metagenomic analysis of bacterial communities of Wadi Namar Lake,Riyadh, Saudi Arabia

Wadi Namar lake is a new touristic attraction area in the south of Riyadh. Human activities around the lake may lead to changes in water quality with subsequent changes in microenvironment components including microbial diversity. The current study was designed to assess possible changes in bacterial communities of the water at Wadi Namar Lake. Therefore, water samples were collected from three different locations along the lake: L1 (no human activities, no plants), L2 (no human activity, some plants) and L3 (human activities, municipal wastes and some plants). The total DNA of the samples was extracted and subjected to 16S rDNA sequencing and metagenomic analysis; water pH, electrical conductivity (EC), total dissolved solids (TDS) as well as the concentration of Na⁺¹, K⁺¹, Cl⁻¹ and total N were analysed. Metagenomic analysis showed variations in relative abundance of 17 phyla, 31 families, 43 genera and 19 species of bacteria between the locations. Proteobacteria was the most abundant phylum in all locations; however, its highest abundance was in L1. Planctomycete phylum was highly abundant in L1 and L3, while its abundance in L2 was low. The phyla Acidobacteria, Candidatus Saccharibacteria, Nitrospirae and Chloroflexi were associated with high TDS, EC, K⁺¹ and Cl⁻¹ concentrations in L3; various human activities around this location had possibly affected microbial diversity. Current study results help in recognising the structure of bacterial communities at Wadi Namar Lake in relation to their surroundings for planning to environment protection and future restoration of affected ecosystems.     

Characterisation of the Physical Composition and Microbial Community Structure of Biofilms within a Model Full-Scale Drinking Water Distribution System

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.