A pilot study of bacteriological population changes through potable water treatment and distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

The influence of the chemical composition of drinking water on cuprosolvency by biofilm bacteria

AIMS: This study investigated the influence of water chemistry on copper solvation (cuprosolvency) by pure culture biofilms of heterotrophic bacteria isolated from copper plumbing. METHODS AND RESULTS: Heterotrophic bacteria isolated from copper plumbing biofilms including Acidovorax delafieldii, Flavobacterium sp., Corynebacterium sp., Pseudomonas sp. and Stenotrophomonas maltophilia were used in laboratory coupon experiments to assess their potential for cuprosolvency. Sterile copper coupons were exposed to pure cultures of bacteria to allow biofilm formation and suspended in drinking waters with different chemical compositions. Sterile coupons not exposed to bacteria were used as controls. After 5 days of incubation, copper release and biofilm accumulation was quantified. The results demonstrated that cuprosolvency in the control experiments was influenced by water pH, total organic carbon (TOC) and conductivity. Cuprosolvency in the presence of biofilms correlated with the chemical composition of the water supplies particularly pH, Langeliers Index, chloride, alkalinity, TOC and soluble phosphate concentrations. CONCLUSIONS: The results suggest water quality may influence cuprosolvency by biofilms present within copper plumbing pipes. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for water chemistry to influence cuprosolvency by biofilms may contribute to the sporadic nature of copper corrosion problems in distribution systems.     

Effects of different nutrients on bacterial growth in a pilot distribution system

The growth of bacterial communities in drinking water distribution systems can lead to the development of problems incompatible with water quality requirements. This study was carried out in order to determine which factors promote bacterial growth in distribution networks. A pilot distribution system was used to perform these experiments. After addition of three different inorganic elements to the network (N, P and S) the results obtained show that they did not contribute to the growth or bacteria either in the circulating water or on the surface of the pipes of the distribution system. However, when organic carbon was added an increase in the number of circulating bacteria was observed though the number of bacteria in the biofilms of the network was constant. These results indicate that in the drinking water of the Barcelona distribution system the factor that controls the growth of bacteria is organic carbon. Moreover, bacteria from the biofilm growing on the surface of the pipes may cause the problems of bacterial growth.     

Drip irrigation biofouling with treated wastewater: bacterial selection revealed by high-throughput sequencing

Clogging of drippers due to the development of biofilms weakens the advantages and impedes the implementation of drip irrigation technology. The objective of this study was to characterise the bacterial community of biofilms that develop in a drip irrigation system supplied with treated wastewater. High-throughput sequencing of 16S rRNA gene amplicons indicated that the bacterial community composition differed between drippers and pipes, mainly due to changes in the abundance of the genus Aquabacterium. Cyanobacteria were found to be involved in the biological fouling of drippers. Moreover, bacterial genera including opportunistic pathogenic bacteria such as Legionella and Pseudomonas were more abundant in dripper and pipe biofilms than in the incoming water. Some genera such as Pseudomonas were mostly recovered from drippers, while others (ie Bacillus, Brevundimonas) mainly occurred in pipes. Variations in the hydraulic conditions and properties of the materials likely explain the shift in bacterial communities observed between pipes and drippers.     

Effects of diverse water pipe materials on bacterial communities and water quality in the annular reactor

To investigate the effects of pipe materials on biofilm accumulation and water quality, an annular reactor with the sample coupons of four pipe materials (steel, copper, stainless steel, and polyvinyl chloride) was operated under hydraulic conditions similar to a real plumbing system for 15 months. The bacterial concentrations were substantially increased in the steel and copper reactors with progression of corrosion, whereas those in stainless steel (STS) and polyvinyl chloride (PVC) reactors were affected mainly by water temperature. The heterotrophic plate count (HPC) of biofilms was about 100 times higher on steel pipe than other pipes throughout the experiment, with the STS pipe showing the lowest bacterial number at the end of the operation. Analysis of the 16S rDNA sequences of 176 cultivated isolates revealed that 66.5% was Proteobacteria and the others included unclassified bacteria, Actinobacteria, and Bacilli. Regardless of the pipe materials, Sphingomonas was the predominant species in all biofilms. PCR-DGGE analysis showed that steel pipe exhibited the highest bacterial diversity among the metallic pipes, and the DGGE profile of biofilm on PVC showed three additional bands not detected from the profiles of the metallic materials. Environmental scanning electron microscopy showed that corrosion level and biofilm accumulation were the least in the STS coupon. These results suggest that the STS pipe is the best material for plumbing systems in terms of the microbiological aspects of water quality.     

