Ultrafiltration impacts many aspects of water quality, including changes in the amount and character of both NOM and bacteria.19 The installation of UF reduced the TCC in the distributed water from 4.8 × 105 ± 1.7 × 105 cells mL−1 to 3.7 × 103 ± 1.2 × 103 cells mL−1, corresponding to a 99% removal of bacteria in the DWDS. This degree of cell removal exposed small relative differences between water sampled at different points within the distribution system, permitting quantification and identification of bacteria from the pipe biofilm that were released into the water as it travelled through the distribution system.

While studies have suggested that the microbiome in distributed drinking water is highly influenced by biofilm on pipe walls,1,20 others have contradicted this hypothesis7,21 and suggested that source water10,22,23 and sand filters24,25 are more influential. In the current study, after day 37, 58% of the bacteria in the distributed water originated from pipe biofilm. While one explanation for this addition of cells to the water could be regrowth, the DWDS sampling points in this study had short residence times (>25 h), and with a growth rate approximated as 0.30 day−1 (or a doubling time of 2.31 days) for distributed water,26 this is an unlikely explanation for the increases in TCC. Nutrient concentrations (DOC, biopolymers, and humic substances, Supplementary Table 3) were reduced by UF; water temperatures ranged from 5.7 to 9 °C (Supplementary Table 2); and 7-day incubation were required to detect heterotrophs (Supplementary Table 2). Taken together, this evidence strongly suggests that the increase in TCC with distance from the treatment plant was due to release of cells from the pipe biofilm into the water.

The short time frame in this study allowed the contribution of cells from the biofilm to be estimated as 0.5% of the total cells present in the water before the change. Applying this estimate for cells released from the biofilm to other systems where the bacterial concentration in the distributed water is high can explain why the contribution from the pipe biofilm to the water microbiome has been difficult to observe. In a year-long sampling campaign by Pinto and colleagues (2014), only water sampled at great distance from the DWTP showed small changes in the water microbiome.27 Henne and colleagues (2012) compared communities from distributed water and biofilm, and the water had a highly homogeneous bacterial community despite observed diversity in the biofilm communities.8 We suggest that the community composition in the distributed water will be clearly associated with processes in the treatment plant, such as the use of sand filters24,25,28 and use of disinfectants29,30,31 unless that treatment (i.e. UF) removes a large percentage of cells. In this case, the bacterial community in the distributed water will contain a majority of cells originating from the pipe biofilm. Given the great diversity in the microbial communities of source water, distributed water and biofilm and other variables governing water quality such as local climate, treatment processes and pipe materials, it is not known if the bacterial community in this study, and the extent to which it was released into the flowing water, reflects what would happen in every DWDS, and additional studies are needed to determine the impact of UF in other systems.

After installation of UF, the percentage of HNA bacteria in the distributed water increased compared to finished water (Supplementary Figure 1). Proctor and colleagues (2018) proposed that HNA bacteria, in contrast to low nucleic acid bacteria, are not as dependent on other bacteria for survival32 and HNA bacteria may survive in distributed water without the biofilm community. The percentage of intact cells also increased in the water as it travelled through the DWDS, and may be a signature for bacterial release from pipe biofilm. Shifts in HNA33 and ICC34 were observed in tap water after overnight stagnation and distributed water, respectively, and may indicate release of biofilm in these contexts.

DNA sequencing studies of bacterial communities in pipe biofilms have shown higher diversity compared to that in the distributed water.8,35 Henne and colleagues (2012) showed higher diversity with lower richness in the biofilm compared to the water phase and suggested that the biofilm community contains evenly distributed members adapted for this specific environment.8 This implies that if only some members of the evenly distributed biofilm community are released into the distributed water there will be a shift in the population towards lower evenness. In the current study, lower diversity (due to both decreased richness and lower evenness) was observed for the community in distributed water after UF installation, compared to those in finished water and before UF installation. This altered community structure in the distributed water can be attributed to interaction with the biofilm, with the similarity between the communities in finished water and before UF installation attributed to the use of diluting feed water for pH adjustment in the first 37 days after UF installation. In this period, lower evenness was observed as increasing dominance in the distributed water of a few specific OTUs, such as genera Nitrospira, and Sphingomonas. Lower diversity in the water microbiome has been observed after flushing, with this uneven detachment of biofilm resulting in a more uneven water community.12,36

