Legionella relative abundance in shower hose biofilms is associated with specific microbiome members.

Legionella are natural inhabitants of building plumbing biofilms, where interactions with other microorganisms influence their survival, proliferation, and death. Here, we investigated the associations of Legionella with bacterial and eukaryotic microbiomes in biofilm samples extracted from 85 shower hoses of a multiunit residential building. Legionella spp. relative abundance in the biofilms ranged between 0-7.8%, of which only 0-0.46% was L. pneumophila. Our data suggest that some microbiome members were associated with high (e.g. Chthonomonas, Vrihiamoeba) or low (e.g. Aquabacterium, Vannella) Legionella relative abundance. The correlations of the different Legionella variants (30 Zero-Radius OTUs detected) showed distinct patterns, suggesting separate ecological niches occupied by different Legionella species. This study provides insights into the ecology of Legionella with respect to: (i) the colonization of a high number of real shower hoses biofilm samples; (ii) the ecological meaning of associations between Legionella and co-occurring bacterial/eukaryotic organisms; (iii) critical points and future directions of microbial-interaction-based-ecological-investigations.

Potential probiotic approaches to control Legionella in engineered aquatic ecosystems.

Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven "probiotic" control approach against Legionella. We focus on: (i) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (ii) exploitative and interference competitive interactions between Legionella and other microorganisms; and (iii) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach.

Variable Legionella Response to Building Occupancy Patterns and Precautionary Flushing.

When stay-at-home orders were issued to slow the spread of COVID-19, building occupancy (and water demand) was drastically decreased in many buildings. There was concern that widespread low water demand may cause unprecedented Legionella occurrence and Legionnaires' disease incidence. In lieu of evidenced-based guidance, many people flushed their water systems as a preventative measure, using highly variable practices. Here, we present field-scale research from a building before, during, and after periods of low occupancy, and controlled stagnation experiments. We document no change, a > 4-log increase, and a > 1.5-log decrease of L. pneumophila during 3- to 7-week periods of low water demand. L. pneumophila increased by > 1-log after precautionary flushing prior to reoccupancy, which was repeated in controlled boiler flushing experiments. These results demonstrate that the impact of low water demand (colloquially called stagnation) is not as straight forward as is generally assumed, and that some flushing practices have potential unintended consequences. In particular, stagnation must be considered in context with other Legionella growth factors like temperature and flow profiles. Boiler flushing practices that dramatically increase the flow rate and rapidly deplete boiler temperature may mobilize Legionella present in biofilms and sediment.

Stagnation leads to short-term fluctuations in the effluent water quality of biofilters: A problem for greywater reuse?

A key characteristic of decentralized greywater treatment and reuse is high variability in both nutrient concentrations and flow. This variability in flow leads to stagnant water in the system and causes short-term fluctuations in the effluent water quality. Automated monitoring tools provide data to understand the mechanisms underlying the dynamics and to adapt control strategies accordingly. We investigated the fluctuations in a building-scale greywater treatment system comprising a membrane bioreactor followed by a biological activated carbon filter. Short-term dynamics in the effluent of the biological activated carbon filter were monitored with automated flow cytometry and turbidity, and the impact of these fluctuations on various hygiene-relevant parameters in the reuse water was evaluated. Continuous biofilm detachment into the stagnant water in the biological activated carbon filter led to temporarily increased turbidity and cell concentrations in the effluent after periods of stagnation. The fluctuations in cell concentrations were consistent with a model assuming higher detachment rates during flow than during times with stagnant water. For this system, total cell concentration and turbidity were strongly correlated. We also showed that the observed increase in cell concentration was not related to either an increase of organic carbon concentration or the concentration of two opportunistic pathogens, P. aeruginosa and L. pneumophila. Our findings demonstrate that turbidity measurements are sensitive to changes in the effluent water quality and can be used to monitor the fluctuations caused by intermittent flow. Intermittent flow did not lead to an increase in opportunistic pathogens, and this study provides no indications that stagnant water in biological activated carbon filters need be prevented.

The value of human data annotation for machine learning based anomaly detection in environmental systems.