Availability of glucose and light modulates the structure and function of a microbial biofilm

We have studied the differences in the organic matter processing and biofilm composition and structure between autoheterotrophic and heterotrophic biofilm communities. Microbial communities grown on artificial biofilms were monitored, following incubation under light and dark conditions and with or without the addition of glucose as a labile organic compound. Glucose addition greatly affected the microbial biofilm composition as shown by differences in 16S rRNA gene fingerprints. A significant increase in β-glucosidase and peptidase enzyme activities were also observed in glucose-amended biofilms incubated in the dark, suggesting an active bacterial community. Light enhanced the algal and bacterial growth, as well as higher extracellular enzyme activity, thereby indicating a tight algal–bacterial coupling in biofilms incubated under illumination. In these biofilms, organic compounds excreted by photosynthetic microorganisms were readily available for bacterial heterotrophs. This algal–bacterial relationship weakened in glucose-amended biofilms grown in the light, probably because heterotrophic bacteria preferentially use external labile compounds. These results suggest that the availability of labile organic matter in the flowing water and the presence of light may alter the biofilm composition and function, therefore affecting the processing capacity of organic matter in the stream ecosystem.     

Impacts of the reduction of nutrient levels on bacterial water quality in distribution systems.

This study evaluated the impacts of reducing nutrient levels on bacterial water quality in drinking water. Two American Water System facilities (sites NJ102a and IN610) with histories of coliform problems were involved, and each water utility received two pilot distribution systems (annular reactors). One reactor simulated the conventional treatment conditions (control), while the other reactor was used to assess the effect of biological filtration and subsequent reduced biodegradable organic matter levels on suspended (water column) and biofilm bacterial concentrations in the distribution systems. Biodegradable organic matter levels were reduced approximately by half after biological treatment. For site NJ102a, the geometric mean of the assimilable organic carbon concentrations was 217 microg/liter in the plant effluent and 91 microg/liter after biological filtration. For both sites, plant effluent biodegradable dissolved organic carbon levels averaged 0.45 mg/liter, versus 0.19 to 0.22 mg/liter following biological treatment. Biological treatment improved the stability of free chlorine residuals, while it had little effect on chloramine consumption patterns. High bacterial levels from the biological filters resulted in higher bacterial concentrations entering the test reactors than entering the control reactors. On average, biofilms in the model systems were reduced by 1 log unit (from 1.4 x 10(5) to 1.4 x 10(4) CFU/cm(2)) and 0.5-log unit (from 2.7 x 10(5) to 7.8 x 10(4) CFU/cm(2)) by biological treatment at sites NJ102a and IN610, respectively. Interestingly, it required several months of biological treatment before there was an observable impact on bacterial water quality in the system, suggesting that the effect of the treatment change was influenced by other factors (i.e., pipe conditions or disinfection, etc.).     

