Shower systems provide unique environments that are conducive to biofilm formation and the proliferation of pathogens. The water heating temperature is a delicate decision that can impact microbial growth, balancing safety and energy consumption. This study investigated the impact of different heating temperatures (39 °C, 45 °C, 51 °C and 58 °C) on the shower hose biofilm (exposed to a final water temperature of 39 °C) using controlled full-scale shower setups. Whole metagenome sequencing and metaproteomics were employed to unveil the microbial composition and protein expression profiles. Overall, the genes and enzymes associated with disinfectant resistance and biofilm formation appeared largely unaffected. However, metagenomic analysis revealed a sharp decline in the number of total (86,371 to 34,550) and unique genes (32,279 to 137) with the increase in hot water temperature, indicating a significant reduction of overall microbial complexity. None of the unique proteins were detected in the proteomics experiments, suggesting smaller variation among biofilms on the proteome level compared to genomic data. Furthermore, out of 43 pathogens detected by metagenomics, only 5 could actually be detected by metaproteomics. Most interestingly, our study indicates that 45 °C heating temperature may represent an optimal balance. It minimizes active biomass (ATP) and reduces the presence of pathogens while saving heating energy. Our study offered new insights into the impact of heating temperature on shower hose biofilm formation and proposed optimal parameters that ensure biosafety while conserving energy.

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Pipe materials appear to play an important role in the development of biofilms in drinking water distribution systems. However, there is controversy as to whether pipe materials shape the composition and diversity of bacterial communities in biofilms. To investigate the long-term effects of pipe materials on biofilms, triplicate samples of mature biofilms on unplasticized polyvinyl chloride (PVC-U), grey cast iron and asbestos cement (pipe age > 40 years) were obtained from three areas of an unchlorinated drinking water distribution system in the Netherlands. Illumina sequencing was performed and 773 OTUs (730 OTUs-814 OTUs) were detected within the biofilms on the three pipe materials, all of which were dominated by Proteobacteria (36.2%-46.1%). Both the alpha and beta diversity results showed that the bacterial communities of the biofilms formed on different pipe materials were highly similar. The neutral community model revealed that the assembly of the biofilm communities was governed by environmental selection rather than neutral processes. Among the 142 shared OTUs between the water and biofilm samples, there were 25 enriched OTUs (e.g., OTU7, assigned as Nitrospira spp.), which accounted for 62.6% of the total sequences, while 16 OTUs were disadvantaged (e.g., OTU14 and OTU40, assigned as Hyphomicrobiaceae), accounting for 2.2% of the sequences. Based on the findings, we propose and discuss a harmonisation process by which biofilms with significant differences due to the pipe material harmonize over time resulting in biofilms with similar bacterial communities. Our findings provide valuable insights into long-term biofilm development, bridging an essential gap in our current understanding of the influence of pipe materials on biofilm communities. These findings also highlight the importance of long-term studies and point to a potentially masked harmonizing process during biofilm development over years/decades.

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Microbial drinking water quality is of great importance to human health. Drinking water distribution systems (DWDSs) are designed as the final barriers for delivering and maintaining the biosafety of drinking water. Though the drinking water produced is usually safe and clean, it is common that the water quality deteriorates during the distribution. Such deteriorations can be linked to the establishment of biofilm in DWDSs, where the majority of the biomass is residing (> 95%). The formed biofilms are reportedly leading causes of the undesired taste, odor, and color of the drinking water, corrosion of the pipes, decay of the disinfectants, and proliferation of pathogenic microbes, giving rise to public health concerns.
