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Ammonia availability due to chloramination can promote the growth of nitrifying organisms, which can deplete chloramine residuals and result in operational problems for drinking water utilities. In this study, we used a metagenomic approach to determine the identity and functional potential of microorganisms involved in nitrogen biotransformation within chloraminated drinking water reservoirs. Spatial changes in the nitrogen species included an increase in nitrate concentrations accompanied by a decrease in ammonium concentrations with increasing distance from the site of chloramination. This nitrifying activity was likely driven by canonical ammonia-oxidizing bacteria (i.e., Nitrosomonas) and nitrite-oxidizing bacteria (i.e., Nitrospira) as well as by complete-ammonia-oxidizing (i.e., comammox) Nitrospira-like bacteria. Functional annotation was used to evaluate genes associated with nitrogen metabolism, and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction, and nitric oxide reduction. Furthermore, we assembled 47 high-quality metagenome-assembled genomes (MAGs) representing a highly diverse assemblage of bacteria. Of these, five MAGs showed high coverage across all samples, which included two Nitrosomonas, Nitrospira, Sphingomonas, and Rhizobiales-like MAGs. Systematic genome-level analyses of these MAGs in relation to nitrogen metabolism suggest that under ammonia-limited conditions, nitrate may be also reduced back to ammonia for assimilation. Alternatively, nitrate may be reduced to nitric oxide and may potentially play a role in regulating biofilm formation. Overall, this study provides insight into the microbial communities and their nitrogen metabolism and, together with the water chemistry data, improves our understanding of nitrogen biotransformation in chloraminated drinking water distribution systems. IMPORTANCE Chloramines are often used as a secondary disinfectant when free chlorine residuals are difficult to maintain. However, chloramination is often associated with the undesirable effect of nitrification, which results in operational problems for many drinking water utilities. The introduction of ammonia during chloramination provides a potential source of nitrogen either through the addition of excess ammonia or through chloramine decay. This promotes the growth of nitrifying microorganisms and provides a nitrogen source (i.e., nitrate) for the growth for other organisms. While the roles of canonical ammonia-oxidizing and nitrite-oxidizing bacteria in chloraminated drinking water systems have been extensively investigated, those studies have largely adopted a targeted gene-centered approach. Further, little is known about the potential long-term cooccurrence of complete-ammonia-oxidizing (i.e., comammox) bacteria and the potential metabolic synergies of nitrifying organisms with their heterotrophic counterparts that are capable of denitrification and nitrogen assimilation. This study leveraged data obtained for genome-resolved metagenomics over a time series to show that while nitrifying bacteria are dominant and likely to play a major role in nitrification, their cooccurrence with heterotrophic organisms suggests that nitric oxide production and nitrate reduction to ammonia may also occur in chloraminated drinking water systems.

Understanding whether the spatiotemporal dynamics of the drinking water microbiome are reproducible in full-scale drinking water systems is an important step in devising engineering strategies for effectively managing and manipulating it. However, direct comparisons across full-scale drinking water systems are challenging because multiple factors, from source water to treatment process choice and configuration, can be unique to each system. This study compared the spatiotemporal dynamics of the drinking water microbiome in two drinking water treatment plants (DWTPs) with identical sequences of treatment strategies. These DWTPs treat source waters from the same river system and treated drinking water is distributed within the same large-scale distribution system (DWDS) with similar disinfectant residual regiments. Dissimilarities in source water communities were tempered by the predisinfection treatments, resulting in highly similar postfiltration microbial communities between the two systems. However, high community turnover due to disinfection resulted in highly dissimilar microbial communities in the finished water between the two systems. Interestingly, however, the degree of similarity of the microbial communities in the two systems increased during transit through the DWDS despite the presence of a disinfectant residual. Overall, our study finds that the drinking water microbiome demonstrated reproducible spatial and temporal dynamics within both, independent but nearly identical, DWTPs and their corresponding DWDSs.