Anomaly detection is the process of identifying unexpected data samples in datasets. Automated anomaly detection is either performed using supervised machine learning models, which require a labelled dataset for their calibration, or unsupervised models, which do not require labels. While academic research has produced a vast array of tools and machine learning models for automated anomaly detection, the research community focused on environmental systems still lacks a comparative analysis that is simultaneously comprehensive, objective, and systematic. This knowledge gap is addressed for the first time in this study, where 15 different supervised and unsupervised anomaly detection models are evaluated on 5 different environmental datasets from engineered and natural aquatic systems. To this end, anomaly detection performance, labelling efforts, as well as the impact of model and algorithm tuning are taken into account. As a result, our analysis reveals the relative strengths and weaknesses of the different approaches in an objective manner without bias for any particular paradigm in machine learning. Most importantly, our results show that expert-based data annotation is extremely valuable for anomaly detection based on machine learning.

Automated flow cytometry as a flexible tool for comparing disinfection characteristics of indigenous bacterial communities and pure cultures.

Bacterial inactivation efficiency of chlorine varies with organisms and environmental conditions. The comparability of different samples/studies, especially comparing indigenous bacterial communities with pure cultures, is impeded by inconsistent experimental conditions and analytical methods used in various studies. We tested a novel 96-well plate FCM experimental and automated analytical approach, where bacterial communities and pure cultures were suspended in the same natural water matrix prior to chlorination directly in the plate. We demonstrated the ability to rapidly monitor the efficiency of 32 different combinations of chlorine concentration and time (i.e. chlorine exposure) on bacterial pure cultures and indigenous aquatic communities, which enabled correct comparison of the data from different samples under the exact same experimental conditions. In this study, the 96-well plate automated FCM approach enabled large sets (896) of independent chlorination experiments to be carried out in a short time period. To our knowledge, this is the largest dataset of chlorination experiments which consumed least time (within 18 h after sampling) until now. Staining with SYBR Green I (SG) and SG combined with propidium iodide (SGPI) was used to assess cellular damage during chlorination. The results showed that with the same chlorine exposure, a higher chlorine concentration with a shorter contact time is favorable for inactivation of bacteria. Our research provides a promising framework to compare disinfection characteristics of various microorganism and can be further developed to diagnose effect of antimicrobial products.

360-Degree Distribution of Biofilm Quantity and Community in an Operational Unchlorinated Drinking Water Distribution Pipe.

In the present study, triplicate rings of 360° pipe surfaces of an operational drinking water distribution pipe were swabbed. Each ring was equally divided into 16 parts for swabbing. The collected swabs were grouped into 3 sections and compared with the biofilm samples sampled by sonication of specimens from the same pipe. The results showed that the biofilm is unevenly distributed over the 16 parts and the 3 sections of the pipe surface. Both the active biomass and the number of observed OTUs increased as the measurements proceeded from the top to the bottom of the pipe. The bacterial community was dominated in all sections by Proteobacteria. At the genus level, Nitrospira spp., Terrimonas spp., and Hyphomicrobium spp. were dominant in all sections. Gaiella spp. and Vicinamibacter spp. dominated in S-I, Blastopirellula spp. and Pirellula spp. dominated in S-II, while Holophaga spp. and Phaeodactylibacter spp. dominated in S-III. When swabbing and pipe specimen sonication were compared, the results showed that the sampling strategy significantly influences the obtained biofilm bacterial community. A consistent multisectional swabbing strategy is proposed for future biofilm sampling; it involves collecting swabs from all sections and comparing the swabs from the same position/section across locations.

Substrate Pre-loading Influences Initial Colonization of GAC Biofilter Biofilms.