In the Netherlands, the control of the biofilm growth in DWDSs is achieved by producing bio-stable drinking water with extremely low nutrients (e.g., AOC < 10 µg C/l). On the other hand, water utilities in many countries usually apply chemical disinfectants (e.g., free chlorine, monochloramine) to control the biofilm growth in DWDSs. Nevertheless, biofilm formation is inevitable, regardless of the strategy. Additionally, there is no standard method to monitor the biofilm growth in DWDSs, which makes the understanding and management of DWDS biofilms more challenging. Efforts have been made to explore the biofilm formation and structure through pilot studies. However, most of these investigations have been conducted in a short time frame (e.g., within weeks to a max of 84 days), where the developed biofilms were far from mature and significantly different from those in the real DWDSs. To uncover how biofilm develops and what roles disinfectants play during the biofilm development, a newly-built pilot system was followed for a 64-weeks period under different disinfection regimes: no disinfectants (NC), free chlorine (FC), and monochloramine (MC) (Chapter 2). The results showed that residual disinfectants presented intensive suppression of the biofilm growth and shaped the biofilm communities. Specifically, MC exhibited stronger suppression of the biofilm activity (i.e., ATP), whereas FC expressed intense selection pressure on the microbes and established more homogenous and less complex biofilm community, with Proteobacteria comprising on average 82% of the relative abundance. The temporal trends highlighted the essential developmental stages in biofilm formation from initial colonization to accumulation and selection and stabilization, which occurred at different rates under each of the conditions, and were associated with significant dynamic changes in biofilm bacterial communities. Reaching stabilization took longest in the MC condition (> 64 weeks), followed by the NC (~ 36 weeks) and FC (~ 19 weeks) conditions. Holistically, the early stages in the biofilm formation in the NC condition were primarily dominated by stochastic processes where colonizers originating from treated water randomly attached to and settled on the pipes, while deterministic processes progressively increased in their relative contributions at the end of the accumulation stage and became predominant at the later stages. In the MC condition, the biofilm succession was governed by stochastic processes during the entire test, even though some deterministic processes occurred during the accumulation stage. Conversely, in the FC condition the biofilm succession was driven by deterministic processes already from the initial development stage.
DWDSs are highly dynamic ecosystems, where the liquid (i.e., bulk water, suspended particles) and solid (i.e., biofilm, loose deposits) phases interact intensively during transport of the water from treatment to consumer. The cells and/or particles that were introduced with the treated water may attach to and/or settle on the pipes, forming biofilm/loose deposits when the hydraulic forces are weak. Conversely, the biofilm/loose deposits might release cells/particles to the bulk water during hydraulic disturbances, affecting the drinking water quality negatively. The hydraulic conditions in DWDSs are very complex and dynamic. They exhibit daily patterns, with high flow rates at high water demand periods (e.g., morning and/or evening hours) and long stagnancy or low flows during the night. However, most monitoring occurs using grab samples at one point in time. Thus, continuous online sampling is required to obtain a representative image of the particles and microbes in drinking water. In Chapter 3, a novel online monitoring and sampling system (OMSS) was developed to investigate the spatiotemporal variations of the planktonic and particle-associated bacteria in an unchlorinated DWDSs. The 16S rRNA gene sequencing combined with SourceTracker2 was used to trace and reveal the origin of the changes in the planktonic and particle-associated bacteria, assigning sampled biofilm and loose deposits as sources. The results showed that, spatially, the particle loads significantly increased from treatment plant within distribution networks, while the trend in the quantity of the particle-associated bacteria was the opposite. Similar to the trend of particle loads, the number of the observed OTUs in both planktonic and particle-associated bacteria increased from the treatment plant within the distribution network. The spatial results implied a dominant role of sedimentation of particles entering the DWDS from the treatment plant, while the observed increases in particles and the associated bacteria primarily originated from the distribution network, which were confirmed by the increased contributions from loose deposits and biofilm determined by SourceTracker2. Temporally, daily peaks in the water quality, including particle-associated bacterial quantity, observed operational taxonomic unit (OTU) number, and contributions of biofilm and loose deposits, were sensitively captured during the high water demand (morning/evening peaks). The temporal results revealed clear dynamic interactions between the liquid (i.e., bulk water, suspended particles) and solid (i.e., biofilm, loose deposits) phases in DWDSs.