Diversity and dynamics of bacterial species in a biofilm at the end of the Seoul water distribution system

To investigate changes in the bacterial species and hygienic safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyse the bacterial population in the biofilm of a semi-pilot galvanized iron pipe model. The presence of sequences from aerobic Sphingomonas sp., anaerobic Rhodobacter sp., and unculturable bacteria indicated that these organisms coexisted after 1 day of model operation, demonstrating the ease of biofilm formation on galvanized iron pipes in the end region of the water distribution system studied. Sequences similar to those of unculturable bacteria, E. coli, and anaerobic bacteria were detected during the course of succession on the biofilm. More complicated band patterns were observed after 70 days of operation. PCR-DGGE illustrated changes in the biofilm during succession as well as the possibilities of anaerobic conditions and faecal contamination of the drinking water system. PCR-DGGE and culture-dependent fatty acid methyl ester (FAME) analysis showed different patterns for the same samples (Lee & Kim 2003); however, PCR-DGGE showed less diversity than did FAME analysis. This study compared the culture-dependent FAME and culture-independent PCR-DGGE methods directly, and their use in promoting the hygienic safety of drinking water.     

Assessment of microbial involvement in the elevation of copper levels in drinking water

A study of bacterial populations in metropolitan Adelaide domestic reticulation pipes was conducted to investigate a possible link between copper in drinking water and biofilms. Biofilm densities from cold water copper pipes at 10 sample sites were measured by viable cell counts. The range detected was from <2 × 10¹ to 3·25 × 10⁷ cfu cm⁻². Five isolates were selected for further experiments as they represented a range of responses to solvated copper and relative tendency for adhesion on glass slides. Drinking water supplied to the Adelaide Hills is high in total organic carbon (TOC; 22·57 mg C l⁻¹) and has a negative Langelier Index (LI; −1·16), whereas Adelaide metropolitan water undergoes filtration and has both a lower TOC and LI (10·72 mg C l⁻¹, LI, −0·49). Copper coupons were exposed to biofilm isolates (24 h), washed and resuspended in Adelaide metropolitan and Adelaide Hills water. Copper coupons not exposed to biofilm isolates were suspended in respective waters as a control. After 5 d of incubation, the copper content of Adelaide Hills water (4·71 ± 0·87 mg Cu l⁻¹), in which the copper coupons were suspended, consistently exceeded values obtained in the metropolitan Adelaide water (1·17 ± 0·249 mg Cu l⁻¹). The concentration of copper in the Adelaide Hills water was influenced by the bacterial species forming the biofilm on the coupon, with Agrobacterium sp. producing significantly higher levels of soluble copper than the control. The experiments reported here indicate that the suspended organic carbon, the aggressivity of the water and the biofilm may independently or synergistically increase the dissolution of copper from pipes into drinking water.     

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.     

Bacterial species in biofilm cultivated from the end of the Seoul water distribution system

AIMS: To investigate changes in the bacterial population and the safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), selective media and bacterial community analyses were applied to a semi-pilot galvanized iron pipe (GIP) model. METHODS AND RESULTS: No total coliforms or faecal streptococci were detected on m-Endo or m-Enterococcus agar. No Salmonella spp. and Shigella spp. were detected on bismuth sulphite agar or Hektoen enteric agar, respectively. The latter two media detected coliforms, where m-Endo was negative. Biofilm formation started within 1 week (ca 104 CFU cm(-2)) and exceeded 105 CFU cm(-2) within 6 weeks. Although the fatty acid methyl ester analysis revealed dynamic changes in bacterial composition, Micrococcus, Bacillus, and Pseudomonas spp. were persistent members of the biofilm community. Micrococcus spp. was detected most frequently and in high numbers. CONCLUSIONS: Coliforms and Enterococcus species can be recovered from biofilms in water distribution systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates the role of biofilms in the chronic deterioration of the water-distribution system in Seoul (Korea).     

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.     

Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Biofouling control in water by various UVC wavelengths and doses