Microbial community composition and stability affect pollutant removal for biological/granular activated carbon (BAC/GAC) processes. Here, we pre-loaded the organic carbon substrates sucrose, lactose, and Lysogeny Broth (LB) medium onto new GAC prior to use and then tested whether this substrate pre-loading promoted development of biofilms with high coverage that remained stable for prolonged operational periods. Temporal dynamics of the biomass and microbial community on the GAC were monitored via flow cytometry (FCM) and sequencing, respectively, in both batch and continuous-flow experiments. In comparison with the non-loaded GAC (control), the initial biofilm biomass on substrate-loaded GAC was 3-114 times higher, but the initial richness was considerably lower (only accounting for 13-28% of the control). The initial community compositions were significantly different between batch and continuous-flow column experiments, even when loaded with the same substrates. In the continuous-flow column experiments, both biomass and microbial community composition became remarkably similar to the control filters after 64 days of operation. From these findings, we conclude that substrate-loaded GAC could enhance initial colonization, affecting both biomass and microbial community composition. However, the biomass and composition did not remain stable during long-term operation due to continuous dispersal and competition from influent bacteria.

Small-Scale Heterogeneity in Drinking Water Biofilms.

Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling (200 individual biofilm sections per hose) on varying scales (µm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real (i.e., uncontrolled) use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with <400 taxa each, and only three taxa accounting for 57%, respectively, 73% of the community. This low diversity was attributed to a strong selective pressure, originating in migrating carbon from the flexible hoses as major carbon source. High-resolution sampling strategy enabled detailed analysis of spatial heterogeneity within an individual drinking water biofilm. This study gives insight into biofilm structure and community composition on cm-to m-scale and is useful for decision-making on sampling strategies in biofilm research and monitoring.

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 °C - 39 °C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 °C and 27 °C), and at high temperatures (39 °C) when the incubator set temperature was higher. This incubator prototype is low-cost (< 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

Detection of microbial disturbances in a drinking water microbial community through continuous acquisition and advanced analysis of flow cytometry data.

Detecting disturbances in microbial communities is an important aspect of managing natural and engineered microbial communities. Here, we implemented a custom-built continuous staining device in combination with real-time flow cytometry (RT-FCM) data acquisition, which, combined with advanced FCM fingerprinting methods, presents a powerful new approach to track and quantify disturbances in aquatic microbial communities. Through this new approach we were able to resolve various natural community and single-species microbial contaminations in a flow-through drinking water reactor. Next to conventional FCM metrics, we applied metrics from a recently developed fingerprinting technique in order to gain additional insight into the microbial dynamics during these contamination events. Importantly, we found that multiple community FCM metrics based on different statistical approaches were required to fully characterize all contaminations. Furthermore we found that for accurate cell concentration measurements and accurate inference from the FCM metrics (coefficient of variation ≤ 5%), at least 1000 cells should be measured, which makes the achievable temporal resolution a function of the prevalent bacterial concentration in the system-of-interest. The integrated RT-FCM acquisition and analysis approach presented herein provides a considerable improvement in the temporal resolution by which microbial disturbances can be observed and simultaneously provides a multi-faceted toolset to characterize such disturbances.

A uniform bacterial growth potential assay for different water types.

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement. This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed. Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 µg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample. The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon.

Flow-cytometric quantification of microbial cells on sand from water biofilters.

Rapid quantification of absolute microbial cell abundances is important for a comprehensive interpretation of microbiome surveys and crucial to support theoretical modelling and the design of engineered systems. In this paper, we propose a protocol specifically optimised for the quantification of microbial abundances in water biofilters using flow cytometry (FCM). We optimised cell detachment from sand biofilter particles for FCM quantification through the evaluation of five chemical dispersants (NaCl, Triton-X100, CaCl2, sodium pyrophosphate (PP), Tween 80 combined with PP), different mechanical pre-treatments (low and high energy sonication and shaking) and two fixation methods (glutaraldehyde and ethanol). The developed protocol was cross-compared using other established and commonly employed methods for biomass quantification in water filter samples (adenosine triphosphate (ATP) quantification, real-time quantitative PCR (qPCR) and volatile solids (VS)). The highest microbial count was obtained by detaching the biofilm from biofilter grains and dispersing clusters into singles cells using Tween 80 and sodium pyrophosphate combined with four steps of high energy sonication (27W, for 80 s each step); glutaraldehyde was shown to be the best fixative solution. The developed protocol was reliable and highly reproducible and produced results that are comparable to data from alternative quantification methods. Indeed, high correlations were found with trends obtained through ATP and qPCR (ρ = 0.98 and ρ = 0.91) measurements. The VS content was confirmed as an inaccurate method to express biomass in sand samples since it correlated poorly with all the other three methods (ρ = 0.005 with FCM, 0.002 with ATP and 0.177 with qPCR). FCM and ATP showed the strongest agreement between absolute counts with a slope of the correlation equal to 0.7, while qPCR seemed to overestimate cell counts by a factor of ten. The rapidity and reproducibility of the method developed make its application ideal for routine quantification of microbial cell abundances on sand from water biofilters and thus useful in revealing the ecological patterns and quantifying the metabolic kinetics involved in such systems.