Driven by increasingly stringent drinking water regulations and challenges to drinking water quality, efforts are underway to further improve water quality. These initiatives include source water switching, upgrading treatment processes, and implementing changes to disinfectant strategies. Such actions change the quality and composition of the treated water that enters the DWDS. This may have transition effects, which in this thesis refers to the water quality deteriorations contributed by the release of cells and particles from biofilm and/or loose deposits due to the irregular changes in supply-water quality. It is largely unknown whether, where and when the transition effects will happen. In Chapter 4, transition effects were investigated through characterizing the particles before (T₀), during (T_3-weeks) and after (T_6-months) introducing additional treatment steps (softening, second rapid sand filtration and adding carbon dioxide) to the existing treatment. The results showed that the upgraded treatment significantly improved the water quality after 6 months’ time. However, significant water quality deterioration was observed at the initial stage (T_3-weeks) when the quality-improved treated water entered into the network. This manifested as a significant increase in total suspended solids (TSS) by 50-260%, active biomass (ATP) by 95-230%, and Mn by 130-250%. Furthermore, pyrosequencing results revealed sharp differences in microbial community composition and structure of the bacteria associated with particles between T₀ and T_3-weeks, implying the potential contributions from biofilm or loose deposits in the DWDS. Interestingly, the domination of Nitrospira spp. and Polaromonas spp. in the distribution system at T_3-weeks, which were detected at rather low relative abundance at treatment plant, further confirmed the potential contributions from biofilm or loose deposits.
Though the study in Chapter 4 confirmed the occurrence of the transition effects, the question how fast/how long the transition effects will occur/last, where the deteriorations originate from, and what actions can be carried out to minimize the transition effects is not clear. The sampling was conducted in a relatively short time frame (i.e., 6 months), with only a few time points (i.e., T₀, T_3-weeks, T_6-months) and without the collection of biofilm and loose deposit samples. Additionally, as what we can see from the results from Chapter 3, it could be imagined that the transition effects might be enhanced during high water demand when shear forces are high. In order to fill the knowledge gaps, the OMSS was applied, accompanied with SourceTracker2, in an unchlorinated DWDS where partial RO was introduced (Chapter 5). The study was conducted before (T_B), immediately after (T₀), one month (T_1M), two month (T_2M), one year (T_1Y) and two years (T_2Y) after the partial RO introduction. Noticeably, significant transition effects in DWDS were captured right after the RO introduction, with increases in the particle loads, bacterial quantity, community diversity, and significant differences between bacterial communities in particles at treatment plant and distribution network. The disturbances lasted one month until T_1M, after which they ceased to be observable around T_2M. The captured deteriorations were confirmed by the increased contributions of loose deposits and biofilm (both the number of the immigrants and their abundance) at T₀ and T_1M determined by SourceTracker2 and neutral community model. While the peak transition window spanned about one month, it took considerably longer, until one year (T_1Y) and two years (T_2Y) later, for the microbial ecology to re-stabilize and for improvements in water quality to become noticeable. In addition, the peaks in the water quality deteriorations were enlarged during the high water demand (morning/evening peaks), which implies that current monitoring could potentially underestimate the extent of the quality deterioration. Remarkably, the observation that loose deposits contributed more to the transition effects than biofilm challenges the traditional standpoint, and provided new insights into the management of the transition effects, where the risks of the transition effects can be largely reduced by conducting flushing before the introduction of treatment changes to remove the loose deposits. In light of the destabilization caused by the changed water quality, flushing with new-quality water might be more rewarding.