UV light irradiation is being increasingly applied as a primary process for water disinfection, effectively used for inactivation of suspended (planktonic) cells. In this study, the use of UV irradiation was evaluated as a pretreatment strategy to control biofouling. The objective of this research was to elucidate the relative effectiveness of various targeted UV wavelengths and a polychromatic spectrum on bacterial inactivation and biofilm control. In a model system using Pseudomonas aeruginosa, the inactivation spectra corresponded to the DNA absorption spectra for all wavelengths between 220 and 280 nm, while wavelengths between 254 nm and 270 nm were the most effective for bacterial inactivation. Similar wavelengths of 254-260-270 nm were also more effective for biofilm control in most cases than targeted 239 and 280 nm. In addition, the prevention of biofilm formation by P. aeruginosa with a full polychromatic lamp was UV dose-dependent. It appears that biofilm control is improved when larger UV doses are given, while higher levels of inactivation are obtained when using a full polychromatic MP lamp. However, no significant differences were found between biofilms produced by bacteria that survived UV irradiation and biofilms produced by control bacteria at the same microbial counts. Moreover, the experiments showed that biofilm prevention depends on the post-treatment incubation time and nutrient availability, in addition to targeted wavelengths, UV spectrum and UV dose.     

Biofilm parameters influencing biocide efficacy

The influence of biofilm areal cell density, species composition, and the presence of abiotic particles on the disinfection and removal of bacterial biofilms by monochloramine was investigated. Mono- and binary population biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown on stainless-steel slides in a continuous flow annular reactor. Biofilms were treated in the reactor with a pulse/step dose of 4 mg/L monochloramine for 2 h. Biofilm samples were disaggregated and assayed for colony formation on R2A agar and for total cell numbers by acridine orange direct counts. These data were used to determine apparent first order rate coefficients for the processes of disinfection and detachment. Disinfection rate coefficients exceeded detachment rate coefficients by as much as an order of magnitude and the two coefficients were poorly correlated (r = 0.272). The overall decay rate coefficient (disinfection plus detachment) depended strongly on the initial biofilm areal cell density. It displayed a parabolic dependence on cell density with a maximum near 10(8) cfu/cm(2). This result points to multiple factors influencing biofilm susceptibility to antimicrobial challenge. Decay rates of K. pneumoniae measured in binary population biofilms were comparable with those measured in monopopulation biofilms (p = 0.61). P. aeruginosa decayed more slowly in biofilsm dominated by K. pneumoniae (p = 0.028), indicating some interaction between species. The presence of kaolin and calcium carbonate particles in the biofilm reduced disinfection efficacy. (c) 1995 John Wiley & Sons, Inc.     

Adaptive responses to antimicrobial agents in biofilms

Bacterial biofilms demonstrate adaptive resistance in response to antimicrobial stress more effectively than corresponding planktonic populations. We propose here that, in biofilms, reaction-diffusion limited penetration may result in only low levels of antimicrobial exposure to deeper regions of the biofilm. Sheltered cells are then able to enter an adapted resistant state if the local time scale for adaptation is faster than that for disinfection. This mechanism is not available to a planktonic population. A mathematical model is presented to illustrate. Results indicate that, for a sufficiently thick biofilm, cells in the biofilm implement adaptive responses more effectively than do freely suspended cells. Effective disinfection requires applied biocide concentration that increases quadratically or exponentially with biofilm thickness.     

Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system

AIMS: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high-quality drinking water. METHODS AND RESULTS: The growth of biofilms was studied using the biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. Vital staining of biofilms with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA-specific 4',6-diamidino-2-phenylindole (DAPI) staining resulted in a significant difference in physiological behaviour of biofilm populations depending on the disinfection technique. Compared with chlorine dioxide disinfection (0.12-0.16 mg l-1), the respiratory activities of the micro-organisms were increased on all materials during u.v. disinfection (u.v.254; 400 J m-2). The biofilm biocoenosis was analysed by in situ hybridization with labelled oligonucleotides specific for some subclasses of Proteobacteria. Using PCR and additional hybridization techniques, the biofilms were also tested for the presence of Legionella spp., atypical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the u.v. disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection. CONCLUSIONS: The results indicated that bacteria are able to regenerate and proliferate more effectively after u.v. irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as the application of u.v. disinfection is used for conditioning of critical water sources for drinking water, the efficiency of u.v. irradiation in natural systems should reach a high standard to avoid adverse impacts on human health.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Influence of pipe materials and VBNC cells on culturable bacteria in a chlorinated drinking water model system

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.     

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.