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.

Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (-80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03-2% in protectant-free cultures to 11-37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific.

Ugly ducklings-the dark side of plastic materials in contact with potable water.

Bath toys pose an interesting link between flexible plastic materials, potable water, external microbial and nutrient contamination, and potentially vulnerable end-users. Here, we characterized biofilm communities inside 19 bath toys used under real conditions. In addition, some determinants for biofilm formation were assessed, using six identical bath toys under controlled conditions with either clean water prior to bathing or dirty water after bathing. All examined bath toys revealed notable biofilms on their inner surface, with average total bacterial numbers of 5.5 × 106 cells/cm2 (clean water controls), 9.5 × 106 cells/cm2 (real bath toys), and 7.3 × 107 cells/cm2 (dirty water controls). Bacterial community compositions were diverse, showing many rare taxa in real bath toys and rather distinct communities in control bath toys, with a noticeable difference between clean and dirty water control biofilms. Fungi were identified in 58% of all real bath toys and in all dirty water control toys. Based on the comparison of clean water and dirty water control bath toys, we argue that bath toy biofilms are influenced by (1) the organic carbon leaching from the flexible plastic material, (2) the chemical and biological tap water quality, (3) additional nutrients from care products and human body fluids in the bath water, as well as, (4) additional bacteria from dirt and/or the end-users' microbiome. The present study gives a detailed characterization of bath toy biofilms and a better understanding of determinants for biofilm formation and development in systems comprising plastic materials in contact with potable water.

Phylogenetic clustering of small low nucleic acid-content bacteria across diverse freshwater ecosystems.

Here we used flow cytometry (FCM) and filtration paired with amplicon sequencing to determine the abundance and composition of small low nucleic acid (LNA)-content bacteria in a variety of freshwater ecosystems. We found that FCM clusters associated with LNA-content bacteria were ubiquitous across several ecosystems, varying from 50 to 90% of aquatic bacteria. Using filter-size separation, we separated small LNA-content bacteria (passing 0.4 µm filter) from large bacteria (captured on 0.4 µm filter) and characterized communities with 16S amplicon sequencing. Small and large bacteria each represented different sub-communities within the ecosystems' community. Moreover, we were able to identify individual operational taxonomical units (OTUs) that appeared exclusively with small bacteria (434 OTUs) or exclusively with large bacteria (441 OTUs). Surprisingly, these exclusive OTUs clustered at the phylum level, with many OTUs appearing exclusively with small bacteria identified as candidate phyla (i.e. lacking cultured representatives) and symbionts. We propose that LNA-content bacteria observed with FCM encompass several previously characterized categories of bacteria (ultramicrobacteria, ultra-small bacteria, candidate phyla radiation) that share many traits including small size and metabolic dependencies on other microorganisms.

Identifying the underlying causes of biological instability in a full-scale drinking water supply system.