To conclude, through conducting studies at both field and pilot scales, the effects of the (changes in) operational conditions on the microbial drinking water quality in DWDSs were comprehensively explored. The findings in the thesis offer novel insights into the drinking water quality management. The knowledge gained from investigating the biofilm succession dynamics under different disinfectant regimes has significantly deepened our understanding of managing drinking water biofilms. These insights serve as valuable information when making informed decisions about the appropriate strategies to employ. The implementation of the developed OMSS is capable of capturing both periodic and aperiodic changes in drinking water quality, making it an essential tool in minimizing assessment deviations and ensuring accurate evaluations of drinking water quality. Moreover, the established methodology holds promise for application in various systems, including those that utilize chlorination. By identifying and characterizing the transition effects resulting from changes in supply water quality, such as treatment upgrades or the introduction of reverse osmosis, the study highlights the significance of considering these effects in water management practices. These observations underscore the importance of addressing the impact of transition effects on drinking water quality and provide practical implications for minimizing their negative consequences.
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The aperiodic changes in the quantity and community of planktonic and particle-associated bacteria have hampered the understanding and management of microbiological water quality in drinking water distribution systems. In this study, online sampling was combined with the microbial fingerprint-based SourceTracker2 to capture and trace the spatiotemporal variations in planktonic and particle-associated bacteria in an unchlorinated distribution system. The results showed that spatially, the particle load significantly increased, while in contrast, the quantity of particle-associated bacteria decreased sharply from the treatment plant to the distribution network. Similar to the trend of particle-associated bacterial diversity, the number of observed OTUs first slightly decreased from the treatment plant to the transportation network and then sharply increased from the transportation network to the distribution network. The SourceTracker2 results revealed that the contribution of particle-associated bacteria from the treatment plant decreased along the distribution distance. The spatial results indicate the dominant role of sedimentation of particles from the treatment plant, while the observed increases in particles and the associated bacteria mainly originated from the distribution network, which were confirmed directly by the increased contributions of loose deposits and biofilm. Temporally, the daily peaks of particle-associated bacterial quantity, observed OTU number, and contributions of loose deposits and biofilms were captured during water demand peaks (e.g., 18–21 h). The temporal results reveal clear linkages between the distribution system harboring bacteria (e.g., within loose deposits and biofilms) and the planktonic and particle-associated bacteria flowing through the distribution system, which are dynamically connected and interact. This study highlights that the spatiotemporal variations in planktonic and particle-associated bacteria are valuable and unneglectable for the widely on-going sampling campaigns required by water quality regulations and/or drinking water microbiological studies.

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The generation and dissemination of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment has become a critical risk to human health. This study is based on a pilot-scale simulated water distribution system to understand the effects of chlorine disinfection treatment (without free chlorine) on ARB and ARGs in biofilms. The hydraulic parameters and pipe materials of the system were simulated based on a drinking water system. The results of the colony counts showed that bacterial multi-antibiotic resistance could be enhanced 13-fold in the biofilms of the pipeline. The use of high-throughput qPCR (HT-qPCR) indicated that the total relative abundance of ARGs in biofilm samples increased significantly (p, 0.05), while the diversity of bacteria was shown to be reduced via taxonomic analysis of the V3–V4 region of 16S rRNA. The prominent types of ARGs were conferred resistance by aminoglycoside and β-lactam after the chlorine disinfection treatment, and antibiotic deactivation was the main mechanism. Phyla Proteobacteria had the highest abundance in both treatment and control groups but decreased from 70.81% (initial biofilm sample) to 26.09% (the sixth-month biofilm sample) in the treatment groups. The results show that the chlorine disinfection plays a role in the risk of development of bacterial antibiotic resistance in pipe networks owing to bacteria in biofilms. This study was the first to investigate the contribution of chlorination without free chlorine to the bacterial community shift and resistome alteration in biofilms at a pilot-test level.