Installation of UF decreased the richness (lower numbers of OTUs) in the distributed water. A rich bacterial community in the water, with many bacteria at low abundance, can be a seed bank for the biofilm community.8 Altered environmental conditions initiated by the UF treatment could trigger cells to enter the biofilm, resulting in the observed decrease in richness.37 This would not appear in the DESeq2 analysis, as this only included OTUs with total read abundance across all the samples >0.1%, and it has been suggested that the rare biosphere represented by OTUs with abundance <0.1% of the community is the dormant microbial seedbank.37

Specific OTUs at class level accounted for much of the observed changes in the water microbiome, including Alphaproteobacteria and Nitrospira, which showed a higher relative abundance in the distributed water after the installation of UF. Higher relative abundance of Alphaproteobacteria has been observed in biofilm compared to the distributed water,8 in water containing biofilm detached by flushing12 and dominating biofilm communities in DWDS7 and water meters.38,39 Observations similar to these seen for Alphaproteobacteria have also been observed for Nitrospira.21,35

Bacteria released from the biofilm were described by 29 OTUs where the absolute read abundance increased in the distributed water compared to the finished water. Two OTUs classified as genus Sphingomonas predominated at DP1 and DP3 relative to DP2 and compared to the rest of the OTUs describing the released biofilm community. Sphingomonas are often detected in bacterial communities from drinking water, with high abundance in biofilms10,38 and a relative abundance in DWDS estimated at up to 85%.7 Sphingomonas possess flagella,40 with this motility perhaps contributing to their release from the biofilm and their proposed role as early colonizers DWDS biofilms.41 Sphingomonadaceae are HNA bacteria (as large bacteria >0.4 µm),32 which supports observed increase of HNA bacteria in distributed water in the current study.

Six of the 29 OTUs released from the biofilm were classified as genus Nitrospira, a group of bacteria that has been found in bacterial communities in drinking water, loose deposits and drinking water biofilms.7,8,21,35 The dominance of this taxa at DP2 might be due to loose deposits containing high amount of biofilm with Nitrospira abundance, which can vary between locations in the distribution, although this was not examined in the current study. Members of this genus can use nitrite as an electron donor instead of organic molecules21,42: nitrite concentrations at DP2 were lower compared to DP1 and DP3. DP2 was consistently warmer, with higher copper concentrations and low-carbon, chloramine-treated water, which may also favour growth of Nitrospira.43

While numerous studies have associated Alphaproteobacteria, Sphingomonas, Nitrospira and Mycobacterium spp. with drinking water and its biofilms, this study showed that members of these classes and genera also move from the pipe biofilm into the drinking water. It does not appear to be a single mode of motility that is used to escape the biofilm: Sphingomonas are almost universally motile via flagella; Nitrospira are generally thought to be nonmotile,44 Mycobacterium spp. use sliding motility45 and Hyphomicrobium are motile as swarmer cells with flagella.46 All modes may be sufficient and, together with random attachment and detachment, account for cell release.9 This could occur for both live and dead cells leaving the biofilm, and it would be interesting to describe this community in the context of cell viability.

In conclusion, although the UF installation modified the type of organic matter and greatly reduced the number of bacterial cells in the distributed water, destabilization of the biofilm, observed as detachment, sloughing or a sudden increases in the number of total cells in distributed water, was not observed during the 114 days of the study. It can take years for changes to be observed in a microbial community in response to an alteration in the environment,47 so the observation of consistently low cell counts over the 0.3 year of the current study does not confirm that this will always be the case and it is not known how this biofilm will adapt over the coming years and seasons to the UF installation. Regions in the DWDS with longer retention times may gradually show increasing cell counts in distributed water from prolonged contact with the biofilm or the dynamics of bacterial release may change. Changes in nutrients, such as those described in this study (both NOM and cells), may, over months and years, change the water and biofilm community composition as they adapt to these new conditions.18 Since this study was conducted during winter, it is also not known to what extent the release of bacterial cells could change with increases in temperature or seasonal changes in water use, which have both been shown to alter the overall numbers of cells in distributed water.34 The impact of having a higher percentage of bacteria in the water that originates from biofilm is also not known. Given that cells originating from biofilms are more likely to form biofilms themselves,48 it would be interesting to see whether shifts in the origin of the bacteria in the distributed water can impact formation of biofilms on new DWDS pipes, water meters or household drinking water plumbing systems.