Changes in bacterial concentration and composition in drinking water during distribution are often attributed to biological (in)stability. Here we assessed temporal biological stability in a full-scale distribution network (DN) supplied with different types of source water: treated and chlorinated surface water and chlorinated groundwater produced at three water treatment plants (WTP). Monitoring was performed weekly during 12 months in two locations in the DN. Flow cytometric total and intact cell concentration (ICC) measurements showed considerable seasonal fluctuations, which were different for two locations. ICC varied between 0.1-3.75 × 105 cells mL-1 and 0.69-4.37 × 105 cells mL-1 at two locations respectively, with ICC increases attributed to temperature-dependent bacterial growth during distribution. Chlorinated water from the different WTP was further analysed with a modified growth potential method, identifying primary and secondary growth limiting compounds. It was observed that bacterial growth in the surface water sample after chlorination was primarily inhibited by phosphorus limitation and secondly by organic carbon limitation, while carbon was limiting in the chlorinated groundwater samples. However, the ratio of available nutrients changed during distribution, and together with disinfection residual decay, this resulted in higher bacterial growth potential detected in the DN than at the WTP. In this study, bacterial growth was found to be higher (i) at higher water temperatures, (ii) in samples with lower chlorine residuals and (iii) in samples with less nutrient (carbon, phosphorus, nitrogen, iron) limitation, while this was significantly different between the samples of different origin. Thus drinking water microbiological quality and biological stability could change during different seasons, and the extent of these changes depends on water temperature, the water source and treatment. Furthermore, differences in primary growth limiting nutrients in different water sources could contribute to biological instability in the network, where mixing occurs.

Biofilms in shower hoses.

Shower hoses offer an excellent bacterial growth environment in close proximity to a critical end-user exposure route within building drinking water plumbing. However, the health risks associated with and processes underlying the development of biofilms in shower hoses are poorly studied. In a global survey, biofilms from 78 shower hoses from 11 countries were characterized in terms of cell concentration (4.1 × 104-5.8 × 108 cells/cm2), metal accumulation (including iron, lead, and copper), and microbiome composition (including presence of potential opportunistic pathogens). In countries using disinfectant, biofilms had on average lower cell concentrations and diversity. Metal accumulation (up to 5 µg-Fe/cm2, 75 ng-Pb/cm2, and 460 ng-Cu/cm2) seemed to be partially responsible for discoloration in biofilms, and likely originated from other pipes upstream in the building. While some genera that may contain potential opportunistic pathogens (Legionella, detected in 21/78 shower hoses) were positively correlated with biofilm cell concentration, others (Mycobacterium, Pseudomonas) had surprisingly non-existent or negative correlations with biofilm cell concentrations. In a controlled study, 15 identical shower hoses were installed for the same time period in the same country, and both stagnant and flowing water samples were collected. Ecological theory of dispersal and selection helped to explain microbiome composition and diversity of different sample types. Shower hose age was related to metal accumulation but not biofilm cell concentration, while frequency of use appeared to influence biofilm cell concentration. This study shows that shower hose biofilms are clearly a critical element of building drinking water plumbing, and a potential target for building drinking water plumbing monitoring.

Short-term organic carbon migration from polymeric materials in contact with chlorinated drinking water.

Polymeric materials are widely used in drinking water distribution systems. These materials could release organic carbon that supports bacterial growth. To date, the available migration assays for polymeric materials have not included the potential influence of chlorination on organic carbon migration behavior. Hence, we established a migration and growth potential protocol specifically for analysis of carbon migration from materials in contact with chlorinated drinking water. Four different materials were tested, including ethylene propylene dienemethylene (EPDM), poly-ethylene (PEX b and PEX c) and poly-butylene (PB). Chlorine consumption rates decreased gradually over time for EPDM, PEXc and PB. In contrast, no free chlorine was detected for PEXb at any time during the 7 migration cycles. Total organic carbon (TOC) and assimilable organic carbon (AOC) was evaluated in both chlorinated and non-chlorinated migrations. TOC concentrations for EPDM and PEXb in chlorinated migrations were significantly higher than non-chlorinated migrations. The AOC results showed pronounced differences among tested materials. AOC concentrations from chlorinated migration waters of EPDM and PB were higher compared to non-chlorinated migrations, whereas the opposite trend was observed for PEXb and PEXc. There was also a considerable difference between tested materials with regards to bacterial growth potential. The results revealed that the materials exposed to chlorine-influenced migration still exhibited a strong biofilm formation potential. The overall results suggested that the choice in material would make a considerable difference in chlorine consumption and carbon migration behavior in drinking water distribution systems.