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Titanium dioxide nanoparticles (TiO₂ NPs) are widely used as nano-agrochemicals. In this study we investigated the influence of soil heterogeneity on bacterial communities exposed to TiO₂ NPs over time. Clay and sandy soils with low- and high-organic matter contents were exposed to environmentally relevant concentration of TiO₂ NPs (1 mg/kg) and soil bacterial communities were sampled after short-term (15 days) and long-term exposure (60 days). After short-term TiO₂ NPs exposure, significant effects regarding the enzyme activity, bacterial community structure and composition, and community functioning were observed in the clay soils with high organic matter (clay-HOM) but not in other soil groups. Response alterations were observed to taxa belonging to Acidobacteria and Verrucomicrobia, and functional pathways related to carbohydrates degradation. These results indicated that soil heterogeneity play more important roles in shaping the bacterial community in soil with low clay fraction and less organic matter, while TiO₂ NPs selection was the main driver in inducing the compositional and functional impacts on the soil bacterial community in the presence of clay soil with high organic matter content. As exposure time increased, the bacterial community recovered after a long-term exposure of 60 days, suggesting that the bacterial evolution and adaptation could overcome the TiO₂ NPs selection after long-term exposure. Our results highlighted the importance of soil heterogeneity including clay fraction and organic matter and exposure duration in assessing the impact of nanoparticle on soil bacterial activity, community and function. By comprehensively evaluating the risks of nanoparticles on soil ecosystem and explicitly and explicitly include spatial and temporal variations, the benefit of nano-agrochemical products has the potential to be promoted in future applications.

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Slow sand filters (SSFs) are widely applied to treat potable water; the removal of contaminants (e.g., particles, organic matter, and microorganism) occurs primarily in the top layer. However, the development of the microbial community and its metabolic function is still poorly understood. In the present study, we analyzed the microbial quantity and community of the influents sampled from the effluent of the last step (rapid sand filtration) and of the top layers of SSFs (Schmutzdecke, 0–2 cm, 4–6 cm, 8–10 cm) sampled near terminal head loss when the Schmutzdecke (SCM) was most developed in two full-scale drinking water treatment plants (DWTPs). The two DWTPs use the same artificially recharged groundwater source. The biomass in the filter, quantified by flow cytometric intact cell counts (ICC) and adenosine triphosphate (ATP), decreased rapidly along the depth till 8–10 cm (>1 log TCC; >75% ATP); the decrease was most pronounced from the SCM to the surface sand layer (0–2 cm), after which the biomass stabilized quickly at lower depths (2–10 cm). Remarkably, beta diversity showed that SSFs layers of the same depth in two DWTPs with distinctive filter age and plant location clustered together, which indicated their insignificant effects in shaping microbial communities in SSFs. The alpha diversity indices followed the trend of the biomass, suggesting more active and diverse communities in SCM layer. PICRUSt-based function prediction revealed significant over-representation of metabolism and degradation of complex organic matters (e.g., butanoate, propanoate, xenobiotic, D-Alanine, chloroalkene, and bisphenol) in SCM layer, the functional importance of which was confirmed by the co-occurrence patterns of the dominant taxa and metabolic functions. Using an island biogeography model, we found that microbial communities in SSFs were strongly assembled by selection (68 OTUs, 50.0% sequences), rather than by simple accumulation of the microbial communities in the influents (120 OTUs, 44.8% sequences). Our findings enhance the understanding of microbial community assembly and of metabolic function in the top layers of SSFs, and constitute a valuable contribution to optimizing the design and operation of biofilters in full-scale DWTPs.