26In addition to containing higher concentrations of organics and bacterial cells, 27 surface waters are often more vulnerable to pollution and microbial contamination 28 with intensive industrial and agricultural activities frequently occurring in areas 29 surrounding the water source. Therefore, surface waters typically require additional 30 treatment, where the choice of treatment strategy is critical for water quality. Using 31 16S rRNA gene profiling, this study provides a unique opportunity to simultaneously 32 investigate and compare two drinking water treatment plants and their 33 corresponding distribution systems. The two treatment plants treat similar surface 34 waters, from the same river system, with the same sequential treatment strategies. 35 Here, the impact of treatment and distribution on the microbial community within and 36 between each system was compared over an eight-month sampling campaign. 37 Overall, reproducible spatial and temporal dynamics within both DWTPs and their 38 corresponding DWDSs were observed. Although source waters showed some 39 dissimilarity in microbial community structure and composition, pre-disinfection 40 treatments (i.e. coagulation, flocculation, sedimentation and filtration) resulted in 41 highly similar microbial communities between the filter effluent samples. This 42 indicated that the same treatments resulted in the development of similar microbial 43 communities. Conversely, post-disinfection (i.e. chlorination and chloramination) 44 resulted in increased dissimilarity between disinfected samples from the two 45 systems, showing alternative responses of the microbial community to disinfection. 46 Lastly, it was observed that within the distribution system the same dominant taxa 47 were selected where samples increased in similarity with increased residence time. 48 Although, differences were found between the two systems, overall treatment and 49 distribution had a similar impact on the microbial community in each system. This 50 study therefore provides valuable information on the impact of treatment and 51 distribution on the drinking water microbiome. 52 53 Keywords: drinking water treatment; drinking water distribution; disinfection; 54 microbial community dynamics; Illumina MiSeq. 55 56 Highlights 57  Source waters show some dissimilarity in microbial community. 58  Treatment processes increases similarity and selects for the same dominant 59 taxa. 60  Differential response to chlorination causing increased dissimilarity and 61 variation. 62  Stabilisation of DWDS microbial community through selection of same 63 dominant taxa. 64  Microbial community dynamics are reproducible between the two systems. 65 66 Abbreviations 67 DWDS, drinking water distribution system; DWTP, drinking water treatment plant; 68 DADA2, Divisive Amplicon Denoising Algorithm; ASV, amplicon sequence variant; 69 AMOVA, analysis of molecular variance; MRA, mean relative abundance; PCoA, 70 Principal coordinate analysis; ANOVA, One-way analysis of varianc...

AbstractNitrification is a common concern in chloraminated drinking water distribution systems. The addition of ammonia promotes the growth of nitrifying organisms, causing the depletion of chloramine residuals and resulting in operational problems for many drinking water utilities. Therefore, a comprehensive understanding of the microbially mediated processes behind nitrogen metabolism together with chemical water quality data, may allow water utilities to better address the undesirable effects caused by nitrification. In this study, a metagenomic approach was applied to characterise the microbial nitrogen metabolism within chloraminated drinking water reservoirs. Samples from two geographically separated but connected chloraminated reservoirs within the same drinking water distribution system (DWDS) were collected within a 2-year sampling campaign. Spatial changes in the nitrogen compounds (ammonium (NH4+), nitrites (NO2−) and nitrates (NO3−)) across the DWDS were observed, where nitrate concentrations increased as the distance from the site of chloramination increased. The observed dominance of Nitrosomonas and Nitrospira-like bacteria, together with the changes in the concentration of nitrogen species, suggests that these bacteria play a significant role in contributing to varying stages of nitrification in both reservoirs. Functionally annotated protein sequences were mined for the genes associated with nitrogen metabolism and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction and nitric oxide reduction. Furthermore, based on the construction of Metagenome Assembled Genomes (MAGs), a highly diverse assemblage of bacteria (i.e., predominately Alpha- and Betaproteobacteria in this study) was observed among the draft genomes. Specifically, 5 MAGs showed high coverage across all samples including two Nitrosomonas, Nitrospira, Sphingomonas and a Rhizobiales-like MAGs. The role of these MAGs in nitrogen metabolism revealed that the fate nitrate may be linked to changes in ammonia concentrations, that is, when ammonia concentrations are low, nitrate may be assimilated back to ammonia for growth. Alternatively, nitrate may be reduced to nitric oxide and potentially used in the regulation of biofilm formation. Therefore, this study provides insight into the genetic network behind microbially mediated nitrogen metabolism and together with the water chemistry data improves our understanding nitrification in chloraminated DWDSs.