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Worldwide, it is common that the drinking water distribution systems (DWDSs) may be subjected to changes of supply water quality due to the needs of upgrading the treatment processes or switching the source water. However, the potential impacts of quality changed supply water on the stabilized ecological niches within DWDSs and the associated water quality deterioration risks were poorly documented. In the present study, such transition effects caused by changing the supply water quality that resulted from destabilization of biofilm and loose deposits in DWDS were investigated by analyzing the physiochemical and microbiological characteristics of suspended particles before (T₀), during (T_3-weeks) and after upgrading the treatments (T_6-months) in an unchlorinated DWDS in the Netherlands. Our results demonstrated that after 6 months’ time the upgraded treatments significantly improved the water quality. Remarkably, water quality deterioration was observed at the initial stage when the quality-improved treated water distributed into the network at T_3-weeks, observed as a spike of total suspended solids (TSS, 50–260%), active biomass (ATP, 95–230%) and inorganic elements (e.g. Mn, 130–250%). Furthermore, pyrosequencing results revealed sharp differences in microbial community composition and structure for the bacteria associated with suspended particles between T₀ and T_3-weeks, which re-stabilized after 6 months at T_6-months. The successful capture of transition effects was especially confirmed by the domination of Nitrospira spp. and Polaromonas spp. in the distribution system at T_3-weeks, which were detected at rather low relative abundance at treatment plant. Though the transitional effects were captured, this study shows that the introduction of softening and additional filtration did not have an effect on the water quality for the consumer which improved considerably after 6-months’ period. The methodology of monitoring suspended particles with MuPFiSs and additional analysis is capable of detecting transitional effects by monitoring the dynamics of suspended particles and its physiochemical and microbiological composition.

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Although water produced by reverse osmosis (RO) filtration has low bacterial growth potential (BGP), post-treatment of RO permeate, which is necessary prior to distribution and human consumption, needs to be examined because of the potential re-introduction of nutrients/contaminants. In this study, drinking water produced from anaerobic groundwater by RO and post-treatment (ion exchange, calcite contactors, and aeration) was compared with that produced by conventional treatment comprising (dry) sand filtration, pellet softening, rapid sand filtration, activated carbon filtration, and UV disinfection. The multi-parametric assessment of biological stability included bacterial quantification, nutrient concentration and composition as well as bacterial community composition and diversity. Results showed that RO permeate remineralised in the laboratory has an extremely low BGP (50 ± 12 × 10³ ICC/mL), which increased to 130 ± 10 × 10³ ICC/mL after site post-treatment. Despite the negative impact of post-treatment, the BGP of the finished RO-treated water was >75% lower than that of conventionally treated water. Organic carbon limited bacterial growth in both RO-treated and conventionally treated waters. The increased BGP in RO-treated water was caused by the re-introduction of nutrients during post-treatment. Similarly, OTUs introduced during post-treatment, assigned to the phyla of Proteobacteria and Bacteroidetes (75–85%), were not present in the source groundwater. Conversely, conventionally treated water shared some OTUs with the source groundwater. It is clear that RO-based treatment achieved an extremely low BGP, which can be further improved by optimising post-treatment, such as using high purity calcite. The multi-parametric approach adopted in this study can offer insights into growth characteristics including limiting nutrients (why) and dominating genera growing (who), which is essential to manage microbiological water quality in water treatment and distribution systems.

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Dialysis water is directly related to the safety of hemodialysis patients, thus its quality is generally ensured by a stepwise water purification cascade. To study the effect of water treatment on the presence of antibiotic resistance genes (ARGs) in dialysis water, this study used propidium monoazide (PMA) in conjunction with high throughput quantitative PCR to analyze the diversity and abundance of ARGs found in viable bacteria from water having undergone various water treatment processes. The results indicated the presence of 35 ARGs in the effluents from the different water treatment steps. Twenty-nine ARGs were found in viable bacteria from the effluent following carbon filtration, the highest among all of the treatment processes, and at 6.96 Log (copies/L) the absolute abundance of the cphA gene was the highest. Two resistance genes, erm (36) and mtrD-02, which belong to the resistance categories macrolides-lincosamides-streptogramin B (MLSB) and other/efflux pump, respectively, were detected in the effluent following reverse osmosis treatment. Both of these genes have demonstrated the potential for horizontal gene transfer. These results indicated that the treated effluent from reverse osmosis, the final treatment step in dialysis-water production, was associated with potential health risks. [Figure not available: see fulltext.].@en